r848  (InvivoGen)

 
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    Name:
    R848
    Description:
    Imidazoquinoline compound
    Catalog Number:
    tlrl-r848-5
    Price:
    None
    Size:
    5 mg
    Category:
    R848 Resiquimod Base Analogs TLR7 8 Ligands TLR Ligands PRR Ligands
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    Structured Review

    InvivoGen r848
    TLR7 signaling and responsiveness is enhanced in DN2. A. CD25 expression is increased over baseline (filled) by trimerized CD40L stimulation (blue line) in rNAV but not DN2 cells. In contrast TLR7 stimulation by <t>R848</t> (red line) increased CD25 expression in both rNAV and DN2 cells. One example out of two experiments is shown. B. The percentage of dual positive phospho-ERK and phospho-MAPKp38 positive CD11c + DN cells is increased after R848 stimulated (bottom) relative to unstimulated (top) and to SWM, rNAV, and DN1 B cells. C. Fluorescence intensity of phospho-ERK and phospho-MAPKp38 is higher in DN2 cells and aNAV cells after R848 stimulation. Histogram coloring is based on median fluorescence intensity as shown by the scale bars. D. Relative induction of pERK (n=5) and pMAPKp38 (n=10), expressed as the fold increase in MFI of R848 stimulated samples, is higher in DN2 cells and aNAV cells. Although induced pY-ERK phosphorylation is lower in HCD B cells (n=7), DN2 and aNAV cells still have significantly higher phosphorylation (repeated measure 1-way ANOVA). E. In SLE patients HLA-DR (n=6) and CD86 (n=5) expression is higher at baseline in DN2 cells (green) than NAV (blue) or SWM (red) but this difference is further enhanced after TLR7 stimulation. Expression of inhibitory receptors CD32b and CD72 is reduced by R848 stimulation in DN2 cells (blue) but not NAV B cells (green) (n=5). HCD B cells demonstrated similar changes except for CD72, which did not decrease in either rNAV or DN2 cells. Red asterisks indicate significant differences from SWM; blue asterisks indicate significant differences from NAV (repeated measure 1-way ANOVA).
    Imidazoquinoline compound
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    r848 - by Bioz Stars, 2020-07
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    Images

    1) Product Images from "Distinct effector B-cells induced by unregulated Toll-like receptor 7 contribute to pathogenic responses in Systemic Lupus Erythematosus"

    Article Title: Distinct effector B-cells induced by unregulated Toll-like receptor 7 contribute to pathogenic responses in Systemic Lupus Erythematosus

    Journal: Immunity

    doi: 10.1016/j.immuni.2018.08.015

    TLR7 signaling and responsiveness is enhanced in DN2. A. CD25 expression is increased over baseline (filled) by trimerized CD40L stimulation (blue line) in rNAV but not DN2 cells. In contrast TLR7 stimulation by R848 (red line) increased CD25 expression in both rNAV and DN2 cells. One example out of two experiments is shown. B. The percentage of dual positive phospho-ERK and phospho-MAPKp38 positive CD11c + DN cells is increased after R848 stimulated (bottom) relative to unstimulated (top) and to SWM, rNAV, and DN1 B cells. C. Fluorescence intensity of phospho-ERK and phospho-MAPKp38 is higher in DN2 cells and aNAV cells after R848 stimulation. Histogram coloring is based on median fluorescence intensity as shown by the scale bars. D. Relative induction of pERK (n=5) and pMAPKp38 (n=10), expressed as the fold increase in MFI of R848 stimulated samples, is higher in DN2 cells and aNAV cells. Although induced pY-ERK phosphorylation is lower in HCD B cells (n=7), DN2 and aNAV cells still have significantly higher phosphorylation (repeated measure 1-way ANOVA). E. In SLE patients HLA-DR (n=6) and CD86 (n=5) expression is higher at baseline in DN2 cells (green) than NAV (blue) or SWM (red) but this difference is further enhanced after TLR7 stimulation. Expression of inhibitory receptors CD32b and CD72 is reduced by R848 stimulation in DN2 cells (blue) but not NAV B cells (green) (n=5). HCD B cells demonstrated similar changes except for CD72, which did not decrease in either rNAV or DN2 cells. Red asterisks indicate significant differences from SWM; blue asterisks indicate significant differences from NAV (repeated measure 1-way ANOVA).
    Figure Legend Snippet: TLR7 signaling and responsiveness is enhanced in DN2. A. CD25 expression is increased over baseline (filled) by trimerized CD40L stimulation (blue line) in rNAV but not DN2 cells. In contrast TLR7 stimulation by R848 (red line) increased CD25 expression in both rNAV and DN2 cells. One example out of two experiments is shown. B. The percentage of dual positive phospho-ERK and phospho-MAPKp38 positive CD11c + DN cells is increased after R848 stimulated (bottom) relative to unstimulated (top) and to SWM, rNAV, and DN1 B cells. C. Fluorescence intensity of phospho-ERK and phospho-MAPKp38 is higher in DN2 cells and aNAV cells after R848 stimulation. Histogram coloring is based on median fluorescence intensity as shown by the scale bars. D. Relative induction of pERK (n=5) and pMAPKp38 (n=10), expressed as the fold increase in MFI of R848 stimulated samples, is higher in DN2 cells and aNAV cells. Although induced pY-ERK phosphorylation is lower in HCD B cells (n=7), DN2 and aNAV cells still have significantly higher phosphorylation (repeated measure 1-way ANOVA). E. In SLE patients HLA-DR (n=6) and CD86 (n=5) expression is higher at baseline in DN2 cells (green) than NAV (blue) or SWM (red) but this difference is further enhanced after TLR7 stimulation. Expression of inhibitory receptors CD32b and CD72 is reduced by R848 stimulation in DN2 cells (blue) but not NAV B cells (green) (n=5). HCD B cells demonstrated similar changes except for CD72, which did not decrease in either rNAV or DN2 cells. Red asterisks indicate significant differences from SWM; blue asterisks indicate significant differences from NAV (repeated measure 1-way ANOVA).

    Techniques Used: Expressing, Fluorescence

    2) Product Images from "Chronic TLR7 and TLR9 signaling drives anemia via differentiation of specialized hemophagocytes"

    Article Title: Chronic TLR7 and TLR9 signaling drives anemia via differentiation of specialized hemophagocytes

    Journal: Science (New York, N.Y.)

    doi: 10.1126/science.aao5213

    TLR7 promotes RPM-like hemophagocyte development in vitro. (A) Representative flow cytometric staining of CMPs cultured with SCF or SCF+R848. (B-D) RNA-Seq analysis of CD11b + F4/80 + ) that were significantly increased in R848-differentiated compared to MCSF-differentiated macrophages. Black line indicates –log p-value calculated using exact hypergeometric probability with a normal approximation. (D) Heat map of increased genes (≥2-fold, FDR≥0.05) in R848-differentiated compared to MCSF-differentiated macrophages in RPM core signature. (E) R848-differentiated macrophages were treated with or without cytochalasin D (CytoD) then allowed to phagocytose CFSE-labeled RBC for 15 min at indicated ratios. Percent of CD11b + F4/80 + macrophages that had phagocytosed RBC is shown. Data are representative of four experiments, n=3 technical replicates per condition/experiment. Mean values+SD (E) are shown.
    Figure Legend Snippet: TLR7 promotes RPM-like hemophagocyte development in vitro. (A) Representative flow cytometric staining of CMPs cultured with SCF or SCF+R848. (B-D) RNA-Seq analysis of CD11b + F4/80 + ) that were significantly increased in R848-differentiated compared to MCSF-differentiated macrophages. Black line indicates –log p-value calculated using exact hypergeometric probability with a normal approximation. (D) Heat map of increased genes (≥2-fold, FDR≥0.05) in R848-differentiated compared to MCSF-differentiated macrophages in RPM core signature. (E) R848-differentiated macrophages were treated with or without cytochalasin D (CytoD) then allowed to phagocytose CFSE-labeled RBC for 15 min at indicated ratios. Percent of CD11b + F4/80 + macrophages that had phagocytosed RBC is shown. Data are representative of four experiments, n=3 technical replicates per condition/experiment. Mean values+SD (E) are shown.

    Techniques Used: In Vitro, Flow Cytometry, Staining, Cell Culture, RNA Sequencing Assay, Labeling

    iHPCs are derived from Ly6C hi monocytes. (A) Splenic monocytes (live singlets, CD11b + F4/80 − Ly6G − Ly6C hi or CCR2 + cells) (black) and iHPCs (live singlets, F4/80 lo Ly6G − Ter-119 + VCAM1 lo or CD31 hi cells) (blue) were assessed for the expression of the cell surface proteins indicated (solid lines) compared to fluorescence minus one (FMO) control stains (dashed lines). Data are representative of three experiments. (B) Bone marrow Ly6C hi monocytes were sorted from WT B6 mice and cultured for 21 hours with media alone (--) or with R848. Spic , Pecam1 , Ccr2, and Ly6c1 transcripts were quantified by qPCR. Data are representative from five experiments with n=3 per experiment. (C, D) Ccr2-DTR − , WT/Ccr2-DTR + , TLR7.1/Ccr2-DTR − , and TLR7.1/Ccr2-DTR + mice (n=5–7 mice per group) were injected with DT every other day for 17 days. (C) Representative flow cytometry of Ter-119 + hemophagocytes pre-gated on live singlets, CD45.2 + Ly6G − Siglec-F − cells from the spleens of TLR7.1/Ccr2-DTR − and TLR7.1/Ccr2-DTR + mice determined by flow cytometry. (D) Frequency and number of the indicated cell populations were quantitated from the spleens of Ccr2-DTR − , Ccr2-DTR + , TLR7.1/Ccr2-DTR − , and TLR7.1/Ccr2-DTR + mice by flow cytometry. Data are combined from three experiments. (E) Bone marrow Ly6C hi monocytes were sorted from WT B6 mice and cultured for 21 hours with media alone (--), R848, LPS, or CpG. Spic , Pecam1 , Ccr2, and Ly6c1 transcripts were quantified by qPCR. Data are representative from two experiments. (F and G) RNA-Seq analysis of RPMs and Ly6C hi monocytes sorted from spleens of WT B6 mice and TLR7.1 mice and iHPCs from spleens of TLR7.1 mice. n=5 (TLR7.1 iHPC), n=4 (WT and TLR7.1 RPM), n=6 (WT and TLR7.1 Mono). (F) PCA of indicated populations. (G) Heat map of DEG between the three populations (RPMs, Ly6C hi monocytes, and iHPCs) sorted from TLR7.1 mice. Mean values+SEM (D), ± SEM (B, E). *p
    Figure Legend Snippet: iHPCs are derived from Ly6C hi monocytes. (A) Splenic monocytes (live singlets, CD11b + F4/80 − Ly6G − Ly6C hi or CCR2 + cells) (black) and iHPCs (live singlets, F4/80 lo Ly6G − Ter-119 + VCAM1 lo or CD31 hi cells) (blue) were assessed for the expression of the cell surface proteins indicated (solid lines) compared to fluorescence minus one (FMO) control stains (dashed lines). Data are representative of three experiments. (B) Bone marrow Ly6C hi monocytes were sorted from WT B6 mice and cultured for 21 hours with media alone (--) or with R848. Spic , Pecam1 , Ccr2, and Ly6c1 transcripts were quantified by qPCR. Data are representative from five experiments with n=3 per experiment. (C, D) Ccr2-DTR − , WT/Ccr2-DTR + , TLR7.1/Ccr2-DTR − , and TLR7.1/Ccr2-DTR + mice (n=5–7 mice per group) were injected with DT every other day for 17 days. (C) Representative flow cytometry of Ter-119 + hemophagocytes pre-gated on live singlets, CD45.2 + Ly6G − Siglec-F − cells from the spleens of TLR7.1/Ccr2-DTR − and TLR7.1/Ccr2-DTR + mice determined by flow cytometry. (D) Frequency and number of the indicated cell populations were quantitated from the spleens of Ccr2-DTR − , Ccr2-DTR + , TLR7.1/Ccr2-DTR − , and TLR7.1/Ccr2-DTR + mice by flow cytometry. Data are combined from three experiments. (E) Bone marrow Ly6C hi monocytes were sorted from WT B6 mice and cultured for 21 hours with media alone (--), R848, LPS, or CpG. Spic , Pecam1 , Ccr2, and Ly6c1 transcripts were quantified by qPCR. Data are representative from two experiments. (F and G) RNA-Seq analysis of RPMs and Ly6C hi monocytes sorted from spleens of WT B6 mice and TLR7.1 mice and iHPCs from spleens of TLR7.1 mice. n=5 (TLR7.1 iHPC), n=4 (WT and TLR7.1 RPM), n=6 (WT and TLR7.1 Mono). (F) PCA of indicated populations. (G) Heat map of DEG between the three populations (RPMs, Ly6C hi monocytes, and iHPCs) sorted from TLR7.1 mice. Mean values+SEM (D), ± SEM (B, E). *p

    Techniques Used: Derivative Assay, Expressing, Fluorescence, Mouse Assay, Cell Culture, Real-time Polymerase Chain Reaction, Injection, Flow Cytometry, Cytometry, RNA Sequencing Assay

    iHPCs differentiate in response to cell-intrinsic TLR7 signals. A) The ratio of TLR7.1 to WT BM-derived cells in mixed bone marrow chimeras of indicated populations after reconstitution. Data are representative of two experiments with n=8–10 per experiment. B) The ratio of WT to Tlr7 −/− BM-derived cells in mixed bone marrow chimeras injected with the TLR7 agonist R848 (right) or PBS (left) for 13 days. Data are representative of two experiments with n=3–5 per group per experiment. (A, B) mean±SEM, (A, B) each symbol represents an individual mouse. *p
    Figure Legend Snippet: iHPCs differentiate in response to cell-intrinsic TLR7 signals. A) The ratio of TLR7.1 to WT BM-derived cells in mixed bone marrow chimeras of indicated populations after reconstitution. Data are representative of two experiments with n=8–10 per experiment. B) The ratio of WT to Tlr7 −/− BM-derived cells in mixed bone marrow chimeras injected with the TLR7 agonist R848 (right) or PBS (left) for 13 days. Data are representative of two experiments with n=3–5 per group per experiment. (A, B) mean±SEM, (A, B) each symbol represents an individual mouse. *p

    Techniques Used: Derivative Assay, Injection

    iHPC differentiation depends on IRF5. (A) Bone marrow Ly6C hi monocytes were sorted from WT and Irf5 −/− mice and cultured for 21 hours with media alone (--) or with R848. Spic , Pecam1 , Ccr2, and Ly6c1 transcripts were quantified by qPCR. Data are representative from two experiments with n=2 to 4 per group per experiment. (B and C) WT and Irf5 −/− mice were injected with R848 i.p. daily for 2 days. Splenocytes were analyzed by flow cytometry. (B) Representative flow plots of WT and Irf5 −/− CD11b + CD31 + iHPCs (gated on live singlets, CD45.2 + F4/80 − Ly6G − Siglec-F − cells). (C) Frequency (left) and number (right) of iHPCs in WT and Irf5 −/− spleens. Data are representative from two experiments with n=4 per group per experiment. Mean values±SEM (A and C) are shown. (A and C) each symbol represents an individual mouse. *p
    Figure Legend Snippet: iHPC differentiation depends on IRF5. (A) Bone marrow Ly6C hi monocytes were sorted from WT and Irf5 −/− mice and cultured for 21 hours with media alone (--) or with R848. Spic , Pecam1 , Ccr2, and Ly6c1 transcripts were quantified by qPCR. Data are representative from two experiments with n=2 to 4 per group per experiment. (B and C) WT and Irf5 −/− mice were injected with R848 i.p. daily for 2 days. Splenocytes were analyzed by flow cytometry. (B) Representative flow plots of WT and Irf5 −/− CD11b + CD31 + iHPCs (gated on live singlets, CD45.2 + F4/80 − Ly6G − Siglec-F − cells). (C) Frequency (left) and number (right) of iHPCs in WT and Irf5 −/− spleens. Data are representative from two experiments with n=4 per group per experiment. Mean values±SEM (A and C) are shown. (A and C) each symbol represents an individual mouse. *p

    Techniques Used: Mouse Assay, Cell Culture, Real-time Polymerase Chain Reaction, Injection, Flow Cytometry, Cytometry

    3) Product Images from "Allogeneic lymphocyte-licensed DCs expand T cells with improved antitumor activity and resistance to oxidative stress and immunosuppressive factors"

    Article Title: Allogeneic lymphocyte-licensed DCs expand T cells with improved antitumor activity and resistance to oxidative stress and immunosuppressive factors

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1038/mtm.2014.1

    Schematic illustration of the rapid expansion protocol (REP) and allosensitized allogeneic lymphocytes (ASAL) expansion protocol (AEP) T-cell expansion protocols and proposed mechanisms of action for the AEP protocol. ( a ) REP: T cells were expanded for 12 days by stimulation with irradiated feeder cells (peripheral blood mononuclear cells (PBMCs) from three donors), OKT-3 and IL-2. ASAL expansion protocol (AEP): Prior to expansion: Monocytes from one donor were differentiated into immature dendritic cells (DCs) with IL-4 and granulocyte-macrophage colony-stimulating factor for 6 days and then matured for 24 hours with IFN-γ, poly(I:C) and R848. ASALs were generated over 7 days by coculturing PBMCs from one donor with irradiated PBMCs from a second donor (autologous to the DCs) at a ratio of 1:1. The ASALs and mDCs can be frozen and stored or used immediately. Expansion phase: T cells, allogeneic to both the ASAL and mDCs, were expanded for 12 days by coculture with irradiated ASAL, mDCs, OKT-3, and IL-2. When comparing the two protocols, the same amount of T cells from the same donors were expanded and analyzed in parallel. ( b ) A proposed illustration why the AEP protocol leads to activation of responder T cells, which are insensitive to immunosuppressive and oxidative stress. ASALs provide “help signals”, which lead to upregulation of CD70, CD80, CD64, and CD40 on allogeneic mDCs. The interaction between ASALSs and mDC also leads to secretion of IL-2 and IFN-γ. Furthermore, CD40L expression on helper cells in the ASAL population can interact with CD40 on mDCs and thereby induce IL-12 secretion and Th1 polarization. CD64 expression on the mDCS ensures proper Fc binding of the OKT-3 antibody. The combination of allogeneic anti-CD3-armed Th1-polarizing mDCs and ASALs leads to T cells, which are insensitive to immunosuppressive and oxidative stress.
    Figure Legend Snippet: Schematic illustration of the rapid expansion protocol (REP) and allosensitized allogeneic lymphocytes (ASAL) expansion protocol (AEP) T-cell expansion protocols and proposed mechanisms of action for the AEP protocol. ( a ) REP: T cells were expanded for 12 days by stimulation with irradiated feeder cells (peripheral blood mononuclear cells (PBMCs) from three donors), OKT-3 and IL-2. ASAL expansion protocol (AEP): Prior to expansion: Monocytes from one donor were differentiated into immature dendritic cells (DCs) with IL-4 and granulocyte-macrophage colony-stimulating factor for 6 days and then matured for 24 hours with IFN-γ, poly(I:C) and R848. ASALs were generated over 7 days by coculturing PBMCs from one donor with irradiated PBMCs from a second donor (autologous to the DCs) at a ratio of 1:1. The ASALs and mDCs can be frozen and stored or used immediately. Expansion phase: T cells, allogeneic to both the ASAL and mDCs, were expanded for 12 days by coculture with irradiated ASAL, mDCs, OKT-3, and IL-2. When comparing the two protocols, the same amount of T cells from the same donors were expanded and analyzed in parallel. ( b ) A proposed illustration why the AEP protocol leads to activation of responder T cells, which are insensitive to immunosuppressive and oxidative stress. ASALs provide “help signals”, which lead to upregulation of CD70, CD80, CD64, and CD40 on allogeneic mDCs. The interaction between ASALSs and mDC also leads to secretion of IL-2 and IFN-γ. Furthermore, CD40L expression on helper cells in the ASAL population can interact with CD40 on mDCs and thereby induce IL-12 secretion and Th1 polarization. CD64 expression on the mDCS ensures proper Fc binding of the OKT-3 antibody. The combination of allogeneic anti-CD3-armed Th1-polarizing mDCs and ASALs leads to T cells, which are insensitive to immunosuppressive and oxidative stress.

    Techniques Used: Irradiation, Generated, Activation Assay, Expressing, Binding Assay

    4) Product Images from "IL-4 Suppresses the Responses to TLR7 and TLR9 Stimulation and Increases the Permissiveness to Retroviral Infection of Murine Conventional Dendritic Cells"

    Article Title: IL-4 Suppresses the Responses to TLR7 and TLR9 Stimulation and Increases the Permissiveness to Retroviral Infection of Murine Conventional Dendritic Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0087668

    IL-4 induces SOCS1 and SOCS2 gene and protein expression in cDCs. A. We analyzed the gene expression of SOCS1, -2 and -3 in cDCs by qPCR after 24 h treatment with IL-4. Results were normalized against untreated cells. Graphs show averages and SE of 6 independent cultures. B. We analyzed the protein expression of SOCS2 in the total cDC lysates after 24 h treatment with IL-4. Results were normalized against GAPDH. C . We analyzed the protein expression of SOCS2 in wild-type (WT) and STAT6-KO cDCs that had been treated or not with IL-4 for 24 h and then stimulated with CpG or R848 for 8 h. Results were normalized against GAPDH. Blot representative of three sets of experiments.
    Figure Legend Snippet: IL-4 induces SOCS1 and SOCS2 gene and protein expression in cDCs. A. We analyzed the gene expression of SOCS1, -2 and -3 in cDCs by qPCR after 24 h treatment with IL-4. Results were normalized against untreated cells. Graphs show averages and SE of 6 independent cultures. B. We analyzed the protein expression of SOCS2 in the total cDC lysates after 24 h treatment with IL-4. Results were normalized against GAPDH. C . We analyzed the protein expression of SOCS2 in wild-type (WT) and STAT6-KO cDCs that had been treated or not with IL-4 for 24 h and then stimulated with CpG or R848 for 8 h. Results were normalized against GAPDH. Blot representative of three sets of experiments.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    IL-4 suppresses TLR7- and TLR9-induced response in splenic DCs ex-vivo . We analyzed the expression of IFN-responsive genes induced by CpG or R848 stimulation in splenic DCs. We cultured the total splenocytes from C57BL/6 mice as 30–50 million cells in 3 ml each well in GM-CSF-complete medium and 2.5 ng/ml of IL-4 overnight and then stimulated with 50 ug CpG or 5 ug R848 for 4 h. Subsequently we sorted the CD11c+ DCs using Miltenyi magnetic bead separation. All the conditions were normalized against the control (GM-CSF medium only) in each experiment. Line graphs show the response calculated as fold change from no IL-4 control of three independent experiments (* p
    Figure Legend Snippet: IL-4 suppresses TLR7- and TLR9-induced response in splenic DCs ex-vivo . We analyzed the expression of IFN-responsive genes induced by CpG or R848 stimulation in splenic DCs. We cultured the total splenocytes from C57BL/6 mice as 30–50 million cells in 3 ml each well in GM-CSF-complete medium and 2.5 ng/ml of IL-4 overnight and then stimulated with 50 ug CpG or 5 ug R848 for 4 h. Subsequently we sorted the CD11c+ DCs using Miltenyi magnetic bead separation. All the conditions were normalized against the control (GM-CSF medium only) in each experiment. Line graphs show the response calculated as fold change from no IL-4 control of three independent experiments (* p

    Techniques Used: Ex Vivo, Expressing, Cell Culture, Mouse Assay

    IL-4 suppression of pro-inflammatory cytokines is STAT6-dependent. We measured by ELISA the levels of IL-6 and IL-12p70 in the supernatants of cDC cultures from wild type and STAT6-KO mice treated or not for 24 h with IL-4 and then stimulated with CpG or R848 for 6 h (IL-6) or 48 h (IL-12p70). Averages and SE of three independent cultures are shown (* p
    Figure Legend Snippet: IL-4 suppression of pro-inflammatory cytokines is STAT6-dependent. We measured by ELISA the levels of IL-6 and IL-12p70 in the supernatants of cDC cultures from wild type and STAT6-KO mice treated or not for 24 h with IL-4 and then stimulated with CpG or R848 for 6 h (IL-6) or 48 h (IL-12p70). Averages and SE of three independent cultures are shown (* p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay

    IL-4 suppresses expression of TLR7- and 9-induced MHC Class I, STAT1 and STAT2 protein expression. A . We treated cDCs with IL-4 or left them untreated for 24 h. We then stimulated the cDCs with CpG or R848 for 24 h and then analyzed MHC class I expression. Histogram bars represent averages and standard errors (SE) of the median fluorescence intensity (MdFI) of seven experiments conducted with seven independent cDC cultures; *p
    Figure Legend Snippet: IL-4 suppresses expression of TLR7- and 9-induced MHC Class I, STAT1 and STAT2 protein expression. A . We treated cDCs with IL-4 or left them untreated for 24 h. We then stimulated the cDCs with CpG or R848 for 24 h and then analyzed MHC class I expression. Histogram bars represent averages and standard errors (SE) of the median fluorescence intensity (MdFI) of seven experiments conducted with seven independent cDC cultures; *p

    Techniques Used: Expressing, Fluorescence

    IL-4 suppresses the expression of TLR7- and TLR9-induced IRF7 expression. A. We analyzed the gene expression of IRF7 by qPCR after stimulation with CpG 1826 (10 ug/ml) or R848 (1 ug/ml) for 6 h in cDCs treated with IL-4 or left untreated for 24 h. All of the conditions were normalized against the control (untreated DCs in medium only) in each experiment. Results are the average of three independent experiments; * p
    Figure Legend Snippet: IL-4 suppresses the expression of TLR7- and TLR9-induced IRF7 expression. A. We analyzed the gene expression of IRF7 by qPCR after stimulation with CpG 1826 (10 ug/ml) or R848 (1 ug/ml) for 6 h in cDCs treated with IL-4 or left untreated for 24 h. All of the conditions were normalized against the control (untreated DCs in medium only) in each experiment. Results are the average of three independent experiments; * p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    IL-4 suppression of TLR7- and TLR9-induced IFN-dependent response in cDCs is STAT6 dependent. A. We analyzed by qPCR the gene expression of the IFN-responsive genes IRF7, ISG15 and Mx-1 induced by 6 h stimulation with CpG or R848 in wild type (WT) (C57BL/6) and STAT6-KO cDCs treated or not for 24 h with IL-4. Results were normalized to WT control cells not treated with IL-4. B. We analyzed the MHC Class I expression on cDCs by flow cytometry. Results are shown as median fluorescence intensity (MdFI). Averages and SE of three independent BMDC cultures are shown. (* p
    Figure Legend Snippet: IL-4 suppression of TLR7- and TLR9-induced IFN-dependent response in cDCs is STAT6 dependent. A. We analyzed by qPCR the gene expression of the IFN-responsive genes IRF7, ISG15 and Mx-1 induced by 6 h stimulation with CpG or R848 in wild type (WT) (C57BL/6) and STAT6-KO cDCs treated or not for 24 h with IL-4. Results were normalized to WT control cells not treated with IL-4. B. We analyzed the MHC Class I expression on cDCs by flow cytometry. Results are shown as median fluorescence intensity (MdFI). Averages and SE of three independent BMDC cultures are shown. (* p

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, Cytometry, Fluorescence

    IL-4 suppresses TLR7- and TLR9-induced production of pro-inflammatory cytokines. A. We analyzed the gene expression of IL-6, TNFα, IL-12p35 and IL-12p40 by qPCR after stimulation with CpG or R848 for 6 h in cDCs treated or not with IL-4 for 24 h. Averages and SE of four independent cDC cultures are shown (* p
    Figure Legend Snippet: IL-4 suppresses TLR7- and TLR9-induced production of pro-inflammatory cytokines. A. We analyzed the gene expression of IL-6, TNFα, IL-12p35 and IL-12p40 by qPCR after stimulation with CpG or R848 for 6 h in cDCs treated or not with IL-4 for 24 h. Averages and SE of four independent cDC cultures are shown (* p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    IL-4 suppresses the expression of TLR7- and 9-induced IFN-responsive gene expression. We treated cDCs at day 6-7 of culture with IL-4 or left them untreated for 24 h. We then stimulated the cDCs with CpG 1826 (10 ug/ml) or R848 (1 ug/ml) for 6 h and then analyzed by qPCR the expression of the IFN-responsive genes ISG15 and Mx-1. All of the conditions were normalized against the control (untreated DCs in medium only) in each experiment. Results are average of three independent experiments, performed with three independent bone marrow-derived cultures from 3 different mice; * p
    Figure Legend Snippet: IL-4 suppresses the expression of TLR7- and 9-induced IFN-responsive gene expression. We treated cDCs at day 6-7 of culture with IL-4 or left them untreated for 24 h. We then stimulated the cDCs with CpG 1826 (10 ug/ml) or R848 (1 ug/ml) for 6 h and then analyzed by qPCR the expression of the IFN-responsive genes ISG15 and Mx-1. All of the conditions were normalized against the control (untreated DCs in medium only) in each experiment. Results are average of three independent experiments, performed with three independent bone marrow-derived cultures from 3 different mice; * p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Derivative Assay, Mouse Assay

    5) Product Images from "Nr4a1-Dependent Ly6Clow Monocytes Monitor Endothelial Cells and Orchestrate Their Disposal"

    Article Title: Nr4a1-Dependent Ly6Clow Monocytes Monitor Endothelial Cells and Orchestrate Their Disposal

    Journal: Cell

    doi: 10.1016/j.cell.2013.03.010

    Retention of Crawling Monocytes in the Kidney Vasculature in Response to TLR7 Agonist Requires Chemokine Receptor Signaling (A) Experiments performed idem as in Figure 3 for Cx3cr1 +/− Rag2 −/− Il2rg −/− or Cx3cr1 −/− Rag2 −/− Il2rg −/− mice. The scale bar represents 40 μm. n = 3 mice per condition. (B) Data idem as in Figures 3 B and 3C. ∗ , p ≤ 0.05; mean ± SEM; n = 3 mice. (C) qPCR for fractalkine ( Cx3cl1 ) messenger RNA in kidney cortex tissue from mice with the indicated genotypes 5hr after painting with R848 or PBS (n = 3–5 mice). ∗ ,p ≤ 0.05; mean ± SEM. (D) Data idem as in Figure 3 D for the stated genotypes and conditions. Right, mice received control or blocking anti-CD11b Ab i.v. injection immediately prior to the experiment. ∗ , p ≤ 0.05; mean ± SEM; n = 3–5 mice per condition. (E) Intravital imaging of peritubular capillaries in Cx3cr1 +/gfp mice 5 hr after R848 painting and immediately after i.v. injection of 10 μg Gr1-APC or Ly6G-PE antibodies. Mean ± SEM, n = 4 mice per condition. (F) GFP − Gr1 + and Ly6G + cells forming clusters in capillaries before and after R848 painting and proportion of clusters in contact with a GFP + cells. n = 5 mice per condition, mean ± SEM. (G) TEM of the superficial kidney cortex from mice 5 hr after PBS or R848 painting. Cells/100 TEM grid squares; mean ± SEM; n = 4 Cx3cr1 +/− mice per condition, 2 Cx3cr1 −/− mice per condition, 2 Nr4a1 −/− mice per condition, 2 Ccr2 −/− mice per condition, and 3 Itgal −/− mice per condition. ∗ , p
    Figure Legend Snippet: Retention of Crawling Monocytes in the Kidney Vasculature in Response to TLR7 Agonist Requires Chemokine Receptor Signaling (A) Experiments performed idem as in Figure 3 for Cx3cr1 +/− Rag2 −/− Il2rg −/− or Cx3cr1 −/− Rag2 −/− Il2rg −/− mice. The scale bar represents 40 μm. n = 3 mice per condition. (B) Data idem as in Figures 3 B and 3C. ∗ , p ≤ 0.05; mean ± SEM; n = 3 mice. (C) qPCR for fractalkine ( Cx3cl1 ) messenger RNA in kidney cortex tissue from mice with the indicated genotypes 5hr after painting with R848 or PBS (n = 3–5 mice). ∗ ,p ≤ 0.05; mean ± SEM. (D) Data idem as in Figure 3 D for the stated genotypes and conditions. Right, mice received control or blocking anti-CD11b Ab i.v. injection immediately prior to the experiment. ∗ , p ≤ 0.05; mean ± SEM; n = 3–5 mice per condition. (E) Intravital imaging of peritubular capillaries in Cx3cr1 +/gfp mice 5 hr after R848 painting and immediately after i.v. injection of 10 μg Gr1-APC or Ly6G-PE antibodies. Mean ± SEM, n = 4 mice per condition. (F) GFP − Gr1 + and Ly6G + cells forming clusters in capillaries before and after R848 painting and proportion of clusters in contact with a GFP + cells. n = 5 mice per condition, mean ± SEM. (G) TEM of the superficial kidney cortex from mice 5 hr after PBS or R848 painting. Cells/100 TEM grid squares; mean ± SEM; n = 4 Cx3cr1 +/− mice per condition, 2 Cx3cr1 −/− mice per condition, 2 Nr4a1 −/− mice per condition, 2 Ccr2 −/− mice per condition, and 3 Itgal −/− mice per condition. ∗ , p

    Techniques Used: Mouse Assay, Real-time Polymerase Chain Reaction, Blocking Assay, Injection, Imaging, Transmission Electron Microscopy

    Focal Necrosis of Endothelial Cells (A) Representative electron micrographs of kidney cortex peritubular capillaries 5 hr after painting with R848 or PBS from Figure 4 G. Single arrow, basal lamina; double arrow, endothelium; ∗ , fluid in the subendothelial space; M, mononuclear cell; PMN, polymorphonuclear cell; E, endothelial cell nucleus. In the PBS-treated healthy peritubular capillary, endothelial cells are flat and close to basal lamina. The R848-treated peritubular capillary shows a swollen necrotic endothelial cell, expanded subendothelial space, mononuclear cell phagocytosing mitochondria, and blebbing necrotic endothelium (images are from Cx3cr −/+ Rag2 −/− IL2rg −/− mice). (B) Similar features to those represented in (A) are shown for Ccr2 −/− mice. (C) Representative healthy endothelium in R848- and PBS-treated kidneys from Itgal −/− , Cx3cr1 −/− , and Nr4a1 −/− mice. Micrographs are representative of experiments analyzed in Figure 4 G. The thick scale represents 5 μm, the thin scale represents 1 μm. Initial magnification was 15,000×. Also see Figure S3 .
    Figure Legend Snippet: Focal Necrosis of Endothelial Cells (A) Representative electron micrographs of kidney cortex peritubular capillaries 5 hr after painting with R848 or PBS from Figure 4 G. Single arrow, basal lamina; double arrow, endothelium; ∗ , fluid in the subendothelial space; M, mononuclear cell; PMN, polymorphonuclear cell; E, endothelial cell nucleus. In the PBS-treated healthy peritubular capillary, endothelial cells are flat and close to basal lamina. The R848-treated peritubular capillary shows a swollen necrotic endothelial cell, expanded subendothelial space, mononuclear cell phagocytosing mitochondria, and blebbing necrotic endothelium (images are from Cx3cr −/+ Rag2 −/− IL2rg −/− mice). (B) Similar features to those represented in (A) are shown for Ccr2 −/− mice. (C) Representative healthy endothelium in R848- and PBS-treated kidneys from Itgal −/− , Cx3cr1 −/− , and Nr4a1 −/− mice. Micrographs are representative of experiments analyzed in Figure 4 G. The thick scale represents 5 μm, the thin scale represents 1 μm. Initial magnification was 15,000×. Also see Figure S3 .

    Techniques Used: Mouse Assay

    Neutrophils Kill Endothelial Cells (A) Endothelial cell microscopic features of chimeric mice described in Figure 6 D expressed as the percentage of mononuclear cells (mono) or PMN-containing fields that present with the indicated lesions. (B) Representative FACS dot plots of peripheral blood cells of mice treated 8 hr earlier with Ly6G-depleting Ab (1A8) or isotype control (2A3). The arrow in the FSC/SSC panel indicates granulocytic cells, the percentage of Lin − CD115 − granulocytes are indicated in red. n = 3 mice per group, mean ± SEM. (C) Presence of intravascular mononuclear (left) and polymorphonuclear cells (right) as quantified by TEM in mice treated with 1A8 or 2A3 8 hr before kidney painting with R848. n = 3 mice per group, mean ± SEM. (D) Endothelial cell microscopic features of granulocyte-depleted and control mice. n = 3 mice per group, mean ± SEM. (E) Representative peritubular capillary containing a monocyte from a 1A8-treated mouse. (F) Proinflammatory cytokine production in vitro by sorted Ly6C low and Ly6C + monocytes after 24 hr stimulation with medium alone or R848 (top) in the absence or presence of a MEK inhibitor (PD) or for medium alone or LPS (bottom) in the absence or presence of the MEK inhibitor (PD) (bottom). Multiplexed ELISA, n = 3 mice per condition. (G) Schematic representation of the molecular and cellular features of the interaction of Ly6C low monocytes with the endothelium in a steady state and TLR7-mediated endothelial “safe disposal.” Also see Figure S3 .
    Figure Legend Snippet: Neutrophils Kill Endothelial Cells (A) Endothelial cell microscopic features of chimeric mice described in Figure 6 D expressed as the percentage of mononuclear cells (mono) or PMN-containing fields that present with the indicated lesions. (B) Representative FACS dot plots of peripheral blood cells of mice treated 8 hr earlier with Ly6G-depleting Ab (1A8) or isotype control (2A3). The arrow in the FSC/SSC panel indicates granulocytic cells, the percentage of Lin − CD115 − granulocytes are indicated in red. n = 3 mice per group, mean ± SEM. (C) Presence of intravascular mononuclear (left) and polymorphonuclear cells (right) as quantified by TEM in mice treated with 1A8 or 2A3 8 hr before kidney painting with R848. n = 3 mice per group, mean ± SEM. (D) Endothelial cell microscopic features of granulocyte-depleted and control mice. n = 3 mice per group, mean ± SEM. (E) Representative peritubular capillary containing a monocyte from a 1A8-treated mouse. (F) Proinflammatory cytokine production in vitro by sorted Ly6C low and Ly6C + monocytes after 24 hr stimulation with medium alone or R848 (top) in the absence or presence of a MEK inhibitor (PD) or for medium alone or LPS (bottom) in the absence or presence of the MEK inhibitor (PD) (bottom). Multiplexed ELISA, n = 3 mice per condition. (G) Schematic representation of the molecular and cellular features of the interaction of Ly6C low monocytes with the endothelium in a steady state and TLR7-mediated endothelial “safe disposal.” Also see Figure S3 .

    Techniques Used: Mouse Assay, FACS, Transmission Electron Microscopy, In Vitro, Enzyme-linked Immunosorbent Assay

    Retention of Crawling Monocytes in the Kidney Vasculature in Response to TLR7 Agonist (A) Representative monocyte tracks and vectors in the kidney cortex after painting with PBS or R848 in Tlr7 +/+ and Tlr7 −/− mice over 5 hr. n = 3 or 4 mice per condition The scale bar represents 40 μm. (B) Track length and speed for monocytes from the experiments described in (A). ∗ , p ≤ 0.05; mean ± SEM. (C) Mean track duration, track displacement, and confinement ratio of crawling monocytes from the experiments described in (A). ∗ , p ≤ 0.05; mean ± SEM. (D) Left, cumulative number of crawling monocytes per frame from experiments described in (A). Middle and right, the same experiment split over two graphs for clarity after PBS, LPS, R848 painting, or i.v. injection of PBS or R848. Data points for the R848 painting are shown twice. ∗ , p ≤ 0.05; n = 3-5 mice per condition. (E and F) Intravital imaging of peritubular capillaries in Cx3cr1 +/gfp mice after i.v. injection of CD11b-PE (magenta), 4.5 hr after R848 painting, and quantification of GFP + cells in the kidney cortex and capillaries at t0 and 4.5 hr after R848 painting. n = 4, mean ± SEM. The scale bar represents 10 μm. Also see Figure S2 and Movies S8 and S9 .
    Figure Legend Snippet: Retention of Crawling Monocytes in the Kidney Vasculature in Response to TLR7 Agonist (A) Representative monocyte tracks and vectors in the kidney cortex after painting with PBS or R848 in Tlr7 +/+ and Tlr7 −/− mice over 5 hr. n = 3 or 4 mice per condition The scale bar represents 40 μm. (B) Track length and speed for monocytes from the experiments described in (A). ∗ , p ≤ 0.05; mean ± SEM. (C) Mean track duration, track displacement, and confinement ratio of crawling monocytes from the experiments described in (A). ∗ , p ≤ 0.05; mean ± SEM. (D) Left, cumulative number of crawling monocytes per frame from experiments described in (A). Middle and right, the same experiment split over two graphs for clarity after PBS, LPS, R848 painting, or i.v. injection of PBS or R848. Data points for the R848 painting are shown twice. ∗ , p ≤ 0.05; n = 3-5 mice per condition. (E and F) Intravital imaging of peritubular capillaries in Cx3cr1 +/gfp mice after i.v. injection of CD11b-PE (magenta), 4.5 hr after R848 painting, and quantification of GFP + cells in the kidney cortex and capillaries at t0 and 4.5 hr after R848 painting. n = 4, mean ± SEM. The scale bar represents 10 μm. Also see Figure S2 and Movies S8 and S9 .

    Techniques Used: Mouse Assay, Injection, Imaging

    Response to R848 and LPS, Related to Figure 3 (A) Application of R848 or LPS to the kidney capsule induces inflammatory cytokines. qPCR for Il1b or Tnf mRNA in sub-capsular kidney cortex of Cx3cr1 −/+ ; Rag2 −/− ; Il2rg −/− mice tissue treated in vivo by painting the kidney capsule with PBS, R848 (200 μg) or LPS (200 μg) for 5 hr n = 2-4 mice per condition. mRNA quantity was normalized to Gapdh and is expressed as fold change over PBS. (B) CD11b expression on circulating Ly6C low monocytes after kidney painting with either R848 (top) or LPS (bottom). Representative histograms are shown (left) and cumulative data mean ± SEM right, n = 3 for R848, n = 2 for LPS.
    Figure Legend Snippet: Response to R848 and LPS, Related to Figure 3 (A) Application of R848 or LPS to the kidney capsule induces inflammatory cytokines. qPCR for Il1b or Tnf mRNA in sub-capsular kidney cortex of Cx3cr1 −/+ ; Rag2 −/− ; Il2rg −/− mice tissue treated in vivo by painting the kidney capsule with PBS, R848 (200 μg) or LPS (200 μg) for 5 hr n = 2-4 mice per condition. mRNA quantity was normalized to Gapdh and is expressed as fold change over PBS. (B) CD11b expression on circulating Ly6C low monocytes after kidney painting with either R848 (top) or LPS (bottom). Representative histograms are shown (left) and cumulative data mean ± SEM right, n = 3 for R848, n = 2 for LPS.

