r837 Invivogen Search Results


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  • 99
    Millipore lps
    Effects of poly(I:C), <t>LPS,</t> R-837 and <t>iE-DAP</t> on the HASMC viability. ( A–C ) HASMC were cultured for 24, 48 and 72 h in the absence of presence of Pam 3 CSK 4 (1 µg/ml), poly(I:C) (10 µg/ml), LPS (100 ng/ml), R-837 (5 µg/ml) and iE-DAP (10 µg/ml). Proliferation was quantified with AlamarBlue and determined using a spectrophotometer at 570 and 620 nm. Data are depicted as percent reduction compared to untreated control and presented as mean ± SEM (n = 7–8). *, p
    Lps, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 55240 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    InvivoGen imiquimod
    pDCs have a transient decrease in the capacity to produce IFNα in vitro. ( A ) Total PBMCs were stimulated with <t>imiquimod</t> for 6 hours, and IFNα production by pDCs was measured by flow cytometry. ( B ) The percentage of pDCs that produced IFNα in response to imiquimod (IQ) stimulation is shown (red). pDC frequency (blue) and HIV-1 VL (black) are included for reference. The percentage of pDCs producing IFNα ( C ) or TNF-α ( D ) in response to imiquimod were compared between the time point before viremia at which the highest pDC frequency occurred and the time point immediately prior. * P
    Imiquimod, supplied by InvivoGen, used in various techniques. Bioz Stars score: 99/100, based on 1371 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    InvivoGen r837
    M45 inhibits IκBα degradation, TNFα and IL-6 production during MCMV infection. (A) NIH-3T3 cells were infected with wt MCMV-GFP, an M45 deletion mutant (ΔM45) or a revertant virus (RM45) at an MOI of 10 and treated 5 h postinfection with the TLR2 agonist Pam 3 CSK 4 (Pam., 0.1 µg/ml), the TLR4 agonist LPS (10 µg/ml), or IL-1β (20 ng/ml) for the indicated times. Levels of the indicated proteins were analyzed by immunoblotting. (B) BMDMs were infected with wt MCMV-GFP, ΔM45, or RM45 at an MOI of 3 and stimulated 8 h postinfection for 16 h with the TLR7 agonist R848 (0.1 µM). TNFα and IL-6 levels in the supernatant were determined by ELISA (mean ± SD). (C) RAW264.7 macrophages were infected with wt MCMV-GFP or ΔM45 at an MOI of 0.1, stimulated 24 h postinfection for 4 hours with TLR agonists Pam 3 CSK 4 (Pam.) or Malp-2 (TLR2), LPS (TLR4), <t>R837</t> (TLR7), or CpG (TLR9), in the presence of brefeldin A. Cells were fixed, permeabilized, and stained with a TNFα-specific antibody. The percentages of TNFα-positive cells within infected (GFP-positive) cell populations were determined by FACS analysis (mean ± SD).
    R837, supplied by InvivoGen, used in various techniques. Bioz Stars score: 91/100, based on 233 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    InvivoGen pam3csk4
    NF-κB signaling blocks NLRC4/caspase-8-dependent apoptosis pathway. Casp1 −/− Casp11 −/− BMDMs were pre-stimulated for with or without various stimuli – <t>Pam3CSK4</t> (1 μg ml −1 ), LPS (1 μg ml −1 ), R837 (2 μg ml −1 ), TNFα (100 ng ml −1 ), IFN-α (100 U ml −1 ), or IFN-β (100 U ml −1 ), then electroporated with flagellin (0.5 μg ml −1 ) or stimulated with FasL (100 ng ml −1 ). ( a ) LDH release after 16 h. Data is represented as mean ± SD; n = 3. ( b ) % YOYO-1 positive BMDMs from live cell imaging taken every 45 min for 16 h.
    Pam3csk4, supplied by InvivoGen, used in various techniques. Bioz Stars score: 99/100, based on 4379 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    InvivoGen r848
    IFNα downregulates the production of CCL23 by <t>R848-treated</t> neutrophils Neutrophils (5 × 10 6 /ml) were cultured with or without 1,000 U/ml IFNα and/or 5 μM R848. After 24 h, supernatants were collected and subjected to CCL23, CCL2, CCL3, and CCL4 measurement by ELISA. Data are depicted as mean ± SEM ( n = 3–19). Asterisks stand for significant increases: ** P
    R848, supplied by InvivoGen, used in various techniques. Bioz Stars score: 99/100, based on 3200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore imiquimod
    Specificity of phloretin with various TLR-specific agonists that selectively activate different TLRs determined by monitoring the inhibition activity of TNF-α production in Raw264.7 cells. Pam 3 CSK 4 (200 ng/mL), Pam 2 CSK 4 (200 ng/mL), poly(I:C) (1 µg/mL), LPS (20 ng/mL), <t>imiquimod</t> (1 µg/mL), and ODN1826 (10 µg/mL) were used to selectively activate respective TLRs. TNF-α secreted into the supernatant was measured by ELISA. Each sample was measured in triplicate. The error bars represent ± SEM. (* p
    Imiquimod, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 175 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    InvivoGen tlr ligands
    Cell-Based <t>TLR</t> Activation Assays. Various CEP adducts and TLR agonists were tested in HEK293 or THP-1 cells. Readouts were NFkB reporter signal or IL-8 secretion (columns), as indicated. In addition, the same wells were analyzed for viability with the CellTiter-Glo kit (axis on right; square symbols) to ensure that any lack of activation was not due to cell toxicity. HEK293 cells were treated with the following reagents: <t>HSA-CTL1,</t> HSA-CTL2, or HSA-CEP: 0, 3.9, 7.8, 15.6, 32.6, 62.5, and 125 and 250 µg/ml; Pam3CSK4∶ 0, 1.5, 3.2, 6.3, 12.5, 25, 50, 100 ng/mL. THP-1 cells were treated with the following reagents: HSA-CTL1, HSA-CTL2, or HSA-CEP: 62.5, 125, and 250 µg/mL; Pam3CSK4∶ 4, 20, and 100 ng/mL; FSL-1∶0.4, 2, and 10 ng/mL; LPS: 4, 20, and 100 ng/mL; R837 or R848∶0.4, 2, and 10 µM; ODN2006G5∶0.2, 1, and 5 µM.
    Tlr Ligands, supplied by InvivoGen, used in various techniques. Bioz Stars score: 99/100, based on 689 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    InvivoGen poly i c
    Expression dynamics of genes involved in innate immune signaling in response to PMA (A) Heatmap showing the proteome fold change of the subset of genes involved in innate immune signaling. Scale indicates the level of expression (Log2-expression values, z-transformed, scaled). (B) Dynamic expression pattern to assess the expression levels of pro-inflammatory cytokine mRNA induced by TLR agonists: CL075-TLR7/8, CpG2006-TLR9, Flagellin-TLR5, FSL1- TLR2/6, LPS 0111:B4, and LPS K12 – TLR4, Pam3CSK4-TLR2/1, Poly I:C-TLR3, R837-TLR7, and R848-TLR7/8. Heatmap depicts Log10 fold change of normalized gene expression for pairwise comparisons of mRNA levels.
    Poly I C, supplied by InvivoGen, used in various techniques. Bioz Stars score: 91/100, based on 189 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    InvivoGen loxoribine
    Expression dynamics of genes involved in innate immune signaling in response to PMA (A) Heatmap showing the proteome fold change of the subset of genes involved in innate immune signaling. Scale indicates the level of expression (Log2-expression values, z-transformed, scaled). (B) Dynamic expression pattern to assess the expression levels of pro-inflammatory cytokine mRNA induced by TLR agonists: CL075-TLR7/8, CpG2006-TLR9, Flagellin-TLR5, FSL1- TLR2/6, LPS 0111:B4, and LPS K12 – TLR4, Pam3CSK4-TLR2/1, Poly I:C-TLR3, R837-TLR7, and R848-TLR7/8. Heatmap depicts Log10 fold change of normalized gene expression for pairwise comparisons of mRNA levels.
    Loxoribine, supplied by InvivoGen, used in various techniques. Bioz Stars score: 97/100, based on 307 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    InvivoGen cl075
    Inhibitory effect of TAC5-a on the TLR signaling pathways. ( A ) The chemical structure of TAC5 and its derivatives represented by different R groups. ( B ) RAW 264.7 cells were treated with various concentrations of compounds for 24 h and the cell viability was measured using an MTT assay. ( C ) The inhibitory effects of TAC5-a, TAC5-c, TAC5-d, and TAC5-e on the TNF-α secretion level in THP1 derived macrophage cells under the influence of different TLR7/8 ligands. R848 and <t>CL075</t> were used to activate TLR7/8, imiquimod (IMQ) to activate TLR7, and TL8-506 to activate TLR8, specifically. ( D – F ) The inhibitory effect of TAC5-a on the TNF-α secretion level in THP1-derived macrophage cells was evaluated following the treatment of cells with different TLR ligands ( D ) Lipopolysaccharide was used to activate TLR4, Pam 3 CSK 4 to activate TLR1/2, and FSL-1 to activate TLR2/6). ( E ) poly(I:C) was used to activate TLR3 and CpG ODN to activate TLR9. ( F ) R848 and CL075 were used to activate TLR7/8, IMQ to activate TLR7, and TL8-506 to activate TLR8. ( G ) The inhibitory effects of TAC5-a on the IL-6 secretion in RAW264.7 cells stimulated with agonists of TLR7/8 (R848 and CL075), TLR7 (IMQ), or TLR8 (TL8-506). All data shown represent the results of five independently conducted experiments; the mean ± SEM of the independent experiments were calculated and used in the two-tailed paired Student’s t -test (* p
    Cl075, supplied by InvivoGen, used in various techniques. Bioz Stars score: 98/100, based on 196 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    InvivoGen cpg
    TCDD decreases BMDC cytokine production following TLR stimulation: Vehicle- and TCDD-BMDCs were harvested on day 10 and stimulated for 24 h with (A) <t>LPS</t> (1 μg/ml) or (B) <t>CpG</t> (0.5μM) or (C) Imiquimod (30 μg/ml) or unstimulated.
    Cpg, supplied by InvivoGen, used in various techniques. Bioz Stars score: 99/100, based on 1755 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    InvivoGen lps
    Reduced uptake of soluble antigen by BMDC in the presence of TLR agonists. Granulocyte macrophage colony-stimulating factor (GM-CSF) BMDC were cultured for 4 h with OVA–Alexa555 (5 μg ml −1 ) in the presence of <t>polyI:C</t> (50 μg ml −1 ), CpG (0.5 μg ml −1 ), R837 (1 μg ml −1 ), <t>LPS</t> (1 μg ml −1 ) or left untreated. Cells were stained with anti-CD11c antibody and samples were acquired by flow cytometry. (A) The mean fluorescent intensity of cells for Alexa555 was determined after gating on the CD11c + cell population. The data of two independent representative experiments were pooled ( n = 4) and the standard deviation is indicated. (B) The frequency of OVA–Alexa555 + cells was determined after gating on the CD11c + cell population. The relative percentage of OVA–Alexa555 + BMDC in samples stimulated with TLR agonists is depicted in relation to the frequency of OVA–Alexa555 + BMDC in the absence of TLR stimulation (unst), which was set to 100%. The results of eight independent experiments were compiled and the standard deviation is indicated. (C) Mice were vaccinated intravenously with GM-CSF BMDC that had been pulsed overnight with OVA in the presence or absence of various TLR ligands. Seven days after vaccination, CFSE-labelled target cells were injected and antigen-specific lysis of peptide-pulsed targets cells versus unpulsed target cells was determined by flow cytometry the following day. The graph compiles data from two independent experiments ( n = 10). For statistical analysis, one-way analysis of variance in combination with Dunnett's (A and B) or Tukey's (C) multiple comparison test was used (** P
    Lps, supplied by InvivoGen, used in various techniques. Bioz Stars score: 94/100, based on 4418 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    InvivoGen s aureus
    Reduced uptake of soluble antigen by BMDC in the presence of TLR agonists. Granulocyte macrophage colony-stimulating factor (GM-CSF) BMDC were cultured for 4 h with OVA–Alexa555 (5 μg ml −1 ) in the presence of <t>polyI:C</t> (50 μg ml −1 ), CpG (0.5 μg ml −1 ), R837 (1 μg ml −1 ), <t>LPS</t> (1 μg ml −1 ) or left untreated. Cells were stained with anti-CD11c antibody and samples were acquired by flow cytometry. (A) The mean fluorescent intensity of cells for Alexa555 was determined after gating on the CD11c + cell population. The data of two independent representative experiments were pooled ( n = 4) and the standard deviation is indicated. (B) The frequency of OVA–Alexa555 + cells was determined after gating on the CD11c + cell population. The relative percentage of OVA–Alexa555 + BMDC in samples stimulated with TLR agonists is depicted in relation to the frequency of OVA–Alexa555 + BMDC in the absence of TLR stimulation (unst), which was set to 100%. The results of eight independent experiments were compiled and the standard deviation is indicated. (C) Mice were vaccinated intravenously with GM-CSF BMDC that had been pulsed overnight with OVA in the presence or absence of various TLR ligands. Seven days after vaccination, CFSE-labelled target cells were injected and antigen-specific lysis of peptide-pulsed targets cells versus unpulsed target cells was determined by flow cytometry the following day. The graph compiles data from two independent experiments ( n = 10). For statistical analysis, one-way analysis of variance in combination with Dunnett's (A and B) or Tukey's (C) multiple comparison test was used (** P
    S Aureus, supplied by InvivoGen, used in various techniques. Bioz Stars score: 92/100, based on 192 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore curdlan
    CD40 ligation on DCs boosts PRR-induced cytokine production. ( A–C ) Spleen-derived DCs were incubated with the indicated doses of CpG, <t>curdlan,</t> or zymosan in the absence or presence of 2 or 10 μg/ml of anti-CD40 antibody. After 20 h, surface
    Curdlan, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 474 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    InvivoGen cpg a
    Activation and apoptosis were not affected in Blimp-1-deficient FLpDCs. (A) Flow cytometric analysis showing the frequency of Annexin V positive cells in 4-OHT treated Ctrl-ER or CKO-ER FLpDCs stimulated with 1 µM <t>CpG-A</t> or medium alone for 6 and 16 h. (B) Flow cytometric analysis of CD86, CD69, and MHCII expression on 4-OHT treated Ctrl-ER or CKO-ER FLpDCs with or without 16 h of 1 µM CpG-A stimulation. The positive frequency of each marker is indicated. Results represent the mean ± SEM and were analyzed by two-tailed unpaired Student’s t -test [ n = 3 in (A,B) ].
    Cpg A, supplied by InvivoGen, used in various techniques. Bioz Stars score: 91/100, based on 248 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    InvivoGen ssrna40
    Activation and apoptosis were not affected in Blimp-1-deficient FLpDCs. (A) Flow cytometric analysis showing the frequency of Annexin V positive cells in 4-OHT treated Ctrl-ER or CKO-ER FLpDCs stimulated with 1 µM <t>CpG-A</t> or medium alone for 6 and 16 h. (B) Flow cytometric analysis of CD86, CD69, and MHCII expression on 4-OHT treated Ctrl-ER or CKO-ER FLpDCs with or without 16 h of 1 µM CpG-A stimulation. The positive frequency of each marker is indicated. Results represent the mean ± SEM and were analyzed by two-tailed unpaired Student’s t -test [ n = 3 in (A,B) ].
    Ssrna40, supplied by InvivoGen, used in various techniques. Bioz Stars score: 96/100, based on 240 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    InvivoGen ultrapure lps
    Plexin-A4 is required for TNF and IL-6 production in macrophages. (A) Expression of Plxna4 mRNA in different immune subpopulations was normalized to Actb mRNA level. T cells, B cells, NK cells, BM pDCs, and splenic mDCs were isolated by FACS sorting. BMMs and BMDCs were, respectively, cultured from BM cells in the presence of L929 cell supernatant and GM-CSF plus IL-4. Macrophages were isolated by adherence for 2 h at 37°C. Results are expressed as mean ± SD. (B) Immunofluorescence staining of plexin-A4 and F4/80 in WT and Plxna4 −/− peritoneal macrophages. (C) WT and Plxna4 −/− peritoneal macrophages were stained with IgG control (gray fill) or antibody against mouse plexin-A4 (black lines). Histograms show gated CD11b + cells. The data shown in A–C are representative of two independent experiments. (D and E) Peritoneal macrophages were left unstimulated (−) or were stimulated with 5 µg/ml <t>Pam3Cys,</t> 10 µg/ml poly(I:C), 1 µg/ml ultrapure <t>LPS,</t> 10 µg/ml R837, or 4 µg/ml CpG-B for 4 h. TNF and IL-6 mRNA (D) and protein (E) were measured by RT-PCR and ELISA, respectively. The results shown in D and E are representative of five independent experiments and are expressed as mean ± SD. *, P
    Ultrapure Lps, supplied by InvivoGen, used in various techniques. Bioz Stars score: 93/100, based on 2107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare fetal bovine serum fbs
    Plexin-A4 is required for TNF and IL-6 production in macrophages. (A) Expression of Plxna4 mRNA in different immune subpopulations was normalized to Actb mRNA level. T cells, B cells, NK cells, BM pDCs, and splenic mDCs were isolated by FACS sorting. BMMs and BMDCs were, respectively, cultured from BM cells in the presence of L929 cell supernatant and GM-CSF plus IL-4. Macrophages were isolated by adherence for 2 h at 37°C. Results are expressed as mean ± SD. (B) Immunofluorescence staining of plexin-A4 and F4/80 in WT and Plxna4 −/− peritoneal macrophages. (C) WT and Plxna4 −/− peritoneal macrophages were stained with IgG control (gray fill) or antibody against mouse plexin-A4 (black lines). Histograms show gated CD11b + cells. The data shown in A–C are representative of two independent experiments. (D and E) Peritoneal macrophages were left unstimulated (−) or were stimulated with 5 µg/ml <t>Pam3Cys,</t> 10 µg/ml poly(I:C), 1 µg/ml ultrapure <t>LPS,</t> 10 µg/ml R837, or 4 µg/ml CpG-B for 4 h. TNF and IL-6 mRNA (D) and protein (E) were measured by RT-PCR and ELISA, respectively. The results shown in D and E are representative of five independent experiments and are expressed as mean ± SD. *, P
    Fetal Bovine Serum Fbs, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 17568 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effects of poly(I:C), LPS, R-837 and iE-DAP on the HASMC viability. ( A–C ) HASMC were cultured for 24, 48 and 72 h in the absence of presence of Pam 3 CSK 4 (1 µg/ml), poly(I:C) (10 µg/ml), LPS (100 ng/ml), R-837 (5 µg/ml) and iE-DAP (10 µg/ml). Proliferation was quantified with AlamarBlue and determined using a spectrophotometer at 570 and 620 nm. Data are depicted as percent reduction compared to untreated control and presented as mean ± SEM (n = 7–8). *, p