    Techniques Used: Real-time Polymerase Chain Reaction, Mouse Assay, In Vivo, Expressing

    TEM of Kidney Capillaries 5 hr after Direct Application of PBS or R848 In Vivo, Related to Figures 4 , 5 , 6 , and 7 Single arrow, basal lamina; double arrow, endothelium; M, shows mononuclear cell; PMN, polymorphonuclear cell; E, endothelial cell nucleus. White boxes indicate the high magnification areas. (A) Glomerular capillaries from Cx3cr1 −/+ ; Rag2 −/− ; Il2rg −/− mice. Left, PBS treated control glomerulus, endothelium is intact and close to basal lamina; Middle, R848 treated glomerular capillary containing mononuclear cell. Endothelium is missing and monocnuclear cell containing phagocytosed material sits directly against basal lamina (see higher magnifications); Right, PMN containing glomerular capillary with thickened area of endothelium. (B) Peritubular capillaries from wild-type B6 mice. Left, PBS treated control peritubular capillary; 3 rd from right, peritubular capillary containing mononuclear cell with necrotic and disrupted endothelium; 2 nd from right, peritubular capillary containing mononuclear cell with necrotic and disrupted endothelium and expanded subendothelial space; 1 st from right, peritubular capillary containing PMN with necrotic and disrupted endothelium and expanded subendothelial space. Images representative of 3 mice per condition. Thick scale bars = 5 μm, thin scale bars = 1 μm. Original magnification x15 000.
    Figure Legend Snippet: TEM of Kidney Capillaries 5 hr after Direct Application of PBS or R848 In Vivo, Related to Figures 4 , 5 , 6 , and 7 Single arrow, basal lamina; double arrow, endothelium; M, shows mononuclear cell; PMN, polymorphonuclear cell; E, endothelial cell nucleus. White boxes indicate the high magnification areas. (A) Glomerular capillaries from Cx3cr1 −/+ ; Rag2 −/− ; Il2rg −/− mice. Left, PBS treated control glomerulus, endothelium is intact and close to basal lamina; Middle, R848 treated glomerular capillary containing mononuclear cell. Endothelium is missing and monocnuclear cell containing phagocytosed material sits directly against basal lamina (see higher magnifications); Right, PMN containing glomerular capillary with thickened area of endothelium. (B) Peritubular capillaries from wild-type B6 mice. Left, PBS treated control peritubular capillary; 3 rd from right, peritubular capillary containing mononuclear cell with necrotic and disrupted endothelium; 2 nd from right, peritubular capillary containing mononuclear cell with necrotic and disrupted endothelium and expanded subendothelial space; 1 st from right, peritubular capillary containing PMN with necrotic and disrupted endothelium and expanded subendothelial space. Images representative of 3 mice per condition. Thick scale bars = 5 μm, thin scale bars = 1 μm. Original magnification x15 000.

    Techniques Used: Transmission Electron Microscopy, In Vivo, Mouse Assay

    6) Product Images from "Nr4a1-Dependent Ly6Clow Monocytes Monitor Endothelial Cells and Orchestrate Their Disposal"

    Article Title: Nr4a1-Dependent Ly6Clow Monocytes Monitor Endothelial Cells and Orchestrate Their Disposal

    Journal: Cell

    doi: 10.1016/j.cell.2013.03.010

    Retention of Crawling Monocytes in the Kidney Vasculature in Response to TLR7 Agonist Requires Chemokine Receptor Signaling (A) Experiments performed idem as in Figure 3 for Cx3cr1 +/− Rag2 −/− Il2rg −/− or Cx3cr1 −/− Rag2 −/− Il2rg −/− mice. The scale bar represents 40 μm. n = 3 mice per condition. (B) Data idem as in Figures 3 B and 3C. ∗ , p ≤ 0.05; mean ± SEM; n = 3 mice. (C) qPCR for fractalkine ( Cx3cl1 ) messenger RNA in kidney cortex tissue from mice with the indicated genotypes 5hr after painting with R848 or PBS (n = 3–5 mice). ∗ ,p ≤ 0.05; mean ± SEM. (D) Data idem as in Figure 3 D for the stated genotypes and conditions. Right, mice received control or blocking anti-CD11b Ab i.v. injection immediately prior to the experiment. ∗ , p ≤ 0.05; mean ± SEM; n = 3–5 mice per condition. (E) Intravital imaging of peritubular capillaries in Cx3cr1 +/gfp mice 5 hr after R848 painting and immediately after i.v. injection of 10 μg Gr1-APC or Ly6G-PE antibodies. Mean ± SEM, n = 4 mice per condition. (F) GFP − Gr1 + and Ly6G + cells forming clusters in capillaries before and after R848 painting and proportion of clusters in contact with a GFP + cells. n = 5 mice per condition, mean ± SEM. (G) TEM of the superficial kidney cortex from mice 5 hr after PBS or R848 painting. Cells/100 TEM grid squares; mean ± SEM; n = 4 Cx3cr1 +/− mice per condition, 2 Cx3cr1 −/− mice per condition, 2 Nr4a1 −/− mice per condition, 2 Ccr2 −/− mice per condition, and 3 Itgal −/− mice per condition. ∗ , p
    Figure Legend Snippet: Retention of Crawling Monocytes in the Kidney Vasculature in Response to TLR7 Agonist Requires Chemokine Receptor Signaling (A) Experiments performed idem as in Figure 3 for Cx3cr1 +/− Rag2 −/− Il2rg −/− or Cx3cr1 −/− Rag2 −/− Il2rg −/− mice. The scale bar represents 40 μm. n = 3 mice per condition. (B) Data idem as in Figures 3 B and 3C. ∗ , p ≤ 0.05; mean ± SEM; n = 3 mice. (C) qPCR for fractalkine ( Cx3cl1 ) messenger RNA in kidney cortex tissue from mice with the indicated genotypes 5hr after painting with R848 or PBS (n = 3–5 mice). ∗ ,p ≤ 0.05; mean ± SEM. (D) Data idem as in Figure 3 D for the stated genotypes and conditions. Right, mice received control or blocking anti-CD11b Ab i.v. injection immediately prior to the experiment. ∗ , p ≤ 0.05; mean ± SEM; n = 3–5 mice per condition. (E) Intravital imaging of peritubular capillaries in Cx3cr1 +/gfp mice 5 hr after R848 painting and immediately after i.v. injection of 10 μg Gr1-APC or Ly6G-PE antibodies. Mean ± SEM, n = 4 mice per condition. (F) GFP − Gr1 + and Ly6G + cells forming clusters in capillaries before and after R848 painting and proportion of clusters in contact with a GFP + cells. n = 5 mice per condition, mean ± SEM. (G) TEM of the superficial kidney cortex from mice 5 hr after PBS or R848 painting. Cells/100 TEM grid squares; mean ± SEM; n = 4 Cx3cr1 +/− mice per condition, 2 Cx3cr1 −/− mice per condition, 2 Nr4a1 −/− mice per condition, 2 Ccr2 −/− mice per condition, and 3 Itgal −/− mice per condition. ∗ , p

    Techniques Used: Mouse Assay, Real-time Polymerase Chain Reaction, Blocking Assay, Injection, Imaging, Transmission Electron Microscopy

    Focal Necrosis of Endothelial Cells (A) Representative electron micrographs of kidney cortex peritubular capillaries 5 hr after painting with R848 or PBS from Figure 4 G. Single arrow, basal lamina; double arrow, endothelium; ∗ , fluid in the subendothelial space; M, mononuclear cell; PMN, polymorphonuclear cell; E, endothelial cell nucleus. In the PBS-treated healthy peritubular capillary, endothelial cells are flat and close to basal lamina. The R848-treated peritubular capillary shows a swollen necrotic endothelial cell, expanded subendothelial space, mononuclear cell phagocytosing mitochondria, and blebbing necrotic endothelium (images are from Cx3cr −/+ Rag2 −/− IL2rg −/− mice). (B) Similar features to those represented in (A) are shown for Ccr2 −/− mice. (C) Representative healthy endothelium in R848- and PBS-treated kidneys from Itgal −/− , Cx3cr1 −/− , and Nr4a1 −/− mice. Micrographs are representative of experiments analyzed in Figure 4 G. The thick scale represents 5 μm, the thin scale represents 1 μm. Initial magnification was 15,000×. Also see Figure S3 .
    Figure Legend Snippet: Focal Necrosis of Endothelial Cells (A) Representative electron micrographs of kidney cortex peritubular capillaries 5 hr after painting with R848 or PBS from Figure 4 G. Single arrow, basal lamina; double arrow, endothelium; ∗ , fluid in the subendothelial space; M, mononuclear cell; PMN, polymorphonuclear cell; E, endothelial cell nucleus. In the PBS-treated healthy peritubular capillary, endothelial cells are flat and close to basal lamina. The R848-treated peritubular capillary shows a swollen necrotic endothelial cell, expanded subendothelial space, mononuclear cell phagocytosing mitochondria, and blebbing necrotic endothelium (images are from Cx3cr −/+ Rag2 −/− IL2rg −/− mice). (B) Similar features to those represented in (A) are shown for Ccr2 −/− mice. (C) Representative healthy endothelium in R848- and PBS-treated kidneys from Itgal −/− , Cx3cr1 −/− , and Nr4a1 −/− mice. Micrographs are representative of experiments analyzed in Figure 4 G. The thick scale represents 5 μm, the thin scale represents 1 μm. Initial magnification was 15,000×. Also see Figure S3 .

    Techniques Used: Mouse Assay

    Neutrophils Kill Endothelial Cells (A) Endothelial cell microscopic features of chimeric mice described in Figure 6 D expressed as the percentage of mononuclear cells (mono) or PMN-containing fields that present with the indicated lesions. (B) Representative FACS dot plots of peripheral blood cells of mice treated 8 hr earlier with Ly6G-depleting Ab (1A8) or isotype control (2A3). The arrow in the FSC/SSC panel indicates granulocytic cells, the percentage of Lin − CD115 − granulocytes are indicated in red. n = 3 mice per group, mean ± SEM. (C) Presence of intravascular mononuclear (left) and polymorphonuclear cells (right) as quantified by TEM in mice treated with 1A8 or 2A3 8 hr before kidney painting with R848. n = 3 mice per group, mean ± SEM. (D) Endothelial cell microscopic features of granulocyte-depleted and control mice. n = 3 mice per group, mean ± SEM. (E) Representative peritubular capillary containing a monocyte from a 1A8-treated mouse. (F) Proinflammatory cytokine production in vitro by sorted Ly6C low and Ly6C + monocytes after 24 hr stimulation with medium alone or R848 (top) in the absence or presence of a MEK inhibitor (PD) or for medium alone or LPS (bottom) in the absence or presence of the MEK inhibitor (PD) (bottom). Multiplexed ELISA, n = 3 mice per condition. (G) Schematic representation of the molecular and cellular features of the interaction of Ly6C low monocytes with the endothelium in a steady state and TLR7-mediated endothelial “safe disposal.” Also see Figure S3 .
    Figure Legend Snippet: Neutrophils Kill Endothelial Cells (A) Endothelial cell microscopic features of chimeric mice described in Figure 6 D expressed as the percentage of mononuclear cells (mono) or PMN-containing fields that present with the indicated lesions. (B) Representative FACS dot plots of peripheral blood cells of mice treated 8 hr earlier with Ly6G-depleting Ab (1A8) or isotype control (2A3). The arrow in the FSC/SSC panel indicates granulocytic cells, the percentage of Lin − CD115 − granulocytes are indicated in red. n = 3 mice per group, mean ± SEM. (C) Presence of intravascular mononuclear (left) and polymorphonuclear cells (right) as quantified by TEM in mice treated with 1A8 or 2A3 8 hr before kidney painting with R848. n = 3 mice per group, mean ± SEM. (D) Endothelial cell microscopic features of granulocyte-depleted and control mice. n = 3 mice per group, mean ± SEM. (E) Representative peritubular capillary containing a monocyte from a 1A8-treated mouse. (F) Proinflammatory cytokine production in vitro by sorted Ly6C low and Ly6C + monocytes after 24 hr stimulation with medium alone or R848 (top) in the absence or presence of a MEK inhibitor (PD) or for medium alone or LPS (bottom) in the absence or presence of the MEK inhibitor (PD) (bottom). Multiplexed ELISA, n = 3 mice per condition. (G) Schematic representation of the molecular and cellular features of the interaction of Ly6C low monocytes with the endothelium in a steady state and TLR7-mediated endothelial “safe disposal.” Also see Figure S3 .

    Techniques Used: Mouse Assay, FACS, Transmission Electron Microscopy, In Vitro, Enzyme-linked Immunosorbent Assay

    Retention of Crawling Monocytes in the Kidney Vasculature in Response to TLR7 Agonist (A) Representative monocyte tracks and vectors in the kidney cortex after painting with PBS or R848 in Tlr7 +/+ and Tlr7 −/− mice over 5 hr. n = 3 or 4 mice per condition The scale bar represents 40 μm. (B) Track length and speed for monocytes from the experiments described in (A). ∗ , p ≤ 0.05; mean ± SEM. (C) Mean track duration, track displacement, and confinement ratio of crawling monocytes from the experiments described in (A). ∗ , p ≤ 0.05; mean ± SEM. (D) Left, cumulative number of crawling monocytes per frame from experiments described in (A). Middle and right, the same experiment split over two graphs for clarity after PBS, LPS, R848 painting, or i.v. injection of PBS or R848. Data points for the R848 painting are shown twice. ∗ , p ≤ 0.05; n = 3-5 mice per condition. (E and F) Intravital imaging of peritubular capillaries in Cx3cr1 +/gfp mice after i.v. injection of CD11b-PE (magenta), 4.5 hr after R848 painting, and quantification of GFP + cells in the kidney cortex and capillaries at t0 and 4.5 hr after R848 painting. n = 4, mean ± SEM. The scale bar represents 10 μm. Also see Figure S2 and Movies S8 and S9 .
    Figure Legend Snippet: Retention of Crawling Monocytes in the Kidney Vasculature in Response to TLR7 Agonist (A) Representative monocyte tracks and vectors in the kidney cortex after painting with PBS or R848 in Tlr7 +/+ and Tlr7 −/− mice over 5 hr. n = 3 or 4 mice per condition The scale bar represents 40 μm. (B) Track length and speed for monocytes from the experiments described in (A). ∗ , p ≤ 0.05; mean ± SEM. (C) Mean track duration, track displacement, and confinement ratio of crawling monocytes from the experiments described in (A). ∗ , p ≤ 0.05; mean ± SEM. (D) Left, cumulative number of crawling monocytes per frame from experiments described in (A). Middle and right, the same experiment split over two graphs for clarity after PBS, LPS, R848 painting, or i.v. injection of PBS or R848. Data points for the R848 painting are shown twice. ∗ , p ≤ 0.05; n = 3-5 mice per condition. (E and F) Intravital imaging of peritubular capillaries in Cx3cr1 +/gfp mice after i.v. injection of CD11b-PE (magenta), 4.5 hr after R848 painting, and quantification of GFP + cells in the kidney cortex and capillaries at t0 and 4.5 hr after R848 painting. n = 4, mean ± SEM. The scale bar represents 10 μm. Also see Figure S2 and Movies S8 and S9 .

    Techniques Used: Mouse Assay, Injection, Imaging

    Response to R848 and LPS, Related to Figure 3 (A) Application of R848 or LPS to the kidney capsule induces inflammatory cytokines. qPCR for Il1b or Tnf mRNA in sub-capsular kidney cortex of Cx3cr1 −/+ ; Rag2 −/− ; Il2rg −/− mice tissue treated in vivo by painting the kidney capsule with PBS, R848 (200 μg) or LPS (200 μg) for 5 hr n = 2-4 mice per condition. mRNA quantity was normalized to Gapdh and is expressed as fold change over PBS. (B) CD11b expression on circulating Ly6C low monocytes after kidney painting with either R848 (top) or LPS (bottom). Representative histograms are shown (left) and cumulative data mean ± SEM right, n = 3 for R848, n = 2 for LPS.
    Figure Legend Snippet: Response to R848 and LPS, Related to Figure 3 (A) Application of R848 or LPS to the kidney capsule induces inflammatory cytokines. qPCR for Il1b or Tnf mRNA in sub-capsular kidney cortex of Cx3cr1 −/+ ; Rag2 −/− ; Il2rg −/− mice tissue treated in vivo by painting the kidney capsule with PBS, R848 (200 μg) or LPS (200 μg) for 5 hr n = 2-4 mice per condition. mRNA quantity was normalized to Gapdh and is expressed as fold change over PBS. (B) CD11b expression on circulating Ly6C low monocytes after kidney painting with either R848 (top) or LPS (bottom). Representative histograms are shown (left) and cumulative data mean ± SEM right, n = 3 for R848, n = 2 for LPS.

    Techniques Used: Real-time Polymerase Chain Reaction, Mouse Assay, In Vivo, Expressing

    TEM of Kidney Capillaries 5 hr after Direct Application of PBS or R848 In Vivo, Related to Figures 4 , 5 , 6 , and 7 Single arrow, basal lamina; double arrow, endothelium; M, shows mononuclear cell; PMN, polymorphonuclear cell; E, endothelial cell nucleus. White boxes indicate the high magnification areas. (A) Glomerular capillaries from Cx3cr1 −/+ ; Rag2 −/− ; Il2rg −/− mice. Left, PBS treated control glomerulus, endothelium is intact and close to basal lamina; Middle, R848 treated glomerular capillary containing mononuclear cell. Endothelium is missing and monocnuclear cell containing phagocytosed material sits directly against basal lamina (see higher magnifications); Right, PMN containing glomerular capillary with thickened area of endothelium. (B) Peritubular capillaries from wild-type B6 mice. Left, PBS treated control peritubular capillary; 3 rd from right, peritubular capillary containing mononuclear cell with necrotic and disrupted endothelium; 2 nd from right, peritubular capillary containing mononuclear cell with necrotic and disrupted endothelium and expanded subendothelial space; 1 st from right, peritubular capillary containing PMN with necrotic and disrupted endothelium and expanded subendothelial space. Images representative of 3 mice per condition. Thick scale bars = 5 μm, thin scale bars = 1 μm. Original magnification x15 000.
    Figure Legend Snippet: TEM of Kidney Capillaries 5 hr after Direct Application of PBS or R848 In Vivo, Related to Figures 4 , 5 , 6 , and 7 Single arrow, basal lamina; double arrow, endothelium; M, shows mononuclear cell; PMN, polymorphonuclear cell; E, endothelial cell nucleus. White boxes indicate the high magnification areas. (A) Glomerular capillaries from Cx3cr1 −/+ ; Rag2 −/− ; Il2rg −/− mice. Left, PBS treated control glomerulus, endothelium is intact and close to basal lamina; Middle, R848 treated glomerular capillary containing mononuclear cell. Endothelium is missing and monocnuclear cell containing phagocytosed material sits directly against basal lamina (see higher magnifications); Right, PMN containing glomerular capillary with thickened area of endothelium. (B) Peritubular capillaries from wild-type B6 mice. Left, PBS treated control peritubular capillary; 3 rd from right, peritubular capillary containing mononuclear cell with necrotic and disrupted endothelium; 2 nd from right, peritubular capillary containing mononuclear cell with necrotic and disrupted endothelium and expanded subendothelial space; 1 st from right, peritubular capillary containing PMN with necrotic and disrupted endothelium and expanded subendothelial space. Images representative of 3 mice per condition. Thick scale bars = 5 μm, thin scale bars = 1 μm. Original magnification x15 000.

    Techniques Used: Transmission Electron Microscopy, In Vivo, Mouse Assay

    7) Product Images from "Nr4a1-Dependent Ly6Clow Monocytes Monitor Endothelial Cells and Orchestrate Their Disposal"

    Article Title: Nr4a1-Dependent Ly6Clow Monocytes Monitor Endothelial Cells and Orchestrate Their Disposal

    Journal: Cell

    doi: 10.1016/j.cell.2013.03.010

    Retention of Crawling Monocytes in the Kidney Vasculature in Response to TLR7 Agonist Requires Chemokine Receptor Signaling (A) Experiments performed idem as in Figure 3 for Cx3cr1 +/− Rag2 −/− Il2rg −/− or Cx3cr1 −/− Rag2 −/− Il2rg −/− mice. The scale bar represents 40 μm. n = 3 mice per condition. (B) Data idem as in Figures 3 B and 3C. ∗ , p ≤ 0.05; mean ± SEM; n = 3 mice. (C) qPCR for fractalkine ( Cx3cl1 ) messenger RNA in kidney cortex tissue from mice with the indicated genotypes 5hr after painting with R848 or PBS (n = 3–5 mice). ∗ ,p ≤ 0.05; mean ± SEM. (D) Data idem as in Figure 3 D for the stated genotypes and conditions. Right, mice received control or blocking anti-CD11b Ab i.v. injection immediately prior to the experiment. ∗ , p ≤ 0.05; mean ± SEM; n = 3–5 mice per condition. (E) Intravital imaging of peritubular capillaries in Cx3cr1 +/gfp mice 5 hr after R848 painting and immediately after i.v. injection of 10 μg Gr1-APC or Ly6G-PE antibodies. Mean ± SEM, n = 4 mice per condition. (F) GFP − Gr1 + and Ly6G + cells forming clusters in capillaries before and after R848 painting and proportion of clusters in contact with a GFP + cells. n = 5 mice per condition, mean ± SEM. (G) TEM of the superficial kidney cortex from mice 5 hr after PBS or R848 painting. Cells/100 TEM grid squares; mean ± SEM; n = 4 Cx3cr1 +/− mice per condition, 2 Cx3cr1 −/− mice per condition, 2 Nr4a1 −/− mice per condition, 2 Ccr2 −/− mice per condition, and 3 Itgal −/− mice per condition. ∗ , p
    Figure Legend Snippet: Retention of Crawling Monocytes in the Kidney Vasculature in Response to TLR7 Agonist Requires Chemokine Receptor Signaling (A) Experiments performed idem as in Figure 3 for Cx3cr1 +/− Rag2 −/− Il2rg −/− or Cx3cr1 −/− Rag2 −/− Il2rg −/− mice. The scale bar represents 40 μm. n = 3 mice per condition. (B) Data idem as in Figures 3 B and 3C. ∗ , p ≤ 0.05; mean ± SEM; n = 3 mice. (C) qPCR for fractalkine ( Cx3cl1 ) messenger RNA in kidney cortex tissue from mice with the indicated genotypes 5hr after painting with R848 or PBS (n = 3–5 mice). ∗ ,p ≤ 0.05; mean ± SEM. (D) Data idem as in Figure 3 D for the stated genotypes and conditions. Right, mice received control or blocking anti-CD11b Ab i.v. injection immediately prior to the experiment. ∗ , p ≤ 0.05; mean ± SEM; n = 3–5 mice per condition. (E) Intravital imaging of peritubular capillaries in Cx3cr1 +/gfp mice 5 hr after R848 painting and immediately after i.v. injection of 10 μg Gr1-APC or Ly6G-PE antibodies. Mean ± SEM, n = 4 mice per condition. (F) GFP − Gr1 + and Ly6G + cells forming clusters in capillaries before and after R848 painting and proportion of clusters in contact with a GFP + cells. n = 5 mice per condition, mean ± SEM. (G) TEM of the superficial kidney cortex from mice 5 hr after PBS or R848 painting. Cells/100 TEM grid squares; mean ± SEM; n = 4 Cx3cr1 +/− mice per condition, 2 Cx3cr1 −/− mice per condition, 2 Nr4a1 −/− mice per condition, 2 Ccr2 −/− mice per condition, and 3 Itgal −/− mice per condition. ∗ , p

    Techniques Used: Mouse Assay, Real-time Polymerase Chain Reaction, Blocking Assay, Injection, Imaging, Transmission Electron Microscopy

    Focal Necrosis of Endothelial Cells (A) Representative electron micrographs of kidney cortex peritubular capillaries 5 hr after painting with R848 or PBS from Figure 4 G. Single arrow, basal lamina; double arrow, endothelium; ∗ , fluid in the subendothelial space; M, mononuclear cell; PMN, polymorphonuclear cell; E, endothelial cell nucleus. In the PBS-treated healthy peritubular capillary, endothelial cells are flat and close to basal lamina. The R848-treated peritubular capillary shows a swollen necrotic endothelial cell, expanded subendothelial space, mononuclear cell phagocytosing mitochondria, and blebbing necrotic endothelium (images are from Cx3cr −/+ Rag2 −/− IL2rg −/− mice). (B) Similar features to those represented in (A) are shown for Ccr2 −/− mice. (C) Representative healthy endothelium in R848- and PBS-treated kidneys from Itgal −/− , Cx3cr1 −/− , and Nr4a1 −/− mice. Micrographs are representative of experiments analyzed in Figure 4 G. The thick scale represents 5 μm, the thin scale represents 1 μm. Initial magnification was 15,000×. Also see Figure S3 .
    Figure Legend Snippet: Focal Necrosis of Endothelial Cells (A) Representative electron micrographs of kidney cortex peritubular capillaries 5 hr after painting with R848 or PBS from Figure 4 G. Single arrow, basal lamina; double arrow, endothelium; ∗ , fluid in the subendothelial space; M, mononuclear cell; PMN, polymorphonuclear cell; E, endothelial cell nucleus. In the PBS-treated healthy peritubular capillary, endothelial cells are flat and close to basal lamina. The R848-treated peritubular capillary shows a swollen necrotic endothelial cell, expanded subendothelial space, mononuclear cell phagocytosing mitochondria, and blebbing necrotic endothelium (images are from Cx3cr −/+ Rag2 −/− IL2rg −/− mice). (B) Similar features to those represented in (A) are shown for Ccr2 −/− mice. (C) Representative healthy endothelium in R848- and PBS-treated kidneys from Itgal −/− , Cx3cr1 −/− , and Nr4a1 −/− mice. Micrographs are representative of experiments analyzed in Figure 4 G. The thick scale represents 5 μm, the thin scale represents 1 μm. Initial magnification was 15,000×. Also see Figure S3 .

    Techniques Used: Mouse Assay

    Neutrophils Kill Endothelial Cells (A) Endothelial cell microscopic features of chimeric mice described in Figure 6 D expressed as the percentage of mononuclear cells (mono) or PMN-containing fields that present with the indicated lesions. (B) Representative FACS dot plots of peripheral blood cells of mice treated 8 hr earlier with Ly6G-depleting Ab (1A8) or isotype control (2A3). The arrow in the FSC/SSC panel indicates granulocytic cells, the percentage of Lin − CD115 − granulocytes are indicated in red. n = 3 mice per group, mean ± SEM. (C) Presence of intravascular mononuclear (left) and polymorphonuclear cells (right) as quantified by TEM in mice treated with 1A8 or 2A3 8 hr before kidney painting with R848. n = 3 mice per group, mean ± SEM. (D) Endothelial cell microscopic features of granulocyte-depleted and control mice. n = 3 mice per group, mean ± SEM. (E) Representative peritubular capillary containing a monocyte from a 1A8-treated mouse. (F) Proinflammatory cytokine production in vitro by sorted Ly6C low and Ly6C + monocytes after 24 hr stimulation with medium alone or R848 (top) in the absence or presence of a MEK inhibitor (PD) or for medium alone or LPS (bottom) in the absence or presence of the MEK inhibitor (PD) (bottom). Multiplexed ELISA, n = 3 mice per condition. (G) Schematic representation of the molecular and cellular features of the interaction of Ly6C low monocytes with the endothelium in a steady state and TLR7-mediated endothelial “safe disposal.” Also see Figure S3 .
    Figure Legend Snippet: Neutrophils Kill Endothelial Cells (A) Endothelial cell microscopic features of chimeric mice described in Figure 6 D expressed as the percentage of mononuclear cells (mono) or PMN-containing fields that present with the indicated lesions. (B) Representative FACS dot plots of peripheral blood cells of mice treated 8 hr earlier with Ly6G-depleting Ab (1A8) or isotype control (2A3). The arrow in the FSC/SSC panel indicates granulocytic cells, the percentage of Lin − CD115 − granulocytes are indicated in red. n = 3 mice per group, mean ± SEM. (C) Presence of intravascular mononuclear (left) and polymorphonuclear cells (right) as quantified by TEM in mice treated with 1A8 or 2A3 8 hr before kidney painting with R848. n = 3 mice per group, mean ± SEM. (D) Endothelial cell microscopic features of granulocyte-depleted and control mice. n = 3 mice per group, mean ± SEM. (E) Representative peritubular capillary containing a monocyte from a 1A8-treated mouse. (F) Proinflammatory cytokine production in vitro by sorted Ly6C low and Ly6C + monocytes after 24 hr stimulation with medium alone or R848 (top) in the absence or presence of a MEK inhibitor (PD) or for medium alone or LPS (bottom) in the absence or presence of the MEK inhibitor (PD) (bottom). Multiplexed ELISA, n = 3 mice per condition. (G) Schematic representation of the molecular and cellular features of the interaction of Ly6C low monocytes with the endothelium in a steady state and TLR7-mediated endothelial “safe disposal.” Also see Figure S3 .

    Techniques Used: Mouse Assay, FACS, Transmission Electron Microscopy, In Vitro, Enzyme-linked Immunosorbent Assay

    Retention of Crawling Monocytes in the Kidney Vasculature in Response to TLR7 Agonist (A) Representative monocyte tracks and vectors in the kidney cortex after painting with PBS or R848 in Tlr7 +/+ and Tlr7 −/− mice over 5 hr. n = 3 or 4 mice per condition The scale bar represents 40 μm. (B) Track length and speed for monocytes from the experiments described in (A). ∗ , p ≤ 0.05; mean ± SEM. (C) Mean track duration, track displacement, and confinement ratio of crawling monocytes from the experiments described in (A). ∗ , p ≤ 0.05; mean ± SEM. (D) Left, cumulative number of crawling monocytes per frame from experiments described in (A). Middle and right, the same experiment split over two graphs for clarity after PBS, LPS, R848 painting, or i.v. injection of PBS or R848. Data points for the R848 painting are shown twice. ∗ , p ≤ 0.05; n = 3-5 mice per condition. (E and F) Intravital imaging of peritubular capillaries in Cx3cr1 +/gfp mice after i.v. injection of CD11b-PE (magenta), 4.5 hr after R848 painting, and quantification of GFP + cells in the kidney cortex and capillaries at t0 and 4.5 hr after R848 painting. n = 4, mean ± SEM. The scale bar represents 10 μm. Also see Figure S2 and Movies S8 and S9 .
    Figure Legend Snippet: Retention of Crawling Monocytes in the Kidney Vasculature in Response to TLR7 Agonist (A) Representative monocyte tracks and vectors in the kidney cortex after painting with PBS or R848 in Tlr7 +/+ and Tlr7 −/− mice over 5 hr. n = 3 or 4 mice per condition The scale bar represents 40 μm. (B) Track length and speed for monocytes from the experiments described in (A). ∗ , p ≤ 0.05; mean ± SEM. (C) Mean track duration, track displacement, and confinement ratio of crawling monocytes from the experiments described in (A). ∗ , p ≤ 0.05; mean ± SEM. (D) Left, cumulative number of crawling monocytes per frame from experiments described in (A). Middle and right, the same experiment split over two graphs for clarity after PBS, LPS, R848 painting, or i.v. injection of PBS or R848. Data points for the R848 painting are shown twice. ∗ , p ≤ 0.05; n = 3-5 mice per condition. (E and F) Intravital imaging of peritubular capillaries in Cx3cr1 +/gfp mice after i.v. injection of CD11b-PE (magenta), 4.5 hr after R848 painting, and quantification of GFP + cells in the kidney cortex and capillaries at t0 and 4.5 hr after R848 painting. n = 4, mean ± SEM. The scale bar represents 10 μm. Also see Figure S2 and Movies S8 and S9 .

    Techniques Used: Mouse Assay, Injection, Imaging

    Response to R848 and LPS, Related to Figure 3 (A) Application of R848 or LPS to the kidney capsule induces inflammatory cytokines. qPCR for Il1b or Tnf mRNA in sub-capsular kidney cortex of Cx3cr1 −/+ ; Rag2 −/− ; Il2rg −/− mice tissue treated in vivo by painting the kidney capsule with PBS, R848 (200 μg) or LPS (200 μg) for 5 hr n = 2-4 mice per condition. mRNA quantity was normalized to Gapdh and is expressed as fold change over PBS. (B) CD11b expression on circulating Ly6C low monocytes after kidney painting with either R848 (top) or LPS (bottom). Representative histograms are shown (left) and cumulative data mean ± SEM right, n = 3 for R848, n = 2 for LPS.
    Figure Legend Snippet: Response to R848 and LPS, Related to Figure 3 (A) Application of R848 or LPS to the kidney capsule induces inflammatory cytokines. qPCR for Il1b or Tnf mRNA in sub-capsular kidney cortex of Cx3cr1 −/+ ; Rag2 −/− ; Il2rg −/− mice tissue treated in vivo by painting the kidney capsule with PBS, R848 (200 μg) or LPS (200 μg) for 5 hr n = 2-4 mice per condition. mRNA quantity was normalized to Gapdh and is expressed as fold change over PBS. (B) CD11b expression on circulating Ly6C low monocytes after kidney painting with either R848 (top) or LPS (bottom). Representative histograms are shown (left) and cumulative data mean ± SEM right, n = 3 for R848, n = 2 for LPS.

    Techniques Used: Real-time Polymerase Chain Reaction, Mouse Assay, In Vivo, Expressing

    TEM of Kidney Capillaries 5 hr after Direct Application of PBS or R848 In Vivo, Related to Figures 4 , 5 , 6 , and 7 Single arrow, basal lamina; double arrow, endothelium; M, shows mononuclear cell; PMN, polymorphonuclear cell; E, endothelial cell nucleus. White boxes indicate the high magnification areas. (A) Glomerular capillaries from Cx3cr1 −/+ ; Rag2 −/− ; Il2rg −/− mice. Left, PBS treated control glomerulus, endothelium is intact and close to basal lamina; Middle, R848 treated glomerular capillary containing mononuclear cell. Endothelium is missing and monocnuclear cell containing phagocytosed material sits directly against basal lamina (see higher magnifications); Right, PMN containing glomerular capillary with thickened area of endothelium. (B) Peritubular capillaries from wild-type B6 mice. Left, PBS treated control peritubular capillary; 3 rd from right, peritubular capillary containing mononuclear cell with necrotic and disrupted endothelium; 2 nd from right, peritubular capillary containing mononuclear cell with necrotic and disrupted endothelium and expanded subendothelial space; 1 st from right, peritubular capillary containing PMN with necrotic and disrupted endothelium and expanded subendothelial space. Images representative of 3 mice per condition. Thick scale bars = 5 μm, thin scale bars = 1 μm. Original magnification x15 000.
    Figure Legend Snippet: TEM of Kidney Capillaries 5 hr after Direct Application of PBS or R848 In Vivo, Related to Figures 4 , 5 , 6 , and 7 Single arrow, basal lamina; double arrow, endothelium; M, shows mononuclear cell; PMN, polymorphonuclear cell; E, endothelial cell nucleus. White boxes indicate the high magnification areas. (A) Glomerular capillaries from Cx3cr1 −/+ ; Rag2 −/− ; Il2rg −/− mice. Left, PBS treated control glomerulus, endothelium is intact and close to basal lamina; Middle, R848 treated glomerular capillary containing mononuclear cell. Endothelium is missing and monocnuclear cell containing phagocytosed material sits directly against basal lamina (see higher magnifications); Right, PMN containing glomerular capillary with thickened area of endothelium. (B) Peritubular capillaries from wild-type B6 mice. Left, PBS treated control peritubular capillary; 3 rd from right, peritubular capillary containing mononuclear cell with necrotic and disrupted endothelium; 2 nd from right, peritubular capillary containing mononuclear cell with necrotic and disrupted endothelium and expanded subendothelial space; 1 st from right, peritubular capillary containing PMN with necrotic and disrupted endothelium and expanded subendothelial space. Images representative of 3 mice per condition. Thick scale bars = 5 μm, thin scale bars = 1 μm. Original magnification x15 000.

    Techniques Used: Transmission Electron Microscopy, In Vivo, Mouse Assay

    8) Product Images from "BDCA2/Fc?RI? Complex Signals through a Novel BCR-Like Pathway in Human Plasmacytoid Dendritic Cells"

    Article Title: BDCA2/Fc?RI? Complex Signals through a Novel BCR-Like Pathway in Human Plasmacytoid Dendritic Cells

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.0050248

    Ligation of the BDCA2/FcɛRIγ Complex Inhibits TLR-Induced Type I IFN Production (A) Transfected Namalwa cells were stimulated with CpG ODN and ligated with anti-BDCA2, anti-BCR, or control mAbs. Shown are the amounts of IFNα and IL-10 secreted by the cells stimulated CpG in a triplicate assay. (B) Purified pDCs were stimulated by CpG (top panel) or R848 (bottom panel) following anti-BDCA2 or control mAb crosslinking. The amounts of the secreted IFNα, TNFα, and IL-6 in a triplicate assay from a representative donor ( n ≥ 3) are shown. (C) Purified pDCs stimulated by CpG first and then treated with anti-BDCA2 or control mAbs at different time intervals. The amounts of the secreted IFNα, TNFα, and IL-6 from a representative donor ( n ≥ 3) are shown. (D) The amounts of type I IFN transcripts were determined by quantitative RT-PCR analysis. The expression is shown as the relative level of transcription compared with unstimulated PBMC.
    Figure Legend Snippet: Ligation of the BDCA2/FcɛRIγ Complex Inhibits TLR-Induced Type I IFN Production (A) Transfected Namalwa cells were stimulated with CpG ODN and ligated with anti-BDCA2, anti-BCR, or control mAbs. Shown are the amounts of IFNα and IL-10 secreted by the cells stimulated CpG in a triplicate assay. (B) Purified pDCs were stimulated by CpG (top panel) or R848 (bottom panel) following anti-BDCA2 or control mAb crosslinking. The amounts of the secreted IFNα, TNFα, and IL-6 in a triplicate assay from a representative donor ( n ≥ 3) are shown. (C) Purified pDCs stimulated by CpG first and then treated with anti-BDCA2 or control mAbs at different time intervals. The amounts of the secreted IFNα, TNFα, and IL-6 from a representative donor ( n ≥ 3) are shown. (D) The amounts of type I IFN transcripts were determined by quantitative RT-PCR analysis. The expression is shown as the relative level of transcription compared with unstimulated PBMC.

    Techniques Used: Ligation, Transfection, Purification, Quantitative RT-PCR, Expressing

    9) Product Images from "Nucleic acids of mammalian origin can act as endogenous ligands for Toll-like receptors and may promote systemic lupus erythematosus"

    Article Title: Nucleic acids of mammalian origin can act as endogenous ligands for Toll-like receptors and may promote systemic lupus erythematosus

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20050914

    IRS specific for TLR7 act independently of TLR9. Splenocytes from C57BL/6 (closed bars) and TLR9-deficient (hatched bars) mice were stimulated for 48 h with R848 (A) or 1018 ISS (B) alone or in the presence of IRS 661 or IRS 954 at different concentrations. IL-6 production was measured by ELISA. Values represent means ± SEM of three independent experiments. **, P
    Figure Legend Snippet: IRS specific for TLR7 act independently of TLR9. Splenocytes from C57BL/6 (closed bars) and TLR9-deficient (hatched bars) mice were stimulated for 48 h with R848 (A) or 1018 ISS (B) alone or in the presence of IRS 661 or IRS 954 at different concentrations. IL-6 production was measured by ELISA. Values represent means ± SEM of three independent experiments. **, P

    Techniques Used: Activated Clotting Time Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay

    IRS are active in vivo to block TLR7 stimulation. BALB/c mice were injected i.p. with inactive control ODN or various amounts of IRS 954 as indicated. 2 h later, mice were injected i.p. route R848. 2 h after the R848 injection, the serum was collected, and IL-12 was measured by immunoassay. Values represent means ± SEM of a group of 10 mice. ***, P
    Figure Legend Snippet: IRS are active in vivo to block TLR7 stimulation. BALB/c mice were injected i.p. with inactive control ODN or various amounts of IRS 954 as indicated. 2 h later, mice were injected i.p. route R848. 2 h after the R848 injection, the serum was collected, and IL-12 was measured by immunoassay. Values represent means ± SEM of a group of 10 mice. ***, P

    Techniques Used: In Vivo, Blocking Assay, Mouse Assay, Injection

    IRS retain similar specificity in human cells. Purified human B cells were stimulated with 0.7 μM 1018 ISS (left) or 1 μM R848 (right) alone or in the presence of IRS 869, IRS 661, or IRS 954 at 0.7 μM and 2.8 μM, respectively. IL-6 production was measured by ELISA and plotted as a percentage of 1018 ISS or R848 alone. Means of maximum levels of IL-6 were 2,904 pg/ml (R848) and 851 pg/ml (1018 ISS). Values represent means ± SEM of 10 (1018 ISS) and 6 (R848) independent donors. ***, P
    Figure Legend Snippet: IRS retain similar specificity in human cells. Purified human B cells were stimulated with 0.7 μM 1018 ISS (left) or 1 μM R848 (right) alone or in the presence of IRS 869, IRS 661, or IRS 954 at 0.7 μM and 2.8 μM, respectively. IL-6 production was measured by ELISA and plotted as a percentage of 1018 ISS or R848 alone. Means of maximum levels of IL-6 were 2,904 pg/ml (R848) and 851 pg/ml (1018 ISS). Values represent means ± SEM of 10 (1018 ISS) and 6 (R848) independent donors. ***, P

    Techniques Used: Purification, Enzyme-linked Immunosorbent Assay

    Identification of inhibitors specific for mouse TLR7 and TLR9 signaling. Splenocytes from BALB/c mice were stimulated for 48 h with (A) 0.7 μM 1018 ISS or (B) 1 μM R848 alone or in the presence of IRS 869 (open squares), IRS 661 (open circles), or IRS 954 (closed triangles) at different concentrations. IL-6 production was measured by ELISA and plotted as a percentage of 1018 ISS (A) or R848 alone (B). Maximum levels of IL-6 varied between 1,663 and 4,266 pg/ml for 1018 ISS and between 924 and 2,721 pg/ml for R848. Values represent the means ± SEM of four independent experiments.
    Figure Legend Snippet: Identification of inhibitors specific for mouse TLR7 and TLR9 signaling. Splenocytes from BALB/c mice were stimulated for 48 h with (A) 0.7 μM 1018 ISS or (B) 1 μM R848 alone or in the presence of IRS 869 (open squares), IRS 661 (open circles), or IRS 954 (closed triangles) at different concentrations. IL-6 production was measured by ELISA and plotted as a percentage of 1018 ISS (A) or R848 alone (B). Maximum levels of IL-6 varied between 1,663 and 4,266 pg/ml for 1018 ISS and between 924 and 2,721 pg/ml for R848. Values represent the means ± SEM of four independent experiments.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    10) Product Images from "A fundamental bimodal role for neuropeptide Y1 receptor in the immune system"

    Article Title: A fundamental bimodal role for neuropeptide Y1 receptor in the immune system

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20051971

    Impaired macrophage function in Y1 − / − mice. (A) Y1 +/+ T cell proliferation in an MLR with Y1 −/− stimulators. Mean counts ± SE of triplicates are shown, representative of three experiments. (B) Serum IL-12 levels in Y1 +/+ (squares) and Y1 −/− (diamonds) mice ( n = 15). (C) Production of IL-12 by Y1 +/+ (white squares) and Y1 −/− (white diamonds) peritoneal macrophages after 4 h of stimulation with LPS. Seven Y1 +/+ and nine Y1 −/− mice were analyzed and the black bars indicate the mean levels. (D) TNF production by Y1 +/+ (white bars) and Y1 −/− (black bars) peritoneal macrophages after 4 h of LPS (100 and 500 μg/ml), R848, and PGN stimulation. Five mice per group were analyzed. Production of TNF (E; left) or IL-12 (F; right) by LPS-stimulated (for 4 h) Y1 +/+ peritoneal macrophages in the presence or absence of a Y1 peptide antagonist as indicated. Mean and SD of triplicates are shown. (D–F) are representative of three independent experiments. p-values as in Fig. 1 .
    Figure Legend Snippet: Impaired macrophage function in Y1 − / − mice. (A) Y1 +/+ T cell proliferation in an MLR with Y1 −/− stimulators. Mean counts ± SE of triplicates are shown, representative of three experiments. (B) Serum IL-12 levels in Y1 +/+ (squares) and Y1 −/− (diamonds) mice ( n = 15). (C) Production of IL-12 by Y1 +/+ (white squares) and Y1 −/− (white diamonds) peritoneal macrophages after 4 h of stimulation with LPS. Seven Y1 +/+ and nine Y1 −/− mice were analyzed and the black bars indicate the mean levels. (D) TNF production by Y1 +/+ (white bars) and Y1 −/− (black bars) peritoneal macrophages after 4 h of LPS (100 and 500 μg/ml), R848, and PGN stimulation. Five mice per group were analyzed. Production of TNF (E; left) or IL-12 (F; right) by LPS-stimulated (for 4 h) Y1 +/+ peritoneal macrophages in the presence or absence of a Y1 peptide antagonist as indicated. Mean and SD of triplicates are shown. (D–F) are representative of three independent experiments. p-values as in Fig. 1 .

    Techniques Used: Mouse Assay

    11) Product Images from "Inhibitor of I?B kinase activity, BAY 11-7082, interferes with interferon regulatory factor 7 nuclear translocation and type I interferon production by plasmacytoid dendritic cells"

    Article Title: Inhibitor of I?B kinase activity, BAY 11-7082, interferes with interferon regulatory factor 7 nuclear translocation and type I interferon production by plasmacytoid dendritic cells

    Journal: Arthritis Research & Therapy

    doi: 10.1186/ar3014

    BAY11 inhibits IFN-α production from human PBMCs . (A) Human PBMCs were incubated for 24 h with BAY11 (10 -9 to 10 -5 M) or vehicle. Viable cells were analyzed by propidium iodide (PI) staining. Similar results were observed in five independent donors and the results of a representative experiment are shown. (B) Human PBMCs were preincubated for 15 minutes with BAY11 or vehicle, followed by addition of 5 μM CpG 2216, 1 μg/ml R848 and 100 μM loxoribine (loxo). After 24 h, the concentrations of IFN-α, TNF-α or IL-12 p40 in the culture supernatants were measured by ELISA. Data are shown as mean ± SEM of four independent donors. Statistical significance was determined using Mann-Whitney test (* P
    Figure Legend Snippet: BAY11 inhibits IFN-α production from human PBMCs . (A) Human PBMCs were incubated for 24 h with BAY11 (10 -9 to 10 -5 M) or vehicle. Viable cells were analyzed by propidium iodide (PI) staining. Similar results were observed in five independent donors and the results of a representative experiment are shown. (B) Human PBMCs were preincubated for 15 minutes with BAY11 or vehicle, followed by addition of 5 μM CpG 2216, 1 μg/ml R848 and 100 μM loxoribine (loxo). After 24 h, the concentrations of IFN-α, TNF-α or IL-12 p40 in the culture supernatants were measured by ELISA. Data are shown as mean ± SEM of four independent donors. Statistical significance was determined using Mann-Whitney test (* P

    Techniques Used: Incubation, Staining, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    12) Product Images from "Vaccinia Virus Protein C6 Is a Virulence Factor that Binds TBK-1 Adaptor Proteins and Inhibits Activation of IRF3 and IRF7"

    Article Title: Vaccinia Virus Protein C6 Is a Virulence Factor that Binds TBK-1 Adaptor Proteins and Inhibits Activation of IRF3 and IRF7

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002247

    C6 prevents IRF7 transactivation stimulated by TBK1-and IKKε-dependent pathways. ( A,E ) Schematic outline of a TBK1/IKKε-dependent ( A ) and –independent ( E ) pathway resulting in the activation of IRF7. ( B–D, F, G ) HEK293 cells were transfected with pFR-firefly luciferase under the control of the Gal4 promoter, a renilla luciferase transfection control, and an IRF7-Gal4 fusion plasmid. C6 expression vector and empty vector (EV) were co-transfected as indicated. In ( B ), cells were infected with Sendai virus (SeV) for 16 h. ( C, D, G ) Expression plasmids (50 ng) for signalling factors were included in the initial transfection as indicated. ( F ) HEK293 cells expressing TLR8 were stimulated with 2.5 µg/ml CL075 or 1 µM R848 for 16 h. ( H ) HEK293 cells were transfected with a firefly luciferase reporter plasmid under the control of an ISRE element, a renilla luciferase transfection control, and a C6 expression plasmid or empty vector. The ISRE was driven by co-transfection of 2 ng of IRF7-4D. In all cases, cells were harvested 24 h after the first transfection, Firefly luciferase activity was measured and normalized to renilla luciferase activity. Data are represented as mean ± SD from one representative of three experiments, each performed in triplicate. ** p
    Figure Legend Snippet: C6 prevents IRF7 transactivation stimulated by TBK1-and IKKε-dependent pathways. ( A,E ) Schematic outline of a TBK1/IKKε-dependent ( A ) and –independent ( E ) pathway resulting in the activation of IRF7. ( B–D, F, G ) HEK293 cells were transfected with pFR-firefly luciferase under the control of the Gal4 promoter, a renilla luciferase transfection control, and an IRF7-Gal4 fusion plasmid. C6 expression vector and empty vector (EV) were co-transfected as indicated. In ( B ), cells were infected with Sendai virus (SeV) for 16 h. ( C, D, G ) Expression plasmids (50 ng) for signalling factors were included in the initial transfection as indicated. ( F ) HEK293 cells expressing TLR8 were stimulated with 2.5 µg/ml CL075 or 1 µM R848 for 16 h. ( H ) HEK293 cells were transfected with a firefly luciferase reporter plasmid under the control of an ISRE element, a renilla luciferase transfection control, and a C6 expression plasmid or empty vector. The ISRE was driven by co-transfection of 2 ng of IRF7-4D. In all cases, cells were harvested 24 h after the first transfection, Firefly luciferase activity was measured and normalized to renilla luciferase activity. Data are represented as mean ± SD from one representative of three experiments, each performed in triplicate. ** p

    Techniques Used: Activation Assay, Transfection, Luciferase, Plasmid Preparation, Expressing, Infection, Cotransfection, Activity Assay

    13) Product Images from "The Imidazoquinoline Toll-Like Receptor-7/8 Agonist Hybrid-2 Potently Induces Cytokine Production by Human Newborn and Adult Leukocytes"

    Article Title: The Imidazoquinoline Toll-Like Receptor-7/8 Agonist Hybrid-2 Potently Induces Cytokine Production by Human Newborn and Adult Leukocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0134640

    Hybrid-2 is more potent than R848 in inducing TNF production by human newborn and adult MoDCs. Stimulation of (A) newborn MoDCs and (B) adult MoDCs for 24 h with Hybrid-2 or R848. Supernatants collected for TNF ELISA. Compound concentrations are shown in μM. Data are shown as mean ± SEM of n = 5. For between-agonist analyses, t test was applied to compare Hybrid-2 to R848. Statistical significance is denoted as follows: *p
    Figure Legend Snippet: Hybrid-2 is more potent than R848 in inducing TNF production by human newborn and adult MoDCs. Stimulation of (A) newborn MoDCs and (B) adult MoDCs for 24 h with Hybrid-2 or R848. Supernatants collected for TNF ELISA. Compound concentrations are shown in μM. Data are shown as mean ± SEM of n = 5. For between-agonist analyses, t test was applied to compare Hybrid-2 to R848. Statistical significance is denoted as follows: *p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Crystal structure of Hybrid-2 and R848 with human TLR8. (A) The C-atoms of Hybrid-2 are shown in green, and the residues on TLR8 are shown in cyan. A water molecule involved in mediating the interaction between the hydroxyl on N 1 -substituent of Hybrid-2 and Asp545 residue is shown as a red sphere. The hydrogen bonds are represented as dashed lines in magenta, and the bond distances are marked in black. Overlay of crystal structures of Hybrid-2 and R848 with TLR8 is shown in (B). R848 is shown in light red and its TLR8 receptor shown in grey. The electrostatic repulsion between the backbone carbonyl of Gly572 residue and ether oxygen of the C2-ethoxymethyl substituent of R848 is shown in magenta.
    Figure Legend Snippet: Crystal structure of Hybrid-2 and R848 with human TLR8. (A) The C-atoms of Hybrid-2 are shown in green, and the residues on TLR8 are shown in cyan. A water molecule involved in mediating the interaction between the hydroxyl on N 1 -substituent of Hybrid-2 and Asp545 residue is shown as a red sphere. The hydrogen bonds are represented as dashed lines in magenta, and the bond distances are marked in black. Overlay of crystal structures of Hybrid-2 and R848 with TLR8 is shown in (B). R848 is shown in light red and its TLR8 receptor shown in grey. The electrostatic repulsion between the backbone carbonyl of Gly572 residue and ether oxygen of the C2-ethoxymethyl substituent of R848 is shown in magenta.

    Techniques Used:

    Hybrid-2 is more potent and effective than R848, para-amine and meta-amine in a whole blood cytokine assay. Human newborn and adult blood was cultured for 6h with TLR 7/8 agonists R848, para-amine, meta-amine and Hybrid-2 and supernatants collected for TNF or IL-1β ELISA. Compound concentrations are shown in μM. Data are shown as mean ± SEM of n = 6–8. For between-agonist analyses, t test was applied to compare Hybrid-2 to the other compounds. For between age-group analyses, t test was applied to compare Hybrid-2 and R848 in newborns and adults. Statistical significance is denoted as follows: *p
    Figure Legend Snippet: Hybrid-2 is more potent and effective than R848, para-amine and meta-amine in a whole blood cytokine assay. Human newborn and adult blood was cultured for 6h with TLR 7/8 agonists R848, para-amine, meta-amine and Hybrid-2 and supernatants collected for TNF or IL-1β ELISA. Compound concentrations are shown in μM. Data are shown as mean ± SEM of n = 6–8. For between-agonist analyses, t test was applied to compare Hybrid-2 to the other compounds. For between age-group analyses, t test was applied to compare Hybrid-2 and R848 in newborns and adults. Statistical significance is denoted as follows: *p

    Techniques Used: Cytokine Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    NF-κB induction by TLR7/8 agonists in HEK-TLR cells. Four TLR7/8 agonists were compared. (A) Structure of R848, (B) Structure of Hybrid-2, (C) Structure of para-amine and (D) Structure of meta-amine. HEK-293 cells transfected with (E) human TLR7 and (F) TLR8 and an NF-κB-driven reporter SEAP gene were stimulated for 18–24 h with TLR agonists. The y-axis shows the level of SEAP activity in the Quanti-blue assay optical density (OD). The x-axis shows the concentration of each compound in mg/ml. Each data point represents the mean ± SD of OD at 650 nm of triplicate culture wells. HEK detection medium alone (negative control) is represented in gray. The TLR7/8 benchmark agonist R848 is represented in black; The TLR 7/8 agonists Para-amine, Meta-amine and Hybrid-2 are represented in blue, green and red respectively. Three stars indicate significance of p
    Figure Legend Snippet: NF-κB induction by TLR7/8 agonists in HEK-TLR cells. Four TLR7/8 agonists were compared. (A) Structure of R848, (B) Structure of Hybrid-2, (C) Structure of para-amine and (D) Structure of meta-amine. HEK-293 cells transfected with (E) human TLR7 and (F) TLR8 and an NF-κB-driven reporter SEAP gene were stimulated for 18–24 h with TLR agonists. The y-axis shows the level of SEAP activity in the Quanti-blue assay optical density (OD). The x-axis shows the concentration of each compound in mg/ml. Each data point represents the mean ± SD of OD at 650 nm of triplicate culture wells. HEK detection medium alone (negative control) is represented in gray. The TLR7/8 benchmark agonist R848 is represented in black; The TLR 7/8 agonists Para-amine, Meta-amine and Hybrid-2 are represented in blue, green and red respectively. Three stars indicate significance of p

    Techniques Used: Transfection, Activity Assay, Concentration Assay, Negative Control

    14) Product Images from "The Imidazoquinoline Toll-Like Receptor-7/8 Agonist Hybrid-2 Potently Induces Cytokine Production by Human Newborn and Adult Leukocytes"

    Article Title: The Imidazoquinoline Toll-Like Receptor-7/8 Agonist Hybrid-2 Potently Induces Cytokine Production by Human Newborn and Adult Leukocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0134640

    Hybrid-2 is more potent than R848 in inducing TNF production by human newborn and adult MoDCs. Stimulation of (A) newborn MoDCs and (B) adult MoDCs for 24 h with Hybrid-2 or R848. Supernatants collected for TNF ELISA. Compound concentrations are shown in μM. Data are shown as mean ± SEM of n = 5. For between-agonist analyses, t test was applied to compare Hybrid-2 to R848. Statistical significance is denoted as follows: *p
    Figure Legend Snippet: Hybrid-2 is more potent than R848 in inducing TNF production by human newborn and adult MoDCs. Stimulation of (A) newborn MoDCs and (B) adult MoDCs for 24 h with Hybrid-2 or R848. Supernatants collected for TNF ELISA. Compound concentrations are shown in μM. Data are shown as mean ± SEM of n = 5. For between-agonist analyses, t test was applied to compare Hybrid-2 to R848. Statistical significance is denoted as follows: *p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Crystal structure of Hybrid-2 and R848 with human TLR8. (A) The C-atoms of Hybrid-2 are shown in green, and the residues on TLR8 are shown in cyan. A water molecule involved in mediating the interaction between the hydroxyl on N 1 -substituent of Hybrid-2 and Asp545 residue is shown as a red sphere. The hydrogen bonds are represented as dashed lines in magenta, and the bond distances are marked in black. Overlay of crystal structures of Hybrid-2 and R848 with TLR8 is shown in (B). R848 is shown in light red and its TLR8 receptor shown in grey. The electrostatic repulsion between the backbone carbonyl of Gly572 residue and ether oxygen of the C2-ethoxymethyl substituent of R848 is shown in magenta.
    Figure Legend Snippet: Crystal structure of Hybrid-2 and R848 with human TLR8. (A) The C-atoms of Hybrid-2 are shown in green, and the residues on TLR8 are shown in cyan. A water molecule involved in mediating the interaction between the hydroxyl on N 1 -substituent of Hybrid-2 and Asp545 residue is shown as a red sphere. The hydrogen bonds are represented as dashed lines in magenta, and the bond distances are marked in black. Overlay of crystal structures of Hybrid-2 and R848 with TLR8 is shown in (B). R848 is shown in light red and its TLR8 receptor shown in grey. The electrostatic repulsion between the backbone carbonyl of Gly572 residue and ether oxygen of the C2-ethoxymethyl substituent of R848 is shown in magenta.

    Techniques Used:

    Hybrid-2 is more potent and effective than R848, para-amine and meta-amine in a whole blood cytokine assay. Human newborn and adult blood was cultured for 6h with TLR 7/8 agonists R848, para-amine, meta-amine and Hybrid-2 and supernatants collected for TNF or IL-1β ELISA. Compound concentrations are shown in μM. Data are shown as mean ± SEM of n = 6–8. For between-agonist analyses, t test was applied to compare Hybrid-2 to the other compounds. For between age-group analyses, t test was applied to compare Hybrid-2 and R848 in newborns and adults. Statistical significance is denoted as follows: *p
    Figure Legend Snippet: Hybrid-2 is more potent and effective than R848, para-amine and meta-amine in a whole blood cytokine assay. Human newborn and adult blood was cultured for 6h with TLR 7/8 agonists R848, para-amine, meta-amine and Hybrid-2 and supernatants collected for TNF or IL-1β ELISA. Compound concentrations are shown in μM. Data are shown as mean ± SEM of n = 6–8. For between-agonist analyses, t test was applied to compare Hybrid-2 to the other compounds. For between age-group analyses, t test was applied to compare Hybrid-2 and R848 in newborns and adults. Statistical significance is denoted as follows: *p

    Techniques Used: Cytokine Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    NF-κB induction by TLR7/8 agonists in HEK-TLR cells. Four TLR7/8 agonists were compared. (A) Structure of R848, (B) Structure of Hybrid-2, (C) Structure of para-amine and (D) Structure of meta-amine. HEK-293 cells transfected with (E) human TLR7 and (F) TLR8 and an NF-κB-driven reporter SEAP gene were stimulated for 18–24 h with TLR agonists. The y-axis shows the level of SEAP activity in the Quanti-blue assay optical density (OD). The x-axis shows the concentration of each compound in mg/ml. Each data point represents the mean ± SD of OD at 650 nm of triplicate culture wells. HEK detection medium alone (negative control) is represented in gray. The TLR7/8 benchmark agonist R848 is represented in black; The TLR 7/8 agonists Para-amine, Meta-amine and Hybrid-2 are represented in blue, green and red respectively. Three stars indicate significance of p
    Figure Legend Snippet: NF-κB induction by TLR7/8 agonists in HEK-TLR cells. Four TLR7/8 agonists were compared. (A) Structure of R848, (B) Structure of Hybrid-2, (C) Structure of para-amine and (D) Structure of meta-amine. HEK-293 cells transfected with (E) human TLR7 and (F) TLR8 and an NF-κB-driven reporter SEAP gene were stimulated for 18–24 h with TLR agonists. The y-axis shows the level of SEAP activity in the Quanti-blue assay optical density (OD). The x-axis shows the concentration of each compound in mg/ml. Each data point represents the mean ± SD of OD at 650 nm of triplicate culture wells. HEK detection medium alone (negative control) is represented in gray. The TLR7/8 benchmark agonist R848 is represented in black; The TLR 7/8 agonists Para-amine, Meta-amine and Hybrid-2 are represented in blue, green and red respectively. Three stars indicate significance of p

    Techniques Used: Transfection, Activity Assay, Concentration Assay, Negative Control

    15) Product Images from "slan/M-DC8+ cells constitute a distinct subset of dendritic cells in human tonsils"

    Article Title: slan/M-DC8+ cells constitute a distinct subset of dendritic cells in human tonsils

    Journal: Oncotarget

    doi:

    Tonsil slan/M-DC8 + DCs, CD1c + DCs, CD14 + CD11b + monocytes/macrophages and CD303 + pDCs produce TNFα but not IL-12p70 a. - h. Tonsil cell suspensions were incubated with or without 100 U ml −1 IFNγ plus either 100 ng ml −1 LPS or 5 μM R848, either for 4 h (to detect TNFα secretion, a-d), or for 12 h, after a 6 h pre-incubation (to detect IL-12p70 secretion, e-h). Graphs show TNFα-secreting slan/M-DC8 + DCs a. , CD1c + DCs b. , CD14 + CD11b + monocytes/macrophages c. and CD303 + pDCs d. , or IL-12p70-secreting slan/M-DC8 + DCs e. , CD1c + DCs f. , CD14 + CD11b + monocytes/macrophages g. and CD303 + pDCs h. . The graphs show the mean of cytokine secreting cells (as percentage of each cell population) calculated from 4 experiments. i. , j. 2.5×10 4 100 μl −1 blood (gray bars), or 5-d TDCM-conditioned (purple bars), slan/M-DC8 + cells were incubated for 24 h with or without 100 U ml −1 IFNγ plus either 100 ng ml −1 LPS or 5 μM R848 to measure the levels of TNFα (i) and IL-12p70 (j) in cell free supernatants by ELISA ( n = 5-7). k. Blood slan/M-DC8 + cells were either immediately stimulated with 100 U ml −1 IFNγ plus 100 ng ml −1 LPS for 24 h, or conditioned in TDCM, in the presence or absence of 10 μg ml −1 anti-IL-10 or IgG2a isotype control mAbs. After 5 d of incubation, TDCM-conditioned slan/M-DC8 + cells were stimulated with IFNγ plus LPS for 24 h. IL-12p70 was then measured in cell-free supernatants by ELISA. Graph show the loss of IL-12p70-producing capacity (in %) by TDCM-conditioned slan/M-DC8 + cells after IFNγ plus LPS stimulation (in the absence or the presence of neutralizing mAbs), as compared to blood slan/M-DC8 + cells incubated with IFNγ plus LPS.
    Figure Legend Snippet: Tonsil slan/M-DC8 + DCs, CD1c + DCs, CD14 + CD11b + monocytes/macrophages and CD303 + pDCs produce TNFα but not IL-12p70 a. - h. Tonsil cell suspensions were incubated with or without 100 U ml −1 IFNγ plus either 100 ng ml −1 LPS or 5 μM R848, either for 4 h (to detect TNFα secretion, a-d), or for 12 h, after a 6 h pre-incubation (to detect IL-12p70 secretion, e-h). Graphs show TNFα-secreting slan/M-DC8 + DCs a. , CD1c + DCs b. , CD14 + CD11b + monocytes/macrophages c. and CD303 + pDCs d. , or IL-12p70-secreting slan/M-DC8 + DCs e. , CD1c + DCs f. , CD14 + CD11b + monocytes/macrophages g. and CD303 + pDCs h. . The graphs show the mean of cytokine secreting cells (as percentage of each cell population) calculated from 4 experiments. i. , j. 2.5×10 4 100 μl −1 blood (gray bars), or 5-d TDCM-conditioned (purple bars), slan/M-DC8 + cells were incubated for 24 h with or without 100 U ml −1 IFNγ plus either 100 ng ml −1 LPS or 5 μM R848 to measure the levels of TNFα (i) and IL-12p70 (j) in cell free supernatants by ELISA ( n = 5-7). k. Blood slan/M-DC8 + cells were either immediately stimulated with 100 U ml −1 IFNγ plus 100 ng ml −1 LPS for 24 h, or conditioned in TDCM, in the presence or absence of 10 μg ml −1 anti-IL-10 or IgG2a isotype control mAbs. After 5 d of incubation, TDCM-conditioned slan/M-DC8 + cells were stimulated with IFNγ plus LPS for 24 h. IL-12p70 was then measured in cell-free supernatants by ELISA. Graph show the loss of IL-12p70-producing capacity (in %) by TDCM-conditioned slan/M-DC8 + cells after IFNγ plus LPS stimulation (in the absence or the presence of neutralizing mAbs), as compared to blood slan/M-DC8 + cells incubated with IFNγ plus LPS.

    Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay

    16) Product Images from "TLR7 and TLR8 expression increases tumor cell proliferation and promotes chemoresistance in human pancreatic cancer"

    Article Title: TLR7 and TLR8 expression increases tumor cell proliferation and promotes chemoresistance in human pancreatic cancer

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2015.3069

    NF-κB and COX-2 gene expression in response to R848 stimulation of TLR7 + and TLR8 + PANC1 cells. (A and B) Stimulation of TLR7 + and TLR8 + PANC1 cells with R848 resulted in significantly increased gene expression levels of NF-κB ( * P
    Figure Legend Snippet: NF-κB and COX-2 gene expression in response to R848 stimulation of TLR7 + and TLR8 + PANC1 cells. (A and B) Stimulation of TLR7 + and TLR8 + PANC1 cells with R848 resulted in significantly increased gene expression levels of NF-κB ( * P

    Techniques Used: Expressing

    17) Product Images from "Dendritic cell-derived VEGF-A plays a role in inflammatory angiogenesis of human secondary lymphoid organs and is driven by the coordinated activation of multiple transcription factors"

    Article Title: Dendritic cell-derived VEGF-A plays a role in inflammatory angiogenesis of human secondary lymphoid organs and is driven by the coordinated activation of multiple transcription factors

    Journal: Oncotarget

    doi: 10.18632/oncotarget.9684

    Human myeloid DCs produce VEGF-A in response to a variety of pro-inflammatory stimuli, provided PGE 2 is present in the microenvironment A. , B. DCs were stimulated for 24 hours with TLR-ligands PAM 3 CSK 4 (TLR1/2, 100 ng/ml), FSL-1 (TLR2/6, 100 ng/ml), Poly I:C (TLR3, 25 μg/ml), LPS (TLR4, 100 ng/ml) and R848 (TLR7 and TLR8, 5 μg/ml), Heat-killed S.aureus (specific for TLR2; 1:10 DC/bacteria ratio), E. coli (specific for TLR4; 1:10 DC/bacteria ratio), β-glucan (10 μg/ml), Curdlan (10 μg/ml), heat-killed C.albicans (specific for C-type lectins; 1:10 DC/fungi ratio), IL-1β (20 ng/ml), TNF-α (20 ng/ml), and necrotic cells (1:2 DC/necrotic cells ratio) in the presence or absence of PGE 2 (10 μM). VEGF-A production was evaluated in cell-free supernatants by ELISA. Data are expressed as mean + SEM ( n = 4); * p
    Figure Legend Snippet: Human myeloid DCs produce VEGF-A in response to a variety of pro-inflammatory stimuli, provided PGE 2 is present in the microenvironment A. , B. DCs were stimulated for 24 hours with TLR-ligands PAM 3 CSK 4 (TLR1/2, 100 ng/ml), FSL-1 (TLR2/6, 100 ng/ml), Poly I:C (TLR3, 25 μg/ml), LPS (TLR4, 100 ng/ml) and R848 (TLR7 and TLR8, 5 μg/ml), Heat-killed S.aureus (specific for TLR2; 1:10 DC/bacteria ratio), E. coli (specific for TLR4; 1:10 DC/bacteria ratio), β-glucan (10 μg/ml), Curdlan (10 μg/ml), heat-killed C.albicans (specific for C-type lectins; 1:10 DC/fungi ratio), IL-1β (20 ng/ml), TNF-α (20 ng/ml), and necrotic cells (1:2 DC/necrotic cells ratio) in the presence or absence of PGE 2 (10 μM). VEGF-A production was evaluated in cell-free supernatants by ELISA. Data are expressed as mean + SEM ( n = 4); * p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    TLR-4-dependent expression of VEGF-A depends on endogenous PGE 2 production A. DCs were stimulated with PAM 3 CSK 4 (100 ng/ml), FSL-1 (100 ng/ml), Poly I:C (25 μg/ml), LPS (100 ng/ml) and R848 (5 μg/ml) for 24 hours. The production of VEGF-A and PGE 2 was evaluated in cell-free supernatants by ELISA or EIA, respectively. Results are expressed as mean + SEM ( n = 4); * p
    Figure Legend Snippet: TLR-4-dependent expression of VEGF-A depends on endogenous PGE 2 production A. DCs were stimulated with PAM 3 CSK 4 (100 ng/ml), FSL-1 (100 ng/ml), Poly I:C (25 μg/ml), LPS (100 ng/ml) and R848 (5 μg/ml) for 24 hours. The production of VEGF-A and PGE 2 was evaluated in cell-free supernatants by ELISA or EIA, respectively. Results are expressed as mean + SEM ( n = 4); * p

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay

    18) Product Images from "Naltrexone Inhibits IL-6 and TNFα Production in Human Immune Cell Subsets following Stimulation with Ligands for Intracellular Toll-Like Receptors"

    Article Title: Naltrexone Inhibits IL-6 and TNFα Production in Human Immune Cell Subsets following Stimulation with Ligands for Intracellular Toll-Like Receptors

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.00809

    Intracellular cytokine staining for TNFα and IL-6 in monocytes and plasmacytoid dendritic cells. 1 × 10 6 peripheral blood mononuclear cells (PBMC) were incubated with either LPS 1 ng/ml (A,C) , R848 1µM (B) , or CpG 1µM (D) and 200 µM naltrexone for 6 h in the presence of brefeldin A for 4 of those hours. After 6 h, PBMC were stained using antibody panel shown in Figure S4 in Supplementary Material and stained for either intracellular IL-6 or TNF-α. Results show the mean fluorescence intensity (MFI) of IL-6 or TNF-α within that subset from 5 donors. Histograms are representative of 5 independent experiments.
    Figure Legend Snippet: Intracellular cytokine staining for TNFα and IL-6 in monocytes and plasmacytoid dendritic cells. 1 × 10 6 peripheral blood mononuclear cells (PBMC) were incubated with either LPS 1 ng/ml (A,C) , R848 1µM (B) , or CpG 1µM (D) and 200 µM naltrexone for 6 h in the presence of brefeldin A for 4 of those hours. After 6 h, PBMC were stained using antibody panel shown in Figure S4 in Supplementary Material and stained for either intracellular IL-6 or TNF-α. Results show the mean fluorescence intensity (MFI) of IL-6 or TNF-α within that subset from 5 donors. Histograms are representative of 5 independent experiments.

    Techniques Used: Staining, Incubation, Fluorescence

    Naltrexone inhibits IL-6 production after toll-like receptor (TLR)7/8 and TLR9 stimulation but not after TLR4 or IL-1 stimulation. 1 × 10 6 peripheral blood mononuclear cells were incubated with (A) 1 ng/ml LPS (TLR4-L), (B) 1µM R848 (TLR7/8-L), (C) 1µM CpG (TLR9-L), (D) 100 ng/ml IL-1 (IL-1R) in the presence or absence of 1–200µM naltrexone for 24 h. Cell-free supernatants were collected and analyzed for IL-6 by ELISA. Data show the mean; SD values are shown and were analyzed using an one-way ANOVA and Tukey’s multiple comparison test ( n = 5 TLR ligand experiments and n = 3 IL-1). * p
    Figure Legend Snippet: Naltrexone inhibits IL-6 production after toll-like receptor (TLR)7/8 and TLR9 stimulation but not after TLR4 or IL-1 stimulation. 1 × 10 6 peripheral blood mononuclear cells were incubated with (A) 1 ng/ml LPS (TLR4-L), (B) 1µM R848 (TLR7/8-L), (C) 1µM CpG (TLR9-L), (D) 100 ng/ml IL-1 (IL-1R) in the presence or absence of 1–200µM naltrexone for 24 h. Cell-free supernatants were collected and analyzed for IL-6 by ELISA. Data show the mean; SD values are shown and were analyzed using an one-way ANOVA and Tukey’s multiple comparison test ( n = 5 TLR ligand experiments and n = 3 IL-1). * p

    Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay

    Naltrexone inhibits IL-6 production in isolated monocytes and B cells after toll-like receptor (TLR)7/8 and TLR9 stimulation, respectively, but has no effect on IL-6 production in isolated monocytes after TLR4 stimulation. (A) CD14+ monocytes were isolated from peripheral blood mononuclear cells (PBMC) using magnetic bead isolation. 1 × 10 5 CD14+ cells were incubated with 1 ng/ml LPS (TLR4-L) or 1µM R848 (TLR7/8-L), in the presence or absence of 200µM naltrexone for 24 h. Cell-free supernatants were collected and analyzed for IL-6 by ELISA. (B) CD19+ B cells were isolated from PBMC using magnetic bead isolation. 10 5 B cells were incubated with 1µM CpG or 3 µg/ml CD40-L and 20 ng/ml IL-4, with or without 200µM naltrexone for 24 h. IL-6 production was measured in cell-free supernatants by ELISA. Data show the mean and SD values ( n = 4).
    Figure Legend Snippet: Naltrexone inhibits IL-6 production in isolated monocytes and B cells after toll-like receptor (TLR)7/8 and TLR9 stimulation, respectively, but has no effect on IL-6 production in isolated monocytes after TLR4 stimulation. (A) CD14+ monocytes were isolated from peripheral blood mononuclear cells (PBMC) using magnetic bead isolation. 1 × 10 5 CD14+ cells were incubated with 1 ng/ml LPS (TLR4-L) or 1µM R848 (TLR7/8-L), in the presence or absence of 200µM naltrexone for 24 h. Cell-free supernatants were collected and analyzed for IL-6 by ELISA. (B) CD19+ B cells were isolated from PBMC using magnetic bead isolation. 10 5 B cells were incubated with 1µM CpG or 3 µg/ml CD40-L and 20 ng/ml IL-4, with or without 200µM naltrexone for 24 h. IL-6 production was measured in cell-free supernatants by ELISA. Data show the mean and SD values ( n = 4).

    Techniques Used: Isolation, Incubation, Enzyme-linked Immunosorbent Assay

    Toll-like receptor ligand (TLR-L) and naltrexone does not affect the viability of peripheral blood mononuclear cells (PBMC). (A) 1 × 10 6 PBMC were incubated with 1–200µM naltrexone for 24 h before percentage viability was assessed using trypan blue exclusion. (B,C) 1 × 10 6 PBMC were incubated with 1 ng/ml LPS (TLR4-L), 1µM R848 (TLR7/8-L), 1µM CpG (TLR9-L), and 200µM naltrexone for 24 h. PBMC were incubated with annexin V and 7AAD before being analyzed by flow cytometry. Figure 4 B shows the gating strategy, and Figure 4 C shows results from 4 donors. AV−7AAD− are viable cells, AV+7AAD− are in early apoptosis, and AV+7AAD+ are in late apoptosis.
    Figure Legend Snippet: Toll-like receptor ligand (TLR-L) and naltrexone does not affect the viability of peripheral blood mononuclear cells (PBMC). (A) 1 × 10 6 PBMC were incubated with 1–200µM naltrexone for 24 h before percentage viability was assessed using trypan blue exclusion. (B,C) 1 × 10 6 PBMC were incubated with 1 ng/ml LPS (TLR4-L), 1µM R848 (TLR7/8-L), 1µM CpG (TLR9-L), and 200µM naltrexone for 24 h. PBMC were incubated with annexin V and 7AAD before being analyzed by flow cytometry. Figure 4 B shows the gating strategy, and Figure 4 C shows results from 4 donors. AV−7AAD− are viable cells, AV+7AAD− are in early apoptosis, and AV+7AAD+ are in late apoptosis.

    Techniques Used: Incubation, Flow Cytometry, Cytometry

    19) Product Images from "HRS plays an important role for TLR7 signaling to orchestrate inflammation and innate immunity upon EV71 infection"

    Article Title: HRS plays an important role for TLR7 signaling to orchestrate inflammation and innate immunity upon EV71 infection

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006585

    HRS interacts with TLR7 and TAB1 in the TLR7 complex during viral infection. ( A and B ) Macrophages derived from THP-1 cells were stimulated with R848 (100 ng/ml) ( A ) or EV71 (MOI = 5) ( B ) for various time periods as indicated. Cell lysates were prepared and immunoprecipitated with rabbit anti-TLR7 or rabbit anti-IgG antibodies. Immunoprecipitates were assayed using Western blotting analysis with specific antibodies as indicated. ( C and D ) Macrophages were stimulated with R848 for 15 min, or infected with EV71 or SeV for 4 h, and then probed with indicated antibodies before confocal microscopy. Bar = 20 μm. ( E ) Macrophages were treated with R848 (100 ng/ml) for 30 min. Cells were probed with indicated antibodies and DAPI stain before confocal microscopy. Bar = 20 μm. ( F ) Mice were infected with EV71 and sacrificed at indicated period. Mice spleens were subjected to immunofluorescence (IF) staining with HRS (Red), TLR7 (Green), and CD68 (Blue) antibodies. Bar = 20 μm.
    Figure Legend Snippet: HRS interacts with TLR7 and TAB1 in the TLR7 complex during viral infection. ( A and B ) Macrophages derived from THP-1 cells were stimulated with R848 (100 ng/ml) ( A ) or EV71 (MOI = 5) ( B ) for various time periods as indicated. Cell lysates were prepared and immunoprecipitated with rabbit anti-TLR7 or rabbit anti-IgG antibodies. Immunoprecipitates were assayed using Western blotting analysis with specific antibodies as indicated. ( C and D ) Macrophages were stimulated with R848 for 15 min, or infected with EV71 or SeV for 4 h, and then probed with indicated antibodies before confocal microscopy. Bar = 20 μm. ( E ) Macrophages were treated with R848 (100 ng/ml) for 30 min. Cells were probed with indicated antibodies and DAPI stain before confocal microscopy. Bar = 20 μm. ( F ) Mice were infected with EV71 and sacrificed at indicated period. Mice spleens were subjected to immunofluorescence (IF) staining with HRS (Red), TLR7 (Green), and CD68 (Blue) antibodies. Bar = 20 μm.

    Techniques Used: Infection, Derivative Assay, Immunoprecipitation, Western Blot, Confocal Microscopy, Staining, Mouse Assay, Immunofluorescence

    HRS promotes the production of interferons mediated by TLR7 signaling. ( A and B ) Macrophages were transfected with siR-HRS or siR-NC and then stimulated with R848 ( A ) or infected with EV71 ( B ) for various time periods as indicated. The proteins in the cell lysates were detected using Western blotting analyses with indicated antibodies. ( C ) Macrophages were transfected with siR-HRS or siR-NC and then stimulated with R848. IFN-β and IFN-λ1 mRNA levels were determined using qPCR. ( D ) RD, 293T, and A549 cells were transfected with pcDNA3.1(-)-Myc or pcDNA3.1(-)-HRS-Myc and infected with EV71, SeV or IAV for 12 h, IFN-β and IFN-λ1 mRNA levels were determined using qPCR. **, P
    Figure Legend Snippet: HRS promotes the production of interferons mediated by TLR7 signaling. ( A and B ) Macrophages were transfected with siR-HRS or siR-NC and then stimulated with R848 ( A ) or infected with EV71 ( B ) for various time periods as indicated. The proteins in the cell lysates were detected using Western blotting analyses with indicated antibodies. ( C ) Macrophages were transfected with siR-HRS or siR-NC and then stimulated with R848. IFN-β and IFN-λ1 mRNA levels were determined using qPCR. ( D ) RD, 293T, and A549 cells were transfected with pcDNA3.1(-)-Myc or pcDNA3.1(-)-HRS-Myc and infected with EV71, SeV or IAV for 12 h, IFN-β and IFN-λ1 mRNA levels were determined using qPCR. **, P

    Techniques Used: Transfection, Infection, Western Blot, Real-time Polymerase Chain Reaction

    TLR7 expression correlates with HRS expression during EV71 infection. ( A ) Stable HEK293T/TLR7/NF-κB reporter cells were transfected with siR-NC, siR-HRS#1 and siR-HRS#2, treated with R848 or TNFα, and infected with EV71 or SeV. NF-κB activities were determined by luciferase activity assays. The HRS and β-actin proteins expressed in the treated cells were detected by Western blotting. The indicated band intensity represents as fold changes to internal control by using Image J software analysis. Results were expressed as fold induction relative to control. ns, non-significant; *, P
    Figure Legend Snippet: TLR7 expression correlates with HRS expression during EV71 infection. ( A ) Stable HEK293T/TLR7/NF-κB reporter cells were transfected with siR-NC, siR-HRS#1 and siR-HRS#2, treated with R848 or TNFα, and infected with EV71 or SeV. NF-κB activities were determined by luciferase activity assays. The HRS and β-actin proteins expressed in the treated cells were detected by Western blotting. The indicated band intensity represents as fold changes to internal control by using Image J software analysis. Results were expressed as fold induction relative to control. ns, non-significant; *, P

    Techniques Used: Expressing, Infection, Transfection, Luciferase, Activity Assay, Western Blot, Software

    HRS is important for TLR7-mediated proinflammatory cytokines production during viral infection. ( A and B ) Macrophages were transfected with siR-HRS or siR-NC and then stimulated with R848 (A) or infected with EV71 ( B ) for various time periods as indicated. Cell extracts were prepared and the proteins in the cell lysates were detected using Western blotting analyses with corresponding antibodies. The indicated band intensity represents as fold changes to internal control GAPDH. ( C and D ) Macrophages were transfected with siR-NC or siR-HRS and stimulated with R848 or infected with EV71 or SeV. The levels of CSF3, IL-1β, and IL-6 mRNAs and proteins were determined using qPCR ( C ) and ELISA ( D ), respectively. **, P
    Figure Legend Snippet: HRS is important for TLR7-mediated proinflammatory cytokines production during viral infection. ( A and B ) Macrophages were transfected with siR-HRS or siR-NC and then stimulated with R848 (A) or infected with EV71 ( B ) for various time periods as indicated. Cell extracts were prepared and the proteins in the cell lysates were detected using Western blotting analyses with corresponding antibodies. The indicated band intensity represents as fold changes to internal control GAPDH. ( C and D ) Macrophages were transfected with siR-NC or siR-HRS and stimulated with R848 or infected with EV71 or SeV. The levels of CSF3, IL-1β, and IL-6 mRNAs and proteins were determined using qPCR ( C ) and ELISA ( D ), respectively. **, P

    Techniques Used: Infection, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    EV71 induces the production of proinflammatory cytokines via stimulating TLR7 signaling and NF-κB activity. ( A ) The monocytic THP-1 cells were treated with individual kinase inhibitors for 6 h, as indicated, and infected with EV71 (MOI = 5) for 12 h. The levels of CSF3 , IL-1β and IL-6 mRNAs were measured by qPCR. ( B ) THP-1 cells were infected with EV71 (MOI = 5) for 12 h and total mRNA extracts were prepared. The mRNA levels of TLRs expressed in the cells were determined by qPCR. ( C ) THP-1 cells were transfected with plasmid transcribing siRNA specific to TLR7 (shTLR7) or its control (shGFP) and then infected with EV71 (MOI = 5) for 12 h. Cytokine mRNA levels were determined by qPCR. (D ) Mouse bone marrow-derived macrophages (BMDMs) isolated from TLR7 wild-type (WT) or TLR7 knock-out (TLR7 -/- ) mice were infected with EV71 (MOI = 5) for 24 h. TLR7 protein was determined by Western blotting. Mouse CSF3 , IL-1β , and IL-6 mRNAs were measured by qPCR. ( E and F ) THP-1 cells were co-transfected with NF-κB reporter plasmid and siRNA specific to TLR7 (shTLR7) or negative control (shGFP) and then infected with EV71 (MOI = 5) for 8 h. NF-κB activity was determined by luciferase activity assay ( E ). The endogenous protein levels were determined by western blotting with indicated antibodies ( F ). ( G ) HEK293T cells were transfected with pFlag2B (vector) or pFlag-TLR7 and infected with EV71 (MOI = 5). TLR7, p65, P-p65, EV71 VP1, and GAPDH proteins expressed in the treated cells were detected by Western blotting. ( H and I ) HEK293T cells were co-transfected with NF-κB reporter and pTLR7, pTLR7 (Y892A) or vector ( H ), or plasmids carrying shRNAs specific to indicated genes ( I ), and then stimulated with R848, or infected with EV71 (MOI = 5) or SeV (HA unit = 5) for 8 h, respectively. NF-κB activities were determined by luciferase activity assay. All qPCR assays used GAPDH mRNA as an internal control. Results were expressed as fold induction relative to control. ns, non-significant; *, P
    Figure Legend Snippet: EV71 induces the production of proinflammatory cytokines via stimulating TLR7 signaling and NF-κB activity. ( A ) The monocytic THP-1 cells were treated with individual kinase inhibitors for 6 h, as indicated, and infected with EV71 (MOI = 5) for 12 h. The levels of CSF3 , IL-1β and IL-6 mRNAs were measured by qPCR. ( B ) THP-1 cells were infected with EV71 (MOI = 5) for 12 h and total mRNA extracts were prepared. The mRNA levels of TLRs expressed in the cells were determined by qPCR. ( C ) THP-1 cells were transfected with plasmid transcribing siRNA specific to TLR7 (shTLR7) or its control (shGFP) and then infected with EV71 (MOI = 5) for 12 h. Cytokine mRNA levels were determined by qPCR. (D ) Mouse bone marrow-derived macrophages (BMDMs) isolated from TLR7 wild-type (WT) or TLR7 knock-out (TLR7 -/- ) mice were infected with EV71 (MOI = 5) for 24 h. TLR7 protein was determined by Western blotting. Mouse CSF3 , IL-1β , and IL-6 mRNAs were measured by qPCR. ( E and F ) THP-1 cells were co-transfected with NF-κB reporter plasmid and siRNA specific to TLR7 (shTLR7) or negative control (shGFP) and then infected with EV71 (MOI = 5) for 8 h. NF-κB activity was determined by luciferase activity assay ( E ). The endogenous protein levels were determined by western blotting with indicated antibodies ( F ). ( G ) HEK293T cells were transfected with pFlag2B (vector) or pFlag-TLR7 and infected with EV71 (MOI = 5). TLR7, p65, P-p65, EV71 VP1, and GAPDH proteins expressed in the treated cells were detected by Western blotting. ( H and I ) HEK293T cells were co-transfected with NF-κB reporter and pTLR7, pTLR7 (Y892A) or vector ( H ), or plasmids carrying shRNAs specific to indicated genes ( I ), and then stimulated with R848, or infected with EV71 (MOI = 5) or SeV (HA unit = 5) for 8 h, respectively. NF-κB activities were determined by luciferase activity assay. All qPCR assays used GAPDH mRNA as an internal control. Results were expressed as fold induction relative to control. ns, non-significant; *, P