    Journal: PLoS ONE

    Article Title: Innate Immune Receptors in Human Airway Smooth Muscle Cells: Activation by TLR1/2, TLR3, TLR4, TLR7 and NOD1 Agonists

    doi: 10.1371/journal.pone.0068701

    Figure Lengend Snippet: Effects of poly(I:C), LPS, R-837 and iE-DAP on the HASMC viability. ( A–C ) HASMC were cultured for 24, 48 and 72 h in the absence of presence of Pam 3 CSK 4 (1 µg/ml), poly(I:C) (10 µg/ml), LPS (100 ng/ml), R-837 (5 µg/ml) and iE-DAP (10 µg/ml). Proliferation was quantified with AlamarBlue and determined using a spectrophotometer at 570 and 620 nm. Data are depicted as percent reduction compared to untreated control and presented as mean ± SEM (n = 7–8). *, p

    Article Snippet: Reagents The following PRR ligands were used: Pam3 CSK4 , FSL-1, poly(I:C), flagellin, R-837, R-848, iE-DAP, MDP and poly(I:C)/LyoVec from Invivogen (San Diego, CA, USA), LPS (E. coli ) from Sigma-Aldrich (St. Louis, MO, USA) and phosphorothioate-modified CpG oligodeoxynucleotide 2006 (CpG), 5′-tcgtcgttttgtcgttttgtcgtt-3′, from DNA Technology A/S (Aarhus, Denmark) (all with endotoxins levels < 0.125 EU/ml).

    Techniques: Cell Culture, Spectrophotometry

    TLR1/2, TLR3, TLR4, TLR7 and NOD1 stimulation induces cell surface marker expression. HASMCs were cultured for 24 h in the absence of presence of Pam 3 CSK 4 (1 µg/ml), FSL-1 (100 ng/ml), poly(I:C) (10 µg/ml), LPS (100 ng/ml), flagellin (1 µg/ml), R-837 (5 µg/ml), R-848 (5 µg/ml), CpG (1 µM), iE-DAP (10 µg/ml), MDP (10 µg/ml) and poly(I:C)/LyoVec (1 µg/ml). Thereafter, the cells were stained with antibodies against ( A ) ICAM-1 and ( B ) HLA-DR and analyzed by FACS. Data are presented as mean fluorescence intensity (MFI) and presented as mean ± SEM (n = 6). *, p

    Journal: PLoS ONE

    Article Title: Innate Immune Receptors in Human Airway Smooth Muscle Cells: Activation by TLR1/2, TLR3, TLR4, TLR7 and NOD1 Agonists

    doi: 10.1371/journal.pone.0068701

    Figure Lengend Snippet: TLR1/2, TLR3, TLR4, TLR7 and NOD1 stimulation induces cell surface marker expression. HASMCs were cultured for 24 h in the absence of presence of Pam 3 CSK 4 (1 µg/ml), FSL-1 (100 ng/ml), poly(I:C) (10 µg/ml), LPS (100 ng/ml), flagellin (1 µg/ml), R-837 (5 µg/ml), R-848 (5 µg/ml), CpG (1 µM), iE-DAP (10 µg/ml), MDP (10 µg/ml) and poly(I:C)/LyoVec (1 µg/ml). Thereafter, the cells were stained with antibodies against ( A ) ICAM-1 and ( B ) HLA-DR and analyzed by FACS. Data are presented as mean fluorescence intensity (MFI) and presented as mean ± SEM (n = 6). *, p

    Article Snippet: Reagents The following PRR ligands were used: Pam3 CSK4 , FSL-1, poly(I:C), flagellin, R-837, R-848, iE-DAP, MDP and poly(I:C)/LyoVec from Invivogen (San Diego, CA, USA), LPS (E. coli ) from Sigma-Aldrich (St. Louis, MO, USA) and phosphorothioate-modified CpG oligodeoxynucleotide 2006 (CpG), 5′-tcgtcgttttgtcgttttgtcgtt-3′, from DNA Technology A/S (Aarhus, Denmark) (all with endotoxins levels < 0.125 EU/ml).

    Techniques: Marker, Expressing, Cell Culture, Staining, FACS, Fluorescence

    TLR1/2, TLR3, TLR4, TLR7 and NOD1 stimulation promotes cytokine and chemokine release. HASMCs were cultured for 24 h in the absence of presence of Pam 3 CSK 4 (TLR1/2; 1 µg/ml), FSL-1 (TLR2/6; 100 ng/ml), poly(I:C) (TLR3; 10 µg/ml), LPS (TLR4; 100 ng/ml), flagellin (TLR5; 1 µg/ml), R-837 (TLR7; 5 µg/ml), R-848 (TLR7/8; 5 µg/ml), CpG (TLR9; 1 µM), iE-DAP (NOD1; 10 µg/ml), MDP (NOD2; 10 µg/ml) and poly(I:C)/LyoVec (RIG-I/MDA-5; 1 µg/ml). Thereafter, the cell-free culture supernatants were recovered and analyzed for levels of ( A ) IL-6 (n = 14), ( B ) IL-8 (n = 14), ( C ) GM-CSF (n = 14), ( D ) eotaxin (n = 7), ( E ) RANTES (n = 7), and ( F ) TGF-β1 (n = 6) using ELISA. Data are presented as mean ± SEM. *, p

    Journal: PLoS ONE

    Article Title: Innate Immune Receptors in Human Airway Smooth Muscle Cells: Activation by TLR1/2, TLR3, TLR4, TLR7 and NOD1 Agonists

    doi: 10.1371/journal.pone.0068701

    Figure Lengend Snippet: TLR1/2, TLR3, TLR4, TLR7 and NOD1 stimulation promotes cytokine and chemokine release. HASMCs were cultured for 24 h in the absence of presence of Pam 3 CSK 4 (TLR1/2; 1 µg/ml), FSL-1 (TLR2/6; 100 ng/ml), poly(I:C) (TLR3; 10 µg/ml), LPS (TLR4; 100 ng/ml), flagellin (TLR5; 1 µg/ml), R-837 (TLR7; 5 µg/ml), R-848 (TLR7/8; 5 µg/ml), CpG (TLR9; 1 µM), iE-DAP (NOD1; 10 µg/ml), MDP (NOD2; 10 µg/ml) and poly(I:C)/LyoVec (RIG-I/MDA-5; 1 µg/ml). Thereafter, the cell-free culture supernatants were recovered and analyzed for levels of ( A ) IL-6 (n = 14), ( B ) IL-8 (n = 14), ( C ) GM-CSF (n = 14), ( D ) eotaxin (n = 7), ( E ) RANTES (n = 7), and ( F ) TGF-β1 (n = 6) using ELISA. Data are presented as mean ± SEM. *, p

    Article Snippet: Reagents The following PRR ligands were used: Pam3 CSK4 , FSL-1, poly(I:C), flagellin, R-837, R-848, iE-DAP, MDP and poly(I:C)/LyoVec from Invivogen (San Diego, CA, USA), LPS (E. coli ) from Sigma-Aldrich (St. Louis, MO, USA) and phosphorothioate-modified CpG oligodeoxynucleotide 2006 (CpG), 5′-tcgtcgttttgtcgttttgtcgtt-3′, from DNA Technology A/S (Aarhus, Denmark) (all with endotoxins levels < 0.125 EU/ml).

    Techniques: Cell Culture, Multiple Displacement Amplification, Enzyme-linked Immunosorbent Assay

    Dose-dependent HASMC activation by Pam 3 CSK 4 , poly(I:C), LPS, R-837 and iE-DAP. Cells were cultured for 24 h with various concentrations of Pam 3 CSK 4 , poly(I:C), LPS, R-837, and iE-DAP followed by measurements of IL-6 ( A ) and IL-8 ( B ) in the cell-free culture supernatants by use of ELISA. All concentration in µg/ml. Data are presented as mean ± SEM (n = 6). *, p

    Journal: PLoS ONE

    Article Title: Innate Immune Receptors in Human Airway Smooth Muscle Cells: Activation by TLR1/2, TLR3, TLR4, TLR7 and NOD1 Agonists

    doi: 10.1371/journal.pone.0068701

    Figure Lengend Snippet: Dose-dependent HASMC activation by Pam 3 CSK 4 , poly(I:C), LPS, R-837 and iE-DAP. Cells were cultured for 24 h with various concentrations of Pam 3 CSK 4 , poly(I:C), LPS, R-837, and iE-DAP followed by measurements of IL-6 ( A ) and IL-8 ( B ) in the cell-free culture supernatants by use of ELISA. All concentration in µg/ml. Data are presented as mean ± SEM (n = 6). *, p

    Article Snippet: Reagents The following PRR ligands were used: Pam3 CSK4 , FSL-1, poly(I:C), flagellin, R-837, R-848, iE-DAP, MDP and poly(I:C)/LyoVec from Invivogen (San Diego, CA, USA), LPS (E. coli ) from Sigma-Aldrich (St. Louis, MO, USA) and phosphorothioate-modified CpG oligodeoxynucleotide 2006 (CpG), 5′-tcgtcgttttgtcgttttgtcgtt-3′, from DNA Technology A/S (Aarhus, Denmark) (all with endotoxins levels < 0.125 EU/ml).

    Techniques: Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay

    PRR activation affects the contractile state. HASMCs were cultured for 24 h in the absence or presence of Pam 3 CSK 4 (1 µg/ml), poly(I:C) (10 µg/ml), LPS (100 ng/ml), R-837 (5 µg/ml) and iE-DAP (10 µg/ml). ( A ) The cells were harvested and analyzed for levels of myosin light chain kinase (MLCK) mRNA using real-time RT-PCR. Data are given in relation to GAPDH as 2 −ΔCt ×10 5 . ( B ) Mean fluorescence intensity (MFI) of the cysteinyl leukotriene 1 receptor (CysLT1R) and ( C ) β 2 -adrenergic receptor (β 2 AR) expression using flow cytometry. Data are presented as mean ± SEM (n = 7). *, p

    Journal: PLoS ONE

    Article Title: Innate Immune Receptors in Human Airway Smooth Muscle Cells: Activation by TLR1/2, TLR3, TLR4, TLR7 and NOD1 Agonists

    doi: 10.1371/journal.pone.0068701

    Figure Lengend Snippet: PRR activation affects the contractile state. HASMCs were cultured for 24 h in the absence or presence of Pam 3 CSK 4 (1 µg/ml), poly(I:C) (10 µg/ml), LPS (100 ng/ml), R-837 (5 µg/ml) and iE-DAP (10 µg/ml). ( A ) The cells were harvested and analyzed for levels of myosin light chain kinase (MLCK) mRNA using real-time RT-PCR. Data are given in relation to GAPDH as 2 −ΔCt ×10 5 . ( B ) Mean fluorescence intensity (MFI) of the cysteinyl leukotriene 1 receptor (CysLT1R) and ( C ) β 2 -adrenergic receptor (β 2 AR) expression using flow cytometry. Data are presented as mean ± SEM (n = 7). *, p

    Article Snippet: Reagents The following PRR ligands were used: Pam3 CSK4 , FSL-1, poly(I:C), flagellin, R-837, R-848, iE-DAP, MDP and poly(I:C)/LyoVec from Invivogen (San Diego, CA, USA), LPS (E. coli ) from Sigma-Aldrich (St. Louis, MO, USA) and phosphorothioate-modified CpG oligodeoxynucleotide 2006 (CpG), 5′-tcgtcgttttgtcgttttgtcgtt-3′, from DNA Technology A/S (Aarhus, Denmark) (all with endotoxins levels < 0.125 EU/ml).

    Techniques: Activation Assay, Cell Culture, Quantitative RT-PCR, Fluorescence, Expressing, Flow Cytometry, Cytometry

    pDCs have a transient decrease in the capacity to produce IFNα in vitro. ( A ) Total PBMCs were stimulated with imiquimod for 6 hours, and IFNα production by pDCs was measured by flow cytometry. ( B ) The percentage of pDCs that produced IFNα in response to imiquimod (IQ) stimulation is shown (red). pDC frequency (blue) and HIV-1 VL (black) are included for reference. The percentage of pDCs producing IFNα ( C ) or TNF-α ( D ) in response to imiquimod were compared between the time point before viremia at which the highest pDC frequency occurred and the time point immediately prior. * P

    Journal: The Journal of Clinical Investigation

    Article Title: Plasmacytoid dendritic cells sense HIV replication before detectable viremia following treatment interruption

    doi: 10.1172/JCI130597

    Figure Lengend Snippet: pDCs have a transient decrease in the capacity to produce IFNα in vitro. ( A ) Total PBMCs were stimulated with imiquimod for 6 hours, and IFNα production by pDCs was measured by flow cytometry. ( B ) The percentage of pDCs that produced IFNα in response to imiquimod (IQ) stimulation is shown (red). pDC frequency (blue) and HIV-1 VL (black) are included for reference. The percentage of pDCs producing IFNα ( C ) or TNF-α ( D ) in response to imiquimod were compared between the time point before viremia at which the highest pDC frequency occurred and the time point immediately prior. * P

    Article Snippet: Cells were stimulated with 2.5 μg/mL imiquimod (InvivoGen) or left in media alone for 6 hours, with GolgiPlug added for the last 4 hours of stimulation.

    Techniques: In Vitro, Flow Cytometry, Produced

    pDC signaling capacity negatively correlates with plasma IFNα2 levels. ( A ) p-IRF7 levels were measured in pDCs by flow cytometry after in vitro imiquimod stimulation of PBMCs obtained at the visit with the lowest IFNα response or from the only remaining sample available after ATI (ATI visit). Spearman’s correlations were determined between the MFI of p-IRF7 and the loss of IFNα-producing capacity, as measured by the fold decrease in pDCs producing IFNα in vitro at the ATI visit compared with levels detected at the preceding visit. ( B and C ) Plasma IFNα2 levels in RV397 participants were measured by SIMOA assay at the ATI visit. Spearman’s correlations were performed between the plasma IFNα2 levels at the ATI visit and the MFI of p-IRF7 in pDCs after in vitro imiquimod stimulation of PBMCs obtained at the ATI visit ( B ) or a pre-ATI visit ( C ). n = 8 participants from RV397 (all of whom received VRC01).

    Journal: The Journal of Clinical Investigation

    Article Title: Plasmacytoid dendritic cells sense HIV replication before detectable viremia following treatment interruption

    doi: 10.1172/JCI130597

    Figure Lengend Snippet: pDC signaling capacity negatively correlates with plasma IFNα2 levels. ( A ) p-IRF7 levels were measured in pDCs by flow cytometry after in vitro imiquimod stimulation of PBMCs obtained at the visit with the lowest IFNα response or from the only remaining sample available after ATI (ATI visit). Spearman’s correlations were determined between the MFI of p-IRF7 and the loss of IFNα-producing capacity, as measured by the fold decrease in pDCs producing IFNα in vitro at the ATI visit compared with levels detected at the preceding visit. ( B and C ) Plasma IFNα2 levels in RV397 participants were measured by SIMOA assay at the ATI visit. Spearman’s correlations were performed between the plasma IFNα2 levels at the ATI visit and the MFI of p-IRF7 in pDCs after in vitro imiquimod stimulation of PBMCs obtained at the ATI visit ( B ) or a pre-ATI visit ( C ). n = 8 participants from RV397 (all of whom received VRC01).

    Article Snippet: Cells were stimulated with 2.5 μg/mL imiquimod (InvivoGen) or left in media alone for 6 hours, with GolgiPlug added for the last 4 hours of stimulation.

    Techniques: Flow Cytometry, In Vitro

    TLR7 pathway is activated on stimulation with its specific ligand imiquimod, R837. Chief downstream regulators, especially NF-ĸB is activated and transclocated to the nucleus on TLR7 activation. a Host immune response on TLR7 activation, using TLR7 ligand. HepG2.2.15 cells were treated with TLR7 agonist, R837 (8μg/ml) for 3 days. mRNA expression of different downstream regulators (NF- КB, JNK, p38 and PI3K) involved in the TLR7 pathway. b Confocal imaging showing nuclear uptake of Nuclear Factor-КB p65 subunit (NF- КB) from cytoplasm in cells where HepG2.2.15 cells are taken as control and HepG2.2.15 cells stimulated with TLR7 ligand, R837 (8μg/ml) for 72 h. c Nuclear Factor-КB p65 subunit (NF-КB) protein expression of HepG2 and HepG2.2.15 cells. d Nuclear Factor-КB p65 subunit (NF- КB) protein expression of HepG2.2.15 cells taken as control and treated with R837 (8μg/ml) for 72 h

    Journal: BMC Infectious Diseases

    Article Title: Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line

    doi: 10.1186/s12879-017-2189-z

    Figure Lengend Snippet: TLR7 pathway is activated on stimulation with its specific ligand imiquimod, R837. Chief downstream regulators, especially NF-ĸB is activated and transclocated to the nucleus on TLR7 activation. a Host immune response on TLR7 activation, using TLR7 ligand. HepG2.2.15 cells were treated with TLR7 agonist, R837 (8μg/ml) for 3 days. mRNA expression of different downstream regulators (NF- КB, JNK, p38 and PI3K) involved in the TLR7 pathway. b Confocal imaging showing nuclear uptake of Nuclear Factor-КB p65 subunit (NF- КB) from cytoplasm in cells where HepG2.2.15 cells are taken as control and HepG2.2.15 cells stimulated with TLR7 ligand, R837 (8μg/ml) for 72 h. c Nuclear Factor-КB p65 subunit (NF-КB) protein expression of HepG2 and HepG2.2.15 cells. d Nuclear Factor-КB p65 subunit (NF- КB) protein expression of HepG2.2.15 cells taken as control and treated with R837 (8μg/ml) for 72 h

    Article Snippet: Analysis of HBV viral properties The synthetic ligand used for TLR7 was Imiquimod-R837 provided by Invivogen.