    Techniques Used: Activity Assay, Infection, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Derivative Assay, Isolation, Knock-Out, Mouse Assay, Western Blot, Negative Control, Luciferase

    HRS interacts with TLR7 to facilitate signaling activation. ( A ) Macrophages were harvested after treated without or with R848 (100 ng/ml) for 30 min. Samples were homogenized and resolved by ultracentrifugation. Fractions were collected from the top of the gradient and analyzed for the distribution of indicated intracellular proteins by Western blotting. ( B ) Macrophages were treated with R848 (100 ng/ml) for indicated time and then applied for cell fraction. Separated cell fractions were subjected to immunoprecipitation (IP) and analyzed by Western blotting. S100K and P100K indicate supernatant and precipitate of cell fractions after 100,000 g ultracentrifugation, respectively. Indicated band intensity represents as fold changes to immunoprecipitated TLR7 by using Image J software analysis. ( C ) HEK293T cells were co-transfected with plasmid expressing Flag-TLR7 and HRS-Myc or FVYE domain deleted HRS-Myc (dFYVE-Myc). Whole cell lysates were prepared and immunoprecipitated and then analyzed by Western blotting. ( D ) HEK293T/TLR7/NF-κB reporter cells were transfected with plasmids expressing HRS or HRS deletion. NF-κB activities were determined using luciferase activity assays. The proteins expressed in the treated cells were detected by Western blotting. *, P
    Figure Legend Snippet: HRS interacts with TLR7 to facilitate signaling activation. ( A ) Macrophages were harvested after treated without or with R848 (100 ng/ml) for 30 min. Samples were homogenized and resolved by ultracentrifugation. Fractions were collected from the top of the gradient and analyzed for the distribution of indicated intracellular proteins by Western blotting. ( B ) Macrophages were treated with R848 (100 ng/ml) for indicated time and then applied for cell fraction. Separated cell fractions were subjected to immunoprecipitation (IP) and analyzed by Western blotting. S100K and P100K indicate supernatant and precipitate of cell fractions after 100,000 g ultracentrifugation, respectively. Indicated band intensity represents as fold changes to immunoprecipitated TLR7 by using Image J software analysis. ( C ) HEK293T cells were co-transfected with plasmid expressing Flag-TLR7 and HRS-Myc or FVYE domain deleted HRS-Myc (dFYVE-Myc). Whole cell lysates were prepared and immunoprecipitated and then analyzed by Western blotting. ( D ) HEK293T/TLR7/NF-κB reporter cells were transfected with plasmids expressing HRS or HRS deletion. NF-κB activities were determined using luciferase activity assays. The proteins expressed in the treated cells were detected by Western blotting. *, P

    Techniques Used: Activation Assay, Western Blot, Immunoprecipitation, Software, Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay

    EV71 infection induces the productions of inflammatory cytokines in vivo and in vitro . ( A ) THP-1 derived macrophages were infected with EV71 (MOI = 5) or UV-inactivated EV71 for 12 h. The amount of 30 cytokines in cell supernatants was measured by Human Cytokine ELISA. Each cytokines concentrations in cell supernatants were reproducible and detectable. Data are shown as fold changes of protein expression compared to mock samples. Proinflammatory cytokines are marked by an asterisk. ( B and C ) CSF3, IL-1β, and IL-6 proteins in sera of EV71-infected patients (n = 40) and healthy individuals (n = 36) ( B ), or in sera of EV71-infected and mock-infected mice (each group, n = 7) ( C ) were measured by ELISA. ( D – F ) THP-1 cells ( D ), macrophages (differentiated from THP-1 cells) ( E ), and PBMCs (isolated from peripheral blood samples of healthy individuals) ( F ) were treated with 100 ng/ml R848 for 12 h or infected with UV-inactivated EV71 (Inact. EV71) or EV71 (MOI = 2) for 24 h. The supernatants of treated cells were collected and CSF3, IL-1β, and IL-6 levels were measured by ELISA. Data are shown as mean ± SD and correspond to a representative experiment out of three performed. ns, non-significant; *, P
    Figure Legend Snippet: EV71 infection induces the productions of inflammatory cytokines in vivo and in vitro . ( A ) THP-1 derived macrophages were infected with EV71 (MOI = 5) or UV-inactivated EV71 for 12 h. The amount of 30 cytokines in cell supernatants was measured by Human Cytokine ELISA. Each cytokines concentrations in cell supernatants were reproducible and detectable. Data are shown as fold changes of protein expression compared to mock samples. Proinflammatory cytokines are marked by an asterisk. ( B and C ) CSF3, IL-1β, and IL-6 proteins in sera of EV71-infected patients (n = 40) and healthy individuals (n = 36) ( B ), or in sera of EV71-infected and mock-infected mice (each group, n = 7) ( C ) were measured by ELISA. ( D – F ) THP-1 cells ( D ), macrophages (differentiated from THP-1 cells) ( E ), and PBMCs (isolated from peripheral blood samples of healthy individuals) ( F ) were treated with 100 ng/ml R848 for 12 h or infected with UV-inactivated EV71 (Inact. EV71) or EV71 (MOI = 2) for 24 h. The supernatants of treated cells were collected and CSF3, IL-1β, and IL-6 levels were measured by ELISA. Data are shown as mean ± SD and correspond to a representative experiment out of three performed. ns, non-significant; *, P

    Techniques Used: Infection, In Vivo, In Vitro, Derivative Assay, Enzyme-linked Immunosorbent Assay, Expressing, Mouse Assay, Isolation

    The sub-cellular localization of HRS during TLR7 signaling activation. (A–G) Macrophages were stimulated without or with R848 for 15 min, and then probed with HRS or organelles markers antibodies against clathrin ( A ), EEA1 ( B ), Rab7 ( C ), Rab11 ( D ), LAMP1 ( E ), Calnexin ( F ) or Rcas1 ( G ) before confocal microscopy, Bar = 20 μm. (H) The intensity of colocalization was calculated by using Image J software analysis. +++, > 80% colocalization; ++, 60%~80% colocalization; +, 40%~60% colocalization; -,
    Figure Legend Snippet: The sub-cellular localization of HRS during TLR7 signaling activation. (A–G) Macrophages were stimulated without or with R848 for 15 min, and then probed with HRS or organelles markers antibodies against clathrin ( A ), EEA1 ( B ), Rab7 ( C ), Rab11 ( D ), LAMP1 ( E ), Calnexin ( F ) or Rcas1 ( G ) before confocal microscopy, Bar = 20 μm. (H) The intensity of colocalization was calculated by using Image J software analysis. +++, > 80% colocalization; ++, 60%~80% colocalization; +, 40%~60% colocalization; -,

    Techniques Used: Activation Assay, Confocal Microscopy, Software

    20) Product Images from "HRS plays an important role for TLR7 signaling to orchestrate inflammation and innate immunity upon EV71 infection"

    Article Title: HRS plays an important role for TLR7 signaling to orchestrate inflammation and innate immunity upon EV71 infection

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006585

    HRS interacts with TLR7 and TAB1 in the TLR7 complex during viral infection. ( A and B ) Macrophages derived from THP-1 cells were stimulated with R848 (100 ng/ml) ( A ) or EV71 (MOI = 5) ( B ) for various time periods as indicated. Cell lysates were prepared and immunoprecipitated with rabbit anti-TLR7 or rabbit anti-IgG antibodies. Immunoprecipitates were assayed using Western blotting analysis with specific antibodies as indicated. ( C and D ) Macrophages were stimulated with R848 for 15 min, or infected with EV71 or SeV for 4 h, and then probed with indicated antibodies before confocal microscopy. Bar = 20 μm. ( E ) Macrophages were treated with R848 (100 ng/ml) for 30 min. Cells were probed with indicated antibodies and DAPI stain before confocal microscopy. Bar = 20 μm. ( F ) Mice were infected with EV71 and sacrificed at indicated period. Mice spleens were subjected to immunofluorescence (IF) staining with HRS (Red), TLR7 (Green), and CD68 (Blue) antibodies. Bar = 20 μm.
    Figure Legend Snippet: HRS interacts with TLR7 and TAB1 in the TLR7 complex during viral infection. ( A and B ) Macrophages derived from THP-1 cells were stimulated with R848 (100 ng/ml) ( A ) or EV71 (MOI = 5) ( B ) for various time periods as indicated. Cell lysates were prepared and immunoprecipitated with rabbit anti-TLR7 or rabbit anti-IgG antibodies. Immunoprecipitates were assayed using Western blotting analysis with specific antibodies as indicated. ( C and D ) Macrophages were stimulated with R848 for 15 min, or infected with EV71 or SeV for 4 h, and then probed with indicated antibodies before confocal microscopy. Bar = 20 μm. ( E ) Macrophages were treated with R848 (100 ng/ml) for 30 min. Cells were probed with indicated antibodies and DAPI stain before confocal microscopy. Bar = 20 μm. ( F ) Mice were infected with EV71 and sacrificed at indicated period. Mice spleens were subjected to immunofluorescence (IF) staining with HRS (Red), TLR7 (Green), and CD68 (Blue) antibodies. Bar = 20 μm.

    Techniques Used: Infection, Derivative Assay, Immunoprecipitation, Western Blot, Confocal Microscopy, Staining, Mouse Assay, Immunofluorescence

    HRS promotes the production of interferons mediated by TLR7 signaling. ( A and B ) Macrophages were transfected with siR-HRS or siR-NC and then stimulated with R848 ( A ) or infected with EV71 ( B ) for various time periods as indicated. The proteins in the cell lysates were detected using Western blotting analyses with indicated antibodies. ( C ) Macrophages were transfected with siR-HRS or siR-NC and then stimulated with R848. IFN-β and IFN-λ1 mRNA levels were determined using qPCR. ( D ) RD, 293T, and A549 cells were transfected with pcDNA3.1(-)-Myc or pcDNA3.1(-)-HRS-Myc and infected with EV71, SeV or IAV for 12 h, IFN-β and IFN-λ1 mRNA levels were determined using qPCR. **, P
    Figure Legend Snippet: HRS promotes the production of interferons mediated by TLR7 signaling. ( A and B ) Macrophages were transfected with siR-HRS or siR-NC and then stimulated with R848 ( A ) or infected with EV71 ( B ) for various time periods as indicated. The proteins in the cell lysates were detected using Western blotting analyses with indicated antibodies. ( C ) Macrophages were transfected with siR-HRS or siR-NC and then stimulated with R848. IFN-β and IFN-λ1 mRNA levels were determined using qPCR. ( D ) RD, 293T, and A549 cells were transfected with pcDNA3.1(-)-Myc or pcDNA3.1(-)-HRS-Myc and infected with EV71, SeV or IAV for 12 h, IFN-β and IFN-λ1 mRNA levels were determined using qPCR. **, P

    Techniques Used: Transfection, Infection, Western Blot, Real-time Polymerase Chain Reaction

    TLR7 expression correlates with HRS expression during EV71 infection. ( A ) Stable HEK293T/TLR7/NF-κB reporter cells were transfected with siR-NC, siR-HRS#1 and siR-HRS#2, treated with R848 or TNFα, and infected with EV71 or SeV. NF-κB activities were determined by luciferase activity assays. The HRS and β-actin proteins expressed in the treated cells were detected by Western blotting. The indicated band intensity represents as fold changes to internal control by using Image J software analysis. Results were expressed as fold induction relative to control. ns, non-significant; *, P
    Figure Legend Snippet: TLR7 expression correlates with HRS expression during EV71 infection. ( A ) Stable HEK293T/TLR7/NF-κB reporter cells were transfected with siR-NC, siR-HRS#1 and siR-HRS#2, treated with R848 or TNFα, and infected with EV71 or SeV. NF-κB activities were determined by luciferase activity assays. The HRS and β-actin proteins expressed in the treated cells were detected by Western blotting. The indicated band intensity represents as fold changes to internal control by using Image J software analysis. Results were expressed as fold induction relative to control. ns, non-significant; *, P

    Techniques Used: Expressing, Infection, Transfection, Luciferase, Activity Assay, Western Blot, Software

    HRS is important for TLR7-mediated proinflammatory cytokines production during viral infection. ( A and B ) Macrophages were transfected with siR-HRS or siR-NC and then stimulated with R848 (A) or infected with EV71 ( B ) for various time periods as indicated. Cell extracts were prepared and the proteins in the cell lysates were detected using Western blotting analyses with corresponding antibodies. The indicated band intensity represents as fold changes to internal control GAPDH. ( C and D ) Macrophages were transfected with siR-NC or siR-HRS and stimulated with R848 or infected with EV71 or SeV. The levels of CSF3, IL-1β, and IL-6 mRNAs and proteins were determined using qPCR ( C ) and ELISA ( D ), respectively. **, P
    Figure Legend Snippet: HRS is important for TLR7-mediated proinflammatory cytokines production during viral infection. ( A and B ) Macrophages were transfected with siR-HRS or siR-NC and then stimulated with R848 (A) or infected with EV71 ( B ) for various time periods as indicated. Cell extracts were prepared and the proteins in the cell lysates were detected using Western blotting analyses with corresponding antibodies. The indicated band intensity represents as fold changes to internal control GAPDH. ( C and D ) Macrophages were transfected with siR-NC or siR-HRS and stimulated with R848 or infected with EV71 or SeV. The levels of CSF3, IL-1β, and IL-6 mRNAs and proteins were determined using qPCR ( C ) and ELISA ( D ), respectively. **, P

    Techniques Used: Infection, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    EV71 induces the production of proinflammatory cytokines via stimulating TLR7 signaling and NF-κB activity. ( A ) The monocytic THP-1 cells were treated with individual kinase inhibitors for 6 h, as indicated, and infected with EV71 (MOI = 5) for 12 h. The levels of CSF3 , IL-1β and IL-6 mRNAs were measured by qPCR. ( B ) THP-1 cells were infected with EV71 (MOI = 5) for 12 h and total mRNA extracts were prepared. The mRNA levels of TLRs expressed in the cells were determined by qPCR. ( C ) THP-1 cells were transfected with plasmid transcribing siRNA specific to TLR7 (shTLR7) or its control (shGFP) and then infected with EV71 (MOI = 5) for 12 h. Cytokine mRNA levels were determined by qPCR. (D ) Mouse bone marrow-derived macrophages (BMDMs) isolated from TLR7 wild-type (WT) or TLR7 knock-out (TLR7 -/- ) mice were infected with EV71 (MOI = 5) for 24 h. TLR7 protein was determined by Western blotting. Mouse CSF3 , IL-1β , and IL-6 mRNAs were measured by qPCR. ( E and F ) THP-1 cells were co-transfected with NF-κB reporter plasmid and siRNA specific to TLR7 (shTLR7) or negative control (shGFP) and then infected with EV71 (MOI = 5) for 8 h. NF-κB activity was determined by luciferase activity assay ( E ). The endogenous protein levels were determined by western blotting with indicated antibodies ( F ). ( G ) HEK293T cells were transfected with pFlag2B (vector) or pFlag-TLR7 and infected with EV71 (MOI = 5). TLR7, p65, P-p65, EV71 VP1, and GAPDH proteins expressed in the treated cells were detected by Western blotting. ( H and I ) HEK293T cells were co-transfected with NF-κB reporter and pTLR7, pTLR7 (Y892A) or vector ( H ), or plasmids carrying shRNAs specific to indicated genes ( I ), and then stimulated with R848, or infected with EV71 (MOI = 5) or SeV (HA unit = 5) for 8 h, respectively. NF-κB activities were determined by luciferase activity assay. All qPCR assays used GAPDH mRNA as an internal control. Results were expressed as fold induction relative to control. ns, non-significant; *, P
    Figure Legend Snippet: EV71 induces the production of proinflammatory cytokines via stimulating TLR7 signaling and NF-κB activity. ( A ) The monocytic THP-1 cells were treated with individual kinase inhibitors for 6 h, as indicated, and infected with EV71 (MOI = 5) for 12 h. The levels of CSF3 , IL-1β and IL-6 mRNAs were measured by qPCR. ( B ) THP-1 cells were infected with EV71 (MOI = 5) for 12 h and total mRNA extracts were prepared. The mRNA levels of TLRs expressed in the cells were determined by qPCR. ( C ) THP-1 cells were transfected with plasmid transcribing siRNA specific to TLR7 (shTLR7) or its control (shGFP) and then infected with EV71 (MOI = 5) for 12 h. Cytokine mRNA levels were determined by qPCR. (D ) Mouse bone marrow-derived macrophages (BMDMs) isolated from TLR7 wild-type (WT) or TLR7 knock-out (TLR7 -/- ) mice were infected with EV71 (MOI = 5) for 24 h. TLR7 protein was determined by Western blotting. Mouse CSF3 , IL-1β , and IL-6 mRNAs were measured by qPCR. ( E and F ) THP-1 cells were co-transfected with NF-κB reporter plasmid and siRNA specific to TLR7 (shTLR7) or negative control (shGFP) and then infected with EV71 (MOI = 5) for 8 h. NF-κB activity was determined by luciferase activity assay ( E ). The endogenous protein levels were determined by western blotting with indicated antibodies ( F ). ( G ) HEK293T cells were transfected with pFlag2B (vector) or pFlag-TLR7 and infected with EV71 (MOI = 5). TLR7, p65, P-p65, EV71 VP1, and GAPDH proteins expressed in the treated cells were detected by Western blotting. ( H and I ) HEK293T cells were co-transfected with NF-κB reporter and pTLR7, pTLR7 (Y892A) or vector ( H ), or plasmids carrying shRNAs specific to indicated genes ( I ), and then stimulated with R848, or infected with EV71 (MOI = 5) or SeV (HA unit = 5) for 8 h, respectively. NF-κB activities were determined by luciferase activity assay. All qPCR assays used GAPDH mRNA as an internal control. Results were expressed as fold induction relative to control. ns, non-significant; *, P

    Techniques Used: Activity Assay, Infection, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Derivative Assay, Isolation, Knock-Out, Mouse Assay, Western Blot, Negative Control, Luciferase

    HRS interacts with TLR7 to facilitate signaling activation. ( A ) Macrophages were harvested after treated without or with R848 (100 ng/ml) for 30 min. Samples were homogenized and resolved by ultracentrifugation. Fractions were collected from the top of the gradient and analyzed for the distribution of indicated intracellular proteins by Western blotting. ( B ) Macrophages were treated with R848 (100 ng/ml) for indicated time and then applied for cell fraction. Separated cell fractions were subjected to immunoprecipitation (IP) and analyzed by Western blotting. S100K and P100K indicate supernatant and precipitate of cell fractions after 100,000 g ultracentrifugation, respectively. Indicated band intensity represents as fold changes to immunoprecipitated TLR7 by using Image J software analysis. ( C ) HEK293T cells were co-transfected with plasmid expressing Flag-TLR7 and HRS-Myc or FVYE domain deleted HRS-Myc (dFYVE-Myc). Whole cell lysates were prepared and immunoprecipitated and then analyzed by Western blotting. ( D ) HEK293T/TLR7/NF-κB reporter cells were transfected with plasmids expressing HRS or HRS deletion. NF-κB activities were determined using luciferase activity assays. The proteins expressed in the treated cells were detected by Western blotting. *, P
    Figure Legend Snippet: HRS interacts with TLR7 to facilitate signaling activation. ( A ) Macrophages were harvested after treated without or with R848 (100 ng/ml) for 30 min. Samples were homogenized and resolved by ultracentrifugation. Fractions were collected from the top of the gradient and analyzed for the distribution of indicated intracellular proteins by Western blotting. ( B ) Macrophages were treated with R848 (100 ng/ml) for indicated time and then applied for cell fraction. Separated cell fractions were subjected to immunoprecipitation (IP) and analyzed by Western blotting. S100K and P100K indicate supernatant and precipitate of cell fractions after 100,000 g ultracentrifugation, respectively. Indicated band intensity represents as fold changes to immunoprecipitated TLR7 by using Image J software analysis. ( C ) HEK293T cells were co-transfected with plasmid expressing Flag-TLR7 and HRS-Myc or FVYE domain deleted HRS-Myc (dFYVE-Myc). Whole cell lysates were prepared and immunoprecipitated and then analyzed by Western blotting. ( D ) HEK293T/TLR7/NF-κB reporter cells were transfected with plasmids expressing HRS or HRS deletion. NF-κB activities were determined using luciferase activity assays. The proteins expressed in the treated cells were detected by Western blotting. *, P

    Techniques Used: Activation Assay, Western Blot, Immunoprecipitation, Software, Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay

    EV71 infection induces the productions of inflammatory cytokines in vivo and in vitro . ( A ) THP-1 derived macrophages were infected with EV71 (MOI = 5) or UV-inactivated EV71 for 12 h. The amount of 30 cytokines in cell supernatants was measured by Human Cytokine ELISA. Each cytokines concentrations in cell supernatants were reproducible and detectable. Data are shown as fold changes of protein expression compared to mock samples. Proinflammatory cytokines are marked by an asterisk. ( B and C ) CSF3, IL-1β, and IL-6 proteins in sera of EV71-infected patients (n = 40) and healthy individuals (n = 36) ( B ), or in sera of EV71-infected and mock-infected mice (each group, n = 7) ( C ) were measured by ELISA. ( D – F ) THP-1 cells ( D ), macrophages (differentiated from THP-1 cells) ( E ), and PBMCs (isolated from peripheral blood samples of healthy individuals) ( F ) were treated with 100 ng/ml R848 for 12 h or infected with UV-inactivated EV71 (Inact. EV71) or EV71 (MOI = 2) for 24 h. The supernatants of treated cells were collected and CSF3, IL-1β, and IL-6 levels were measured by ELISA. Data are shown as mean ± SD and correspond to a representative experiment out of three performed. ns, non-significant; *, P
    Figure Legend Snippet: EV71 infection induces the productions of inflammatory cytokines in vivo and in vitro . ( A ) THP-1 derived macrophages were infected with EV71 (MOI = 5) or UV-inactivated EV71 for 12 h. The amount of 30 cytokines in cell supernatants was measured by Human Cytokine ELISA. Each cytokines concentrations in cell supernatants were reproducible and detectable. Data are shown as fold changes of protein expression compared to mock samples. Proinflammatory cytokines are marked by an asterisk. ( B and C ) CSF3, IL-1β, and IL-6 proteins in sera of EV71-infected patients (n = 40) and healthy individuals (n = 36) ( B ), or in sera of EV71-infected and mock-infected mice (each group, n = 7) ( C ) were measured by ELISA. ( D – F ) THP-1 cells ( D ), macrophages (differentiated from THP-1 cells) ( E ), and PBMCs (isolated from peripheral blood samples of healthy individuals) ( F ) were treated with 100 ng/ml R848 for 12 h or infected with UV-inactivated EV71 (Inact. EV71) or EV71 (MOI = 2) for 24 h. The supernatants of treated cells were collected and CSF3, IL-1β, and IL-6 levels were measured by ELISA. Data are shown as mean ± SD and correspond to a representative experiment out of three performed. ns, non-significant; *, P

    Techniques Used: Infection, In Vivo, In Vitro, Derivative Assay, Enzyme-linked Immunosorbent Assay, Expressing, Mouse Assay, Isolation

    The sub-cellular localization of HRS during TLR7 signaling activation. (A–G) Macrophages were stimulated without or with R848 for 15 min, and then probed with HRS or organelles markers antibodies against clathrin ( A ), EEA1 ( B ), Rab7 ( C ), Rab11 ( D ), LAMP1 ( E ), Calnexin ( F ) or Rcas1 ( G ) before confocal microscopy, Bar = 20 μm. (H) The intensity of colocalization was calculated by using Image J software analysis. +++, > 80% colocalization; ++, 60%~80% colocalization; +, 40%~60% colocalization; -,
    Figure Legend Snippet: The sub-cellular localization of HRS during TLR7 signaling activation. (A–G) Macrophages were stimulated without or with R848 for 15 min, and then probed with HRS or organelles markers antibodies against clathrin ( A ), EEA1 ( B ), Rab7 ( C ), Rab11 ( D ), LAMP1 ( E ), Calnexin ( F ) or Rcas1 ( G ) before confocal microscopy, Bar = 20 μm. (H) The intensity of colocalization was calculated by using Image J software analysis. +++, > 80% colocalization; ++, 60%~80% colocalization; +, 40%~60% colocalization; -,

    Techniques Used: Activation Assay, Confocal Microscopy, Software

    21) Product Images from "Human Blood CD1c+ Dendritic Cells Promote Th1 and Th17 Effector Function in Memory CD4+ T Cells"

    Article Title: Human Blood CD1c+ Dendritic Cells Promote Th1 and Th17 Effector Function in Memory CD4+ T Cells

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.00971

    Changes in pattern recognition receptor expression after activation of CD1c + dendritic cells (DC) and CD141 + DC. Normalized log 2 gene expression was calculated from data for CD1c + DC and CD141 + DC untreated or activated with poly I:C or R848 in vivo obtained from GEO GSE99666. Boxes represent 25th–75th percentiles ± minimum and maximum values with line at the median from two to three individual donors. The expression threshold determined by the bi-modal distribution of detected versus non-detected probes was 3.6 (dotted line).
    Figure Legend Snippet: Changes in pattern recognition receptor expression after activation of CD1c + dendritic cells (DC) and CD141 + DC. Normalized log 2 gene expression was calculated from data for CD1c + DC and CD141 + DC untreated or activated with poly I:C or R848 in vivo obtained from GEO GSE99666. Boxes represent 25th–75th percentiles ± minimum and maximum values with line at the median from two to three individual donors. The expression threshold determined by the bi-modal distribution of detected versus non-detected probes was 3.6 (dotted line).

    Techniques Used: Expressing, Activation Assay, In Vivo

    CD1c + dendritic cells (DC) produce high levels of IL-23 and IL-12p70. Production of (A) IL-12p70, (B) IL-23, (C) IL-1β, and (D) IL-6 by CD1c + DC (red) and monocytes (blue) following stimulation with LPS, R848, and/or poly I:C (PIC). Data points are eight individual donors with the mean + SEM shown. Cytokine levels are expressed as picogram per milliliter per 5 × 10 4 cells.
    Figure Legend Snippet: CD1c + dendritic cells (DC) produce high levels of IL-23 and IL-12p70. Production of (A) IL-12p70, (B) IL-23, (C) IL-1β, and (D) IL-6 by CD1c + DC (red) and monocytes (blue) following stimulation with LPS, R848, and/or poly I:C (PIC). Data points are eight individual donors with the mean + SEM shown. Cytokine levels are expressed as picogram per milliliter per 5 × 10 4 cells.

    Techniques Used:

    Activated CD1c + dendritic cells (DC) induce Th1 and Th17 cytokines in memory CD4 + T cells. (A) Proliferation of memory CD4 + T cells cultured alone, or with CD1c + DC or monocytes untreated or activated with R848 + LPS. Shown are the percentages of dividing cells measured by carboxyfluorescein succinimidyl ester (CFSE) dilution after 7 days of culture from one representative of three donors. (B) Production of interferon-γ and IL-17A from proliferating memory CD4 + T cells after restimulation with phorbol 12-myristate 13-acetate/ionomycin at day 7. (C) Cytokine production in the supernatants of memory CD4 + T cells after 7 days culture with CD1c + DC or monocytes untreated or activated with R848 + LPS. Data points are seven to eight individual donors with the mean + SEM shown.
    Figure Legend Snippet: Activated CD1c + dendritic cells (DC) induce Th1 and Th17 cytokines in memory CD4 + T cells. (A) Proliferation of memory CD4 + T cells cultured alone, or with CD1c + DC or monocytes untreated or activated with R848 + LPS. Shown are the percentages of dividing cells measured by carboxyfluorescein succinimidyl ester (CFSE) dilution after 7 days of culture from one representative of three donors. (B) Production of interferon-γ and IL-17A from proliferating memory CD4 + T cells after restimulation with phorbol 12-myristate 13-acetate/ionomycin at day 7. (C) Cytokine production in the supernatants of memory CD4 + T cells after 7 days culture with CD1c + DC or monocytes untreated or activated with R848 + LPS. Data points are seven to eight individual donors with the mean + SEM shown.

    Techniques Used: Cell Culture

    Cytokine production by optimally activated dendritic cells (DC) subsets. (A) Cytokine production by CD1c + DC and CD141 + DC untreated or activated with R848 + poly I:C, and plasmacytoid DC activated with R848 + CpG. (B) Cytokine production by CD1c + DC and CD141 + DC after activation with combined R848, poly I:C, and LPS. Data points are individual donors with mean ± SEM shown. Cytokine levels are expressed as picogram per milliliter per 5 × 10 4 cells. p values are shown.
    Figure Legend Snippet: Cytokine production by optimally activated dendritic cells (DC) subsets. (A) Cytokine production by CD1c + DC and CD141 + DC untreated or activated with R848 + poly I:C, and plasmacytoid DC activated with R848 + CpG. (B) Cytokine production by CD1c + DC and CD141 + DC after activation with combined R848, poly I:C, and LPS. Data points are individual donors with mean ± SEM shown. Cytokine levels are expressed as picogram per milliliter per 5 × 10 4 cells. p values are shown.

    Techniques Used: Activation Assay

    Production of Th1 and Th17 cytokines by memory CD4 + T cells activated with human dendritic cells (DC) subsets. Memory CD4 + T cells were cultured with CD1c + DC or CD141 + DC activated with R848 + poly I:C, or plasmacytoid DC activated with R848 + CpG and cytokines measured in the culture supernatants after 7 days. Data points are four individual donors with mean + SEM shown.
    Figure Legend Snippet: Production of Th1 and Th17 cytokines by memory CD4 + T cells activated with human dendritic cells (DC) subsets. Memory CD4 + T cells were cultured with CD1c + DC or CD141 + DC activated with R848 + poly I:C, or plasmacytoid DC activated with R848 + CpG and cytokines measured in the culture supernatants after 7 days. Data points are four individual donors with mean + SEM shown.

    Techniques Used: Cell Culture

    22) Product Images from "Human Blood CD1c+ Dendritic Cells Promote Th1 and Th17 Effector Function in Memory CD4+ T Cells"

    Article Title: Human Blood CD1c+ Dendritic Cells Promote Th1 and Th17 Effector Function in Memory CD4+ T Cells

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.00971

    Changes in pattern recognition receptor expression after activation of CD1c + dendritic cells (DC) and CD141 + DC. Normalized log 2 gene expression was calculated from data for CD1c + DC and CD141 + DC untreated or activated with poly I:C or R848 in vivo obtained from GEO GSE99666. Boxes represent 25th–75th percentiles ± minimum and maximum values with line at the median from two to three individual donors. The expression threshold determined by the bi-modal distribution of detected versus non-detected probes was 3.6 (dotted line).
    Figure Legend Snippet: Changes in pattern recognition receptor expression after activation of CD1c + dendritic cells (DC) and CD141 + DC. Normalized log 2 gene expression was calculated from data for CD1c + DC and CD141 + DC untreated or activated with poly I:C or R848 in vivo obtained from GEO GSE99666. Boxes represent 25th–75th percentiles ± minimum and maximum values with line at the median from two to three individual donors. The expression threshold determined by the bi-modal distribution of detected versus non-detected probes was 3.6 (dotted line).

    Techniques Used: Expressing, Activation Assay, In Vivo

    CD1c + dendritic cells (DC) produce high levels of IL-23 and IL-12p70. Production of (A) IL-12p70, (B) IL-23, (C) IL-1β, and (D) IL-6 by CD1c + DC (red) and monocytes (blue) following stimulation with LPS, R848, and/or poly I:C (PIC). Data points are eight individual donors with the mean + SEM shown. Cytokine levels are expressed as picogram per milliliter per 5 × 10 4 cells.
    Figure Legend Snippet: CD1c + dendritic cells (DC) produce high levels of IL-23 and IL-12p70. Production of (A) IL-12p70, (B) IL-23, (C) IL-1β, and (D) IL-6 by CD1c + DC (red) and monocytes (blue) following stimulation with LPS, R848, and/or poly I:C (PIC). Data points are eight individual donors with the mean + SEM shown. Cytokine levels are expressed as picogram per milliliter per 5 × 10 4 cells.

    Techniques Used:

    Activated CD1c + dendritic cells (DC) induce Th1 and Th17 cytokines in memory CD4 + T cells. (A) Proliferation of memory CD4 + T cells cultured alone, or with CD1c + DC or monocytes untreated or activated with R848 + LPS. Shown are the percentages of dividing cells measured by carboxyfluorescein succinimidyl ester (CFSE) dilution after 7 days of culture from one representative of three donors. (B) Production of interferon-γ and IL-17A from proliferating memory CD4 + T cells after restimulation with phorbol 12-myristate 13-acetate/ionomycin at day 7. (C) Cytokine production in the supernatants of memory CD4 + T cells after 7 days culture with CD1c + DC or monocytes untreated or activated with R848 + LPS. Data points are seven to eight individual donors with the mean + SEM shown.
    Figure Legend Snippet: Activated CD1c + dendritic cells (DC) induce Th1 and Th17 cytokines in memory CD4 + T cells. (A) Proliferation of memory CD4 + T cells cultured alone, or with CD1c + DC or monocytes untreated or activated with R848 + LPS. Shown are the percentages of dividing cells measured by carboxyfluorescein succinimidyl ester (CFSE) dilution after 7 days of culture from one representative of three donors. (B) Production of interferon-γ and IL-17A from proliferating memory CD4 + T cells after restimulation with phorbol 12-myristate 13-acetate/ionomycin at day 7. (C) Cytokine production in the supernatants of memory CD4 + T cells after 7 days culture with CD1c + DC or monocytes untreated or activated with R848 + LPS. Data points are seven to eight individual donors with the mean + SEM shown.

    Techniques Used: Cell Culture

    Cytokine production by optimally activated dendritic cells (DC) subsets. (A) Cytokine production by CD1c + DC and CD141 + DC untreated or activated with R848 + poly I:C, and plasmacytoid DC activated with R848 + CpG. (B) Cytokine production by CD1c + DC and CD141 + DC after activation with combined R848, poly I:C, and LPS. Data points are individual donors with mean ± SEM shown. Cytokine levels are expressed as picogram per milliliter per 5 × 10 4 cells. p values are shown.
    Figure Legend Snippet: Cytokine production by optimally activated dendritic cells (DC) subsets. (A) Cytokine production by CD1c + DC and CD141 + DC untreated or activated with R848 + poly I:C, and plasmacytoid DC activated with R848 + CpG. (B) Cytokine production by CD1c + DC and CD141 + DC after activation with combined R848, poly I:C, and LPS. Data points are individual donors with mean ± SEM shown. Cytokine levels are expressed as picogram per milliliter per 5 × 10 4 cells. p values are shown.

    Techniques Used: Activation Assay

    Production of Th1 and Th17 cytokines by memory CD4 + T cells activated with human dendritic cells (DC) subsets. Memory CD4 + T cells were cultured with CD1c + DC or CD141 + DC activated with R848 + poly I:C, or plasmacytoid DC activated with R848 + CpG and cytokines measured in the culture supernatants after 7 days. Data points are four individual donors with mean + SEM shown.
    Figure Legend Snippet: Production of Th1 and Th17 cytokines by memory CD4 + T cells activated with human dendritic cells (DC) subsets. Memory CD4 + T cells were cultured with CD1c + DC or CD141 + DC activated with R848 + poly I:C, or plasmacytoid DC activated with R848 + CpG and cytokines measured in the culture supernatants after 7 days. Data points are four individual donors with mean + SEM shown.