    Techniques: Activation Assay, Expressing, Imaging

    Enhanced type I interferon responses in female Cd200 −/− mice. A IFN-α concentrations in the sera of male and female WT (open bars) and Cd200 −/− (gray bars) mice were measured by ELISA. Naïve mice (n = 4) and mice at 4 days post-infection with coronavirus (MHV-EFLM) (n = 7) were used. B Male and female WT (open bars) and Cd200 −/− (gray bars) mice were injected intraperitoneally with the TLR7 ligand imiquimod (50 µg/mouse). One-hour later sera were collected, and the concentrations of IFNα were measured by ELISA (n = 6). Dotted lines in (A,B) indicate the detection limit of the ELISA. C Male and female WT (open bars) and Cd200 −/− (gray bars) mice were injected intraperitoneally with the TLR7 ligand imiquimod (50 µg/mouse). Twenty-four hours later livers were collected and IFNβ expression was determined by qRT-PCR (n = 6). Mean ± SEM is shown. Statistical significance was calculated with Mann-Whitney test. ns = not significant. * = p

    Journal: PLoS Pathogens

    Article Title: CD200 Receptor Controls Sex-Specific TLR7 Responses to Viral Infection

    doi: 10.1371/journal.ppat.1002710

    Figure Lengend Snippet: Enhanced type I interferon responses in female Cd200 −/− mice. A IFN-α concentrations in the sera of male and female WT (open bars) and Cd200 −/− (gray bars) mice were measured by ELISA. Naïve mice (n = 4) and mice at 4 days post-infection with coronavirus (MHV-EFLM) (n = 7) were used. B Male and female WT (open bars) and Cd200 −/− (gray bars) mice were injected intraperitoneally with the TLR7 ligand imiquimod (50 µg/mouse). One-hour later sera were collected, and the concentrations of IFNα were measured by ELISA (n = 6). Dotted lines in (A,B) indicate the detection limit of the ELISA. C Male and female WT (open bars) and Cd200 −/− (gray bars) mice were injected intraperitoneally with the TLR7 ligand imiquimod (50 µg/mouse). Twenty-four hours later livers were collected and IFNβ expression was determined by qRT-PCR (n = 6). Mean ± SEM is shown. Statistical significance was calculated with Mann-Whitney test. ns = not significant. * = p

    Article Snippet: In additional experiments we injected the mice intraperitoneally with the TLR7 agonist imiquimod (Invivogen; 50 µg in 200 µl PBS).

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay, Infection, Injection, Expressing, Quantitative RT-PCR, MANN-WHITNEY

    Stimulation of CD200R directly inhibits TLR mediated NFκB activity. A HEK 293 T cells were transiently transfected with TLR7, NF-κB luciferase reporter and LAIR-1-CD200R chimera or signaling-defective LAIR-1-CD200R-FFF constructs. Twenty-four hours later cells were stimulated with control or anti-LAIR-1 antibody. Forty-eight hours after transfection cells were stimulated with the TLR7 ligand imiquimod (3.0 µg/ml). Seventy-two hours after transfection cells were harvested, luciferase activity, and total protein content were determined. Protein normalized, luciferase activity from 3 independent experiments is shown. B HEK 293 T cells with stable expression of TLR7 were transiently transfected with a NF-κB luciferase reporter construct or an IFNβ luciferase reporter construct and a LAIR-1-CD200R chimera. TLR7 stimulation and CD200R crosslinking was performed as in (A). Protein normalized with luciferase activity from 3 independent experiments is shown. Mean ± SEM is shown. Statistical significance was calculated with Mann-Whitney test. ns = not significant. * = p

    Journal: PLoS Pathogens

    Article Title: CD200 Receptor Controls Sex-Specific TLR7 Responses to Viral Infection

    doi: 10.1371/journal.ppat.1002710

    Figure Lengend Snippet: Stimulation of CD200R directly inhibits TLR mediated NFκB activity. A HEK 293 T cells were transiently transfected with TLR7, NF-κB luciferase reporter and LAIR-1-CD200R chimera or signaling-defective LAIR-1-CD200R-FFF constructs. Twenty-four hours later cells were stimulated with control or anti-LAIR-1 antibody. Forty-eight hours after transfection cells were stimulated with the TLR7 ligand imiquimod (3.0 µg/ml). Seventy-two hours after transfection cells were harvested, luciferase activity, and total protein content were determined. Protein normalized, luciferase activity from 3 independent experiments is shown. B HEK 293 T cells with stable expression of TLR7 were transiently transfected with a NF-κB luciferase reporter construct or an IFNβ luciferase reporter construct and a LAIR-1-CD200R chimera. TLR7 stimulation and CD200R crosslinking was performed as in (A). Protein normalized with luciferase activity from 3 independent experiments is shown. Mean ± SEM is shown. Statistical significance was calculated with Mann-Whitney test. ns = not significant. * = p

    Article Snippet: In additional experiments we injected the mice intraperitoneally with the TLR7 agonist imiquimod (Invivogen; 50 µg in 200 µl PBS).

    Techniques: Activity Assay, Transfection, Luciferase, Field Flow Fractionation, Construct, Expressing, MANN-WHITNEY

    Inhibitory effect of SARS-CoV PLPro on TLR7 agonist-induced activation of type I IFN signaling. ISRE-driven luciferase reporter activity and the mRNA levels of PKR and IRF7 were determined 4 h post-IMQ treatment. ISRE-driven firefly luciferase activity was normalized by Renilla luciferase activity ( A ). Relative mRNA levels of PKR ( B ) and IRF7 ( C ) were normalized by GAPDH mRNA, presented as a relative ratio. In addition, the activated status of STAT1 was examined using Western blot with anti-phospho-STAT1 (Tyr701) antibodies ( D ). * p -Value

    Journal: International Journal of Molecular Sciences

    Article Title: SARS Coronavirus Papain-Like Protease Inhibits the TLR7 Signaling Pathway through Removing Lys63-Linked Polyubiquitination of TRAF3 and TRAF6

    doi: 10.3390/ijms17050678

    Figure Lengend Snippet: Inhibitory effect of SARS-CoV PLPro on TLR7 agonist-induced activation of type I IFN signaling. ISRE-driven luciferase reporter activity and the mRNA levels of PKR and IRF7 were determined 4 h post-IMQ treatment. ISRE-driven firefly luciferase activity was normalized by Renilla luciferase activity ( A ). Relative mRNA levels of PKR ( B ) and IRF7 ( C ) were normalized by GAPDH mRNA, presented as a relative ratio. In addition, the activated status of STAT1 was examined using Western blot with anti-phospho-STAT1 (Tyr701) antibodies ( D ). * p -Value

    Article Snippet: To monitor the activation of the IMQ-induced TLR7 signaling pathway, PLPro-expressing and vector control cells were treated with 10 μM IMQ (InvivoGen, San Diego, CA, USA) and harvested 0, 30 and 60 min post-treatment.

    Techniques: Activation Assay, Luciferase, Activity Assay, Western Blot

    Detection of IMQ-induced AP-1-mediated production of IL-6 and IL-8 in the vector control and PLPro-expressing cells. AP-1-driven firefly luciferase activity was normalized by Renilla luciferase activity ( A ). Relative mRNA levels of IL-6 ( B ) and IL-8 ( C ) were normalized by GAPDH mRNA, presented as a relative ratio. In addition, the activated status of p38 MAPK and AP-1 was examined using Western blot with anti-phospho-p38 MAPK and anti-phospho-c-Jun antibodies ( D ). * p -Value

    Journal: International Journal of Molecular Sciences

    Article Title: SARS Coronavirus Papain-Like Protease Inhibits the TLR7 Signaling Pathway through Removing Lys63-Linked Polyubiquitination of TRAF3 and TRAF6

    doi: 10.3390/ijms17050678

    Figure Lengend Snippet: Detection of IMQ-induced AP-1-mediated production of IL-6 and IL-8 in the vector control and PLPro-expressing cells. AP-1-driven firefly luciferase activity was normalized by Renilla luciferase activity ( A ). Relative mRNA levels of IL-6 ( B ) and IL-8 ( C ) were normalized by GAPDH mRNA, presented as a relative ratio. In addition, the activated status of p38 MAPK and AP-1 was examined using Western blot with anti-phospho-p38 MAPK and anti-phospho-c-Jun antibodies ( D ). * p -Value

    Article Snippet: To monitor the activation of the IMQ-induced TLR7 signaling pathway, PLPro-expressing and vector control cells were treated with 10 μM IMQ (InvivoGen, San Diego, CA, USA) and harvested 0, 30 and 60 min post-treatment.

    Techniques: Plasmid Preparation, Expressing, Luciferase, Activity Assay, Western Blot

    Detecting the Lys48- and Lys63-linked ubiquitination of TRAF3 and TRAF6 measured by the immunoprecipitation assay. Vector control and PLPro-expressing cells were treated with(out) IMQ for 1 day; cell lysates were immunoprecipitated with anti-TRAF3 ( A ) or anti-TRAF6 ( B ) followed by Western blotting probed with either anti-Lys48 ubiquitin or anti-Lys63 ubiquitin antibodies. Phospho-TBK1 levels were detected by Western blot ( C ). The relative band intensity of phospho-TBK1 was normalized by TBK1, compared to the mock vector control cells, and quantified using ImageJ based on triplicate replicates of each experiment ( D ).

    Journal: International Journal of Molecular Sciences

    Article Title: SARS Coronavirus Papain-Like Protease Inhibits the TLR7 Signaling Pathway through Removing Lys63-Linked Polyubiquitination of TRAF3 and TRAF6

    doi: 10.3390/ijms17050678

    Figure Lengend Snippet: Detecting the Lys48- and Lys63-linked ubiquitination of TRAF3 and TRAF6 measured by the immunoprecipitation assay. Vector control and PLPro-expressing cells were treated with(out) IMQ for 1 day; cell lysates were immunoprecipitated with anti-TRAF3 ( A ) or anti-TRAF6 ( B ) followed by Western blotting probed with either anti-Lys48 ubiquitin or anti-Lys63 ubiquitin antibodies. Phospho-TBK1 levels were detected by Western blot ( C ). The relative band intensity of phospho-TBK1 was normalized by TBK1, compared to the mock vector control cells, and quantified using ImageJ based on triplicate replicates of each experiment ( D ).

    Article Snippet: To monitor the activation of the IMQ-induced TLR7 signaling pathway, PLPro-expressing and vector control cells were treated with 10 μM IMQ (InvivoGen, San Diego, CA, USA) and harvested 0, 30 and 60 min post-treatment.

    Techniques: Immunoprecipitation, Plasmid Preparation, Expressing, Western Blot

    Inhibition of IMQ-induced TNF-α production via NF-κB signaling by SARS-CoV PLPro. Both types of cells were treated with(out) IMQ for 4 h, and then, NF-κB-driven luciferase reporter activity and the TNF-α mRNA level were determined using the dual luciferase reporter assay ( A ) and quantitative PCR ( B ), respectively. For determining NF-κB activation, the lysates were also analyzed using Western blot with anti-phospho-NF-κB p65 antibodies ( C ). ** p -Value

    Journal: International Journal of Molecular Sciences

    Article Title: SARS Coronavirus Papain-Like Protease Inhibits the TLR7 Signaling Pathway through Removing Lys63-Linked Polyubiquitination of TRAF3 and TRAF6

    doi: 10.3390/ijms17050678

    Figure Lengend Snippet: Inhibition of IMQ-induced TNF-α production via NF-κB signaling by SARS-CoV PLPro. Both types of cells were treated with(out) IMQ for 4 h, and then, NF-κB-driven luciferase reporter activity and the TNF-α mRNA level were determined using the dual luciferase reporter assay ( A ) and quantitative PCR ( B ), respectively. For determining NF-κB activation, the lysates were also analyzed using Western blot with anti-phospho-NF-κB p65 antibodies ( C ). ** p -Value

    Article Snippet: To monitor the activation of the IMQ-induced TLR7 signaling pathway, PLPro-expressing and vector control cells were treated with 10 μM IMQ (InvivoGen, San Diego, CA, USA) and harvested 0, 30 and 60 min post-treatment.

    Techniques: Inhibition, Luciferase, Activity Assay, Reporter Assay, Real-time Polymerase Chain Reaction, Activation Assay, Western Blot

    M45 inhibits IκBα degradation, TNFα and IL-6 production during MCMV infection. (A) NIH-3T3 cells were infected with wt MCMV-GFP, an M45 deletion mutant (ΔM45) or a revertant virus (RM45) at an MOI of 10 and treated 5 h postinfection with the TLR2 agonist Pam 3 CSK 4 (Pam., 0.1 µg/ml), the TLR4 agonist LPS (10 µg/ml), or IL-1β (20 ng/ml) for the indicated times. Levels of the indicated proteins were analyzed by immunoblotting. (B) BMDMs were infected with wt MCMV-GFP, ΔM45, or RM45 at an MOI of 3 and stimulated 8 h postinfection for 16 h with the TLR7 agonist R848 (0.1 µM). TNFα and IL-6 levels in the supernatant were determined by ELISA (mean ± SD). (C) RAW264.7 macrophages were infected with wt MCMV-GFP or ΔM45 at an MOI of 0.1, stimulated 24 h postinfection for 4 hours with TLR agonists Pam 3 CSK 4 (Pam.) or Malp-2 (TLR2), LPS (TLR4), R837 (TLR7), or CpG (TLR9), in the presence of brefeldin A. Cells were fixed, permeabilized, and stained with a TNFα-specific antibody. The percentages of TNFα-positive cells within infected (GFP-positive) cell populations were determined by FACS analysis (mean ± SD).

    Journal: PLoS Pathogens

    Article Title: Viral Mediated Redirection of NEMO/IKK? to Autophagosomes Curtails the Inflammatory Cascade

    doi: 10.1371/journal.ppat.1002517

    Figure Lengend Snippet: M45 inhibits IκBα degradation, TNFα and IL-6 production during MCMV infection. (A) NIH-3T3 cells were infected with wt MCMV-GFP, an M45 deletion mutant (ΔM45) or a revertant virus (RM45) at an MOI of 10 and treated 5 h postinfection with the TLR2 agonist Pam 3 CSK 4 (Pam., 0.1 µg/ml), the TLR4 agonist LPS (10 µg/ml), or IL-1β (20 ng/ml) for the indicated times. Levels of the indicated proteins were analyzed by immunoblotting. (B) BMDMs were infected with wt MCMV-GFP, ΔM45, or RM45 at an MOI of 3 and stimulated 8 h postinfection for 16 h with the TLR7 agonist R848 (0.1 µM). TNFα and IL-6 levels in the supernatant were determined by ELISA (mean ± SD). (C) RAW264.7 macrophages were infected with wt MCMV-GFP or ΔM45 at an MOI of 0.1, stimulated 24 h postinfection for 4 hours with TLR agonists Pam 3 CSK 4 (Pam.) or Malp-2 (TLR2), LPS (TLR4), R837 (TLR7), or CpG (TLR9), in the presence of brefeldin A. Cells were fixed, permeabilized, and stained with a TNFα-specific antibody. The percentages of TNFα-positive cells within infected (GFP-positive) cell populations were determined by FACS analysis (mean ± SD).

    Article Snippet: The following receptor ligands were used: LPS, LTA-SA, CpG (ODN 1668 or ODN 1826), R837 (Invivogen), R848 (Enzo LifeSciences), IL-1β, Pam3 CSK4 (Imgenex), Malp-2 (Alexis Biochemicals), TNFα (Promokine).

    Techniques: Infection, Mutagenesis, Enzyme-linked Immunosorbent Assay, Staining, FACS

    NF-κB signaling blocks NLRC4/caspase-8-dependent apoptosis pathway. Casp1 −/− Casp11 −/− BMDMs were pre-stimulated for with or without various stimuli – Pam3CSK4 (1 μg ml −1 ), LPS (1 μg ml −1 ), R837 (2 μg ml −1 ), TNFα (100 ng ml −1 ), IFN-α (100 U ml −1 ), or IFN-β (100 U ml −1 ), then electroporated with flagellin (0.5 μg ml −1 ) or stimulated with FasL (100 ng ml −1 ). ( a ) LDH release after 16 h. Data is represented as mean ± SD; n = 3. ( b ) % YOYO-1 positive BMDMs from live cell imaging taken every 45 min for 16 h.

    Journal: Scientific Reports

    Article Title: ASC- and caspase-8-dependent apoptotic pathway diverges from the NLRC4 inflammasome in macrophages

    doi: 10.1038/s41598-018-21998-3

    Figure Lengend Snippet: NF-κB signaling blocks NLRC4/caspase-8-dependent apoptosis pathway. Casp1 −/− Casp11 −/− BMDMs were pre-stimulated for with or without various stimuli – Pam3CSK4 (1 μg ml −1 ), LPS (1 μg ml −1 ), R837 (2 μg ml −1 ), TNFα (100 ng ml −1 ), IFN-α (100 U ml −1 ), or IFN-β (100 U ml −1 ), then electroporated with flagellin (0.5 μg ml −1 ) or stimulated with FasL (100 ng ml −1 ). ( a ) LDH release after 16 h. Data is represented as mean ± SD; n = 3. ( b ) % YOYO-1 positive BMDMs from live cell imaging taken every 45 min for 16 h.

    Article Snippet: Ultra-pure flagellin ( Psuedomonas aeruginosa ), Pam3CSK4, R837 (Imiquimod), ultra-pure LPS ( E. coli O111:B4), Poly(dA:dT), Nigericin were purchased from Invivogen, Mega FasL from AdipoGen, IFN-α and IFN-β from PBL Assay Science, TNFα from Genentech, ATP and bovine cytochrome-c from Sigma, YOYO-1 dye from Thermofisher Scientific, Nuclear-ID DNA stain from Enzo Life Sciences.