    Techniques Used: Cell Culture

    23) Product Images from "Snapin promotes HIV‐1 transmission from dendritic cells by dampening TLR8 signaling"

    Article Title: Snapin promotes HIV‐1 transmission from dendritic cells by dampening TLR8 signaling

    Journal: The EMBO Journal

    doi: 10.15252/embj.201695364

    Enhanced signaling through TLR 8 in Snapin knockdown cells DCs were transfected with Snapin or Ctrl siRNAs and activated with ssRNA40. mRNA was harvested 6 h post‐activation for IL‐6 and IFNβ1 qPCR. Results were relative to a GAPDH and lyovec control. DCs treated as in (A) and supernatant analyzed using IL‐6 or TNFα ELISA. Data are representative of 4–6 independent experiments. DCs transfected with Ctrl or Snapin siRNAs and stimulated with R848 over time. Cells were lysed and immunoblotted for phospho‐p38 (pp38), phospho‐Stat1 (p‐STAT1), STAT1, phospho‐IκB‐α, and LAMP1. Immunoblot quantification is shown in the right panel. DCs transfected with Ctrl or Snapin siRNA and activated with ssRNA40 over time. Immunoblot using anti‐phospho‐p38 (upper panel), phospho‐Erk (middle panel), and β‐tubulin (lower panel) is shown. Immunoblot quantification is shown in the right panel. mRNA of TNFA and ifnb1 (left and middle panel) or TNFα protein level in DCs transfected with Ctrl or Snapin siRNAs and left uninfected or infected with Sendai virus. Data information: Data are representative of three independent experiments from different donors. Results are presented as mean ± SEM. * P
    Figure Legend Snippet: Enhanced signaling through TLR 8 in Snapin knockdown cells DCs were transfected with Snapin or Ctrl siRNAs and activated with ssRNA40. mRNA was harvested 6 h post‐activation for IL‐6 and IFNβ1 qPCR. Results were relative to a GAPDH and lyovec control. DCs treated as in (A) and supernatant analyzed using IL‐6 or TNFα ELISA. Data are representative of 4–6 independent experiments. DCs transfected with Ctrl or Snapin siRNAs and stimulated with R848 over time. Cells were lysed and immunoblotted for phospho‐p38 (pp38), phospho‐Stat1 (p‐STAT1), STAT1, phospho‐IκB‐α, and LAMP1. Immunoblot quantification is shown in the right panel. DCs transfected with Ctrl or Snapin siRNA and activated with ssRNA40 over time. Immunoblot using anti‐phospho‐p38 (upper panel), phospho‐Erk (middle panel), and β‐tubulin (lower panel) is shown. Immunoblot quantification is shown in the right panel. mRNA of TNFA and ifnb1 (left and middle panel) or TNFα protein level in DCs transfected with Ctrl or Snapin siRNAs and left uninfected or infected with Sendai virus. Data information: Data are representative of three independent experiments from different donors. Results are presented as mean ± SEM. * P

    Techniques Used: Transfection, Activation Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Infection

    24) Product Images from "TLR7/8 activation in neutrophils impairs immune complex phagocytosis through shedding of FcgRIIA"

    Article Title: TLR7/8 activation in neutrophils impairs immune complex phagocytosis through shedding of FcgRIIA

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20161512

    TLR7/8 activation induces shedding of FcgRIIA. (A) Neutrophils were activated with R848 and cell surface expression of FcgRs analyzed by flow cytometry. The results are presented as FcgR levels as compared with no R848 added (percentage of control). The experiment was repeated 5 (FcgRI), 7 (FcgRIII), and 25 (FcgRIIA) times; combined results are shown and compared using paired Student’s t test (FcgRIIA, P
    Figure Legend Snippet: TLR7/8 activation induces shedding of FcgRIIA. (A) Neutrophils were activated with R848 and cell surface expression of FcgRs analyzed by flow cytometry. The results are presented as FcgR levels as compared with no R848 added (percentage of control). The experiment was repeated 5 (FcgRI), 7 (FcgRIII), and 25 (FcgRIIA) times; combined results are shown and compared using paired Student’s t test (FcgRIIA, P

    Techniques Used: Activation Assay, Expressing, Flow Cytometry, Cytometry

    Proteolytic cleavage of monocyte FcgRIIA inhibits clearance of ICs. (A) Monocytes were incubated with R848 or neutrophil supernatant before addition of RNP-ICs or beads. Phagocytosis was determined by flow cytometry. The experiment was repeated four (beads, RNase, RNP-IC+R848) or seven (PMN sup) times; combined results are shown and compared using paired Student’s t test (RNP-IC, P = 0.0003; Beads, P = 0.0007). (B) ICs were added to PBMCs with or without prior treatment with neutrophil supernatant (A). After phagocytosis for 30 min, remaining cell-free ICs were analyzed for C5a-inducing ability upon addition of 1% normal human serum. The experiment was repeated three times; combined results are shown and compared using paired t test (P = 0.0084). C) C5a serum levels were measured in healthy controls (HC, n = 9) and SLE patients ( n = 36) by ELISA. Combined results are shown and analyzed using Mann-Whitney U test (P = 0.047). (D and E) Serum levels of C5a in SLE patients were related to ability of serum to induce shedding of FcgRIIA on healthy control neutrophils. In D, combined results from 35 SLE patients are shown and analyzed using Spearman’s correlation test (r = −0.42; P = 0.011). In E, combined results are shown from SLE patients inducing shedding ( n = 15) or not ( n = 20), and compared using Mann-Whitney U test (P = 0.0281). *, P
    Figure Legend Snippet: Proteolytic cleavage of monocyte FcgRIIA inhibits clearance of ICs. (A) Monocytes were incubated with R848 or neutrophil supernatant before addition of RNP-ICs or beads. Phagocytosis was determined by flow cytometry. The experiment was repeated four (beads, RNase, RNP-IC+R848) or seven (PMN sup) times; combined results are shown and compared using paired Student’s t test (RNP-IC, P = 0.0003; Beads, P = 0.0007). (B) ICs were added to PBMCs with or without prior treatment with neutrophil supernatant (A). After phagocytosis for 30 min, remaining cell-free ICs were analyzed for C5a-inducing ability upon addition of 1% normal human serum. The experiment was repeated three times; combined results are shown and compared using paired t test (P = 0.0084). C) C5a serum levels were measured in healthy controls (HC, n = 9) and SLE patients ( n = 36) by ELISA. Combined results are shown and analyzed using Mann-Whitney U test (P = 0.047). (D and E) Serum levels of C5a in SLE patients were related to ability of serum to induce shedding of FcgRIIA on healthy control neutrophils. In D, combined results from 35 SLE patients are shown and analyzed using Spearman’s correlation test (r = −0.42; P = 0.011). In E, combined results are shown from SLE patients inducing shedding ( n = 15) or not ( n = 20), and compared using Mann-Whitney U test (P = 0.0281). *, P

    Techniques Used: Incubation, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    FcgRIIA shedding requires serine proteases. (A) Cell surface levels of FcgRIIA (IV.3) was analyzed by flow cytometry upon R848 activation in the presence of a pan protease inhibitor or inhibitors of matrix metalloproteases (GM6001, 10 µM), cysteine proteases (E-64, 1 µM), serine proteases (AEBSF, 100 µM), neutrophil elastase (Elastase inhibitor IV, 25 µM), cathepsin G (chymostatin, 10 µg/ml), or furin (chloromethylketone (CMK, 25 µM). The experiment was repeated three (E-64), four (Pan Prot.inh., P
    Figure Legend Snippet: FcgRIIA shedding requires serine proteases. (A) Cell surface levels of FcgRIIA (IV.3) was analyzed by flow cytometry upon R848 activation in the presence of a pan protease inhibitor or inhibitors of matrix metalloproteases (GM6001, 10 µM), cysteine proteases (E-64, 1 µM), serine proteases (AEBSF, 100 µM), neutrophil elastase (Elastase inhibitor IV, 25 µM), cathepsin G (chymostatin, 10 µg/ml), or furin (chloromethylketone (CMK, 25 µM). The experiment was repeated three (E-64), four (Pan Prot.inh., P

    Techniques Used: Flow Cytometry, Cytometry, Activation Assay, Protease Inhibitor

    FcgRIIA shedding requires reactive oxygen species. (A) Neutrophils were activated with R848, and FcgRIIA and CD66b levels were analyzed by flow cytometry. The experiment was repeated eight times; combined results are shown and compared using paired Student’s t test (P
    Figure Legend Snippet: FcgRIIA shedding requires reactive oxygen species. (A) Neutrophils were activated with R848, and FcgRIIA and CD66b levels were analyzed by flow cytometry. The experiment was repeated eight times; combined results are shown and compared using paired Student’s t test (P

    Techniques Used: Flow Cytometry, Cytometry

    TLR7/8-activated neutrophils shed FcgRIIA from monocytes and pDCs. (A and B) PBMCs were coincubated with neutrophils (PMNs) in the presence of R848 and a pan-protease inhibitor. Levels of FcgRIIA on (A) monocytes (CD14 + ) and (B) pDCs (CD304 + ) were determined by flow cytometry and expressed as FcgRIIA (percentage of control) as compared with PBMCs incubated in medium in absence of neutrophils. In A, the experiment was repeated 11 times with the exception of the proteinase inhibitor ( n = 5); combined results are shown and compared using paired t test (PMN+PBMC, P
    Figure Legend Snippet: TLR7/8-activated neutrophils shed FcgRIIA from monocytes and pDCs. (A and B) PBMCs were coincubated with neutrophils (PMNs) in the presence of R848 and a pan-protease inhibitor. Levels of FcgRIIA on (A) monocytes (CD14 + ) and (B) pDCs (CD304 + ) were determined by flow cytometry and expressed as FcgRIIA (percentage of control) as compared with PBMCs incubated in medium in absence of neutrophils. In A, the experiment was repeated 11 times with the exception of the proteinase inhibitor ( n = 5); combined results are shown and compared using paired t test (PMN+PBMC, P

    Techniques Used: Protease Inhibitor, Flow Cytometry, Cytometry, Incubation

    25) Product Images from "K63-Linked Polyubiquitination on TRAF6 Regulates LPS-Mediated MAPK Activation, Cytokine Production, and Bacterial Clearance in Toll-Like Receptor 7/8 Primed Murine Macrophages"

    Article Title: K63-Linked Polyubiquitination on TRAF6 Regulates LPS-Mediated MAPK Activation, Cytokine Production, and Bacterial Clearance in Toll-Like Receptor 7/8 Primed Murine Macrophages

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00279

    Small-molecule enhancer of rapamycin (SMER) 3 decreases MAPKs phosphorylation in R848-primed bone marrow-derived macrophages (BMDMs). BMDMs were preexposed to either R848 (100 ng/mL) or R848 and the E3 ubiquitin ligase inhibitor SMER3 (5 µM) for 24 h, cells were washed and subsequently challenged with LPS for different time periods. Whole cell extracts were subjected to SDS-PAGE and Western blot analysis performed using antibodies against TRAF6 and phosphorylated forms of TGF-β-activated kinase (TAK) 1, ERK1/2, and p38. TRAF6/β-actin or phosphorylated protein/total protein values of TRAF6 (A,B) , densitometry analysis expressed as fold increase of the ratio of phosphorylated TAK1 (C,D) , phosphorylated ERK1/2 (E,F) , and phosphorylated p38 (G,H) . Densitometry results shown are mean of five independent experiments. Representative results of Western blots are shown out of a total of five independent experiments. A p value
    Figure Legend Snippet: Small-molecule enhancer of rapamycin (SMER) 3 decreases MAPKs phosphorylation in R848-primed bone marrow-derived macrophages (BMDMs). BMDMs were preexposed to either R848 (100 ng/mL) or R848 and the E3 ubiquitin ligase inhibitor SMER3 (5 µM) for 24 h, cells were washed and subsequently challenged with LPS for different time periods. Whole cell extracts were subjected to SDS-PAGE and Western blot analysis performed using antibodies against TRAF6 and phosphorylated forms of TGF-β-activated kinase (TAK) 1, ERK1/2, and p38. TRAF6/β-actin or phosphorylated protein/total protein values of TRAF6 (A,B) , densitometry analysis expressed as fold increase of the ratio of phosphorylated TAK1 (C,D) , phosphorylated ERK1/2 (E,F) , and phosphorylated p38 (G,H) . Densitometry results shown are mean of five independent experiments. Representative results of Western blots are shown out of a total of five independent experiments. A p value

    Techniques Used: Derivative Assay, SDS Page, Western Blot

    Proposed model of toll-like receptor (TLR) 7/8 priming of bone marrow-derived macrophages, increased activation of MAPKs, and production of inflammatory cytokines on subsequent exposure to TLR2/4 ligands. TLR7/8 is localized in the endosome where it recognizes ssRNA viruses or synthetic ligand R848. TLR2 and 4 are localized on the plasma membrane where they recognize the bacterial ligands PAM or LPS respectively. TLR7/8 priming leads to increased K63-linked polyubiquitination of proteins involved in TLR signaling and specifically TRAF6. Cells preexposed to viral ligands (TLR7/8) respond to subsequent stimulation with TLR2/4 ligands with K63-linked polyubiquitination of TRAF6, which leads to an enhanced activation of MAPKs and production of pro-inflammatory cytokines. Enhanced activation and cytokine storm are inhibited in the presence of an ubiquitin ligase inhibitor [small-molecule enhancer of rapamycin (SMER) 3]. SMER3 inhibits the K63 polyubiquitination of proteins, specifically the TRAF6 activity resulting in a decreased production of pro-inflammatory cytokines.
    Figure Legend Snippet: Proposed model of toll-like receptor (TLR) 7/8 priming of bone marrow-derived macrophages, increased activation of MAPKs, and production of inflammatory cytokines on subsequent exposure to TLR2/4 ligands. TLR7/8 is localized in the endosome where it recognizes ssRNA viruses or synthetic ligand R848. TLR2 and 4 are localized on the plasma membrane where they recognize the bacterial ligands PAM or LPS respectively. TLR7/8 priming leads to increased K63-linked polyubiquitination of proteins involved in TLR signaling and specifically TRAF6. Cells preexposed to viral ligands (TLR7/8) respond to subsequent stimulation with TLR2/4 ligands with K63-linked polyubiquitination of TRAF6, which leads to an enhanced activation of MAPKs and production of pro-inflammatory cytokines. Enhanced activation and cytokine storm are inhibited in the presence of an ubiquitin ligase inhibitor [small-molecule enhancer of rapamycin (SMER) 3]. SMER3 inhibits the K63 polyubiquitination of proteins, specifically the TRAF6 activity resulting in a decreased production of pro-inflammatory cytokines.

    Techniques Used: Derivative Assay, Activation Assay, Activity Assay

    R848-primed bone marrow-derived macrophages (BMDMs) exhibit reduced bacterial clearance and produce significantly higher cytokines as compared with unprimed BMDMs on subsequent exposure to live E. coli . BMDMs were preexposed to either R848 (100 ng/mL) or R848 and small-molecule enhancer of rapamycin (SMER) 3 (5 µM) for 24 h, cells were washed and subsequently challenged with live bacteria E. coli with an MOI 10:1 (bacteria:BMDMs). Culture supernatants and cell lysates were collected 3 h after infection. For bacterial clearance assay, after 3 h of infection, BMDMs were lysed in PBS containing 0.2% TritonX-100, and a serial dilution of lysate was streaked in duplicate on LB agar plates, and incubated overnight at 37°C. CFUs were counted the next day. Data represent mean ± SEM from five different experiments and * represents p value
    Figure Legend Snippet: R848-primed bone marrow-derived macrophages (BMDMs) exhibit reduced bacterial clearance and produce significantly higher cytokines as compared with unprimed BMDMs on subsequent exposure to live E. coli . BMDMs were preexposed to either R848 (100 ng/mL) or R848 and small-molecule enhancer of rapamycin (SMER) 3 (5 µM) for 24 h, cells were washed and subsequently challenged with live bacteria E. coli with an MOI 10:1 (bacteria:BMDMs). Culture supernatants and cell lysates were collected 3 h after infection. For bacterial clearance assay, after 3 h of infection, BMDMs were lysed in PBS containing 0.2% TritonX-100, and a serial dilution of lysate was streaked in duplicate on LB agar plates, and incubated overnight at 37°C. CFUs were counted the next day. Data represent mean ± SEM from five different experiments and * represents p value

    Techniques Used: Derivative Assay, Infection, Serial Dilution, Incubation

    Distinct increase of MAPK phosphorylation in R848-primed bone marrow-derived macrophages (BMDMs). BMDMs were preexposed to R848 (100 ng/mL) or medium alone for 24 h, washed and subsequently challenged with LPS (500 ng/mL) or PAM (500 ng/mL) for different time periods as indicated. Whole cell extracts were prepared and subjected to SDS-PAGE and Western blot analysis using phosphorylated antibodies against ERK1/2, p38, and c-Jun NH(2)-terminal kinase (JNK). Equal loading was determined using antibodies against total ERK, total p38, and total JNK. Representative results of Western blots are shown out of a total of five independent experiments. Densitometry analysis is expressed as fold increase of the ratio phosphorylated protein/total protein. Expression of pERK in the absence or presence of LPS (A,B) and PAM (C,D) , expression of pp38 in the absence or presence of LPS (E,F) and PAM (G,H) , and expression of pJNK in the absence or presence of LPS (I,J) and PAM (K,L) . Densitometry results shown are mean of five independent experiments. A p value
    Figure Legend Snippet: Distinct increase of MAPK phosphorylation in R848-primed bone marrow-derived macrophages (BMDMs). BMDMs were preexposed to R848 (100 ng/mL) or medium alone for 24 h, washed and subsequently challenged with LPS (500 ng/mL) or PAM (500 ng/mL) for different time periods as indicated. Whole cell extracts were prepared and subjected to SDS-PAGE and Western blot analysis using phosphorylated antibodies against ERK1/2, p38, and c-Jun NH(2)-terminal kinase (JNK). Equal loading was determined using antibodies against total ERK, total p38, and total JNK. Representative results of Western blots are shown out of a total of five independent experiments. Densitometry analysis is expressed as fold increase of the ratio phosphorylated protein/total protein. Expression of pERK in the absence or presence of LPS (A,B) and PAM (C,D) , expression of pp38 in the absence or presence of LPS (E,F) and PAM (G,H) , and expression of pJNK in the absence or presence of LPS (I,J) and PAM (K,L) . Densitometry results shown are mean of five independent experiments. A p value

    Techniques Used: Derivative Assay, SDS Page, Western Blot, Expressing

    R848-primed bone marrow-derived macrophages (BMDMs) exhibit increased expression and activation of toll-like receptor signaling molecules. BMDMs were preexposed to R848 (100 ng/mL) or cultured in medium for 24 h, washed and subsequently challenged with LPS (500 ng/mL) or PAM (500 ng/mL) for different time periods. Whole cell extracts were prepared and subjected to SDS-PAGE and Western blot analysis using specific antibodies against TRAF6, pTAK1, and β-actin for equal loading was performed. Representative results of Western blots are shown out of a total of five independent experiments. Densitometry analysis expressed as fold increase of the ratio of specific protein/β-actin. TRAF6 in the absence or presence of LPS (A,B) and PAM (C,D) . Phosphorylated TGF-β-activated kinase 1 in the absence or presence of LPS (E,F) and PAM (G,H) . Densitometry results shown are mean of five independent experiments. A p value
    Figure Legend Snippet: R848-primed bone marrow-derived macrophages (BMDMs) exhibit increased expression and activation of toll-like receptor signaling molecules. BMDMs were preexposed to R848 (100 ng/mL) or cultured in medium for 24 h, washed and subsequently challenged with LPS (500 ng/mL) or PAM (500 ng/mL) for different time periods. Whole cell extracts were prepared and subjected to SDS-PAGE and Western blot analysis using specific antibodies against TRAF6, pTAK1, and β-actin for equal loading was performed. Representative results of Western blots are shown out of a total of five independent experiments. Densitometry analysis expressed as fold increase of the ratio of specific protein/β-actin. TRAF6 in the absence or presence of LPS (A,B) and PAM (C,D) . Phosphorylated TGF-β-activated kinase 1 in the absence or presence of LPS (E,F) and PAM (G,H) . Densitometry results shown are mean of five independent experiments. A p value

    Techniques Used: Derivative Assay, Expressing, Activation Assay, Cell Culture, SDS Page, Western Blot

    R848-primed bone marrow-derived macrophages (BMDMs) exhibit increased K63-specific polyubiquitination and upregulated expression of K63-linked polyubiquitinated TRAF6. BMDMs were preexposed to R848 (100 ng/mL) or medium alone for 24 h, washed and subsequently challenged with LPS (500 ng/mL) for different time periods as indicated. Whole cell extracts were subjected to SDS-PAGE, and Western blot analysis was performed using antibodies against polyubiquitin (A) or K63-linked polyubiquitin (B) . Representative results are shown out of a total of five independent experiments. Unprimed or R848-primed BMDMs were challenged with LPS (500 ng/mL) for 30 and 180 min. Cell lysates were prepared and immunoprecipitated (IP) for TRAF6 protein using TRAF6 antibody. IP protein was immunoblotted with K63-specific polyubiquitin antibody to detect K63-linked polyubiquitination on TRAF6 (C) . Representative result is shown out of a total of three independent experiments.
    Figure Legend Snippet: R848-primed bone marrow-derived macrophages (BMDMs) exhibit increased K63-specific polyubiquitination and upregulated expression of K63-linked polyubiquitinated TRAF6. BMDMs were preexposed to R848 (100 ng/mL) or medium alone for 24 h, washed and subsequently challenged with LPS (500 ng/mL) for different time periods as indicated. Whole cell extracts were subjected to SDS-PAGE, and Western blot analysis was performed using antibodies against polyubiquitin (A) or K63-linked polyubiquitin (B) . Representative results are shown out of a total of five independent experiments. Unprimed or R848-primed BMDMs were challenged with LPS (500 ng/mL) for 30 and 180 min. Cell lysates were prepared and immunoprecipitated (IP) for TRAF6 protein using TRAF6 antibody. IP protein was immunoblotted with K63-specific polyubiquitin antibody to detect K63-linked polyubiquitination on TRAF6 (C) . Representative result is shown out of a total of three independent experiments.

    Techniques Used: Derivative Assay, Expressing, SDS Page, Western Blot, Immunoprecipitation

    Pretreatment of R848-primed bone marrow-derived macrophages (BMDMs) with small-molecule enhancer of rapamycin (SMER) 3 reduces the expression of polyubiquitinated and K63-linked polyubiquitinated proteins and inhibits the production of inflammatory cytokines. BMDMs were preexposed to either R848 (100 ng/mL) or R848 and SMER3 (5 µM) for 24 h, cells were washed and subsequently challenged with LPS (500 ng/mL) for different time periods. Whole cell extracts were prepared and subjected to SDS-PAGE and Western blot analysis to assess the expression of polyubiquitin (A) and K63-linked polyubiquitin proteins (B) . Representative results of Western blot are shown out of a total of four independent experiments. Culture supernatants were collected after 24 h to measure IL-6 (C) , TNF-α (D) , IL-1β (E) , and IL-10 (F) production via enzyme-linked immunosorbent assay (ELISA). ELISA results shown are mean of four independent experiments. A p value
    Figure Legend Snippet: Pretreatment of R848-primed bone marrow-derived macrophages (BMDMs) with small-molecule enhancer of rapamycin (SMER) 3 reduces the expression of polyubiquitinated and K63-linked polyubiquitinated proteins and inhibits the production of inflammatory cytokines. BMDMs were preexposed to either R848 (100 ng/mL) or R848 and SMER3 (5 µM) for 24 h, cells were washed and subsequently challenged with LPS (500 ng/mL) for different time periods. Whole cell extracts were prepared and subjected to SDS-PAGE and Western blot analysis to assess the expression of polyubiquitin (A) and K63-linked polyubiquitin proteins (B) . Representative results of Western blot are shown out of a total of four independent experiments. Culture supernatants were collected after 24 h to measure IL-6 (C) , TNF-α (D) , IL-1β (E) , and IL-10 (F) production via enzyme-linked immunosorbent assay (ELISA). ELISA results shown are mean of four independent experiments. A p value

    Techniques Used: Derivative Assay, Expressing, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay

    Increased production of inflammatory cytokines by R848-primed bone marrow-derived macrophages (BMDMs) in response to toll-like receptor (TLR) 4 (LPS) and TLR2 (PAM) ligands. Murine BMDMs were cultured at a density of 2 × 10 6 cells/well and were preexposed to R848 (100 ng/mL) or medium alone for 24 h, washed and subsequently challenged with LPS (500 ng/mL) or PAM (500 ng/mL) for further 24 h. Conditioned media were analyzed for IL-6 (A,B) , TNF-α ( C,D ), IL-1β ( E,F ), and IL-10 ( G,H ) via enzyme-linked immunosorbent assay. Conditioned media were assayed for nitric oxide (NO) levels by nitrite quantification via Griess reaction (I,J) . Results shown are a mean of five independent experiments. A p value
    Figure Legend Snippet: Increased production of inflammatory cytokines by R848-primed bone marrow-derived macrophages (BMDMs) in response to toll-like receptor (TLR) 4 (LPS) and TLR2 (PAM) ligands. Murine BMDMs were cultured at a density of 2 × 10 6 cells/well and were preexposed to R848 (100 ng/mL) or medium alone for 24 h, washed and subsequently challenged with LPS (500 ng/mL) or PAM (500 ng/mL) for further 24 h. Conditioned media were analyzed for IL-6 (A,B) , TNF-α ( C,D ), IL-1β ( E,F ), and IL-10 ( G,H ) via enzyme-linked immunosorbent assay. Conditioned media were assayed for nitric oxide (NO) levels by nitrite quantification via Griess reaction (I,J) . Results shown are a mean of five independent experiments. A p value

    Techniques Used: Derivative Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    26) Product Images from "BAFF augments IgA2 and IL‐10 production by TLR7/8 stimulated total peripheral blood B cells"

    Article Title: BAFF augments IgA2 and IL‐10 production by TLR7/8 stimulated total peripheral blood B cells

    Journal: European Journal of Immunology

    doi: 10.1002/eji.201646861

    BAFF and Retinoic acid differentially affect IL‐10 and IL‐6 production. IL‐10 (A, C) and IL‐6 (B,D) production by total peripheral blood (A,B) or naïve (C,D) B cells left unstimulated or stimulated for 6 days by CpG‐ODN or R848 in combination with T‐cell independent B‐cell class switch inducing factors as measured by Cytometric Bead Array on a flow cytometer in the supernatant of 6‐day‐old cultures. Data shown as mean + SEM, A and B: n = 8–14 donors per group, combined graph of five separate experiments, C and D: 3–5 donors per group, combined graph of two separate experiments * p
    Figure Legend Snippet: BAFF and Retinoic acid differentially affect IL‐10 and IL‐6 production. IL‐10 (A, C) and IL‐6 (B,D) production by total peripheral blood (A,B) or naïve (C,D) B cells left unstimulated or stimulated for 6 days by CpG‐ODN or R848 in combination with T‐cell independent B‐cell class switch inducing factors as measured by Cytometric Bead Array on a flow cytometer in the supernatant of 6‐day‐old cultures. Data shown as mean + SEM, A and B: n = 8–14 donors per group, combined graph of five separate experiments, C and D: 3–5 donors per group, combined graph of two separate experiments * p

    Techniques Used: Flow Cytometry, Cytometry

    BAFF and Retinoic acid differentially affect IgA1 and IgA2 production in mature stimulated B cells. IgA1 (A,C) and IgA2 (B,D) production by total peripheral blood (A,B) or naïve (C,D) B cells left unstimulated or stimulated for 6 days by CpG‐ODN or R848 in combination with T cell independent B cell class switch inducing factors as measured by IgA1 and IgA2 specific ELISA. Data shown as mean + SEM, A and B: n = 8–14 donors per group, combined graph of five separate experiments * p
    Figure Legend Snippet: BAFF and Retinoic acid differentially affect IgA1 and IgA2 production in mature stimulated B cells. IgA1 (A,C) and IgA2 (B,D) production by total peripheral blood (A,B) or naïve (C,D) B cells left unstimulated or stimulated for 6 days by CpG‐ODN or R848 in combination with T cell independent B cell class switch inducing factors as measured by IgA1 and IgA2 specific ELISA. Data shown as mean + SEM, A and B: n = 8–14 donors per group, combined graph of five separate experiments * p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Retinoic acid affects homing marker expression on mature CpG‐ODN stimulated B cells, Percentages of homing molecules CCR10 + (A,C) and β7 + (B,D) on total peripheral blood (A,B) and naïve (C,D) B cells after 6 days culture with T cell independent B cell class switch inducing factors in combination with CpG‐ODN or R848 as measured by flow cytometry. Data shown as mean + SEM, A and B: n = 5 donors per group, combined graph of three separate experiments, Repeated measures ANOVA, Tukey post‐hoc test, * p
    Figure Legend Snippet: Retinoic acid affects homing marker expression on mature CpG‐ODN stimulated B cells, Percentages of homing molecules CCR10 + (A,C) and β7 + (B,D) on total peripheral blood (A,B) and naïve (C,D) B cells after 6 days culture with T cell independent B cell class switch inducing factors in combination with CpG‐ODN or R848 as measured by flow cytometry. Data shown as mean + SEM, A and B: n = 5 donors per group, combined graph of three separate experiments, Repeated measures ANOVA, Tukey post‐hoc test, * p

    Techniques Used: Marker, Expressing, Flow Cytometry, Cytometry

    BAFF and retinoic acid differentially affect viability of stimulated mature B cells, (A) Representative flow cytometry plots for viability staining (CpG‐ODN), (B) Percentages of viable B cells (6 days of culture of total peripheral blood B cells) after stimulation with CpG‐ODN or R848 in combination with T cell independent B cell class switch inducing factors (mean +SEM, n = 5 donors per group, combined graph of three separate experiments, Repeated measures ANOVA, Tukey post‐hoc test, * p
    Figure Legend Snippet: BAFF and retinoic acid differentially affect viability of stimulated mature B cells, (A) Representative flow cytometry plots for viability staining (CpG‐ODN), (B) Percentages of viable B cells (6 days of culture of total peripheral blood B cells) after stimulation with CpG‐ODN or R848 in combination with T cell independent B cell class switch inducing factors (mean +SEM, n = 5 donors per group, combined graph of three separate experiments, Repeated measures ANOVA, Tukey post‐hoc test, * p

    Techniques Used: Flow Cytometry, Cytometry, Staining

    Retinoic acid increases the percentage of CD38 on naive and mature B cells, (A) Representative flow cytometry plots for CD38 staining, (B) Percentage of CD38 + B cells after 6 days of culture of total peripheral blood B cells after stimulation with CpG‐ODN or R848 in combination with T cell independent B cell class switch inducing factors (mean +SEM, n = 5 donors per group, combined graph of three separate experiments, Repeated measures ANOVA, Tukey post‐hoc test, * p
    Figure Legend Snippet: Retinoic acid increases the percentage of CD38 on naive and mature B cells, (A) Representative flow cytometry plots for CD38 staining, (B) Percentage of CD38 + B cells after 6 days of culture of total peripheral blood B cells after stimulation with CpG‐ODN or R848 in combination with T cell independent B cell class switch inducing factors (mean +SEM, n = 5 donors per group, combined graph of three separate experiments, Repeated measures ANOVA, Tukey post‐hoc test, * p

    Techniques Used: Flow Cytometry, Cytometry, Staining

    27) Product Images from "BAFF augments IgA2 and IL‐10 production by TLR7/8 stimulated total peripheral blood B cells"

    Article Title: BAFF augments IgA2 and IL‐10 production by TLR7/8 stimulated total peripheral blood B cells

    Journal: European Journal of Immunology

    doi: 10.1002/eji.201646861

    BAFF and Retinoic acid differentially affect IL‐10 and IL‐6 production. IL‐10 (A, C) and IL‐6 (B,D) production by total peripheral blood (A,B) or naïve (C,D) B cells left unstimulated or stimulated for 6 days by CpG‐ODN or R848 in combination with T‐cell independent B‐cell class switch inducing factors as measured by Cytometric Bead Array on a flow cytometer in the supernatant of 6‐day‐old cultures. Data shown as mean + SEM, A and B: n = 8–14 donors per group, combined graph of five separate experiments, C and D: 3–5 donors per group, combined graph of two separate experiments * p
    Figure Legend Snippet: BAFF and Retinoic acid differentially affect IL‐10 and IL‐6 production. IL‐10 (A, C) and IL‐6 (B,D) production by total peripheral blood (A,B) or naïve (C,D) B cells left unstimulated or stimulated for 6 days by CpG‐ODN or R848 in combination with T‐cell independent B‐cell class switch inducing factors as measured by Cytometric Bead Array on a flow cytometer in the supernatant of 6‐day‐old cultures. Data shown as mean + SEM, A and B: n = 8–14 donors per group, combined graph of five separate experiments, C and D: 3–5 donors per group, combined graph of two separate experiments * p

    Techniques Used: Flow Cytometry, Cytometry

    BAFF and Retinoic acid differentially affect IgA1 and IgA2 production in mature stimulated B cells. IgA1 (A,C) and IgA2 (B,D) production by total peripheral blood (A,B) or naïve (C,D) B cells left unstimulated or stimulated for 6 days by CpG‐ODN or R848 in combination with T cell independent B cell class switch inducing factors as measured by IgA1 and IgA2 specific ELISA. Data shown as mean + SEM, A and B: n = 8–14 donors per group, combined graph of five separate experiments * p
    Figure Legend Snippet: BAFF and Retinoic acid differentially affect IgA1 and IgA2 production in mature stimulated B cells. IgA1 (A,C) and IgA2 (B,D) production by total peripheral blood (A,B) or naïve (C,D) B cells left unstimulated or stimulated for 6 days by CpG‐ODN or R848 in combination with T cell independent B cell class switch inducing factors as measured by IgA1 and IgA2 specific ELISA. Data shown as mean + SEM, A and B: n = 8–14 donors per group, combined graph of five separate experiments * p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Retinoic acid affects homing marker expression on mature CpG‐ODN stimulated B cells, Percentages of homing molecules CCR10 + (A,C) and β7 + (B,D) on total peripheral blood (A,B) and naïve (C,D) B cells after 6 days culture with T cell independent B cell class switch inducing factors in combination with CpG‐ODN or R848 as measured by flow cytometry. Data shown as mean + SEM, A and B: n = 5 donors per group, combined graph of three separate experiments, Repeated measures ANOVA, Tukey post‐hoc test, * p
    Figure Legend Snippet: Retinoic acid affects homing marker expression on mature CpG‐ODN stimulated B cells, Percentages of homing molecules CCR10 + (A,C) and β7 + (B,D) on total peripheral blood (A,B) and naïve (C,D) B cells after 6 days culture with T cell independent B cell class switch inducing factors in combination with CpG‐ODN or R848 as measured by flow cytometry. Data shown as mean + SEM, A and B: n = 5 donors per group, combined graph of three separate experiments, Repeated measures ANOVA, Tukey post‐hoc test, * p

    Techniques Used: Marker, Expressing, Flow Cytometry, Cytometry

    BAFF and retinoic acid differentially affect viability of stimulated mature B cells, (A) Representative flow cytometry plots for viability staining (CpG‐ODN), (B) Percentages of viable B cells (6 days of culture of total peripheral blood B cells) after stimulation with CpG‐ODN or R848 in combination with T cell independent B cell class switch inducing factors (mean +SEM, n = 5 donors per group, combined graph of three separate experiments, Repeated measures ANOVA, Tukey post‐hoc test, * p
    Figure Legend Snippet: BAFF and retinoic acid differentially affect viability of stimulated mature B cells, (A) Representative flow cytometry plots for viability staining (CpG‐ODN), (B) Percentages of viable B cells (6 days of culture of total peripheral blood B cells) after stimulation with CpG‐ODN or R848 in combination with T cell independent B cell class switch inducing factors (mean +SEM, n = 5 donors per group, combined graph of three separate experiments, Repeated measures ANOVA, Tukey post‐hoc test, * p

    Techniques Used: Flow Cytometry, Cytometry, Staining

    Retinoic acid increases the percentage of CD38 on naive and mature B cells, (A) Representative flow cytometry plots for CD38 staining, (B) Percentage of CD38 + B cells after 6 days of culture of total peripheral blood B cells after stimulation with CpG‐ODN or R848 in combination with T cell independent B cell class switch inducing factors (mean +SEM, n = 5 donors per group, combined graph of three separate experiments, Repeated measures ANOVA, Tukey post‐hoc test, * p
    Figure Legend Snippet: Retinoic acid increases the percentage of CD38 on naive and mature B cells, (A) Representative flow cytometry plots for CD38 staining, (B) Percentage of CD38 + B cells after 6 days of culture of total peripheral blood B cells after stimulation with CpG‐ODN or R848 in combination with T cell independent B cell class switch inducing factors (mean +SEM, n = 5 donors per group, combined graph of three separate experiments, Repeated measures ANOVA, Tukey post‐hoc test, * p

    Techniques Used: Flow Cytometry, Cytometry, Staining

    28) Product Images from "In vivo characterization of the physicochemical properties of TLR agonist delivery that enhance vaccine immunogenicity"

    Article Title: In vivo characterization of the physicochemical properties of TLR agonist delivery that enhance vaccine immunogenicity

    Journal: Nature biotechnology

    doi: 10.1038/nbt.3371

    Persistent, locally restricted innate immune activation is necessary and sufficient for eliciting protective CD8 and Th1-type CD4 T cell responses (a–c) CpG ODN 1826 (3.1 nmoles, 20 μg), R848 (62.5 nmoles, 20 μg) or PP-7/8a (62.5 nmoles, 120 μg) were delivered subcutaneously into both hind footpads of C57BL/6 mice. (a) Supernatant of ex vivo cultured lymph node cell suspensions (n = 4) and (b) serum (n = 5) were assessed for IL-12p40 by ELISA at serial timepoints. (c) Percent body weight change (n = 3) following subcutaneous administration of different vaccine adjuvants (significance is shown for comparison with naïve; two-way ANOVA with Bonferroni correction). (d) Relationship between biodistribution and local and systemic innate immune activation. (e, f) C57BL/6 mice received subcutaneous administration of protein antigen (either 50 μg of OVA, or 20 μg of SIV Gag p41) formulated with adjuvant at days 0 and 14. At day 24, tetramer + CD8 T cell responses were assessed from whole blood by flow cytometry (n = 6). (g, h) Mice were challenged intravenously at day 28 with either (g) LM -OVA or (h) LM -Gag, and bacterial burden in spleens (n = 6) was evaluated on day 31 and 30, respectively. (i–l) C57BL/6 mice received subcutaneous immunizations of 20 μg of MML with or without adjuvant on days 0, 21 and 42. (i) Splenocytes were isolated on day 70 and stimulated in vitro with an MML peptide pool. CD4 T cells in the mixed splenocyte cultures were evaluated for Th1 characteristic cytokine (IFNγ, IL-2 and TNFα) production (n = 4). (j) Mice were challenged intradermally in both ears with L. major at day 70. Ear lesion diameters (n = 6) were measured for 12 weeks (significance is shown for comparison with protein alone). All data are representative of two or more independent experiments, except the Leishmania ear lesion kinetic is from a single study. Data on log scale are reported as geometric mean with 95% CI. Unless stated otherwise, comparison of multiple groups for statistical significance was determined using Kruskal-Wallis ANOVA with Dunn’s post test; ns, not significant (P > 0.05); *, P
    Figure Legend Snippet: Persistent, locally restricted innate immune activation is necessary and sufficient for eliciting protective CD8 and Th1-type CD4 T cell responses (a–c) CpG ODN 1826 (3.1 nmoles, 20 μg), R848 (62.5 nmoles, 20 μg) or PP-7/8a (62.5 nmoles, 120 μg) were delivered subcutaneously into both hind footpads of C57BL/6 mice. (a) Supernatant of ex vivo cultured lymph node cell suspensions (n = 4) and (b) serum (n = 5) were assessed for IL-12p40 by ELISA at serial timepoints. (c) Percent body weight change (n = 3) following subcutaneous administration of different vaccine adjuvants (significance is shown for comparison with naïve; two-way ANOVA with Bonferroni correction). (d) Relationship between biodistribution and local and systemic innate immune activation. (e, f) C57BL/6 mice received subcutaneous administration of protein antigen (either 50 μg of OVA, or 20 μg of SIV Gag p41) formulated with adjuvant at days 0 and 14. At day 24, tetramer + CD8 T cell responses were assessed from whole blood by flow cytometry (n = 6). (g, h) Mice were challenged intravenously at day 28 with either (g) LM -OVA or (h) LM -Gag, and bacterial burden in spleens (n = 6) was evaluated on day 31 and 30, respectively. (i–l) C57BL/6 mice received subcutaneous immunizations of 20 μg of MML with or without adjuvant on days 0, 21 and 42. (i) Splenocytes were isolated on day 70 and stimulated in vitro with an MML peptide pool. CD4 T cells in the mixed splenocyte cultures were evaluated for Th1 characteristic cytokine (IFNγ, IL-2 and TNFα) production (n = 4). (j) Mice were challenged intradermally in both ears with L. major at day 70. Ear lesion diameters (n = 6) were measured for 12 weeks (significance is shown for comparison with protein alone). All data are representative of two or more independent experiments, except the Leishmania ear lesion kinetic is from a single study. Data on log scale are reported as geometric mean with 95% CI. Unless stated otherwise, comparison of multiple groups for statistical significance was determined using Kruskal-Wallis ANOVA with Dunn’s post test; ns, not significant (P > 0.05); *, P

    Techniques Used: Activation Assay, Mouse Assay, Ex Vivo, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Isolation, In Vitro

    29) Product Images from "Small-molecule inhibition of TLR8 through stabilization of its resting state"

    Article Title: Small-molecule inhibition of TLR8 through stabilization of its resting state

    Journal: Nature chemical biology

    doi: 10.1038/nchembio.2518

    Crystal structure of the TLR8/CU-CPT8m complex (a) Front (top) and side (bottom) views of the unliganded (left, PDB ID 3W3G), TLR8/ CU-CPT8m (middle) and TLR8/R848 (right, PDB ID 3W3N) complexes. TLR8 and its dimerization partner TLR8* are colored green and cyan, respectively. The distances between the C-termini of the two protomers of TLR8 dimer (TLR8/ CU-CPT8m ) is similar to that of the unliganded dimer (right). Superimposition of the TLR8 structure complexed with CU-CPT8m onto the corresponding unliganded TLR8 segment (a.a. 32–816) produces root-mean-square deviation (RMSD) values of 2.4 Å. The ligand molecules are illustrated by space-filling representations. The C, O and N atoms of the ligands are colored yellow, red, and blue, respectively. (b) Close-up view of antagonist binding site of unliganded TLR8 (left) and TLR8/ CU-CPT8m (right). Water molecules are indicated by red filled circles. (c) Schematic representation of interactions between CU-CPT8m and the TLR8 protein. The hydrophobic pocket and hydrogen bonds are shown as dashed gray arcs and dashed red lines, respectively.
    Figure Legend Snippet: Crystal structure of the TLR8/CU-CPT8m complex (a) Front (top) and side (bottom) views of the unliganded (left, PDB ID 3W3G), TLR8/ CU-CPT8m (middle) and TLR8/R848 (right, PDB ID 3W3N) complexes. TLR8 and its dimerization partner TLR8* are colored green and cyan, respectively. The distances between the C-termini of the two protomers of TLR8 dimer (TLR8/ CU-CPT8m ) is similar to that of the unliganded dimer (right). Superimposition of the TLR8 structure complexed with CU-CPT8m onto the corresponding unliganded TLR8 segment (a.a. 32–816) produces root-mean-square deviation (RMSD) values of 2.4 Å. The ligand molecules are illustrated by space-filling representations. The C, O and N atoms of the ligands are colored yellow, red, and blue, respectively. (b) Close-up view of antagonist binding site of unliganded TLR8 (left) and TLR8/ CU-CPT8m (right). Water molecules are indicated by red filled circles. (c) Schematic representation of interactions between CU-CPT8m and the TLR8 protein. The hydrophobic pocket and hydrogen bonds are shown as dashed gray arcs and dashed red lines, respectively.

    Techniques Used: Binding Assay

    CU-CPT8m potently and selectively inhibited TLR8 (a) Chemical structures of CU-CPT8m and 6 (negative control), concentration-response curve and dose-dependent cytotoxicity of CU-CPT8m in HEK-Blue TLR8 cell line. Data was normalized to a DMSO control (data are mean ± SD ; n = 3 independent experiments). (b) ITC thermogram of CU-CPT8m titrated into hTLR8 to determine binding affinity and stoichiometry (representative of one independent experiment). The raw data are presented on top and the integrated peak areas are shown and fitted below. Mean K d = 0.22 μM; stoichiometric binding N = 0.5. (c) Specificity test for CU-CPT8m (1 μM) with TLR-specific agonists used to selectively activate different HEK-Blue TLR-overexpressing cells in the presence or absence of 1 μM CU-CPT8m (data are mean ± SD ; n = 3 independent experiments). (d) TNF-α and IL-8 mRNA level in R848 treated HEK-Blue TLR8 cells in the presence and absence of 1 μM CU-CPT8m or the negative control, 6 (10 μM). Data are the average quantification of two independent experiments. (e) Dose-dependent response of CU-CPT8m on TLR8-mediated TNF-α production in THP-1 cells with indicated concentration of CU-CPT8m or 6 . Data are mean ± SD ; n = 3 independent experiments. (f) Dose-dependent response of CU-CPT8m or 6 on TLR8-mediated TNF-α production in PBMC cells induced by 1 μg/mL R848. Data are mean ± SD ; n = 3 independent experiments.
    Figure Legend Snippet: CU-CPT8m potently and selectively inhibited TLR8 (a) Chemical structures of CU-CPT8m and 6 (negative control), concentration-response curve and dose-dependent cytotoxicity of CU-CPT8m in HEK-Blue TLR8 cell line. Data was normalized to a DMSO control (data are mean ± SD ; n = 3 independent experiments). (b) ITC thermogram of CU-CPT8m titrated into hTLR8 to determine binding affinity and stoichiometry (representative of one independent experiment). The raw data are presented on top and the integrated peak areas are shown and fitted below. Mean K d = 0.22 μM; stoichiometric binding N = 0.5. (c) Specificity test for CU-CPT8m (1 μM) with TLR-specific agonists used to selectively activate different HEK-Blue TLR-overexpressing cells in the presence or absence of 1 μM CU-CPT8m (data are mean ± SD ; n = 3 independent experiments). (d) TNF-α and IL-8 mRNA level in R848 treated HEK-Blue TLR8 cells in the presence and absence of 1 μM CU-CPT8m or the negative control, 6 (10 μM). Data are the average quantification of two independent experiments. (e) Dose-dependent response of CU-CPT8m on TLR8-mediated TNF-α production in THP-1 cells with indicated concentration of CU-CPT8m or 6 . Data are mean ± SD ; n = 3 independent experiments. (f) Dose-dependent response of CU-CPT8m or 6 on TLR8-mediated TNF-α production in PBMC cells induced by 1 μg/mL R848. Data are mean ± SD ; n = 3 independent experiments.

    Techniques Used: Negative Control, Concentration Assay, Binding Assay

    Proposed antagonistic mechanism of CU-CPT compounds (top) and schematic representation of domain arrangement in each TLR8 forms (bottom) LRR8, LRR11-13, LRR15-16, and LRR17-18 are colored yellow, green, blue, and purple, respectively. In the bottom panel, the antagonist and agonist are illustrated by yellow and orange circles. Interactions between ligands and protruding loop regions are shown by dashed arrows. TLR8 utilized LRR11-13 in common for both agonist and antagonist binding on one side of the interface, while on the other side LRR17*-18* and LRR15*-16* for agonist and antagonist binding, respectively. Binding of agonist (e.g. R848 ) brings two TLR8 C-termini to a closer distance to initiate downstream signaling; while binding of antagonists (e.g. CU-CPT8m , CU-CPT9b ) at the antagonist binding site stabilizes inactive TLR8 dimer with C-termini further apart, preventing TLR8 from activation.
    Figure Legend Snippet: Proposed antagonistic mechanism of CU-CPT compounds (top) and schematic representation of domain arrangement in each TLR8 forms (bottom) LRR8, LRR11-13, LRR15-16, and LRR17-18 are colored yellow, green, blue, and purple, respectively. In the bottom panel, the antagonist and agonist are illustrated by yellow and orange circles. Interactions between ligands and protruding loop regions are shown by dashed arrows. TLR8 utilized LRR11-13 in common for both agonist and antagonist binding on one side of the interface, while on the other side LRR17*-18* and LRR15*-16* for agonist and antagonist binding, respectively. Binding of agonist (e.g. R848 ) brings two TLR8 C-termini to a closer distance to initiate downstream signaling; while binding of antagonists (e.g. CU-CPT8m , CU-CPT9b ) at the antagonist binding site stabilizes inactive TLR8 dimer with C-termini further apart, preventing TLR8 from activation.

    Techniques Used: Cycling Probe Technology, Binding Assay, Activation Assay

    TLR8 inhibitors consistently recognize an allosteric pocket on the protein-protein interface, stabilizing the inactive TLR8 dimer (a) Chemical structure of CU-CPT9a and CU-CPT9b. (b) Close-up view of antagonist binding site (left) and its schematic representation of TLR8/ CU-CPT9b (right). The C, O and N atoms of the ligands are colored yellow, red, and blue, respectively. Water molecules mediating the ligand recognition are indicated by red filled circles and hydrogen bonds by dashed lines. (c) Dose-dependent dimerization of TLR8. Elution profiles of gel filtration chromatography of TLR8 with CU-CPT9b (left) and R848 (right) at various concentrations. Retention volume and normalized absorbance at 280 nm (A 280 ) are shown on the left, and retention volume of TLR8 peak is plotted against its molar ratio (ligand/TLR8) on the right (representative of one independent experiment).
    Figure Legend Snippet: TLR8 inhibitors consistently recognize an allosteric pocket on the protein-protein interface, stabilizing the inactive TLR8 dimer (a) Chemical structure of CU-CPT9a and CU-CPT9b. (b) Close-up view of antagonist binding site (left) and its schematic representation of TLR8/ CU-CPT9b (right). The C, O and N atoms of the ligands are colored yellow, red, and blue, respectively. Water molecules mediating the ligand recognition are indicated by red filled circles and hydrogen bonds by dashed lines. (c) Dose-dependent dimerization of TLR8. Elution profiles of gel filtration chromatography of TLR8 with CU-CPT9b (left) and R848 (right) at various concentrations. Retention volume and normalized absorbance at 280 nm (A 280 ) are shown on the left, and retention volume of TLR8 peak is plotted against its molar ratio (ligand/TLR8) on the right (representative of one independent experiment).

    Techniques Used: Binding Assay, Filtration, Chromatography

    30) Product Images from "Conventional DCs from Male and Female Lupus-Prone B6.NZM Sle1/Sle2/Sle3 Mice Express an IFN Signature and Have a Higher Immunometabolism That Are Enhanced by Estrogen"

    Article Title: Conventional DCs from Male and Female Lupus-Prone B6.NZM Sle1/Sle2/Sle3 Mice Express an IFN Signature and Have a Higher Immunometabolism That Are Enhanced by Estrogen

    Journal: Journal of Immunology Research

    doi: 10.1155/2018/1601079

    Estrogen enhances the upregulation of ISGs in response to TLR stimulation. B6 (black closed symbols) or TCSle (gray open symbols) female (circle) and male (triangle) mice were cultured with GM-CSF in standard phenol red/media conditions or media depleted of phenol red and void of steroids (charcoal-treated FBS: 0 E2) supplemented with 0.03 nM, 0.1 nM, or 50 nM E2. On day 7, cDCs were stimulated with CpG B 1826 (10 μ g/mL) (a–c) or R848 (1 μ g/mL) (d–f). Six hours post stimulation, cDCs were harvested for qRT-PCR analysis. ISGs were normalized to the housekeeping gene cyclophilin . Standard female B6 control condition without stimulation (not shown) was set to 1 in each experiment. Mean + SE values are from 6 independent experiments, using one mouse of each strain and sex per experiment. Two-way ANOVA analysis with Tukey multiple comparisons was used to calculate the significance of the effects of E2 treatment within each group of mice and the results are represented by brackets below the graph. A black star indicates statistical significance in all the fours curves representing both sexes and strains of cDCs. Two-way ANOVA analysis with Tukey multiple comparisons was used to compare differences between B6 and TCSle, and results are shown in a box surrounding the symbol Δ for significance between B6 and TCSle males or the symbol O for significance between B6 and TCSle females. ∗ , Δ, and O represent p
    Figure Legend Snippet: Estrogen enhances the upregulation of ISGs in response to TLR stimulation. B6 (black closed symbols) or TCSle (gray open symbols) female (circle) and male (triangle) mice were cultured with GM-CSF in standard phenol red/media conditions or media depleted of phenol red and void of steroids (charcoal-treated FBS: 0 E2) supplemented with 0.03 nM, 0.1 nM, or 50 nM E2. On day 7, cDCs were stimulated with CpG B 1826 (10 μ g/mL) (a–c) or R848 (1 μ g/mL) (d–f). Six hours post stimulation, cDCs were harvested for qRT-PCR analysis. ISGs were normalized to the housekeeping gene cyclophilin . Standard female B6 control condition without stimulation (not shown) was set to 1 in each experiment. Mean + SE values are from 6 independent experiments, using one mouse of each strain and sex per experiment. Two-way ANOVA analysis with Tukey multiple comparisons was used to calculate the significance of the effects of E2 treatment within each group of mice and the results are represented by brackets below the graph. A black star indicates statistical significance in all the fours curves representing both sexes and strains of cDCs. Two-way ANOVA analysis with Tukey multiple comparisons was used to compare differences between B6 and TCSle, and results are shown in a box surrounding the symbol Δ for significance between B6 and TCSle males or the symbol O for significance between B6 and TCSle females. ∗ , Δ, and O represent p

    Techniques Used: Mouse Assay, Cell Culture, Quantitative RT-PCR

    E2 enhances the higher energy metabolism of TCSle cDCs. cDCs from B6 (darker color) or TCSle (lighter color) female (red/orange) and male (blue/azur) mice were cultured in standard conditions or in hormone-depleted conditions supplemented with 0.03 nM, 0.1 nM, or 50 nM E2 (a–k). On day 7, cDC were stimulated with CpG B 1826 (10 μ g/mL) or R848 (1 μ g/mL). Supernatants were harvested and analyzed using the Griess reaction 24 hours after stimulation (a–e). Six hours post stimulation, cDCs were harvested for qRT-PCR analysis (f–j). Nos2 (f–j) and Pdk1 (k) genes were normalized to the housekeeping gene cyclophilin . Standard female B6 condition was set to 1. Mean + SE values are from 3 independent experiments, using one mouse per strain per experiment. Two-way ANOVA analysis with Tukey multiple comparisons was used to determine the significant activation by CpG or R848 within each group of mice, represented by brackets below the graph. Black ∗ indicates significance within all 4 groups while individual colors/symbols represent significance within a single group (a–k). Two-way ANOVA analysis with Tukey multiple comparisons, used to compare differences between females and males, is represented by brackets and ∗ above the graph (a–e). Tukey multiple comparisons, used to compare differences between B6 and TCSle, is represented by a box surrounding red Δ symbol for B6 and TCSle females or a blue O symbol for B6 and TCSle males (a–j). Two-way ANOVA analysis with Tukey multiple comparisons is used to determine the effects of E2 treatment on cDC differentiation within each group of mice represented by brackets and symbols below the graph (j). ∗ , Δ, and O represent p
    Figure Legend Snippet: E2 enhances the higher energy metabolism of TCSle cDCs. cDCs from B6 (darker color) or TCSle (lighter color) female (red/orange) and male (blue/azur) mice were cultured in standard conditions or in hormone-depleted conditions supplemented with 0.03 nM, 0.1 nM, or 50 nM E2 (a–k). On day 7, cDC were stimulated with CpG B 1826 (10 μ g/mL) or R848 (1 μ g/mL). Supernatants were harvested and analyzed using the Griess reaction 24 hours after stimulation (a–e). Six hours post stimulation, cDCs were harvested for qRT-PCR analysis (f–j). Nos2 (f–j) and Pdk1 (k) genes were normalized to the housekeeping gene cyclophilin . Standard female B6 condition was set to 1. Mean + SE values are from 3 independent experiments, using one mouse per strain per experiment. Two-way ANOVA analysis with Tukey multiple comparisons was used to determine the significant activation by CpG or R848 within each group of mice, represented by brackets below the graph. Black ∗ indicates significance within all 4 groups while individual colors/symbols represent significance within a single group (a–k). Two-way ANOVA analysis with Tukey multiple comparisons, used to compare differences between females and males, is represented by brackets and ∗ above the graph (a–e). Tukey multiple comparisons, used to compare differences between B6 and TCSle, is represented by a box surrounding red Δ symbol for B6 and TCSle females or a blue O symbol for B6 and TCSle males (a–j). Two-way ANOVA analysis with Tukey multiple comparisons is used to determine the effects of E2 treatment on cDC differentiation within each group of mice represented by brackets and symbols below the graph (j). ∗ , Δ, and O represent p

    Techniques Used: Mouse Assay, Cell Culture, Quantitative RT-PCR, Activation Assay

    Female and male TCSle cDCs have a higher immunometabolism. Bone marrow-derived cDCs from B6 and TCSle female and male mice were cultured in standard conditions. On day 7, cDCs were stimulated with CpG (10 μ g/mL) or R848 (1 μ g/mL). cDCs were harvested 6 hours post stimulation for qRT-PCR analysis. Pdk1 was normalized to the housekeeping gene cyclophilin (a). Standard female unstimulated B6 condition was set to 1 in each experiment. Supernatants were harvested at the 24-hour time point and analyzed using the Griess reaction to measure nitrites (b). Dotted lines at 18 and 40 represent female B6 levels after CpG and R848 stimulation. Mean + SE values are from 3 independent experiments, using one mouse of each strain and sex per experiment (a, b). Two-way ANOVA analysis with Tukey multiple comparisons was used to determine significant activation by CpG or R848 within each group of mice represented by brackets and ∗ below the graph. Two-way ANOVA analysis with Tukey multiple comparisons, used to compare differences between B6 and TCSle, is represented by brackets and ∗ above the bars. ∗ p
    Figure Legend Snippet: Female and male TCSle cDCs have a higher immunometabolism. Bone marrow-derived cDCs from B6 and TCSle female and male mice were cultured in standard conditions. On day 7, cDCs were stimulated with CpG (10 μ g/mL) or R848 (1 μ g/mL). cDCs were harvested 6 hours post stimulation for qRT-PCR analysis. Pdk1 was normalized to the housekeeping gene cyclophilin (a). Standard female unstimulated B6 condition was set to 1 in each experiment. Supernatants were harvested at the 24-hour time point and analyzed using the Griess reaction to measure nitrites (b). Dotted lines at 18 and 40 represent female B6 levels after CpG and R848 stimulation. Mean + SE values are from 3 independent experiments, using one mouse of each strain and sex per experiment (a, b). Two-way ANOVA analysis with Tukey multiple comparisons was used to determine significant activation by CpG or R848 within each group of mice represented by brackets and ∗ below the graph. Two-way ANOVA analysis with Tukey multiple comparisons, used to compare differences between B6 and TCSle, is represented by brackets and ∗ above the bars. ∗ p

    Techniques Used: Derivative Assay, Mouse Assay, Cell Culture, Quantitative RT-PCR, Activation Assay

    Estrogen enhances CXCL10 chemokine production not predicted by Cxcl10 RNA. Bone marrow precursors from B6 (black closed symbols) or TCSle (gray open symbols) female (circle) and male (triangle) mice were cultured in standard conditions or hormone-depleted conditions supplemented with 0.03 nM, 0.1 nM, or 50 nM E2 (a, b, e, f). TCSle cDCs were cultured in standard conditions supplemented with Fulvestrant (Fulvi 1 μ M or 100 nM in DMSO) and Tamoxifen (Tam 10 nM or 100 nM in ethanol) (c, d). On day 7, cDCs were stimulated with CpG (10 μ g/mL) (a, c, e) or R848 (1 μ g/mL) (b, d, f). Supernatants were harvested 24 hours post stimulation and analyzed by ELISA for CXCL10 protein levels (a–d). Total RNA was isolated 6 hours post stimulation and analyzed by qRT-PCR (e, f). Mean + SE values are from 3 (female cDCs) or 4 (male cDCs) independent experiments or (c and d) 2 independent experiments using one mouse per strain per experiment (a, b, e, f). Two-way ANOVA analysis with Tukey multiple comparisons was used to calculate the significance of the effects of E2 treatment within each group of mice, represented by brackets below the graph. Black ∗ indicates significance within all 4 groups while symbols represent significance within a single group. Two-way ANOVA analysis with Tukey multiple comparisons was used to compare differences between B6 and TCSle, and results are shown in a box surrounding the symbol Δ for significance between B6 and TCSle males or the symbol O for significance between B6 and TCSle females. ∗ , Δ, and O represent p
    Figure Legend Snippet: Estrogen enhances CXCL10 chemokine production not predicted by Cxcl10 RNA. Bone marrow precursors from B6 (black closed symbols) or TCSle (gray open symbols) female (circle) and male (triangle) mice were cultured in standard conditions or hormone-depleted conditions supplemented with 0.03 nM, 0.1 nM, or 50 nM E2 (a, b, e, f). TCSle cDCs were cultured in standard conditions supplemented with Fulvestrant (Fulvi 1 μ M or 100 nM in DMSO) and Tamoxifen (Tam 10 nM or 100 nM in ethanol) (c, d). On day 7, cDCs were stimulated with CpG (10 μ g/mL) (a, c, e) or R848 (1 μ g/mL) (b, d, f). Supernatants were harvested 24 hours post stimulation and analyzed by ELISA for CXCL10 protein levels (a–d). Total RNA was isolated 6 hours post stimulation and analyzed by qRT-PCR (e, f). Mean + SE values are from 3 (female cDCs) or 4 (male cDCs) independent experiments or (c and d) 2 independent experiments using one mouse per strain per experiment (a, b, e, f). Two-way ANOVA analysis with Tukey multiple comparisons was used to calculate the significance of the effects of E2 treatment within each group of mice, represented by brackets below the graph. Black ∗ indicates significance within all 4 groups while symbols represent significance within a single group. Two-way ANOVA analysis with Tukey multiple comparisons was used to compare differences between B6 and TCSle, and results are shown in a box surrounding the symbol Δ for significance between B6 and TCSle males or the symbol O for significance between B6 and TCSle females. ∗ , Δ, and O represent p

    Techniques Used: Mouse Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Isolation, Quantitative RT-PCR

    E2 enhances both IFN-dependent and IFN-independent cytokine production. Bone marrow precursors from B6 (black closed symbols) or TCSle (gray open symbols) female (circle) and male (triangle) mice were cultured with GM-CSF in standard phenol red/media conditions or media depleted in phenol red and void of steroids (charcoal-treated FBS: 0 E2) supplemented with 0.03 nM, 0.1 nM, or 50 nM E2. On day 7, cDCs were stimulated with CpG (10 μ g/mL) (a, c, e) or R848 (1 μ g/mL) (b, d, f). Supernatants were harvested and analyzed by ELISA 24 hours (IL-12p70 (a, b)) or 6 hours (IL-6 (c, d) and TNF- α (e, f)) post stimulation. Mean + SE values are from 3 (female cDCs) or 4 (male cDCs) independent experiments, using one mouse of each strain and sex per experiment. Two-way ANOVA analysis with Tukey multiple comparisons, used to compare differences between B6 and TCSle, did not reveal significance between the strains. Two-way ANOVA analysis with Tukey multiple comparisons was used to calculate the significance of the effects of E2 treatment within each group of mice, represented by brackets below the graph. Black ∗ indicates significance within all 4 groups while symbols represent significance within a single group. ∗ , Δ, and O represent p
    Figure Legend Snippet: E2 enhances both IFN-dependent and IFN-independent cytokine production. Bone marrow precursors from B6 (black closed symbols) or TCSle (gray open symbols) female (circle) and male (triangle) mice were cultured with GM-CSF in standard phenol red/media conditions or media depleted in phenol red and void of steroids (charcoal-treated FBS: 0 E2) supplemented with 0.03 nM, 0.1 nM, or 50 nM E2. On day 7, cDCs were stimulated with CpG (10 μ g/mL) (a, c, e) or R848 (1 μ g/mL) (b, d, f). Supernatants were harvested and analyzed by ELISA 24 hours (IL-12p70 (a, b)) or 6 hours (IL-6 (c, d) and TNF- α (e, f)) post stimulation. Mean + SE values are from 3 (female cDCs) or 4 (male cDCs) independent experiments, using one mouse of each strain and sex per experiment. Two-way ANOVA analysis with Tukey multiple comparisons, used to compare differences between B6 and TCSle, did not reveal significance between the strains. Two-way ANOVA analysis with Tukey multiple comparisons was used to calculate the significance of the effects of E2 treatment within each group of mice, represented by brackets below the graph. Black ∗ indicates significance within all 4 groups while symbols represent significance within a single group. ∗ , Δ, and O represent p

    Techniques Used: Mouse Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    Estrogen enhances the upregulation of cDC activation markers in response to TLR stimulation. Bone marrow precursors from B6 (black closed symbols) or TCSle (gray open symbols) female (circle) and male (triangle) mice were cultured with GM-CSF in standard phenol red/media conditions or media depleted in phenol red and void of steroids (charcoal-treated FBS: 0 E2) supplemented with 0.03 nM, 0.1 nM, or 50 nM E2. On day 7, cDCs were stimulated with CpG (10 μ g/mL) (b, e, h) or R848 (1 μ g/mL) (c, f, i). cDCs were harvested 24 hours post stimulation, stained, and gated on singlets, live cells, and CD11c + CD11b + (gating shown in Figure 1(c) ) before analyzing costimulatory molecules (a–i). Representative histogram plots of CD40, CD86, and CD80 on unstimulated (black line) or CpG-stimulated (gray histogram) female TCSle cDCs from standard conditions (j). Mean + SE values are from 3 (female cDCs) or 4 (male cDCs) independent experiments, using one mouse of each strain and sex per experiment. Two-way ANOVA analysis with Tukey multiple comparisons was used to calculate the significance of the effects of E2 treatment within each group of mice, represented by brackets below the graph. Black ∗ indicates significance within all 4 groups while symbols represent significance within a single group. Two-way ANOVA analysis with Tukey multiple comparisons was used to compare differences between B6 and TCSle, and results are shown in a box surrounding the symbol Δ for significance between B6 and TCSle males or the symbol O for significance between B6 and TCSle females. ∗ , Δ, and O represent p
    Figure Legend Snippet: Estrogen enhances the upregulation of cDC activation markers in response to TLR stimulation. Bone marrow precursors from B6 (black closed symbols) or TCSle (gray open symbols) female (circle) and male (triangle) mice were cultured with GM-CSF in standard phenol red/media conditions or media depleted in phenol red and void of steroids (charcoal-treated FBS: 0 E2) supplemented with 0.03 nM, 0.1 nM, or 50 nM E2. On day 7, cDCs were stimulated with CpG (10 μ g/mL) (b, e, h) or R848 (1 μ g/mL) (c, f, i). cDCs were harvested 24 hours post stimulation, stained, and gated on singlets, live cells, and CD11c + CD11b + (gating shown in Figure 1(c) ) before analyzing costimulatory molecules (a–i). Representative histogram plots of CD40, CD86, and CD80 on unstimulated (black line) or CpG-stimulated (gray histogram) female TCSle cDCs from standard conditions (j). Mean + SE values are from 3 (female cDCs) or 4 (male cDCs) independent experiments, using one mouse of each strain and sex per experiment. Two-way ANOVA analysis with Tukey multiple comparisons was used to calculate the significance of the effects of E2 treatment within each group of mice, represented by brackets below the graph. Black ∗ indicates significance within all 4 groups while symbols represent significance within a single group. Two-way ANOVA analysis with Tukey multiple comparisons was used to compare differences between B6 and TCSle, and results are shown in a box surrounding the symbol Δ for significance between B6 and TCSle males or the symbol O for significance between B6 and TCSle females. ∗ , Δ, and O represent p

    Techniques Used: Activation Assay, Mouse Assay, Cell Culture, Staining

    31) Product Images from "Optimisation ofex vivo memory B cell expansion/differentiation for interrogation of rare peripheral memory B cell subset responses"

    Article Title: Optimisation ofex vivo memory B cell expansion/differentiation for interrogation of rare peripheral memory B cell subset responses

    Journal: Wellcome Open Research

    doi: 10.12688/wellcomeopenres.11386.2

    CD40 stimulation in combination with IL-21 TLR stimulation of memory B cells over a 10 day culture period induces high levels of Ig secretion. ( A ) 2×10 3 memory B cells were cultured for 5 days with HV13280 cells at a ratio of 4:1 with the addition of a checkerboard of different stimulants suggested to play a role in memory B cell expansion/differentiation (see Table 1 ). Total Ig (IgG + IgA) was measured in the culture supernatant by ELISA and the top readout for each crossover is shown. The full checkerboard screen can be seen in Supplementary Figure 2 . ( B and C ) Total Ig (IgG + IgA) measured by ELISA in culture supernatant of memory B cells stimulated with HV13280 cells, IL-21, R848 and CpG at ( B ) day 5 and ( C ) day 10 of culture. ( D ) Composition of the stimulation mixtures used in B C. Data is representative of one donor.
    Figure Legend Snippet: CD40 stimulation in combination with IL-21 TLR stimulation of memory B cells over a 10 day culture period induces high levels of Ig secretion. ( A ) 2×10 3 memory B cells were cultured for 5 days with HV13280 cells at a ratio of 4:1 with the addition of a checkerboard of different stimulants suggested to play a role in memory B cell expansion/differentiation (see Table 1 ). Total Ig (IgG + IgA) was measured in the culture supernatant by ELISA and the top readout for each crossover is shown. The full checkerboard screen can be seen in Supplementary Figure 2 . ( B and C ) Total Ig (IgG + IgA) measured by ELISA in culture supernatant of memory B cells stimulated with HV13280 cells, IL-21, R848 and CpG at ( B ) day 5 and ( C ) day 10 of culture. ( D ) Composition of the stimulation mixtures used in B C. Data is representative of one donor.

    Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay

    A Design of Experiments approach identifies IL-21, CpG and CD40 stimulation as having significant first order effects on IgG, IgM IgA secretion by differentiated memory B cells. 2×10 3 memory B cells were cultured with all possible combinations of HV13280 cells, IL-21, R848 and CpG at three set concentrations for 10 days, after which total Ig (IgG, IgA IgM) was measured in culture supernatant by ELISA. ( A – D ) Using a Matlab script, the effect of ( A ) IL-21, ( B ) R848, ( C ) CpG and ( D ) CD40 stimulation at the chosen concentrations on IgG, IgM and IgA secretion could be determined. ( E ) P-values showing the effects of each expansion stimulus on IgG, IgM and IgA secretion into culture supernatant over the 10 day culture. Data shows a summary of three independent donors.
    Figure Legend Snippet: A Design of Experiments approach identifies IL-21, CpG and CD40 stimulation as having significant first order effects on IgG, IgM IgA secretion by differentiated memory B cells. 2×10 3 memory B cells were cultured with all possible combinations of HV13280 cells, IL-21, R848 and CpG at three set concentrations for 10 days, after which total Ig (IgG, IgA IgM) was measured in culture supernatant by ELISA. ( A – D ) Using a Matlab script, the effect of ( A ) IL-21, ( B ) R848, ( C ) CpG and ( D ) CD40 stimulation at the chosen concentrations on IgG, IgM and IgA secretion could be determined. ( E ) P-values showing the effects of each expansion stimulus on IgG, IgM and IgA secretion into culture supernatant over the 10 day culture. Data shows a summary of three independent donors.

    Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay

    32) Product Images from "Optimisation ofex vivo memory B cell expansion/differentiation for interrogation of rare peripheral memory B cell subset responses"

    Article Title: Optimisation ofex vivo memory B cell expansion/differentiation for interrogation of rare peripheral memory B cell subset responses

    Journal: Wellcome Open Research

    doi: 10.12688/wellcomeopenres.11386.2

    CD40 stimulation in combination with IL-21 TLR stimulation of memory B cells over a 10 day culture period induces high levels of Ig secretion. ( A ) 2×10 3 memory B cells were cultured for 5 days with HV13280 cells at a ratio of 4:1 with the addition of a checkerboard of different stimulants suggested to play a role in memory B cell expansion/differentiation (see Table 1 ). Total Ig (IgG + IgA) was measured in the culture supernatant by ELISA and the top readout for each crossover is shown. The full checkerboard screen can be seen in Supplementary Figure 2 . ( B and C ) Total Ig (IgG + IgA) measured by ELISA in culture supernatant of memory B cells stimulated with HV13280 cells, IL-21, R848 and CpG at ( B ) day 5 and ( C ) day 10 of culture. ( D ) Composition of the stimulation mixtures used in B C. Data is representative of one donor.
    Figure Legend Snippet: CD40 stimulation in combination with IL-21 TLR stimulation of memory B cells over a 10 day culture period induces high levels of Ig secretion. ( A ) 2×10 3 memory B cells were cultured for 5 days with HV13280 cells at a ratio of 4:1 with the addition of a checkerboard of different stimulants suggested to play a role in memory B cell expansion/differentiation (see Table 1 ). Total Ig (IgG + IgA) was measured in the culture supernatant by ELISA and the top readout for each crossover is shown. The full checkerboard screen can be seen in Supplementary Figure 2 . ( B and C ) Total Ig (IgG + IgA) measured by ELISA in culture supernatant of memory B cells stimulated with HV13280 cells, IL-21, R848 and CpG at ( B ) day 5 and ( C ) day 10 of culture. ( D ) Composition of the stimulation mixtures used in B C. Data is representative of one donor.

    Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay

    A Design of Experiments approach identifies IL-21, CpG and CD40 stimulation as having significant first order effects on IgG, IgM IgA secretion by differentiated memory B cells. 2×10 3 memory B cells were cultured with all possible combinations of HV13280 cells, IL-21, R848 and CpG at three set concentrations for 10 days, after which total Ig (IgG, IgA IgM) was measured in culture supernatant by ELISA. ( A – D ) Using a Matlab script, the effect of ( A ) IL-21, ( B ) R848, ( C ) CpG and ( D ) CD40 stimulation at the chosen concentrations on IgG, IgM and IgA secretion could be determined. ( E ) P-values showing the effects of each expansion stimulus on IgG, IgM and IgA secretion into culture supernatant over the 10 day culture. Data shows a summary of three independent donors.
    Figure Legend Snippet: A Design of Experiments approach identifies IL-21, CpG and CD40 stimulation as having significant first order effects on IgG, IgM IgA secretion by differentiated memory B cells. 2×10 3 memory B cells were cultured with all possible combinations of HV13280 cells, IL-21, R848 and CpG at three set concentrations for 10 days, after which total Ig (IgG, IgA IgM) was measured in culture supernatant by ELISA. ( A – D ) Using a Matlab script, the effect of ( A ) IL-21, ( B ) R848, ( C ) CpG and ( D ) CD40 stimulation at the chosen concentrations on IgG, IgM and IgA secretion could be determined. ( E ) P-values showing the effects of each expansion stimulus on IgG, IgM and IgA secretion into culture supernatant over the 10 day culture. Data shows a summary of three independent donors.

    Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay

    33) Product Images from "Toll‐Like Receptor 9 Deficiency Breaks Tolerance to RNA‐Associated Antigens and Up‐Regulates Toll‐Like Receptor 7 Protein in Sle1 Mice"

    Article Title: Toll‐Like Receptor 9 Deficiency Breaks Tolerance to RNA‐Associated Antigens and Up‐Regulates Toll‐Like Receptor 7 Protein in Sle1 Mice

    Journal: Arthritis & Rheumatology (Hoboken, N.j.)

    doi: 10.1002/art.40535

    Infiltration of dendritic cells (DCs) with increased Toll‐like receptor 7 (TLR‐7) protein expression into Sle1 TLR‐9 −/− mouse kidneys. A , Analysis of renal CD45+CD11b+Gr‐1 −/low subsets using antibodies to CD11c, major histocompatibility complex class II (MHCII), F4/80, and CD11b. The magenta boxed area includes the CD11c+MHCII− precursor population. The black boxed area includes the CD11c+MHCII+ cells. CD11c+MHCII+ cells were further subdivided into F4/80+ macrophages (Mϕ; green encircled area) and F4/80 −/low DCs (blue encircled area). B , Cumulative frequencies of renal CD11b+Gr‐1− subsets identified in A in Sle1 and Sle1 TLR‐9 −/− mice. Bars show the mean ± SEM. Data are from 3 independent cohorts of 4.5–6.5‐month‐old mice with a total of 9–16 mice per group. C , Proliferation of CD4+TCRvα2+ Sle1 OT‐II cells exposed to ovalbumin‐pulsed F4/80+ macrophages or F4/80 −/low DCs, with or without prior stimulation with R848. Circles represent cells sorted from an individual mouse (n = 6), except for Sle1 F4/80 −/low DCs that were pooled to a total of 3 samples due to low cell numbers. D , Analysis of TLR‐7 protein expression, using flow cytometry, in the CD11b+Gr‐1− subsets identified in A in Sle1 and Sle1 TLR‐9 −/− mice. Symbols represent individual samples; horizontal lines and error bars show the mean ± SEM. E , Correlation of TLR‐7 expression in Sle1 and Sle1 TLR‐9 −/− mice with percentages of renal macrophage and DC infiltration. Data in D and E are from 3 independent experiments with a total of 8 mice (ages 5.7–6.4 months) per group. Each experiment was conducted with a TLR‐7 −/− and a fluorescence minus one (FMO) control (a total of 3 each). Parametric data were assessed by one‐way analysis of variance (with Bonferroni adjustment for multiple comparisons) or Student's t ‐test, and nonparametric data were assessed by Kruskal‐Wallis test (with Dunn's multiple comparison test) or Mann‐Whitney test. Correlations were determined using Spearman's rank correlation for nonparametric data and Pearson's correlation for parametric data. * = P
    Figure Legend Snippet: Infiltration of dendritic cells (DCs) with increased Toll‐like receptor 7 (TLR‐7) protein expression into Sle1 TLR‐9 −/− mouse kidneys. A , Analysis of renal CD45+CD11b+Gr‐1 −/low subsets using antibodies to CD11c, major histocompatibility complex class II (MHCII), F4/80, and CD11b. The magenta boxed area includes the CD11c+MHCII− precursor population. The black boxed area includes the CD11c+MHCII+ cells. CD11c+MHCII+ cells were further subdivided into F4/80+ macrophages (Mϕ; green encircled area) and F4/80 −/low DCs (blue encircled area). B , Cumulative frequencies of renal CD11b+Gr‐1− subsets identified in A in Sle1 and Sle1 TLR‐9 −/− mice. Bars show the mean ± SEM. Data are from 3 independent cohorts of 4.5–6.5‐month‐old mice with a total of 9–16 mice per group. C , Proliferation of CD4+TCRvα2+ Sle1 OT‐II cells exposed to ovalbumin‐pulsed F4/80+ macrophages or F4/80 −/low DCs, with or without prior stimulation with R848. Circles represent cells sorted from an individual mouse (n = 6), except for Sle1 F4/80 −/low DCs that were pooled to a total of 3 samples due to low cell numbers. D , Analysis of TLR‐7 protein expression, using flow cytometry, in the CD11b+Gr‐1− subsets identified in A in Sle1 and Sle1 TLR‐9 −/− mice. Symbols represent individual samples; horizontal lines and error bars show the mean ± SEM. E , Correlation of TLR‐7 expression in Sle1 and Sle1 TLR‐9 −/− mice with percentages of renal macrophage and DC infiltration. Data in D and E are from 3 independent experiments with a total of 8 mice (ages 5.7–6.4 months) per group. Each experiment was conducted with a TLR‐7 −/− and a fluorescence minus one (FMO) control (a total of 3 each). Parametric data were assessed by one‐way analysis of variance (with Bonferroni adjustment for multiple comparisons) or Student's t ‐test, and nonparametric data were assessed by Kruskal‐Wallis test (with Dunn's multiple comparison test) or Mann‐Whitney test. Correlations were determined using Spearman's rank correlation for nonparametric data and Pearson's correlation for parametric data. * = P

    Techniques Used: Expressing, Mouse Assay, Flow Cytometry, Cytometry, Fluorescence, MANN-WHITNEY

    Regulation of antibody production by Toll‐like receptor 9 (TLR‐9) in Sle1 mice. A and B , B cell (B220+) activation ( A ) and proliferation ( B ) in splenocytes from young (8–10‐week‐old) Sle1 and Sle1 TLR‐9 −/− mice that were left untreated (media), stimulated with R848 (0.01 μg/ml), or stimulated with lipopolysaccharide (LPS; 1 μg/ml). B cell activation was measured by flow cytometry after 24 hours of stimulation and is shown as the percentage of CD69+ cells. B cell proliferation was measured according to the 5,6‐carboxyfluorescein succinimidyl ester dilution after 72 hours of stimulation. C , TLR‐7 expression, measured by intracellular flow cytometry, in mouse splenic B220+ B cells. TLR‐7–deficient Sle1 mice ( Sle1 TLR‐7 −/− ) (n = 4) and fluorescence minus one (FMO) samples were used as negative controls. MFI = median fluorescence intensity. D , Levels of IgG subtypes, measured by Luminex, in Sle1 and Sle1 TLR‐9 −/− mouse culture supernatants collected after 96 hours of incubation. Cultures were left untreated, stimulated with R848, or stimulated with LPS. E , Expression of surface IgG (sIgG) (IgG1/IgG2a/IgG2b/IgG3) on freshly isolated B220+CD19+ splenocytes from Sle1 and Sle1 TLR‐9 −/− mice. F and G , TLR‐7 expression and frequencies of splenic CD138+ plasma/plasmablasts ( F ) and CD11b+ dendritic cells (DCs) ( G ) in Sle1 and Sle1 TLR‐9 −/− mice. In A , B , and D , bars show the mean ± SEM from 2–3 independent experiments (n = 9–15 mice per group). Data were assessed by multiple t ‐tests, and statistical significance was corrected using the Holm‐Sidak method. In C , E , F , and G , data are from 1 representative experiment with 8–10‐week‐old mice (n = 8 Sle1 and 5 Sle1 TLR‐9 −/− mice). Circles represent individual mice; horizontal lines and error bars show the mean ± SEM. Significance was determined by Student's t ‐test. * = P
    Figure Legend Snippet: Regulation of antibody production by Toll‐like receptor 9 (TLR‐9) in Sle1 mice. A and B , B cell (B220+) activation ( A ) and proliferation ( B ) in splenocytes from young (8–10‐week‐old) Sle1 and Sle1 TLR‐9 −/− mice that were left untreated (media), stimulated with R848 (0.01 μg/ml), or stimulated with lipopolysaccharide (LPS; 1 μg/ml). B cell activation was measured by flow cytometry after 24 hours of stimulation and is shown as the percentage of CD69+ cells. B cell proliferation was measured according to the 5,6‐carboxyfluorescein succinimidyl ester dilution after 72 hours of stimulation. C , TLR‐7 expression, measured by intracellular flow cytometry, in mouse splenic B220+ B cells. TLR‐7–deficient Sle1 mice ( Sle1 TLR‐7 −/− ) (n = 4) and fluorescence minus one (FMO) samples were used as negative controls. MFI = median fluorescence intensity. D , Levels of IgG subtypes, measured by Luminex, in Sle1 and Sle1 TLR‐9 −/− mouse culture supernatants collected after 96 hours of incubation. Cultures were left untreated, stimulated with R848, or stimulated with LPS. E , Expression of surface IgG (sIgG) (IgG1/IgG2a/IgG2b/IgG3) on freshly isolated B220+CD19+ splenocytes from Sle1 and Sle1 TLR‐9 −/− mice. F and G , TLR‐7 expression and frequencies of splenic CD138+ plasma/plasmablasts ( F ) and CD11b+ dendritic cells (DCs) ( G ) in Sle1 and Sle1 TLR‐9 −/− mice. In A , B , and D , bars show the mean ± SEM from 2–3 independent experiments (n = 9–15 mice per group). Data were assessed by multiple t ‐tests, and statistical significance was corrected using the Holm‐Sidak method. In C , E , F , and G , data are from 1 representative experiment with 8–10‐week‐old mice (n = 8 Sle1 and 5 Sle1 TLR‐9 −/− mice). Circles represent individual mice; horizontal lines and error bars show the mean ± SEM. Significance was determined by Student's t ‐test. * = P

    Techniques Used: Mouse Assay, Activation Assay, Flow Cytometry, Cytometry, Expressing, Fluorescence, Luminex, Incubation, Isolation

    34) Product Images from "An autologous dendritic cell vaccine polarizes a Th-1 response which is tumoricidal to patient-derived breast cancer cells"

    Article Title: An autologous dendritic cell vaccine polarizes a Th-1 response which is tumoricidal to patient-derived breast cancer cells

    Journal: Cancer Immunology, Immunotherapy

    doi: 10.1007/s00262-018-2238-5

    DCs from patients with breast cancer, pulsed with tumour-specific lysate and matured with Ampligen®, an IFN-containing cocktail and R848 and or with IFN-containing cocktail only express higher levels of co-stimulatory molecules compared to immature DCs or DCs pulsed with tumour-specific lysate only. Immature DCs were differentiated from monocytes, incubated in CellGenix DC complete medium with or without 100 µg/mL tumour-specific lysate for 6 h at 37 °C. The cells were then matured with or without or in combination with 100 μg/ml Ampligen®, an IFN-containing cocktail (10 ng/mL IFN-α, 25 ng/mL IFN-γ, 1 µg/mL CD40L and 10 ng/mL IL-1β), and/or 2.5 µg/mL R848 for 42 h at 37 °C. The monocytes, immature DCs and mature DCs were subjected to a haematoxylin and eosin stain ( a ) or were stained with CD14 PE-CY7 (monocytes), CD40 FITC (immature and mature DCs) and or CD83 APC (mature DCs; A ) for confocal microscopy. The maturation phenotype was also determined by flow cytometry ( b ). Arrows ( → ) show dendrites being expressed on the surface of mature DCs. Data were analysed for statistical significance by one-way Anova with Dunnett’s post-test, where **, *** and **** indicate p
    Figure Legend Snippet: DCs from patients with breast cancer, pulsed with tumour-specific lysate and matured with Ampligen®, an IFN-containing cocktail and R848 and or with IFN-containing cocktail only express higher levels of co-stimulatory molecules compared to immature DCs or DCs pulsed with tumour-specific lysate only. Immature DCs were differentiated from monocytes, incubated in CellGenix DC complete medium with or without 100 µg/mL tumour-specific lysate for 6 h at 37 °C. The cells were then matured with or without or in combination with 100 μg/ml Ampligen®, an IFN-containing cocktail (10 ng/mL IFN-α, 25 ng/mL IFN-γ, 1 µg/mL CD40L and 10 ng/mL IL-1β), and/or 2.5 µg/mL R848 for 42 h at 37 °C. The monocytes, immature DCs and mature DCs were subjected to a haematoxylin and eosin stain ( a ) or were stained with CD14 PE-CY7 (monocytes), CD40 FITC (immature and mature DCs) and or CD83 APC (mature DCs; A ) for confocal microscopy. The maturation phenotype was also determined by flow cytometry ( b ). Arrows ( → ) show dendrites being expressed on the surface of mature DCs. Data were analysed for statistical significance by one-way Anova with Dunnett’s post-test, where **, *** and **** indicate p

    Techniques Used: Incubation, H&E Stain, Staining, Confocal Microscopy, Flow Cytometry, Cytometry

    The TCRs of CD8 + T-cells primed with tumour-specific lysate/Ampligen®/IFN-cocktail and R848-matured DCs can recognise HER-2 and MUC-1-specific tetramers. Immature DCs were differentiated from monocytes as indicated previously. DCs were matured and effector cells were generated as indicated in the methods. The cells were stained with CD8 FITC, CD3 PerCP/Cy5.5, HER-2 APC, MUC-1 PE and Zombie NIR according to the manufacturer’s instructions (MBL, USA). The levels of HER-2 and MUC-1 recognised by the TCR on CD8 + T-cells were determined by flow cytometry. Data were analysed for statistical significance by one-way Anova with Dunnett’s post-test where *, *** indicate p
    Figure Legend Snippet: The TCRs of CD8 + T-cells primed with tumour-specific lysate/Ampligen®/IFN-cocktail and R848-matured DCs can recognise HER-2 and MUC-1-specific tetramers. Immature DCs were differentiated from monocytes as indicated previously. DCs were matured and effector cells were generated as indicated in the methods. The cells were stained with CD8 FITC, CD3 PerCP/Cy5.5, HER-2 APC, MUC-1 PE and Zombie NIR according to the manufacturer’s instructions (MBL, USA). The levels of HER-2 and MUC-1 recognised by the TCR on CD8 + T-cells were determined by flow cytometry. Data were analysed for statistical significance by one-way Anova with Dunnett’s post-test where *, *** indicate p

    Techniques Used: Generated, Staining, Flow Cytometry, Cytometry

    DCs pulsed with tumour-specific lysate and matured with Ampligen®, an IFN-containing cocktail and R848 express higher levels of the Th1 effector cytokines, IL-12p70, compared to the immature DCs (iDCs) or DCs pulsed with tumour-specific lysate only. The level of IL-12p70 from the culture supernatants of the immature DCs or matured DCs was determined using the ELIZAPRO IL-12p70 detection kit from Mabtech as indicated by the manufacturer. Data were analysed for statistical significance by one-way Anova with Dunnett’s post-test or Wilcoxon signed rank paired t test, *, ***, **** indicate p
    Figure Legend Snippet: DCs pulsed with tumour-specific lysate and matured with Ampligen®, an IFN-containing cocktail and R848 express higher levels of the Th1 effector cytokines, IL-12p70, compared to the immature DCs (iDCs) or DCs pulsed with tumour-specific lysate only. The level of IL-12p70 from the culture supernatants of the immature DCs or matured DCs was determined using the ELIZAPRO IL-12p70 detection kit from Mabtech as indicated by the manufacturer. Data were analysed for statistical significance by one-way Anova with Dunnett’s post-test or Wilcoxon signed rank paired t test, *, ***, **** indicate p

    Techniques Used:

    PBMCs from breast cancer patients co-cultured with tumour-specific lysate pulsed and Ampligen®/IFN-containing cocktail/R848-matured DCs results in cytotoxic T-lymphocyte-mediated killing of primary breast cancer cells in vitro. Matured DCs were prepared as indicated in the methods. The matured DCs were then co-cultured with PBMC at a ratio of 1:10 for 7 days at 37 °C. The primary breast cancer cells were incubated with or without the primed PBMCs (effector cells) at a ratio of 1:10 ( a and c ) or the primary cells were incubated with the effector T-cells at various ratios indicated ( b and d ) for 4 h at 37 °C. Cytotoxicity ( a and b ) was determined using the LDH assay (Cytotoxicity Detection Kit Plus LDH; Roche, Germany) and cell death ( c and d ) of the primary breast cancer cells was measured by flow cytometry. Data were analysed for statistical significance by one-way Anova with Dunnett’s post-test where *, ***indicate p
    Figure Legend Snippet: PBMCs from breast cancer patients co-cultured with tumour-specific lysate pulsed and Ampligen®/IFN-containing cocktail/R848-matured DCs results in cytotoxic T-lymphocyte-mediated killing of primary breast cancer cells in vitro. Matured DCs were prepared as indicated in the methods. The matured DCs were then co-cultured with PBMC at a ratio of 1:10 for 7 days at 37 °C. The primary breast cancer cells were incubated with or without the primed PBMCs (effector cells) at a ratio of 1:10 ( a and c ) or the primary cells were incubated with the effector T-cells at various ratios indicated ( b and d ) for 4 h at 37 °C. Cytotoxicity ( a and b ) was determined using the LDH assay (Cytotoxicity Detection Kit Plus LDH; Roche, Germany) and cell death ( c and d ) of the primary breast cancer cells was measured by flow cytometry. Data were analysed for statistical significance by one-way Anova with Dunnett’s post-test where *, ***indicate p

    Techniques Used: Cell Culture, In Vitro, Incubation, Lactate Dehydrogenase Assay, Flow Cytometry, Cytometry

    35) Product Images from "Circulating and Hepatic BDCA1+, BDCA2+, and BDCA3+ Dendritic Cells Are Differentially Subverted in Patients With Chronic HBV Infection"

    Article Title: Circulating and Hepatic BDCA1+, BDCA2+, and BDCA3+ Dendritic Cells Are Differentially Subverted in Patients With Chronic HBV Infection

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.00112

    Altered maturation of intrahepatic BDCA2+ pDCs and BDCA3+ cDC1 in chronic HBV patients after TLRs stimulation. LMNC suspensions from non-viral infected controls or HBV patients were stimulated for 22 h with a mixture of TLRLs (MIX:polyI:C+R848+CPG A ) and the expression of the maturation markers CD40, CD80, and CD86 was measured by flow cytometry. (A) Percentages or MFI of CD40, CD80, and CD86 molecules on cDC2, pDCs, and cDC1. Open symbols, CTRL ( n = 15–16); filled symbols, HBV ( n = 10–12). P -values were calculated using the 2-way-RM ANOVA test (straight line, * P ≤ 0.05) and Mann–Whitney test (dashed lines). Bar indicated mean. (B) Spearman correlations between CD80 on intrahepatic cDC1 from HBV patients after MIX stimulation with HBV DNA ( n = 9).
    Figure Legend Snippet: Altered maturation of intrahepatic BDCA2+ pDCs and BDCA3+ cDC1 in chronic HBV patients after TLRs stimulation. LMNC suspensions from non-viral infected controls or HBV patients were stimulated for 22 h with a mixture of TLRLs (MIX:polyI:C+R848+CPG A ) and the expression of the maturation markers CD40, CD80, and CD86 was measured by flow cytometry. (A) Percentages or MFI of CD40, CD80, and CD86 molecules on cDC2, pDCs, and cDC1. Open symbols, CTRL ( n = 15–16); filled symbols, HBV ( n = 10–12). P -values were calculated using the 2-way-RM ANOVA test (straight line, * P ≤ 0.05) and Mann–Whitney test (dashed lines). Bar indicated mean. (B) Spearman correlations between CD80 on intrahepatic cDC1 from HBV patients after MIX stimulation with HBV DNA ( n = 9).