    Techniques: Live Cell Imaging

    ASC and caspase-8 are required for caspase-1-independent NLRC4 activated cell death in BMDMs. ( a – d ) BMDMs with or without Pam3CSK4 (1 μg ml −1 ) pre-stimulation were electroporated with flagellin (0.5 μg ml −1 ) or cytochrome-c (50 μg ml −1 ). ( a ) and ( c ) LDH release after 16 h. Data is represented as mean ± SD; n = 3. ( b ) and ( d ) % YOYO-1 positive BMDMs from two mice/genotype after flagellin electroporation. Live cell images taken every 30 min up to 16 h. ( e ) Immunoblot of caspase-8, caspase-1, caspase-3 and GSDMD in combined cell extract (ext) and supernatant (sup) from BMDMs 3 hrs after flagellin electroporation (no pre-stimulation). Pro-forms (pro) and cleaved forms are represented in blots. ( f ) BMDCs and thioglycollate-elicited peritoneal macrophages with no pre-stimulation were electroporated with flagellin (0.5 μg ml −1 ), cytochrome-c (50 μg ml −1 ), or FasL (100 ng ml −1 ) and measured for LDH release after 16 h. Data is represented as mean ± SD; n = 3.

    Journal: Scientific Reports

    Article Title: ASC- and caspase-8-dependent apoptotic pathway diverges from the NLRC4 inflammasome in macrophages

    doi: 10.1038/s41598-018-21998-3

    Figure Lengend Snippet: ASC and caspase-8 are required for caspase-1-independent NLRC4 activated cell death in BMDMs. ( a – d ) BMDMs with or without Pam3CSK4 (1 μg ml −1 ) pre-stimulation were electroporated with flagellin (0.5 μg ml −1 ) or cytochrome-c (50 μg ml −1 ). ( a ) and ( c ) LDH release after 16 h. Data is represented as mean ± SD; n = 3. ( b ) and ( d ) % YOYO-1 positive BMDMs from two mice/genotype after flagellin electroporation. Live cell images taken every 30 min up to 16 h. ( e ) Immunoblot of caspase-8, caspase-1, caspase-3 and GSDMD in combined cell extract (ext) and supernatant (sup) from BMDMs 3 hrs after flagellin electroporation (no pre-stimulation). Pro-forms (pro) and cleaved forms are represented in blots. ( f ) BMDCs and thioglycollate-elicited peritoneal macrophages with no pre-stimulation were electroporated with flagellin (0.5 μg ml −1 ), cytochrome-c (50 μg ml −1 ), or FasL (100 ng ml −1 ) and measured for LDH release after 16 h. Data is represented as mean ± SD; n = 3.

    Article Snippet: Ultra-pure flagellin ( Psuedomonas aeruginosa ), Pam3CSK4, R837 (Imiquimod), ultra-pure LPS ( E. coli O111:B4), Poly(dA:dT), Nigericin were purchased from Invivogen, Mega FasL from AdipoGen, IFN-α and IFN-β from PBL Assay Science, TNFα from Genentech, ATP and bovine cytochrome-c from Sigma, YOYO-1 dye from Thermofisher Scientific, Nuclear-ID DNA stain from Enzo Life Sciences.

    Techniques: Mouse Assay, Electroporation

    Caspase-1 does not compete with ASC for interaction with NLRC4 in enzymatically inactive Casp1 C284A/C284A BMDMs. ( a ) Immunoblot of pro-caspase-1 and actin from wt, Casp1 −/− , Casp1 C284A/C284A BMDMs. ( b ) BMDMs with Pam3CSK4 (1 μg ml −1 ) pre-stimulation were electroporated with flagellin (0.5 μg ml −1 ). IL-1β release and LDH release measured after 16 h. Data is expressed as mean ± SD; n = 3. ( c ) % YOYO-1 positive BMDMs from two mice/genotype after stimulation. BMDMs with no pre-stimulation were electroporated with flagellin (0.5 μg ml −1 ). Live cell images taken every 45 min for 16 h.

    Journal: Scientific Reports

    Article Title: ASC- and caspase-8-dependent apoptotic pathway diverges from the NLRC4 inflammasome in macrophages

    doi: 10.1038/s41598-018-21998-3

    Figure Lengend Snippet: Caspase-1 does not compete with ASC for interaction with NLRC4 in enzymatically inactive Casp1 C284A/C284A BMDMs. ( a ) Immunoblot of pro-caspase-1 and actin from wt, Casp1 −/− , Casp1 C284A/C284A BMDMs. ( b ) BMDMs with Pam3CSK4 (1 μg ml −1 ) pre-stimulation were electroporated with flagellin (0.5 μg ml −1 ). IL-1β release and LDH release measured after 16 h. Data is expressed as mean ± SD; n = 3. ( c ) % YOYO-1 positive BMDMs from two mice/genotype after stimulation. BMDMs with no pre-stimulation were electroporated with flagellin (0.5 μg ml −1 ). Live cell images taken every 45 min for 16 h.

    Article Snippet: Ultra-pure flagellin ( Psuedomonas aeruginosa ), Pam3CSK4, R837 (Imiquimod), ultra-pure LPS ( E. coli O111:B4), Poly(dA:dT), Nigericin were purchased from Invivogen, Mega FasL from AdipoGen, IFN-α and IFN-β from PBL Assay Science, TNFα from Genentech, ATP and bovine cytochrome-c from Sigma, YOYO-1 dye from Thermofisher Scientific, Nuclear-ID DNA stain from Enzo Life Sciences.

    Techniques: Mouse Assay

    NLRC4 activates a caspase-1-independent cell death pathway in absence of TLR signaling. ( a – c ) BMDMs with or without Pam3CSK4 (1 μg ml −1 ) pre-stimulation were electroporated with flagellin (0.5 μg ml −1 ). ( a ) LDH release measured after 16 h. Data is represented as mean ± SD; n = 3. ( b ) % YOYO-1 positive BMDMs from two mice/genotype after flagellin electroporation. Live cell images taken every 30 min up to 16 h. ( c ) Transmission electron microscopy BMDMs 6 h after flagellin electroporation. Representative images captured at 1000× magnification or 2000× magnification. Black arrows (→) indicate free nuclei. White asterisks (*) indicate chromatin condensation.

    Journal: Scientific Reports

    Article Title: ASC- and caspase-8-dependent apoptotic pathway diverges from the NLRC4 inflammasome in macrophages

    doi: 10.1038/s41598-018-21998-3

    Figure Lengend Snippet: NLRC4 activates a caspase-1-independent cell death pathway in absence of TLR signaling. ( a – c ) BMDMs with or without Pam3CSK4 (1 μg ml −1 ) pre-stimulation were electroporated with flagellin (0.5 μg ml −1 ). ( a ) LDH release measured after 16 h. Data is represented as mean ± SD; n = 3. ( b ) % YOYO-1 positive BMDMs from two mice/genotype after flagellin electroporation. Live cell images taken every 30 min up to 16 h. ( c ) Transmission electron microscopy BMDMs 6 h after flagellin electroporation. Representative images captured at 1000× magnification or 2000× magnification. Black arrows (→) indicate free nuclei. White asterisks (*) indicate chromatin condensation.

    Article Snippet: Ultra-pure flagellin ( Psuedomonas aeruginosa ), Pam3CSK4, R837 (Imiquimod), ultra-pure LPS ( E. coli O111:B4), Poly(dA:dT), Nigericin were purchased from Invivogen, Mega FasL from AdipoGen, IFN-α and IFN-β from PBL Assay Science, TNFα from Genentech, ATP and bovine cytochrome-c from Sigma, YOYO-1 dye from Thermofisher Scientific, Nuclear-ID DNA stain from Enzo Life Sciences.

    Techniques: Mouse Assay, Electroporation, Transmission Assay, Electron Microscopy

    IFNα downregulates the production of CCL23 by R848-treated neutrophils Neutrophils (5 × 10 6 /ml) were cultured with or without 1,000 U/ml IFNα and/or 5 μM R848. After 24 h, supernatants were collected and subjected to CCL23, CCL2, CCL3, and CCL4 measurement by ELISA. Data are depicted as mean ± SEM ( n = 3–19). Asterisks stand for significant increases: ** P

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Human Neutrophils Produce CCL23 in Response to Various TLR-Agonists and TNFα

    doi: 10.3389/fcimb.2017.00176

    Figure Lengend Snippet: IFNα downregulates the production of CCL23 by R848-treated neutrophils Neutrophils (5 × 10 6 /ml) were cultured with or without 1,000 U/ml IFNα and/or 5 μM R848. After 24 h, supernatants were collected and subjected to CCL23, CCL2, CCL3, and CCL4 measurement by ELISA. Data are depicted as mean ± SEM ( n = 3–19). Asterisks stand for significant increases: ** P

    Article Snippet: Cells were then suspended at 5 × 106 /ml in RPMI 1640 medium supplemented with 10% low endotoxin FBS ( < 0.5 EU/ml; from BioWhittaker-Lonza, Basel, Switzerland) and then plated either in 6/24-well tissue culture plates or in polystyrene flasks (from Greiner Bio-One, Kremsmünster, Austria) for culture at 37°C, 5% CO2 atmosphere, in the presence or the absence of: 0.2–50 μM R848, 5–50 μM R837, 1 μg/ml Pam3CSK4 (Invivogen, San Diego, CA, USA), 10 ng/ml TNFα (Peprotech, Rocky Hill, NJ, USA), 0.1–10 μg/ml ultrapure LPS from E. coli 0111:B4 strain (Alexis, Enzo Life Sciences, Farmingdale, NY, USA), 1,000 U/ml pegylated IFNα-2a (Pegasys, Roche, Basel, Switzerland), 100 U/ml IFNγ (R & D Systems, Minneapolis, MN, USA), 10 ng/ml GM-CSF (Miltenyi Biotec), 1,000 U/ml G-CSF (Myelostim, Italfarmaco Spa, Milano, Italy), 10–100 ng/ml IL-18 (MBL International, Nagoya, Japan), 10–100 ng/ml IL-33 (Peprotech), 10 nM fMLF (Sigma, Saint Louis, MO, USA), 500 μg/ml particulate β-glucan (InvivoGen), or 500 μg/ml curdlan (Sigma).

    Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

    Kinetics of CCL23, CCL2, CCL3, and CCL4 mRNA expression in neutrophils and autologousCD14 + -monocytes incubated with R848 . Neutrophils and autologous CD14 + -monocytes were cultured with or without 5 μM R848 for up to 24 h to evaluate their CCL23 (A) , CCL2 (B) , CCL3 (C) , and CCL4 (D) mRNA expression by RT-qPCR. Gene expression is depicted as mean normalized expression (MNE) units after GAPDH mRNA normalization (mean ± SEM, n = 5). Asterisks stand for significant increase: * P

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Human Neutrophils Produce CCL23 in Response to Various TLR-Agonists and TNFα

    doi: 10.3389/fcimb.2017.00176

    Figure Lengend Snippet: Kinetics of CCL23, CCL2, CCL3, and CCL4 mRNA expression in neutrophils and autologousCD14 + -monocytes incubated with R848 . Neutrophils and autologous CD14 + -monocytes were cultured with or without 5 μM R848 for up to 24 h to evaluate their CCL23 (A) , CCL2 (B) , CCL3 (C) , and CCL4 (D) mRNA expression by RT-qPCR. Gene expression is depicted as mean normalized expression (MNE) units after GAPDH mRNA normalization (mean ± SEM, n = 5). Asterisks stand for significant increase: * P

    Article Snippet: Cells were then suspended at 5 × 106 /ml in RPMI 1640 medium supplemented with 10% low endotoxin FBS ( < 0.5 EU/ml; from BioWhittaker-Lonza, Basel, Switzerland) and then plated either in 6/24-well tissue culture plates or in polystyrene flasks (from Greiner Bio-One, Kremsmünster, Austria) for culture at 37°C, 5% CO2 atmosphere, in the presence or the absence of: 0.2–50 μM R848, 5–50 μM R837, 1 μg/ml Pam3CSK4 (Invivogen, San Diego, CA, USA), 10 ng/ml TNFα (Peprotech, Rocky Hill, NJ, USA), 0.1–10 μg/ml ultrapure LPS from E. coli 0111:B4 strain (Alexis, Enzo Life Sciences, Farmingdale, NY, USA), 1,000 U/ml pegylated IFNα-2a (Pegasys, Roche, Basel, Switzerland), 100 U/ml IFNγ (R & D Systems, Minneapolis, MN, USA), 10 ng/ml GM-CSF (Miltenyi Biotec), 1,000 U/ml G-CSF (Myelostim, Italfarmaco Spa, Milano, Italy), 10–100 ng/ml IL-18 (MBL International, Nagoya, Japan), 10–100 ng/ml IL-33 (Peprotech), 10 nM fMLF (Sigma, Saint Louis, MO, USA), 500 μg/ml particulate β-glucan (InvivoGen), or 500 μg/ml curdlan (Sigma).

    Techniques: Expressing, Incubation, Cell Culture, Quantitative RT-PCR

    Production of CCL23 by neutrophils and monocytes incubated with R848 . Neutrophils and CD14 + -monocytes were cultured at 5 × 10 6 cells/ml with or without 5 μM R848 for up to 48 h to evaluate their capacity to produce and release CCL23, as detected by ELISA ( n = 4–19) (A) . In (B) , neutrophils were cultured with or without 5 μM R848 for 24 h and then further incubated for 4 h with or without 10 nM fMLF or 10 ng/ml TNFα to evaluate released and cell-associated CCL23 ( n = 4). Asterisks stand for significant increases: *** P

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Human Neutrophils Produce CCL23 in Response to Various TLR-Agonists and TNFα

    doi: 10.3389/fcimb.2017.00176

    Figure Lengend Snippet: Production of CCL23 by neutrophils and monocytes incubated with R848 . Neutrophils and CD14 + -monocytes were cultured at 5 × 10 6 cells/ml with or without 5 μM R848 for up to 48 h to evaluate their capacity to produce and release CCL23, as detected by ELISA ( n = 4–19) (A) . In (B) , neutrophils were cultured with or without 5 μM R848 for 24 h and then further incubated for 4 h with or without 10 nM fMLF or 10 ng/ml TNFα to evaluate released and cell-associated CCL23 ( n = 4). Asterisks stand for significant increases: *** P

    Article Snippet: Cells were then suspended at 5 × 106 /ml in RPMI 1640 medium supplemented with 10% low endotoxin FBS ( < 0.5 EU/ml; from BioWhittaker-Lonza, Basel, Switzerland) and then plated either in 6/24-well tissue culture plates or in polystyrene flasks (from Greiner Bio-One, Kremsmünster, Austria) for culture at 37°C, 5% CO2 atmosphere, in the presence or the absence of: 0.2–50 μM R848, 5–50 μM R837, 1 μg/ml Pam3CSK4 (Invivogen, San Diego, CA, USA), 10 ng/ml TNFα (Peprotech, Rocky Hill, NJ, USA), 0.1–10 μg/ml ultrapure LPS from E. coli 0111:B4 strain (Alexis, Enzo Life Sciences, Farmingdale, NY, USA), 1,000 U/ml pegylated IFNα-2a (Pegasys, Roche, Basel, Switzerland), 100 U/ml IFNγ (R & D Systems, Minneapolis, MN, USA), 10 ng/ml GM-CSF (Miltenyi Biotec), 1,000 U/ml G-CSF (Myelostim, Italfarmaco Spa, Milano, Italy), 10–100 ng/ml IL-18 (MBL International, Nagoya, Japan), 10–100 ng/ml IL-33 (Peprotech), 10 nM fMLF (Sigma, Saint Louis, MO, USA), 500 μg/ml particulate β-glucan (InvivoGen), or 500 μg/ml curdlan (Sigma).

    Techniques: Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay

    Other agonists, besides R848, trigger the production of CCL23 by human neutrophils . Neutrophils (5 × 10 6 /ml) were cultured with or without 5–50 μM R837, 5 μM R848, 5 μM CL075, 0.1–10 μg/ml LPS, 1 μg/ml Pam3CSK4, 10 ng/ml TNFα, 10 nM fMLF, 10 ng/ml G-CSF, 10 ng/ml GM-CSF, 500 μg/ml β-Glucan, 500 μg/ml Curdlan, 100 ng/ml IL-18, and 100 ng/ml IL-33 for 24 h. Then, extracellular supernatants were collected to evaluate CCL23 (A) and CXCL8 (B) production by ELISA (mean ± SEM, n = 3–5). Asterisks stand for significant increases as compared to untreated cells: * P

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Human Neutrophils Produce CCL23 in Response to Various TLR-Agonists and TNFα

    doi: 10.3389/fcimb.2017.00176

    Figure Lengend Snippet: Other agonists, besides R848, trigger the production of CCL23 by human neutrophils . Neutrophils (5 × 10 6 /ml) were cultured with or without 5–50 μM R837, 5 μM R848, 5 μM CL075, 0.1–10 μg/ml LPS, 1 μg/ml Pam3CSK4, 10 ng/ml TNFα, 10 nM fMLF, 10 ng/ml G-CSF, 10 ng/ml GM-CSF, 500 μg/ml β-Glucan, 500 μg/ml Curdlan, 100 ng/ml IL-18, and 100 ng/ml IL-33 for 24 h. Then, extracellular supernatants were collected to evaluate CCL23 (A) and CXCL8 (B) production by ELISA (mean ± SEM, n = 3–5). Asterisks stand for significant increases as compared to untreated cells: * P

    Article Snippet: Cells were then suspended at 5 × 106 /ml in RPMI 1640 medium supplemented with 10% low endotoxin FBS ( < 0.5 EU/ml; from BioWhittaker-Lonza, Basel, Switzerland) and then plated either in 6/24-well tissue culture plates or in polystyrene flasks (from Greiner Bio-One, Kremsmünster, Austria) for culture at 37°C, 5% CO2 atmosphere, in the presence or the absence of: 0.2–50 μM R848, 5–50 μM R837, 