    Techniques Used: Infection, Expressing, Flow Cytometry, Cytometry, MANN-WHITNEY

    Improvement of cytokine secretion by LMNCs in chronic HBV patients upon TLR triggering. LMNCs (1 × 10 6 cells/ml) from non-viral infected controls or HBV patients were stimulated for 22 h with a mixture of TLRL (MIX:polyI:C+R848+CPG A ) and the culture supernatants were examined for the presence of IL-12p70, IFNβ, IFNλ1, IFNλ2, TNFα, IP10, MCP1, IL-10, and TGF-ß1 by Luminex technology. (A) Quantification of cytokine secretion in the supernatants. Open symbols, HD ( n = 16); filled symbols, HBV ( n = 11–12). P -values were calculated using Mann–Whitney test. (B) Spearman correlation between the IL-12p70 secretion by intrahepatic LMNCs from HBV patients with plasmatic HBsAg levels ( n = 10). (C) Cytokine's secretion by liver cell suspensions were reported to the absolute number of DCs present in samples before stimulation with the mix of TLRLs (amount of cytokine/10 5 DC) as shown for cDC2+cDC1 (IL-12p70), pDCs (IFNβ) and cDC1+pDCs (IFNλ1, IFNλ2). Open symbols, HD ( n = 16–17); filled symbols, HBV ( n = 28–30). P -values were calculated using Mann–Whitney test. P -values were calculated using Man-Whitney test (dashed lines).
    Figure Legend Snippet: Improvement of cytokine secretion by LMNCs in chronic HBV patients upon TLR triggering. LMNCs (1 × 10 6 cells/ml) from non-viral infected controls or HBV patients were stimulated for 22 h with a mixture of TLRL (MIX:polyI:C+R848+CPG A ) and the culture supernatants were examined for the presence of IL-12p70, IFNβ, IFNλ1, IFNλ2, TNFα, IP10, MCP1, IL-10, and TGF-ß1 by Luminex technology. (A) Quantification of cytokine secretion in the supernatants. Open symbols, HD ( n = 16); filled symbols, HBV ( n = 11–12). P -values were calculated using Mann–Whitney test. (B) Spearman correlation between the IL-12p70 secretion by intrahepatic LMNCs from HBV patients with plasmatic HBsAg levels ( n = 10). (C) Cytokine's secretion by liver cell suspensions were reported to the absolute number of DCs present in samples before stimulation with the mix of TLRLs (amount of cytokine/10 5 DC) as shown for cDC2+cDC1 (IL-12p70), pDCs (IFNβ) and cDC1+pDCs (IFNλ1, IFNλ2). Open symbols, HD ( n = 16–17); filled symbols, HBV ( n = 28–30). P -values were calculated using Mann–Whitney test. P -values were calculated using Man-Whitney test (dashed lines).

    Techniques Used: Infection, Luminex, MANN-WHITNEY

    Defective maturation of circulating BDCA1+ cDC2, BDCA2+ pDCs, and BDCA3+ cDC1 from chronic HBV patients upon TLR triggering. PBMCs from HD or HBV patients were stimulated for 22 h with or without TLRLs (polyI:C, R848 or CPG A (ODN2336)) alone or mixed together (MIX:polyI:C+R848+CPG A ) and the expression of the maturation markers was measured by flow cytometry. Percentages of CD40, CD80, and CD86 molecules on (A) cDC2, (B) pDCs, and (C) cDC1. Open symbols, HD ( n = 13–18); filled symbols, HBV ( n = 22–26). P -values were calculated using the 2-way-RM ANOVA test (straight line) * P ≤ 0.05, ** P
    Figure Legend Snippet: Defective maturation of circulating BDCA1+ cDC2, BDCA2+ pDCs, and BDCA3+ cDC1 from chronic HBV patients upon TLR triggering. PBMCs from HD or HBV patients were stimulated for 22 h with or without TLRLs (polyI:C, R848 or CPG A (ODN2336)) alone or mixed together (MIX:polyI:C+R848+CPG A ) and the expression of the maturation markers was measured by flow cytometry. Percentages of CD40, CD80, and CD86 molecules on (A) cDC2, (B) pDCs, and (C) cDC1. Open symbols, HD ( n = 13–18); filled symbols, HBV ( n = 22–26). P -values were calculated using the 2-way-RM ANOVA test (straight line) * P ≤ 0.05, ** P

    Techniques Used: Expressing, Flow Cytometry, Cytometry

    Impairment of intracellular IL-12p70, TNFα, IFNα, and IFNλ1 (IL-29) production by blood DC subsets from chronic HBV patients upon TLR triggering. Whole blood samples were stimulated for 5-h with or without polyI:C, R848, CPG A (ODN2336) alone or mixed together (MIX:polyI:C+R848+CPG A ) and the production of cytokines by each DC subset was measured by intracellular staining by flow cytometry. Percentages of cytokine-expressing cells within (A) cDC2, (B) pDCs, and (C) cDC1. Open symbols, HD ( n = 20–24); filled symbols, HBV ( n = 20–26). (D) Cytokine's secretion by PBMCs were reported to the absolute number of DCs present in sample before stimulation with TLRLs by calculating the cytokine production per DC subsets in each sample (amount of cytokine/10 5 DCs) for cDC2+cDC1 (IL-12p70), pDCs (IFNα2, IFNβ) and cDC1+pDCs (IFNλ1, IFNλ2). Results are expressed in ng or pg/10 5 DC subsets. Open symbols, HD ( n = 20–24); filled symbols, HBV patients ( n = 20–26). P -values were calculated using the 2-way-RM ANOVA test (straight line) * P ≤ 0.05, ** P
    Figure Legend Snippet: Impairment of intracellular IL-12p70, TNFα, IFNα, and IFNλ1 (IL-29) production by blood DC subsets from chronic HBV patients upon TLR triggering. Whole blood samples were stimulated for 5-h with or without polyI:C, R848, CPG A (ODN2336) alone or mixed together (MIX:polyI:C+R848+CPG A ) and the production of cytokines by each DC subset was measured by intracellular staining by flow cytometry. Percentages of cytokine-expressing cells within (A) cDC2, (B) pDCs, and (C) cDC1. Open symbols, HD ( n = 20–24); filled symbols, HBV ( n = 20–26). (D) Cytokine's secretion by PBMCs were reported to the absolute number of DCs present in sample before stimulation with TLRLs by calculating the cytokine production per DC subsets in each sample (amount of cytokine/10 5 DCs) for cDC2+cDC1 (IL-12p70), pDCs (IFNα2, IFNβ) and cDC1+pDCs (IFNλ1, IFNλ2). Results are expressed in ng or pg/10 5 DC subsets. Open symbols, HD ( n = 20–24); filled symbols, HBV patients ( n = 20–26). P -values were calculated using the 2-way-RM ANOVA test (straight line) * P ≤ 0.05, ** P

    Techniques Used: Staining, Flow Cytometry, Cytometry, Expressing

    36) Product Images from "Effect of in vivo Hydroxychloroquine and ex vivo Anti-BDCA2 mAb Treatment on pDC IFNα Production From Patients Affected With Cutaneous Lupus Erythematosus"

    Article Title: Effect of in vivo Hydroxychloroquine and ex vivo Anti-BDCA2 mAb Treatment on pDC IFNα Production From Patients Affected With Cutaneous Lupus Erythematosus

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.00275

    HCQ-mediated inhibition of IFNα production from pDCs in response to CpG-A and ssRNA, but not to R848, can be detected after isolation of PBMC from whole blood pre-treated with HCQ. Whole blood samples from healthy donors were treated with HCQ (1000 ng/ml) or not for 1 h prior to PBMC isolation and then stimulated with CpG-A (10 μM), R848 (1 μM) or ssRNA (4 μg/ml) for 6h. IFNα was analyzed by intracellular cytokine staining. (A) Representative dot plots of IFNα+ cells within a BDCA4+ and CD123+ gate. (B) Percentages of IFNα-producing pDCs detected as shown in A ) after CpG-A stimulation ( n = 5 donors), R848 stimulation ( n = 3 donors), or ssRNA ( n = 5 donors) from PBMC isolated from whole blood pre-treated with HCQ or not. Statistical significance was assessed using two-tailed paired Student's t-test (* p
    Figure Legend Snippet: HCQ-mediated inhibition of IFNα production from pDCs in response to CpG-A and ssRNA, but not to R848, can be detected after isolation of PBMC from whole blood pre-treated with HCQ. Whole blood samples from healthy donors were treated with HCQ (1000 ng/ml) or not for 1 h prior to PBMC isolation and then stimulated with CpG-A (10 μM), R848 (1 μM) or ssRNA (4 μg/ml) for 6h. IFNα was analyzed by intracellular cytokine staining. (A) Representative dot plots of IFNα+ cells within a BDCA4+ and CD123+ gate. (B) Percentages of IFNα-producing pDCs detected as shown in A ) after CpG-A stimulation ( n = 5 donors), R848 stimulation ( n = 3 donors), or ssRNA ( n = 5 donors) from PBMC isolated from whole blood pre-treated with HCQ or not. Statistical significance was assessed using two-tailed paired Student's t-test (* p

    Techniques Used: Inhibition, Isolation, Staining, Two Tailed Test

    24F4A further reduces pDC IFNα production after CpG-A, R848 or ssRNA stimulations of PBMC isolated from CLE patients with blood IFN-high signature or concomitant SLE diagnostic. (A) Distribution of the blood IFN signature scores in healthy individuals ( n = 52) and CLE patients ( n = 30). (B) Comparison of SLEDAI2-K disease scores (left) or CLASI skin scores (right) between CLE patients with blood IFN-low and blood IFN-high signature scores. Statistical significance was assessed using Mann-Whitney test (* p
    Figure Legend Snippet: 24F4A further reduces pDC IFNα production after CpG-A, R848 or ssRNA stimulations of PBMC isolated from CLE patients with blood IFN-high signature or concomitant SLE diagnostic. (A) Distribution of the blood IFN signature scores in healthy individuals ( n = 52) and CLE patients ( n = 30). (B) Comparison of SLEDAI2-K disease scores (left) or CLASI skin scores (right) between CLE patients with blood IFN-low and blood IFN-high signature scores. Statistical significance was assessed using Mann-Whitney test (* p

    Techniques Used: Isolation, Diagnostic Assay, MANN-WHITNEY

    Effect of HCQ and BIIB059 on IFNα release from human whole blood stimulated with CpG-A, R848, or ssRNA. Whole blood from human healthy donors was stimulated or not with CpG-A (10 μM), R848 (1 μM), or ssRNA (4 μg/ml) complexed with pARG in presence or absence of HCQ (550 ng/ml), isotype control mAb (10 μg/ml), 24F4A (10 μg/ml) or a combination of both HCQ and 24F4A. Secreted IFNα was measured 18 h after stimulation in the serum using ELISA. Same color and/or shape-coded data points represent data obtained from a given donor ( n = at least 6 donors). Statistical tests were performed using paired one-way Anova (ns, non-significant p ≥ 0.05, * p
    Figure Legend Snippet: Effect of HCQ and BIIB059 on IFNα release from human whole blood stimulated with CpG-A, R848, or ssRNA. Whole blood from human healthy donors was stimulated or not with CpG-A (10 μM), R848 (1 μM), or ssRNA (4 μg/ml) complexed with pARG in presence or absence of HCQ (550 ng/ml), isotype control mAb (10 μg/ml), 24F4A (10 μg/ml) or a combination of both HCQ and 24F4A. Secreted IFNα was measured 18 h after stimulation in the serum using ELISA. Same color and/or shape-coded data points represent data obtained from a given donor ( n = at least 6 donors). Statistical tests were performed using paired one-way Anova (ns, non-significant p ≥ 0.05, * p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    The percentage of IFNα-producing pDCs upon stimulation with CpG-A, but not R848 or ssRNA, is negatively correlated with whole blood HCQ concentrations from CLE patients. (A) Association of the percentage of IFNα-producing pDCs identified by flow cytometry from PBMC after CpG-A ( n = 22 donors), R848 ( n = 22 donors), or ssRNA ( n = 24 donors) stimulations with the HCQ concentrations in whole blood from CLE patients. Patients with non-detectable blood HCQ levels are shown at 0 ng/ml blood HCQ (B) Association of the change (ratio) between 3-month apart clinical visits in the percentage of IFNα-producing pDCs identified by flow cytometry from PBMC after CpG-A ( n = 21 donors), R848 ( n = 21 donors), or ssRNA ( n = 22 donors) stimulation with the change in the blood HCQ concentration of CLE patients. Data points from patients with increase in blood HCQ concentration > 600 ng/ml between the two visits are highlighted in red. Data points from patients with decrease in blood HCQ concentration > 500 ng/ml between two visits are highlighted in green. Statistical association was assessed using Pearson's (P) and Spearman's rank (S) correlations.
    Figure Legend Snippet: The percentage of IFNα-producing pDCs upon stimulation with CpG-A, but not R848 or ssRNA, is negatively correlated with whole blood HCQ concentrations from CLE patients. (A) Association of the percentage of IFNα-producing pDCs identified by flow cytometry from PBMC after CpG-A ( n = 22 donors), R848 ( n = 22 donors), or ssRNA ( n = 24 donors) stimulations with the HCQ concentrations in whole blood from CLE patients. Patients with non-detectable blood HCQ levels are shown at 0 ng/ml blood HCQ (B) Association of the change (ratio) between 3-month apart clinical visits in the percentage of IFNα-producing pDCs identified by flow cytometry from PBMC after CpG-A ( n = 21 donors), R848 ( n = 21 donors), or ssRNA ( n = 22 donors) stimulation with the change in the blood HCQ concentration of CLE patients. Data points from patients with increase in blood HCQ concentration > 600 ng/ml between the two visits are highlighted in red. Data points from patients with decrease in blood HCQ concentration > 500 ng/ml between two visits are highlighted in green. Statistical association was assessed using Pearson's (P) and Spearman's rank (S) correlations.

    Techniques Used: Flow Cytometry, Cytometry, Concentration Assay

    24F4A further reduces pDC IFNα production after CpG-A, R848 or ssRNA stimulations of PBMC isolated from CLE patients regardless blood HCQ levels. (A) Concentrations of HCQ in whole blood from the CLE patient cohort studied ( n = 30). (B) Representative dot plots of IFNα+ cells within a BDCA4+ and CD123+ gate after CpG-A, R848 or ssRNA stimulation with or without pre-treatment with 24F4A (10 μg/ml 30 min) from CLE patient without detectable blood HCQ (top panel) or a CLE patient with a blood HCQ concentration of more than 500ng/ml (bottom panel). (C) Effect of 24F4A on the percentage of IFNα-producing pDCs induced by CpG-A, R848 and ssRNA stimulations and detected by flow cytometry in PBMC from CLE patients without detectable level of blood HCQ ( n ≥ 6 donors), with blood HCQ concentrations lower than 500 ng/ml ( n ≥ 8 donors) or > 500ng/ml ( n ≥ 6 donors). Statistical significance was assessed with a two-tailed paired Student's t-test using log2-transformed values (* p
    Figure Legend Snippet: 24F4A further reduces pDC IFNα production after CpG-A, R848 or ssRNA stimulations of PBMC isolated from CLE patients regardless blood HCQ levels. (A) Concentrations of HCQ in whole blood from the CLE patient cohort studied ( n = 30). (B) Representative dot plots of IFNα+ cells within a BDCA4+ and CD123+ gate after CpG-A, R848 or ssRNA stimulation with or without pre-treatment with 24F4A (10 μg/ml 30 min) from CLE patient without detectable blood HCQ (top panel) or a CLE patient with a blood HCQ concentration of more than 500ng/ml (bottom panel). (C) Effect of 24F4A on the percentage of IFNα-producing pDCs induced by CpG-A, R848 and ssRNA stimulations and detected by flow cytometry in PBMC from CLE patients without detectable level of blood HCQ ( n ≥ 6 donors), with blood HCQ concentrations lower than 500 ng/ml ( n ≥ 8 donors) or > 500ng/ml ( n ≥ 6 donors). Statistical significance was assessed with a two-tailed paired Student's t-test using log2-transformed values (* p

    Techniques Used: Isolation, Concentration Assay, Flow Cytometry, Cytometry, Two Tailed Test, Transformation Assay

    37) Product Images from "Effect of in vivo Hydroxychloroquine and ex vivo Anti-BDCA2 mAb Treatment on pDC IFNα Production From Patients Affected With Cutaneous Lupus Erythematosus"

    Article Title: Effect of in vivo Hydroxychloroquine and ex vivo Anti-BDCA2 mAb Treatment on pDC IFNα Production From Patients Affected With Cutaneous Lupus Erythematosus

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.00275

    HCQ-mediated inhibition of IFNα production from pDCs in response to CpG-A and ssRNA, but not to R848, can be detected after isolation of PBMC from whole blood pre-treated with HCQ. Whole blood samples from healthy donors were treated with HCQ (1000 ng/ml) or not for 1 h prior to PBMC isolation and then stimulated with CpG-A (10 μM), R848 (1 μM) or ssRNA (4 μg/ml) for 6h. IFNα was analyzed by intracellular cytokine staining. (A) Representative dot plots of IFNα+ cells within a BDCA4+ and CD123+ gate. (B) Percentages of IFNα-producing pDCs detected as shown in A ) after CpG-A stimulation ( n = 5 donors), R848 stimulation ( n = 3 donors), or ssRNA ( n = 5 donors) from PBMC isolated from whole blood pre-treated with HCQ or not. Statistical significance was assessed using two-tailed paired Student's t-test (* p
    Figure Legend Snippet: HCQ-mediated inhibition of IFNα production from pDCs in response to CpG-A and ssRNA, but not to R848, can be detected after isolation of PBMC from whole blood pre-treated with HCQ. Whole blood samples from healthy donors were treated with HCQ (1000 ng/ml) or not for 1 h prior to PBMC isolation and then stimulated with CpG-A (10 μM), R848 (1 μM) or ssRNA (4 μg/ml) for 6h. IFNα was analyzed by intracellular cytokine staining. (A) Representative dot plots of IFNα+ cells within a BDCA4+ and CD123+ gate. (B) Percentages of IFNα-producing pDCs detected as shown in A ) after CpG-A stimulation ( n = 5 donors), R848 stimulation ( n = 3 donors), or ssRNA ( n = 5 donors) from PBMC isolated from whole blood pre-treated with HCQ or not. Statistical significance was assessed using two-tailed paired Student's t-test (* p

    Techniques Used: Inhibition, Isolation, Staining, Two Tailed Test

    24F4A further reduces pDC IFNα production after CpG-A, R848 or ssRNA stimulations of PBMC isolated from CLE patients with blood IFN-high signature or concomitant SLE diagnostic. (A) Distribution of the blood IFN signature scores in healthy individuals ( n = 52) and CLE patients ( n = 30). (B) Comparison of SLEDAI2-K disease scores (left) or CLASI skin scores (right) between CLE patients with blood IFN-low and blood IFN-high signature scores. Statistical significance was assessed using Mann-Whitney test (* p
    Figure Legend Snippet: 24F4A further reduces pDC IFNα production after CpG-A, R848 or ssRNA stimulations of PBMC isolated from CLE patients with blood IFN-high signature or concomitant SLE diagnostic. (A) Distribution of the blood IFN signature scores in healthy individuals ( n = 52) and CLE patients ( n = 30). (B) Comparison of SLEDAI2-K disease scores (left) or CLASI skin scores (right) between CLE patients with blood IFN-low and blood IFN-high signature scores. Statistical significance was assessed using Mann-Whitney test (* p

    Techniques Used: Isolation, Diagnostic Assay, MANN-WHITNEY

    Effect of HCQ and BIIB059 on IFNα release from human whole blood stimulated with CpG-A, R848, or ssRNA. Whole blood from human healthy donors was stimulated or not with CpG-A (10 μM), R848 (1 μM), or ssRNA (4 μg/ml) complexed with pARG in presence or absence of HCQ (550 ng/ml), isotype control mAb (10 μg/ml), 24F4A (10 μg/ml) or a combination of both HCQ and 24F4A. Secreted IFNα was measured 18 h after stimulation in the serum using ELISA. Same color and/or shape-coded data points represent data obtained from a given donor ( n = at least 6 donors). Statistical tests were performed using paired one-way Anova (ns, non-significant p ≥ 0.05, * p
    Figure Legend Snippet: Effect of HCQ and BIIB059 on IFNα release from human whole blood stimulated with CpG-A, R848, or ssRNA. Whole blood from human healthy donors was stimulated or not with CpG-A (10 μM), R848 (1 μM), or ssRNA (4 μg/ml) complexed with pARG in presence or absence of HCQ (550 ng/ml), isotype control mAb (10 μg/ml), 24F4A (10 μg/ml) or a combination of both HCQ and 24F4A. Secreted IFNα was measured 18 h after stimulation in the serum using ELISA. Same color and/or shape-coded data points represent data obtained from a given donor ( n = at least 6 donors). Statistical tests were performed using paired one-way Anova (ns, non-significant p ≥ 0.05, * p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    The percentage of IFNα-producing pDCs upon stimulation with CpG-A, but not R848 or ssRNA, is negatively correlated with whole blood HCQ concentrations from CLE patients. (A) Association of the percentage of IFNα-producing pDCs identified by flow cytometry from PBMC after CpG-A ( n = 22 donors), R848 ( n = 22 donors), or ssRNA ( n = 24 donors) stimulations with the HCQ concentrations in whole blood from CLE patients. Patients with non-detectable blood HCQ levels are shown at 0 ng/ml blood HCQ (B) Association of the change (ratio) between 3-month apart clinical visits in the percentage of IFNα-producing pDCs identified by flow cytometry from PBMC after CpG-A ( n = 21 donors), R848 ( n = 21 donors), or ssRNA ( n = 22 donors) stimulation with the change in the blood HCQ concentration of CLE patients. Data points from patients with increase in blood HCQ concentration > 600 ng/ml between the two visits are highlighted in red. Data points from patients with decrease in blood HCQ concentration > 500 ng/ml between two visits are highlighted in green. Statistical association was assessed using Pearson's (P) and Spearman's rank (S) correlations.
    Figure Legend Snippet: The percentage of IFNα-producing pDCs upon stimulation with CpG-A, but not R848 or ssRNA, is negatively correlated with whole blood HCQ concentrations from CLE patients. (A) Association of the percentage of IFNα-producing pDCs identified by flow cytometry from PBMC after CpG-A ( n = 22 donors), R848 ( n = 22 donors), or ssRNA ( n = 24 donors) stimulations with the HCQ concentrations in whole blood from CLE patients. Patients with non-detectable blood HCQ levels are shown at 0 ng/ml blood HCQ (B) Association of the change (ratio) between 3-month apart clinical visits in the percentage of IFNα-producing pDCs identified by flow cytometry from PBMC after CpG-A ( n = 21 donors), R848 ( n = 21 donors), or ssRNA ( n = 22 donors) stimulation with the change in the blood HCQ concentration of CLE patients. Data points from patients with increase in blood HCQ concentration > 600 ng/ml between the two visits are highlighted in red. Data points from patients with decrease in blood HCQ concentration > 500 ng/ml between two visits are highlighted in green. Statistical association was assessed using Pearson's (P) and Spearman's rank (S) correlations.

    Techniques Used: Flow Cytometry, Cytometry, Concentration Assay

    24F4A further reduces pDC IFNα production after CpG-A, R848 or ssRNA stimulations of PBMC isolated from CLE patients regardless blood HCQ levels. (A) Concentrations of HCQ in whole blood from the CLE patient cohort studied ( n = 30). (B) Representative dot plots of IFNα+ cells within a BDCA4+ and CD123+ gate after CpG-A, R848 or ssRNA stimulation with or without pre-treatment with 24F4A (10 μg/ml 30 min) from CLE patient without detectable blood HCQ (top panel) or a CLE patient with a blood HCQ concentration of more than 500ng/ml (bottom panel). (C) Effect of 24F4A on the percentage of IFNα-producing pDCs induced by CpG-A, R848 and ssRNA stimulations and detected by flow cytometry in PBMC from CLE patients without detectable level of blood HCQ ( n ≥ 6 donors), with blood HCQ concentrations lower than 500 ng/ml ( n ≥ 8 donors) or > 500ng/ml ( n ≥ 6 donors). Statistical significance was assessed with a two-tailed paired Student's t-test using log2-transformed values (* p
    Figure Legend Snippet: 24F4A further reduces pDC IFNα production after CpG-A, R848 or ssRNA stimulations of PBMC isolated from CLE patients regardless blood HCQ levels. (A) Concentrations of HCQ in whole blood from the CLE patient cohort studied ( n = 30). (B) Representative dot plots of IFNα+ cells within a BDCA4+ and CD123+ gate after CpG-A, R848 or ssRNA stimulation with or without pre-treatment with 24F4A (10 μg/ml 30 min) from CLE patient without detectable blood HCQ (top panel) or a CLE patient with a blood HCQ concentration of more than 500ng/ml (bottom panel). (C) Effect of 24F4A on the percentage of IFNα-producing pDCs induced by CpG-A, R848 and ssRNA stimulations and detected by flow cytometry in PBMC from CLE patients without detectable level of blood HCQ ( n ≥ 6 donors), with blood HCQ concentrations lower than 500 ng/ml ( n ≥ 8 donors) or > 500ng/ml ( n ≥ 6 donors). Statistical significance was assessed with a two-tailed paired Student's t-test using log2-transformed values (* p

    Techniques Used: Isolation, Concentration Assay, Flow Cytometry, Cytometry, Two Tailed Test, Transformation Assay

    38) Product Images from "Effect of in vivo Hydroxychloroquine and ex vivo Anti-BDCA2 mAb Treatment on pDC IFNα Production From Patients Affected With Cutaneous Lupus Erythematosus"

    Article Title: Effect of in vivo Hydroxychloroquine and ex vivo Anti-BDCA2 mAb Treatment on pDC IFNα Production From Patients Affected With Cutaneous Lupus Erythematosus

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.00275

    HCQ-mediated inhibition of IFNα production from pDCs in response to CpG-A and ssRNA, but not to R848, can be detected after isolation of PBMC from whole blood pre-treated with HCQ. Whole blood samples from healthy donors were treated with HCQ (1000 ng/ml) or not for 1 h prior to PBMC isolation and then stimulated with CpG-A (10 μM), R848 (1 μM) or ssRNA (4 μg/ml) for 6h. IFNα was analyzed by intracellular cytokine staining. (A) Representative dot plots of IFNα+ cells within a BDCA4+ and CD123+ gate. (B) Percentages of IFNα-producing pDCs detected as shown in A ) after CpG-A stimulation ( n = 5 donors), R848 stimulation ( n = 3 donors), or ssRNA ( n = 5 donors) from PBMC isolated from whole blood pre-treated with HCQ or not. Statistical significance was assessed using two-tailed paired Student's t-test (* p
    Figure Legend Snippet: HCQ-mediated inhibition of IFNα production from pDCs in response to CpG-A and ssRNA, but not to R848, can be detected after isolation of PBMC from whole blood pre-treated with HCQ. Whole blood samples from healthy donors were treated with HCQ (1000 ng/ml) or not for 1 h prior to PBMC isolation and then stimulated with CpG-A (10 μM), R848 (1 μM) or ssRNA (4 μg/ml) for 6h. IFNα was analyzed by intracellular cytokine staining. (A) Representative dot plots of IFNα+ cells within a BDCA4+ and CD123+ gate. (B) Percentages of IFNα-producing pDCs detected as shown in A ) after CpG-A stimulation ( n = 5 donors), R848 stimulation ( n = 3 donors), or ssRNA ( n = 5 donors) from PBMC isolated from whole blood pre-treated with HCQ or not. Statistical significance was assessed using two-tailed paired Student's t-test (* p

    Techniques Used: Inhibition, Isolation, Staining, Two Tailed Test

    24F4A further reduces pDC IFNα production after CpG-A, R848 or ssRNA stimulations of PBMC isolated from CLE patients with blood IFN-high signature or concomitant SLE diagnostic. (A) Distribution of the blood IFN signature scores in healthy individuals ( n = 52) and CLE patients ( n = 30). (B) Comparison of SLEDAI2-K disease scores (left) or CLASI skin scores (right) between CLE patients with blood IFN-low and blood IFN-high signature scores. Statistical significance was assessed using Mann-Whitney test (* p
    Figure Legend Snippet: 24F4A further reduces pDC IFNα production after CpG-A, R848 or ssRNA stimulations of PBMC isolated from CLE patients with blood IFN-high signature or concomitant SLE diagnostic. (A) Distribution of the blood IFN signature scores in healthy individuals ( n = 52) and CLE patients ( n = 30). (B) Comparison of SLEDAI2-K disease scores (left) or CLASI skin scores (right) between CLE patients with blood IFN-low and blood IFN-high signature scores. Statistical significance was assessed using Mann-Whitney test (* p

    Techniques Used: Isolation, Diagnostic Assay, MANN-WHITNEY

    Effect of HCQ and BIIB059 on IFNα release from human whole blood stimulated with CpG-A, R848, or ssRNA. Whole blood from human healthy donors was stimulated or not with CpG-A (10 μM), R848 (1 μM), or ssRNA (4 μg/ml) complexed with pARG in presence or absence of HCQ (550 ng/ml), isotype control mAb (10 μg/ml), 24F4A (10 μg/ml) or a combination of both HCQ and 24F4A. Secreted IFNα was measured 18 h after stimulation in the serum using ELISA. Same color and/or shape-coded data points represent data obtained from a given donor ( n = at least 6 donors). Statistical tests were performed using paired one-way Anova (ns, non-significant p ≥ 0.05, * p
    Figure Legend Snippet: Effect of HCQ and BIIB059 on IFNα release from human whole blood stimulated with CpG-A, R848, or ssRNA. Whole blood from human healthy donors was stimulated or not with CpG-A (10 μM), R848 (1 μM), or ssRNA (4 μg/ml) complexed with pARG in presence or absence of HCQ (550 ng/ml), isotype control mAb (10 μg/ml), 24F4A (10 μg/ml) or a combination of both HCQ and 24F4A. Secreted IFNα was measured 18 h after stimulation in the serum using ELISA. Same color and/or shape-coded data points represent data obtained from a given donor ( n = at least 6 donors). Statistical tests were performed using paired one-way Anova (ns, non-significant p ≥ 0.05, * p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    The percentage of IFNα-producing pDCs upon stimulation with CpG-A, but not R848 or ssRNA, is negatively correlated with whole blood HCQ concentrations from CLE patients. (A) Association of the percentage of IFNα-producing pDCs identified by flow cytometry from PBMC after CpG-A ( n = 22 donors), R848 ( n = 22 donors), or ssRNA ( n = 24 donors) stimulations with the HCQ concentrations in whole blood from CLE patients. Patients with non-detectable blood HCQ levels are shown at 0 ng/ml blood HCQ (B) Association of the change (ratio) between 3-month apart clinical visits in the percentage of IFNα-producing pDCs identified by flow cytometry from PBMC after CpG-A ( n = 21 donors), R848 ( n = 21 donors), or ssRNA ( n = 22 donors) stimulation with the change in the blood HCQ concentration of CLE patients. Data points from patients with increase in blood HCQ concentration > 600 ng/ml between the two visits are highlighted in red. Data points from patients with decrease in blood HCQ concentration > 500 ng/ml between two visits are highlighted in green. Statistical association was assessed using Pearson's (P) and Spearman's rank (S) correlations.
    Figure Legend Snippet: The percentage of IFNα-producing pDCs upon stimulation with CpG-A, but not R848 or ssRNA, is negatively correlated with whole blood HCQ concentrations from CLE patients. (A) Association of the percentage of IFNα-producing pDCs identified by flow cytometry from PBMC after CpG-A ( n = 22 donors), R848 ( n = 22 donors), or ssRNA ( n = 24 donors) stimulations with the HCQ concentrations in whole blood from CLE patients. Patients with non-detectable blood HCQ levels are shown at 0 ng/ml blood HCQ (B) Association of the change (ratio) between 3-month apart clinical visits in the percentage of IFNα-producing pDCs identified by flow cytometry from PBMC after CpG-A ( n = 21 donors), R848 ( n = 21 donors), or ssRNA ( n = 22 donors) stimulation with the change in the blood HCQ concentration of CLE patients. Data points from patients with increase in blood HCQ concentration > 600 ng/ml between the two visits are highlighted in red. Data points from patients with decrease in blood HCQ concentration > 500 ng/ml between two visits are highlighted in green. Statistical association was assessed using Pearson's (P) and Spearman's rank (S) correlations.

    Techniques Used: Flow Cytometry, Cytometry, Concentration Assay

    24F4A further reduces pDC IFNα production after CpG-A, R848 or ssRNA stimulations of PBMC isolated from CLE patients regardless blood HCQ levels. (A) Concentrations of HCQ in whole blood from the CLE patient cohort studied ( n = 30). (B) Representative dot plots of IFNα+ cells within a BDCA4+ and CD123+ gate after CpG-A, R848 or ssRNA stimulation with or without pre-treatment with 24F4A (10 μg/ml 30 min) from CLE patient without detectable blood HCQ (top panel) or a CLE patient with a blood HCQ concentration of more than 500ng/ml (bottom panel). (C) Effect of 24F4A on the percentage of IFNα-producing pDCs induced by CpG-A, R848 and ssRNA stimulations and detected by flow cytometry in PBMC from CLE patients without detectable level of blood HCQ ( n ≥ 6 donors), with blood HCQ concentrations lower than 500 ng/ml ( n ≥ 8 donors) or > 500ng/ml ( n ≥ 6 donors). Statistical significance was assessed with a two-tailed paired Student's t-test using log2-transformed values (* p
    Figure Legend Snippet: 24F4A further reduces pDC IFNα production after CpG-A, R848 or ssRNA stimulations of PBMC isolated from CLE patients regardless blood HCQ levels. (A) Concentrations of HCQ in whole blood from the CLE patient cohort studied ( n = 30). (B) Representative dot plots of IFNα+ cells within a BDCA4+ and CD123+ gate after CpG-A, R848 or ssRNA stimulation with or without pre-treatment with 24F4A (10 μg/ml 30 min) from CLE patient without detectable blood HCQ (top panel) or a CLE patient with a blood HCQ concentration of more than 500ng/ml (bottom panel). (C) Effect of 24F4A on the percentage of IFNα-producing pDCs induced by CpG-A, R848 and ssRNA stimulations and detected by flow cytometry in PBMC from CLE patients without detectable level of blood HCQ ( n ≥ 6 donors), with blood HCQ concentrations lower than 500 ng/ml ( n ≥ 8 donors) or > 500ng/ml ( n ≥ 6 donors). Statistical significance was assessed with a two-tailed paired Student's t-test using log2-transformed values (* p

    Techniques Used: Isolation, Concentration Assay, Flow Cytometry, Cytometry, Two Tailed Test, Transformation Assay

    39) Product Images from "Effect of in vivo Hydroxychloroquine and ex vivo Anti-BDCA2 mAb Treatment on pDC IFNα Production From Patients Affected With Cutaneous Lupus Erythematosus"

    Article Title: Effect of in vivo Hydroxychloroquine and ex vivo Anti-BDCA2 mAb Treatment on pDC IFNα Production From Patients Affected With Cutaneous Lupus Erythematosus

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.00275

    HCQ-mediated inhibition of IFNα production from pDCs in response to CpG-A and ssRNA, but not to R848, can be detected after isolation of PBMC from whole blood pre-treated with HCQ. Whole blood samples from healthy donors were treated with HCQ (1000 ng/ml) or not for 1 h prior to PBMC isolation and then stimulated with CpG-A (10 μM), R848 (1 μM) or ssRNA (4 μg/ml) for 6h. IFNα was analyzed by intracellular cytokine staining. (A) Representative dot plots of IFNα+ cells within a BDCA4+ and CD123+ gate. (B) Percentages of IFNα-producing pDCs detected as shown in A ) after CpG-A stimulation ( n = 5 donors), R848 stimulation ( n = 3 donors), or ssRNA ( n = 5 donors) from PBMC isolated from whole blood pre-treated with HCQ or not. Statistical significance was assessed using two-tailed paired Student's t-test (* p
    Figure Legend Snippet: HCQ-mediated inhibition of IFNα production from pDCs in response to CpG-A and ssRNA, but not to R848, can be detected after isolation of PBMC from whole blood pre-treated with HCQ. Whole blood samples from healthy donors were treated with HCQ (1000 ng/ml) or not for 1 h prior to PBMC isolation and then stimulated with CpG-A (10 μM), R848 (1 μM) or ssRNA (4 μg/ml) for 6h. IFNα was analyzed by intracellular cytokine staining. (A) Representative dot plots of IFNα+ cells within a BDCA4+ and CD123+ gate. (B) Percentages of IFNα-producing pDCs detected as shown in A ) after CpG-A stimulation ( n = 5 donors), R848 stimulation ( n = 3 donors), or ssRNA ( n = 5 donors) from PBMC isolated from whole blood pre-treated with HCQ or not. Statistical significance was assessed using two-tailed paired Student's t-test (* p

    Techniques Used: Inhibition, Isolation, Staining, Two Tailed Test

    24F4A further reduces pDC IFNα production after CpG-A, R848 or ssRNA stimulations of PBMC isolated from CLE patients with blood IFN-high signature or concomitant SLE diagnostic. (A) Distribution of the blood IFN signature scores in healthy individuals ( n = 52) and CLE patients ( n = 30). (B) Comparison of SLEDAI2-K disease scores (left) or CLASI skin scores (right) between CLE patients with blood IFN-low and blood IFN-high signature scores. Statistical significance was assessed using Mann-Whitney test (* p
    Figure Legend Snippet: 24F4A further reduces pDC IFNα production after CpG-A, R848 or ssRNA stimulations of PBMC isolated from CLE patients with blood IFN-high signature or concomitant SLE diagnostic. (A) Distribution of the blood IFN signature scores in healthy individuals ( n = 52) and CLE patients ( n = 30). (B) Comparison of SLEDAI2-K disease scores (left) or CLASI skin scores (right) between CLE patients with blood IFN-low and blood IFN-high signature scores. Statistical significance was assessed using Mann-Whitney test (* p

    Techniques Used: Isolation, Diagnostic Assay, MANN-WHITNEY

    Effect of HCQ and BIIB059 on IFNα release from human whole blood stimulated with CpG-A, R848, or ssRNA. Whole blood from human healthy donors was stimulated or not with CpG-A (10 μM), R848 (1 μM), or ssRNA (4 μg/ml) complexed with pARG in presence or absence of HCQ (550 ng/ml), isotype control mAb (10 μg/ml), 24F4A (10 μg/ml) or a combination of both HCQ and 24F4A. Secreted IFNα was measured 18 h after stimulation in the serum using ELISA. Same color and/or shape-coded data points represent data obtained from a given donor ( n = at least 6 donors). Statistical tests were performed using paired one-way Anova (ns, non-significant p ≥ 0.05, * p
    Figure Legend Snippet: Effect of HCQ and BIIB059 on IFNα release from human whole blood stimulated with CpG-A, R848, or ssRNA. Whole blood from human healthy donors was stimulated or not with CpG-A (10 μM), R848 (1 μM), or ssRNA (4 μg/ml) complexed with pARG in presence or absence of HCQ (550 ng/ml), isotype control mAb (10 μg/ml), 24F4A (10 μg/ml) or a combination of both HCQ and 24F4A. Secreted IFNα was measured 18 h after stimulation in the serum using ELISA. Same color and/or shape-coded data points represent data obtained from a given donor ( n = at least 6 donors). Statistical tests were performed using paired one-way Anova (ns, non-significant p ≥ 0.05, * p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    The percentage of IFNα-producing pDCs upon stimulation with CpG-A, but not R848 or ssRNA, is negatively correlated with whole blood HCQ concentrations from CLE patients. (A) Association of the percentage of IFNα-producing pDCs identified by flow cytometry from PBMC after CpG-A ( n = 22 donors), R848 ( n = 22 donors), or ssRNA ( n = 24 donors) stimulations with the HCQ concentrations in whole blood from CLE patients. Patients with non-detectable blood HCQ levels are shown at 0 ng/ml blood HCQ (B) Association of the change (ratio) between 3-month apart clinical visits in the percentage of IFNα-producing pDCs identified by flow cytometry from PBMC after CpG-A ( n = 21 donors), R848 ( n = 21 donors), or ssRNA ( n = 22 donors) stimulation with the change in the blood HCQ concentration of CLE patients. Data points from patients with increase in blood HCQ concentration > 600 ng/ml between the two visits are highlighted in red. Data points from patients with decrease in blood HCQ concentration > 500 ng/ml between two visits are highlighted in green. Statistical association was assessed using Pearson's (P) and Spearman's rank (S) correlations.
    Figure Legend Snippet: The percentage of IFNα-producing pDCs upon stimulation with CpG-A, but not R848 or ssRNA, is negatively correlated with whole blood HCQ concentrations from CLE patients. (A) Association of the percentage of IFNα-producing pDCs identified by flow cytometry from PBMC after CpG-A ( n = 22 donors), R848 ( n = 22 donors), or ssRNA ( n = 24 donors) stimulations with the HCQ concentrations in whole blood from CLE patients. Patients with non-detectable blood HCQ levels are shown at 0 ng/ml blood HCQ (B) Association of the change (ratio) between 3-month apart clinical visits in the percentage of IFNα-producing pDCs identified by flow cytometry from PBMC after CpG-A ( n = 21 donors), R848 ( n = 21 donors), or ssRNA ( n = 22 donors) stimulation with the change in the blood HCQ concentration of CLE patients. Data points from patients with increase in blood HCQ concentration > 600 ng/ml between the two visits are highlighted in red. Data points from patients with decrease in blood HCQ concentration > 500 ng/ml between two visits are highlighted in green. Statistical association was assessed using Pearson's (P) and Spearman's rank (S) correlations.

    Techniques Used: Flow Cytometry, Cytometry, Concentration Assay

    24F4A further reduces pDC IFNα production after CpG-A, R848 or ssRNA stimulations of PBMC isolated from CLE patients regardless blood HCQ levels. (A) Concentrations of HCQ in whole blood from the CLE patient cohort studied ( n = 30). (B) Representative dot plots of IFNα+ cells within a BDCA4+ and CD123+ gate after CpG-A, R848 or ssRNA stimulation with or without pre-treatment with 24F4A (10 μg/ml 30 min) from CLE patient without detectable blood HCQ (top panel) or a CLE patient with a blood HCQ concentration of more than 500ng/ml (bottom panel). (C) Effect of 24F4A on the percentage of IFNα-producing pDCs induced by CpG-A, R848 and ssRNA stimulations and detected by flow cytometry in PBMC from CLE patients without detectable level of blood HCQ ( n ≥ 6 donors), with blood HCQ concentrations lower than 500 ng/ml ( n ≥ 8 donors) or > 500ng/ml ( n ≥ 6 donors). Statistical significance was assessed with a two-tailed paired Student's t-test using log2-transformed values (* p
    Figure Legend Snippet: 24F4A further reduces pDC IFNα production after CpG-A, R848 or ssRNA stimulations of PBMC isolated from CLE patients regardless blood HCQ levels. (A) Concentrations of HCQ in whole blood from the CLE patient cohort studied ( n = 30). (B) Representative dot plots of IFNα+ cells within a BDCA4+ and CD123+ gate after CpG-A, R848 or ssRNA stimulation with or without pre-treatment with 24F4A (10 μg/ml 30 min) from CLE patient without detectable blood HCQ (top panel) or a CLE patient with a blood HCQ concentration of more than 500ng/ml (bottom panel). (C) Effect of 24F4A on the percentage of IFNα-producing pDCs induced by CpG-A, R848 and ssRNA stimulations and detected by flow cytometry in PBMC from CLE patients without detectable level of blood HCQ ( n ≥ 6 donors), with blood HCQ concentrations lower than 500 ng/ml ( n ≥ 8 donors) or > 500ng/ml ( n ≥ 6 donors). Statistical significance was assessed with a two-tailed paired Student's t-test using log2-transformed values (* p

    Techniques Used: Isolation, Concentration Assay, Flow Cytometry, Cytometry, Two Tailed Test, Transformation Assay

    40) Product Images from "Effect of in vivo Hydroxychloroquine and ex vivo Anti-BDCA2 mAb Treatment on pDC IFNα Production From Patients Affected With Cutaneous Lupus Erythematosus"

    Article Title: Effect of in vivo Hydroxychloroquine and ex vivo Anti-BDCA2 mAb Treatment on pDC IFNα Production From Patients Affected With Cutaneous Lupus Erythematosus

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.00275

    HCQ-mediated inhibition of IFNα production from pDCs in response to CpG-A and ssRNA, but not to R848, can be detected after isolation of PBMC from whole blood pre-treated with HCQ. Whole blood samples from healthy donors were treated with HCQ (1000 ng/ml) or not for 1 h prior to PBMC isolation and then stimulated with CpG-A (10 μM), R848 (1 μM) or ssRNA (4 μg/ml) for 6h. IFNα was analyzed by intracellular cytokine staining. (A) Representative dot plots of IFNα+ cells within a BDCA4+ and CD123+ gate. (B) Percentages of IFNα-producing pDCs detected as shown in A ) after CpG-A stimulation ( n = 5 donors), R848 stimulation ( n = 3 donors), or ssRNA ( n = 5 donors) from PBMC isolated from whole blood pre-treated with HCQ or not. Statistical significance was assessed using two-tailed paired Student's t-test (* p
    Figure Legend Snippet: HCQ-mediated inhibition of IFNα production from pDCs in response to CpG-A and ssRNA, but not to R848, can be detected after isolation of PBMC from whole blood pre-treated with HCQ. Whole blood samples from healthy donors were treated with HCQ (1000 ng/ml) or not for 1 h prior to PBMC isolation and then stimulated with CpG-A (10 μM), R848 (1 μM) or ssRNA (4 μg/ml) for 6h. IFNα was analyzed by intracellular cytokine staining. (A) Representative dot plots of IFNα+ cells within a BDCA4+ and CD123+ gate. (B) Percentages of IFNα-producing pDCs detected as shown in A ) after CpG-A stimulation ( n = 5 donors), R848 stimulation ( n = 3 donors), or ssRNA ( n = 5 donors) from PBMC isolated from whole blood pre-treated with HCQ or not. Statistical significance was assessed using two-tailed paired Student's t-test (* p

    Techniques Used: Inhibition, Isolation, Staining, Two Tailed Test

    24F4A further reduces pDC IFNα production after CpG-A, R848 or ssRNA stimulations of PBMC isolated from CLE patients with blood IFN-high signature or concomitant SLE diagnostic. (A) Distribution of the blood IFN signature scores in healthy individuals ( n = 52) and CLE patients ( n = 30). (B) Comparison of SLEDAI2-K disease scores (left) or CLASI skin scores (right) between CLE patients with blood IFN-low and blood IFN-high signature scores. Statistical significance was assessed using Mann-Whitney test (* p
    Figure Legend Snippet: 24F4A further reduces pDC IFNα production after CpG-A, R848 or ssRNA stimulations of PBMC isolated from CLE patients with blood IFN-high signature or concomitant SLE diagnostic. (A) Distribution of the blood IFN signature scores in healthy individuals ( n = 52) and CLE patients ( n = 30). (B) Comparison of SLEDAI2-K disease scores (left) or CLASI skin scores (right) between CLE patients with blood IFN-low and blood IFN-high signature scores. Statistical significance was assessed using Mann-Whitney test (* p

    Techniques Used: Isolation, Diagnostic Assay, MANN-WHITNEY

    Effect of HCQ and BIIB059 on IFNα release from human whole blood stimulated with CpG-A, R848, or ssRNA. Whole blood from human healthy donors was stimulated or not with CpG-A (10 μM), R848 (1 μM), or ssRNA (4 μg/ml) complexed with pARG in presence or absence of HCQ (550 ng/ml), isotype control mAb (10 μg/ml), 24F4A (10 μg/ml) or a combination of both HCQ and 24F4A. Secreted IFNα was measured 18 h after stimulation in the serum using ELISA. Same color and/or shape-coded data points represent data obtained from a given donor ( n = at least 6 donors). Statistical tests were performed using paired one-way Anova (ns, non-significant p ≥ 0.05, * p
    Figure Legend Snippet: Effect of HCQ and BIIB059 on IFNα release from human whole blood stimulated with CpG-A, R848, or ssRNA. Whole blood from human healthy donors was stimulated or not with CpG-A (10 μM), R848 (1 μM), or ssRNA (4 μg/ml) complexed with pARG in presence or absence of HCQ (550 ng/ml), isotype control mAb (10 μg/ml), 24F4A (10 μg/ml) or a combination of both HCQ and 24F4A. Secreted IFNα was measured 18 h after stimulation in the serum using ELISA. Same color and/or shape-coded data points represent data obtained from a given donor ( n = at least 6 donors). Statistical tests were performed using paired one-way Anova (ns, non-significant p ≥ 0.05, * p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    The percentage of IFNα-producing pDCs upon stimulation with CpG-A, but not R848 or ssRNA, is negatively correlated with whole blood HCQ concentrations from CLE patients. (A) Association of the percentage of IFNα-producing pDCs identified by flow cytometry from PBMC after CpG-A ( n = 22 donors), R848 ( n = 22 donors), or ssRNA ( n = 24 donors) stimulations with the HCQ concentrations in whole blood from CLE patients. Patients with non-detectable blood HCQ levels are shown at 0 ng/ml blood HCQ (B) Association of the change (ratio) between 3-month apart clinical visits in the percentage of IFNα-producing pDCs identified by flow cytometry from PBMC after CpG-A ( n = 21 donors), R848 ( n = 21 donors), or ssRNA ( n = 22 donors) stimulation with the change in the blood HCQ concentration of CLE patients. Data points from patients with increase in blood HCQ concentration > 600 ng/ml between the two visits are highlighted in red. Data points from patients with decrease in blood HCQ concentration > 500 ng/ml between two visits are highlighted in green. Statistical association was assessed using Pearson's (P) and Spearman's rank (S) correlations.
    Figure Legend Snippet: The percentage of IFNα-producing pDCs upon stimulation with CpG-A, but not R848 or ssRNA, is negatively correlated with whole blood HCQ concentrations from CLE patients. (A) Association of the percentage of IFNα-producing pDCs identified by flow cytometry from PBMC after CpG-A ( n = 22 donors), R848 ( n = 22 donors), or ssRNA ( n = 24 donors) stimulations with the HCQ concentrations in whole blood from CLE patients. Patients with non-detectable blood HCQ levels are shown at 0 ng/ml blood HCQ (B) Association of the change (ratio) between 3-month apart clinical visits in the percentage of IFNα-producing pDCs identified by flow cytometry from PBMC after CpG-A ( n = 21 donors), R848 ( n = 21 donors), or ssRNA ( n = 22 donors) stimulation with the change in the blood HCQ concentration of CLE patients. Data points from patients with increase in blood HCQ concentration > 600 ng/ml between the two visits are highlighted in red. Data points from patients with decrease in blood HCQ concentration > 500 ng/ml between two visits are highlighted in green. Statistical association was assessed using Pearson's (P) and Spearman's rank (S) correlations.

    Techniques Used: Flow Cytometry, Cytometry, Concentration Assay

    24F4A further reduces pDC IFNα production after CpG-A, R848 or ssRNA stimulations of PBMC isolated from CLE patients regardless blood HCQ levels. (A) Concentrations of HCQ in whole blood from the CLE patient cohort studied ( n = 30). (B) Representative dot plots of IFNα+ cells within a BDCA4+ and CD123+ gate after CpG-A, R848 or ssRNA stimulation with or without pre-treatment with 24F4A (10 μg/ml 30 min) from CLE patient without detectable blood HCQ (top panel) or a CLE patient with a blood HCQ concentration of more than 500ng/ml (bottom panel). (C) Effect of 24F4A on the percentage of IFNα-producing pDCs induced by CpG-A, R848 and ssRNA stimulations and detected by flow cytometry in PBMC from CLE patients without detectable level of blood HCQ ( n ≥ 6 donors), with blood HCQ concentrations lower than 500 ng/ml ( n ≥ 8 donors) or > 500ng/ml ( n ≥ 6 donors). Statistical significance was assessed with a two-tailed paired Student's t-test using log2-transformed values (* p
    Figure Legend Snippet: 24F4A further reduces pDC IFNα production after CpG-A, R848 or ssRNA stimulations of PBMC isolated from CLE patients regardless blood HCQ levels. (A) Concentrations of HCQ in whole blood from the CLE patient cohort studied ( n = 30). (B) Representative dot plots of IFNα+ cells within a BDCA4+ and CD123+ gate after CpG-A, R848 or ssRNA stimulation with or without pre-treatment with 24F4A (10 μg/ml 30 min) from CLE patient without detectable blood HCQ (top panel) or a CLE patient with a blood HCQ concentration of more than 500ng/ml (bottom panel). (C) Effect of 24F4A on the percentage of IFNα-producing pDCs induced by CpG-A, R848 and ssRNA stimulations and detected by flow cytometry in PBMC from CLE patients without detectable level of blood HCQ ( n ≥ 6 donors), with blood HCQ concentrations lower than 500 ng/ml ( n ≥ 8 donors) or > 500ng/ml ( n ≥ 6 donors). Statistical significance was assessed with a two-tailed paired Student's t-test using log2-transformed values (* p

    Techniques Used: Isolation, Concentration Assay, Flow Cytometry, Cytometry, Two Tailed Test, Transformation Assay

    Related Articles

    Flow Cytometry:

    Article Title: Nr4a1-Dependent Ly6Clow Monocytes Monitor Endothelial Cells and Orchestrate Their Disposal
    Article Snippet: .. After 8 hr, neutrophil depletion was assesses by flow cytometry and the mice were subjected to kidney painting with R848 as below. ..

    Intravital Microscopy:

    Article Title: Nr4a1-Dependent Ly6Clow Monocytes Monitor Endothelial Cells and Orchestrate Their Disposal
    Article Snippet: .. Monocyte Retention by Capillaries via TLR7, Related to Figure 3 Analysis of time-lapse intravital microscopy of peritubular capillaries in the superficial renal cortex of Cx3cr1 gfp/+ , Tlr7 +/+ , or Tlr7 −/− mice 0–5 hr after direct treatment of kidney capsule with R848 or PBS. ..

    Gel Permeation Chromatography:

    Article Title: BCR-signalling synergizes with TLR-signalling for induction of AID and immunoglobulin class-switching through the non-canonical NF-?B pathway
    Article Snippet: .. CSR sIgδ+ B cells were cultured at 105 cell ml−1 in RPMI–FBS supplemented with 50 μM β-mercaptoethanol in the presence of the following stimuli at the indicated dose, unless otherwise specified: TLR1/2 ligand Pam3 CSK4 (100 ng ml−1 , Invivogen), TLR4 ligand monophophoryl lipid A (lipid A, 1 μg ml−1 , Sigma-Aldrich), TLR7 ligand R-848 (30 ng ml−1 , Invivogen) or TLR9 ligand CpG ODN1826 with a phosphorothioate backbone (CpG, 1 μM , sequence 5′-TCCATGACGTTCCTGACGTT-3′; the sequence of the control GpC ODN 1745 was 5′-TCCATGAGCTTCCTGAGTCT-3′; Operon), LPS deproteinized by chloroform extraction (3 μg ml−1 , from E. coli , serotype 055:B5, Sigma-Aldrich), mCD154 (1 U ml−1 ), BAFF (250 ng ml−1 , Alexis) or APRIL (250 ng ml−1 , Alexis). mCD154 was obtained by expression of mouse CD154 on membranes of Sf21 insect cells . ..

    Cytometry:

    Article Title: Nr4a1-Dependent Ly6Clow Monocytes Monitor Endothelial Cells and Orchestrate Their Disposal
    Article Snippet: .. After 8 hr, neutrophil depletion was assesses by flow cytometry and the mice were subjected to kidney painting with R848 as below. ..

    Cell Culture:

    Article Title: BCR-signalling synergizes with TLR-signalling for induction of AID and immunoglobulin class-switching through the non-canonical NF-?B pathway
    Article Snippet: .. CSR sIgδ+ B cells were cultured at 105 cell ml−1 in RPMI–FBS supplemented with 50 μM β-mercaptoethanol in the presence of the following stimuli at the indicated dose, unless otherwise specified: TLR1/2 ligand Pam3 CSK4 (100 ng ml−1 , Invivogen), TLR4 ligand monophophoryl lipid A (lipid A, 1 μg ml−1 , Sigma-Aldrich), TLR7 ligand R-848 (30 ng ml−1 , Invivogen) or TLR9 ligand CpG ODN1826 with a phosphorothioate backbone (CpG, 1 μM , sequence 5′-TCCATGACGTTCCTGACGTT-3′; the sequence of the control GpC ODN 1745 was 5′-TCCATGAGCTTCCTGAGTCT-3′; Operon), LPS deproteinized by chloroform extraction (3 μg ml−1 , from E. coli , serotype 055:B5, Sigma-Aldrich), mCD154 (1 U ml−1 ), BAFF (250 ng ml−1 , Alexis) or APRIL (250 ng ml−1 , Alexis). mCD154 was obtained by expression of mouse CD154 on membranes of Sf21 insect cells . ..

    Mouse Assay:

    Article Title: Nr4a1-Dependent Ly6Clow Monocytes Monitor Endothelial Cells and Orchestrate Their Disposal
    Article Snippet: .. Monocyte Retention by Capillaries via TLR7, Related to Figure 3 Analysis of time-lapse intravital microscopy of peritubular capillaries in the superficial renal cortex of Cx3cr1 gfp/+ , Tlr7 +/+ , or Tlr7 −/− mice 0–5 hr after direct treatment of kidney capsule with R848 or PBS. ..

    Article Title: Nr4a1-Dependent Ly6Clow Monocytes Monitor Endothelial Cells and Orchestrate Their Disposal
    Article Snippet: .. After 8 hr, neutrophil depletion was assesses by flow cytometry and the mice were subjected to kidney painting with R848 as below. ..

    Real-time Polymerase Chain Reaction:

    Article Title: IL-4 Suppresses the Responses to TLR7 and TLR9 Stimulation and Increases the Permissiveness to Retroviral Infection of Murine Conventional Dendritic Cells
    Article Snippet: .. We analyzed the gene expression of TLR7 and TLR9 by qPCR in the RAG-KO cDCs after 24 h treatment with IL-4 and then stimulated with CpG or R848 for 6 h. All of the conditions were normalized against the control (untreated DCs in medium only) in each experiment. ..

    other:

    Article Title: Chronic TLR7 and TLR9 signaling drives anemia via differentiation of specialized hemophagocytes
    Article Snippet: CMPs were sorted as above and then placed in culture at 10,000 cells/well in either 10 μg/ml of R848 or 40 ng/ml of MCSF.

    Article Title: Nr4a1-Dependent Ly6Clow Monocytes Monitor Endothelial Cells and Orchestrate Their Disposal
    Article Snippet: MEK inhibitor PD98059 (PD; Enzo Lifescience), R848 (InvivoGEN) and LPS from E. Coli 0111:B4 (#L4391; Sigma).

    Expressing:

    Article Title: BCR-signalling synergizes with TLR-signalling for induction of AID and immunoglobulin class-switching through the non-canonical NF-?B pathway
    Article Snippet: .. CSR sIgδ+ B cells were cultured at 105 cell ml−1 in RPMI–FBS supplemented with 50 μM β-mercaptoethanol in the presence of the following stimuli at the indicated dose, unless otherwise specified: TLR1/2 ligand Pam3 CSK4 (100 ng ml−1 , Invivogen), TLR4 ligand monophophoryl lipid A (lipid A, 1 μg ml−1 , Sigma-Aldrich), TLR7 ligand R-848 (30 ng ml−1 , Invivogen) or TLR9 ligand CpG ODN1826 with a phosphorothioate backbone (CpG, 1 μM , sequence 5′-TCCATGACGTTCCTGACGTT-3′; the sequence of the control GpC ODN 1745 was 5′-TCCATGAGCTTCCTGAGTCT-3′; Operon), LPS deproteinized by chloroform extraction (3 μg ml−1 , from E. coli , serotype 055:B5, Sigma-Aldrich), mCD154 (1 U ml−1 ), BAFF (250 ng ml−1 , Alexis) or APRIL (250 ng ml−1 , Alexis). mCD154 was obtained by expression of mouse CD154 on membranes of Sf21 insect cells . ..

    Article Title: IL-4 Suppresses the Responses to TLR7 and TLR9 Stimulation and Increases the Permissiveness to Retroviral Infection of Murine Conventional Dendritic Cells
    Article Snippet: .. We analyzed the gene expression of TLR7 and TLR9 by qPCR in the RAG-KO cDCs after 24 h treatment with IL-4 and then stimulated with CpG or R848 for 6 h. All of the conditions were normalized against the control (untreated DCs in medium only) in each experiment. ..

    Sequencing:

    Article Title: BCR-signalling synergizes with TLR-signalling for induction of AID and immunoglobulin class-switching through the non-canonical NF-?B pathway
    Article Snippet: .. CSR sIgδ+ B cells were cultured at 105 cell ml−1 in RPMI–FBS supplemented with 50 μM β-mercaptoethanol in the presence of the following stimuli at the indicated dose, unless otherwise specified: TLR1/2 ligand Pam3 CSK4 (100 ng ml−1 , Invivogen), TLR4 ligand monophophoryl lipid A (lipid A, 1 μg ml−1 , Sigma-Aldrich), TLR7 ligand R-848 (30 ng ml−1 , Invivogen) or TLR9 ligand CpG ODN1826 with a phosphorothioate backbone (CpG, 1 μM , sequence 5′-TCCATGACGTTCCTGACGTT-3′; the sequence of the control GpC ODN 1745 was 5′-TCCATGAGCTTCCTGAGTCT-3′; Operon), LPS deproteinized by chloroform extraction (3 μg ml−1 , from E. coli , serotype 055:B5, Sigma-Aldrich), mCD154 (1 U ml−1 ), BAFF (250 ng ml−1 , Alexis) or APRIL (250 ng ml−1 , Alexis). mCD154 was obtained by expression of mouse CD154 on membranes of Sf21 insect cells . ..

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    InvivoGen r848
    TLR7 signaling and responsiveness is enhanced in DN2. A. CD25 expression is increased over baseline (filled) by trimerized CD40L stimulation (blue line) in rNAV but not DN2 cells. In contrast TLR7 stimulation by <t>R848</t> (red line) increased CD25 expression in both rNAV and DN2 cells. One example out of two experiments is shown. B. The percentage of dual positive phospho-ERK and phospho-MAPKp38 positive CD11c + DN cells is increased after R848 stimulated (bottom) relative to unstimulated (top) and to SWM, rNAV, and DN1 B cells. C. Fluorescence intensity of phospho-ERK and phospho-MAPKp38 is higher in DN2 cells and aNAV cells after R848 stimulation. Histogram coloring is based on median fluorescence intensity as shown by the scale bars. D. Relative induction of pERK (n=5) and pMAPKp38 (n=10), expressed as the fold increase in MFI of R848 stimulated samples, is higher in DN2 cells and aNAV cells. Although induced pY-ERK phosphorylation is lower in HCD B cells (n=7), DN2 and aNAV cells still have significantly higher phosphorylation (repeated measure 1-way ANOVA). E. In SLE patients HLA-DR (n=6) and CD86 (n=5) expression is higher at baseline in DN2 cells (green) than NAV (blue) or SWM (red) but this difference is further enhanced after TLR7 stimulation. Expression of inhibitory receptors CD32b and CD72 is reduced by R848 stimulation in DN2 cells (blue) but not NAV B cells (green) (n=5). HCD B cells demonstrated similar changes except for CD72, which did not decrease in either rNAV or DN2 cells. Red asterisks indicate significant differences from SWM; blue asterisks indicate significant differences from NAV (repeated measure 1-way ANOVA).
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    TLR7 signaling and responsiveness is enhanced in DN2. A. CD25 expression is increased over baseline (filled) by trimerized CD40L stimulation (blue line) in rNAV but not DN2 cells. In contrast TLR7 stimulation by R848 (red line) increased CD25 expression in both rNAV and DN2 cells. One example out of two experiments is shown. B. The percentage of dual positive phospho-ERK and phospho-MAPKp38 positive CD11c + DN cells is increased after R848 stimulated (bottom) relative to unstimulated (top) and to SWM, rNAV, and DN1 B cells. C. Fluorescence intensity of phospho-ERK and phospho-MAPKp38 is higher in DN2 cells and aNAV cells after R848 stimulation. Histogram coloring is based on median fluorescence intensity as shown by the scale bars. D. Relative induction of pERK (n=5) and pMAPKp38 (n=10), expressed as the fold increase in MFI of R848 stimulated samples, is higher in DN2 cells and aNAV cells. Although induced pY-ERK phosphorylation is lower in HCD B cells (n=7), DN2 and aNAV cells still have significantly higher phosphorylation (repeated measure 1-way ANOVA). E. In SLE patients HLA-DR (n=6) and CD86 (n=5) expression is higher at baseline in DN2 cells (green) than NAV (blue) or SWM (red) but this difference is further enhanced after TLR7 stimulation. Expression of inhibitory receptors CD32b and CD72 is reduced by R848 stimulation in DN2 cells (blue) but not NAV B cells (green) (n=5). HCD B cells demonstrated similar changes except for CD72, which did not decrease in either rNAV or DN2 cells. Red asterisks indicate significant differences from SWM; blue asterisks indicate significant differences from NAV (repeated measure 1-way ANOVA).

    Journal: Immunity

    Article Title: Distinct effector B-cells induced by unregulated Toll-like receptor 7 contribute to pathogenic responses in Systemic Lupus Erythematosus

    doi: 10.1016/j.immuni.2018.08.015

    Figure Lengend Snippet: TLR7 signaling and responsiveness is enhanced in DN2. A. CD25 expression is increased over baseline (filled) by trimerized CD40L stimulation (blue line) in rNAV but not DN2 cells. In contrast TLR7 stimulation by R848 (red line) increased CD25 expression in both rNAV and DN2 cells. One example out of two experiments is shown. B. The percentage of dual positive phospho-ERK and phospho-MAPKp38 positive CD11c + DN cells is increased after R848 stimulated (bottom) relative to unstimulated (top) and to SWM, rNAV, and DN1 B cells. C. Fluorescence intensity of phospho-ERK and phospho-MAPKp38 is higher in DN2 cells and aNAV cells after R848 stimulation. Histogram coloring is based on median fluorescence intensity as shown by the scale bars. D. Relative induction of pERK (n=5) and pMAPKp38 (n=10), expressed as the fold increase in MFI of R848 stimulated samples, is higher in DN2 cells and aNAV cells. Although induced pY-ERK phosphorylation is lower in HCD B cells (n=7), DN2 and aNAV cells still have significantly higher phosphorylation (repeated measure 1-way ANOVA). E. In SLE patients HLA-DR (n=6) and CD86 (n=5) expression is higher at baseline in DN2 cells (green) than NAV (blue) or SWM (red) but this difference is further enhanced after TLR7 stimulation. Expression of inhibitory receptors CD32b and CD72 is reduced by R848 stimulation in DN2 cells (blue) but not NAV B cells (green) (n=5). HCD B cells demonstrated similar changes except for CD72, which did not decrease in either rNAV or DN2 cells. Red asterisks indicate significant differences from SWM; blue asterisks indicate significant differences from NAV (repeated measure 1-way ANOVA).

    Article Snippet: To test antibody production, sorted B cells were plated at 20,000–30,000 cells/well in 200 μl RPMI plus 10% FBS stimulated with 1.25 μg/ml R848 (Invivogen), 10 μg/ml goat F(ab’)2 anti-human IgG (Southern Biotech), and the following recombinant cytokines (R & D); 50 ng/ml IL-2, 100 ng/ml IL-21, 250 ng/ml IL-10, and 100 ng/ml BAFF.

    Techniques: Expressing, Fluorescence

    TLR-induced cytokine responses in blood cells from TOSV-infected patients and healthy controls. Blood cells were left unstimulated or stimulated with poly(A:U) (TLR3 ligand; 20 µg/mL) or Resiquimod-R848 (TLR7/8 ligand; 10 µg/mL). After 24 h, supernatants were collected and analyzed for cytokine levels. White squares indicate healthy controls and black triangles indicate TOSV-infected patients. N = 8 TOSV-infected patients and n = 7 healthy controls were evaluated. Data were analyzed using the non-parametric Mann–Whitney U test. * indicates p ≤ 0.05.

    Journal: Viruses

    Article Title: Meningitis Caused by Toscana Virus Is Associated with Strong Antiviral Response in the CNS and Altered Frequency of Blood Antigen-Presenting Cells

    doi: 10.3390/v7112909

    Figure Lengend Snippet: TLR-induced cytokine responses in blood cells from TOSV-infected patients and healthy controls. Blood cells were left unstimulated or stimulated with poly(A:U) (TLR3 ligand; 20 µg/mL) or Resiquimod-R848 (TLR7/8 ligand; 10 µg/mL). After 24 h, supernatants were collected and analyzed for cytokine levels. White squares indicate healthy controls and black triangles indicate TOSV-infected patients. N = 8 TOSV-infected patients and n = 7 healthy controls were evaluated. Data were analyzed using the non-parametric Mann–Whitney U test. * indicates p ≤ 0.05.

    Article Snippet: Stimulation of Blood Cells with Specific TLR Ligands Whole blood samples were diluted 1:1 with RPMI medium and separate aliquots (200 µL) of diluted blood distributed into a 96-well flat bottom plate for cell cultures were left unstimulated or stimulated either with poly(A:U) (TLR3 ligand; 20 µg/mL; InvivoGen, San Diego, CA, USA) or Resiquimod-R848 (TLR 7/8 ligand; 10 µg/mL, InvivoGen).

    Techniques: Infection, MANN-WHITNEY

    TLR7 promotes RPM-like hemophagocyte development in vitro. (A) Representative flow cytometric staining of CMPs cultured with SCF or SCF+R848. (B-D) RNA-Seq analysis of CD11b + F4/80 + ) that were significantly increased in R848-differentiated compared to MCSF-differentiated macrophages. Black line indicates –log p-value calculated using exact hypergeometric probability with a normal approximation. (D) Heat map of increased genes (≥2-fold, FDR≥0.05) in R848-differentiated compared to MCSF-differentiated macrophages in RPM core signature. (E) R848-differentiated macrophages were treated with or without cytochalasin D (CytoD) then allowed to phagocytose CFSE-labeled RBC for 15 min at indicated ratios. Percent of CD11b + F4/80 + macrophages that had phagocytosed RBC is shown. Data are representative of four experiments, n=3 technical replicates per condition/experiment. Mean values+SD (E) are shown.

    Journal: Science (New York, N.Y.)

    Article Title: Chronic TLR7 and TLR9 signaling drives anemia via differentiation of specialized hemophagocytes

    doi: 10.1126/science.aao5213

    Figure Lengend Snippet: TLR7 promotes RPM-like hemophagocyte development in vitro. (A) Representative flow cytometric staining of CMPs cultured with SCF or SCF+R848. (B-D) RNA-Seq analysis of CD11b + F4/80 + ) that were significantly increased in R848-differentiated compared to MCSF-differentiated macrophages. Black line indicates –log p-value calculated using exact hypergeometric probability with a normal approximation. (D) Heat map of increased genes (≥2-fold, FDR≥0.05) in R848-differentiated compared to MCSF-differentiated macrophages in RPM core signature. (E) R848-differentiated macrophages were treated with or without cytochalasin D (CytoD) then allowed to phagocytose CFSE-labeled RBC for 15 min at indicated ratios. Percent of CD11b + F4/80 + macrophages that had phagocytosed RBC is shown. Data are representative of four experiments, n=3 technical replicates per condition/experiment. Mean values+SD (E) are shown.

    Article Snippet: CMPs were sorted as above and then placed in culture at 10,000 cells/well in either 10 μg/ml of R848 or 40 ng/ml of MCSF.

    Techniques: In Vitro, Flow Cytometry, Staining, Cell Culture, RNA Sequencing Assay, Labeling

    iHPCs are derived from Ly6C hi monocytes. (A) Splenic monocytes (live singlets, CD11b + F4/80 − Ly6G − Ly6C hi or CCR2 + cells) (black) and iHPCs (live singlets, F4/80 lo Ly6G − Ter-119 + VCAM1 lo or CD31 hi cells) (blue) were assessed for the expression of the cell surface proteins indicated (solid lines) compared to fluorescence minus one (FMO) control stains (dashed lines). Data are representative of three experiments. (B) Bone marrow Ly6C hi monocytes were sorted from WT B6 mice and cultured for 21 hours with media alone (--) or with R848. Spic , Pecam1 , Ccr2, and Ly6c1 transcripts were quantified by qPCR. Data are representative from five experiments with n=3 per experiment. (C, D) Ccr2-DTR − , WT/Ccr2-DTR + , TLR7.1/Ccr2-DTR − , and TLR7.1/Ccr2-DTR + mice (n=5–7 mice per group) were injected with DT every other day for 17 days. (C) Representative flow cytometry of Ter-119 + hemophagocytes pre-gated on live singlets, CD45.2 + Ly6G − Siglec-F − cells from the spleens of TLR7.1/Ccr2-DTR − and TLR7.1/Ccr2-DTR + mice determined by flow cytometry. (D) Frequency and number of the indicated cell populations were quantitated from the spleens of Ccr2-DTR − , Ccr2-DTR + , TLR7.1/Ccr2-DTR − , and TLR7.1/Ccr2-DTR + mice by flow cytometry. Data are combined from three experiments. (E) Bone marrow Ly6C hi monocytes were sorted from WT B6 mice and cultured for 21 hours with media alone (--), R848, LPS, or CpG. Spic , Pecam1 , Ccr2, and Ly6c1 transcripts were quantified by qPCR. Data are representative from two experiments. (F and G) RNA-Seq analysis of RPMs and Ly6C hi monocytes sorted from spleens of WT B6 mice and TLR7.1 mice and iHPCs from spleens of TLR7.1 mice. n=5 (TLR7.1 iHPC), n=4 (WT and TLR7.1 RPM), n=6 (WT and TLR7.1 Mono). (F) PCA of indicated populations. (G) Heat map of DEG between the three populations (RPMs, Ly6C hi monocytes, and iHPCs) sorted from TLR7.1 mice. Mean values+SEM (D), ± SEM (B, E). *p

    Journal: Science (New York, N.Y.)

    Article Title: Chronic TLR7 and TLR9 signaling drives anemia via differentiation of specialized hemophagocytes

    doi: 10.1126/science.aao5213

    Figure Lengend Snippet: iHPCs are derived from Ly6C hi monocytes. (A) Splenic monocytes (live singlets, CD11b + F4/80 − Ly6G − Ly6C hi or CCR2 + cells) (black) and iHPCs (live singlets, F4/80 lo Ly6G − Ter-119 + VCAM1 lo or CD31 hi cells) (blue) were assessed for the expression of the cell surface proteins indicated (solid lines) compared to fluorescence minus one (FMO) control stains (dashed lines). Data are representative of three experiments. (B) Bone marrow Ly6C hi monocytes were sorted from WT B6 mice and cultured for 21 hours with media alone (--) or with R848. Spic , Pecam1 , Ccr2, and Ly6c1 transcripts were quantified by qPCR. Data are representative from five experiments with n=3 per experiment. (C, D) Ccr2-DTR − , WT/Ccr2-DTR + , TLR7.1/Ccr2-DTR − , and TLR7.1/Ccr2-DTR + mice (n=5–7 mice per group) were injected with DT every other day for 17 days. (C) Representative flow cytometry of Ter-119 + hemophagocytes pre-gated on live singlets, CD45.2 + Ly6G − Siglec-F − cells from the spleens of TLR7.1/Ccr2-DTR − and TLR7.1/Ccr2-DTR + mice determined by flow cytometry. (D) Frequency and number of the indicated cell populations were quantitated from the spleens of Ccr2-DTR − , Ccr2-DTR + , TLR7.1/Ccr2-DTR − , and TLR7.1/Ccr2-DTR + mice by flow cytometry. Data are combined from three experiments. (E) Bone marrow Ly6C hi monocytes were sorted from WT B6 mice and cultured for 21 hours with media alone (--), R848, LPS, or CpG. Spic , Pecam1 , Ccr2, and Ly6c1 transcripts were quantified by qPCR. Data are representative from two experiments. (F and G) RNA-Seq analysis of RPMs and Ly6C hi monocytes sorted from spleens of WT B6 mice and TLR7.1 mice and iHPCs from spleens of TLR7.1 mice. n=5 (TLR7.1 iHPC), n=4 (WT and TLR7.1 RPM), n=6 (WT and TLR7.1 Mono). (F) PCA of indicated populations. (G) Heat map of DEG between the three populations (RPMs, Ly6C hi monocytes, and iHPCs) sorted from TLR7.1 mice. Mean values+SEM (D), ± SEM (B, E). *p

    Article Snippet: CMPs were sorted as above and then placed in culture at 10,000 cells/well in either 10 μg/ml of R848 or 40 ng/ml of MCSF.

    Techniques: Derivative Assay, Expressing, Fluorescence, Mouse Assay, Cell Culture, Real-time Polymerase Chain Reaction, Injection, Flow Cytometry, Cytometry, RNA Sequencing Assay

    iHPCs differentiate in response to cell-intrinsic TLR7 signals. A) The ratio of TLR7.1 to WT BM-derived cells in mixed bone marrow chimeras of indicated populations after reconstitution. Data are representative of two experiments with n=8–10 per experiment. B) The ratio of WT to Tlr7 −/− BM-derived cells in mixed bone marrow chimeras injected with the TLR7 agonist R848 (right) or PBS (left) for 13 days. Data are representative of two experiments with n=3–5 per group per experiment. (A, B) mean±SEM, (A, B) each symbol represents an individual mouse. *p

    Journal: Science (New York, N.Y.)

    Article Title: Chronic TLR7 and TLR9 signaling drives anemia via differentiation of specialized hemophagocytes

    doi: 10.1126/science.aao5213

    Figure Lengend Snippet: iHPCs differentiate in response to cell-intrinsic TLR7 signals. A) The ratio of TLR7.1 to WT BM-derived cells in mixed bone marrow chimeras of indicated populations after reconstitution. Data are representative of two experiments with n=8–10 per experiment. B) The ratio of WT to Tlr7 −/− BM-derived cells in mixed bone marrow chimeras injected with the TLR7 agonist R848 (right) or PBS (left) for 13 days. Data are representative of two experiments with n=3–5 per group per experiment. (A, B) mean±SEM, (A, B) each symbol represents an individual mouse. *p

    Article Snippet: CMPs were sorted as above and then placed in culture at 10,000 cells/well in either 10 μg/ml of R848 or 40 ng/ml of MCSF.

    Techniques: Derivative Assay, Injection

    iHPC differentiation depends on IRF5. (A) Bone marrow Ly6C hi monocytes were sorted from WT and Irf5 −/− mice and cultured for 21 hours with media alone (--) or with R848. Spic , Pecam1 , Ccr2, and Ly6c1 transcripts were quantified by qPCR. Data are representative from two experiments with n=2 to 4 per group per experiment. (B and C) WT and Irf5 −/− mice were injected with R848 i.p. daily for 2 days. Splenocytes were analyzed by flow cytometry. (B) Representative flow plots of WT and Irf5 −/− CD11b + CD31 + iHPCs (gated on live singlets, CD45.2 + F4/80 − Ly6G − Siglec-F − cells). (C) Frequency (left) and number (right) of iHPCs in WT and Irf5 −/− spleens. Data are representative from two experiments with n=4 per group per experiment. Mean values±SEM (A and C) are shown. (A and C) each symbol represents an individual mouse. *p

    Journal: Science (New York, N.Y.)

    Article Title: Chronic TLR7 and TLR9 signaling drives anemia via differentiation of specialized hemophagocytes

    doi: 10.1126/science.aao5213

    Figure Lengend Snippet: iHPC differentiation depends on IRF5. (A) Bone marrow Ly6C hi monocytes were sorted from WT and Irf5 −/− mice and cultured for 21 hours with media alone (--) or with R848. Spic , Pecam1 , Ccr2, and Ly6c1 transcripts were quantified by qPCR. Data are representative from two experiments with n=2 to 4 per group per experiment. (B and C) WT and Irf5 −/− mice were injected with R848 i.p. daily for 2 days. Splenocytes were analyzed by flow cytometry. (B) Representative flow plots of WT and Irf5 −/− CD11b + CD31 + iHPCs (gated on live singlets, CD45.2 + F4/80 − Ly6G − Siglec-F − cells). (C) Frequency (left) and number (right) of iHPCs in WT and Irf5 −/− spleens. Data are representative from two experiments with n=4 per group per experiment. Mean values±SEM (A and C) are shown. (A and C) each symbol represents an individual mouse. *p

    Article Snippet: CMPs were sorted as above and then placed in culture at 10,000 cells/well in either 10 μg/ml of R848 or 40 ng/ml of MCSF.

    Techniques: Mouse Assay, Cell Culture, Real-time Polymerase Chain Reaction, Injection, Flow Cytometry, Cytometry

    BCR crosslinking enables TLRs to induce efficient CSR. ( a ) Proportions of surface IgG1 + (sIgG1 + ) B cells after stimulation of CFSE-labelled sIgδ + B cells with TLR1/2 ligand Pam 3 CSK 4 , TLR4 ligand lipid A, TLR7 ligand R-848 or TLR9 ligand CpG at indicated doses, in the absence or presence of anti-δ mAb/dex at indicated doses, plus IL-4 (3 ng ml −1 ). ( b ) Proportions of sIgG1 + B cells at each cell division after stimulation of sIgδ + B cells with LPS or mCD154 at indicated doses, in the absence or presence of anti-δ mAb/dex (100 ng ml −1 ), plus IL-4 (3 ng ml −1 ). ( c ) Proportions of sIgG1 + B cells within each B cell division after stimulation of CFSE-labelled sIgδ + B cells with Pam 3 CSK 4 (100 ng ml −1 ), lipid A (1 μg ml −1 ), R-848 (30 ng ml −1 ), CpG (1 μ M ), LPS (3 μg ml −1 ) or mCD154 (1 U ml −1 ), in the absence (teal) or presence (plum) of anti-δ mAb/dex (100 ng ml −1 ), plus IL-4 (3 ng ml −1 ). Data are representative of four independent experiments.

    Journal: Nature Communications

    Article Title: BCR-signalling synergizes with TLR-signalling for induction of AID and immunoglobulin class-switching through the non-canonical NF-?B pathway

    doi: 10.1038/ncomms1769

    Figure Lengend Snippet: BCR crosslinking enables TLRs to induce efficient CSR. ( a ) Proportions of surface IgG1 + (sIgG1 + ) B cells after stimulation of CFSE-labelled sIgδ + B cells with TLR1/2 ligand Pam 3 CSK 4 , TLR4 ligand lipid A, TLR7 ligand R-848 or TLR9 ligand CpG at indicated doses, in the absence or presence of anti-δ mAb/dex at indicated doses, plus IL-4 (3 ng ml −1 ). ( b ) Proportions of sIgG1 + B cells at each cell division after stimulation of sIgδ + B cells with LPS or mCD154 at indicated doses, in the absence or presence of anti-δ mAb/dex (100 ng ml −1 ), plus IL-4 (3 ng ml −1 ). ( c ) Proportions of sIgG1 + B cells within each B cell division after stimulation of CFSE-labelled sIgδ + B cells with Pam 3 CSK 4 (100 ng ml −1 ), lipid A (1 μg ml −1 ), R-848 (30 ng ml −1 ), CpG (1 μ M ), LPS (3 μg ml −1 ) or mCD154 (1 U ml −1 ), in the absence (teal) or presence (plum) of anti-δ mAb/dex (100 ng ml −1 ), plus IL-4 (3 ng ml −1 ). Data are representative of four independent experiments.

    Article Snippet: CSR sIgδ+ B cells were cultured at 105 cell ml−1 in RPMI–FBS supplemented with 50 μM β-mercaptoethanol in the presence of the following stimuli at the indicated dose, unless otherwise specified: TLR1/2 ligand Pam3 CSK4 (100 ng ml−1 , Invivogen), TLR4 ligand monophophoryl lipid A (lipid A, 1 μg ml−1 , Sigma-Aldrich), TLR7 ligand R-848 (30 ng ml−1 , Invivogen) or TLR9 ligand CpG ODN1826 with a phosphorothioate backbone (CpG, 1 μM , sequence 5′-TCCATGACGTTCCTGACGTT-3′; the sequence of the control GpC ODN 1745 was 5′-TCCATGAGCTTCCTGAGTCT-3′; Operon), LPS deproteinized by chloroform extraction (3 μg ml−1 , from E. coli , serotype 055:B5, Sigma-Aldrich), mCD154 (1 U ml−1 ), BAFF (250 ng ml−1 , Alexis) or APRIL (250 ng ml−1 , Alexis). mCD154 was obtained by expression of mouse CD154 on membranes of Sf21 insect cells .

    Techniques: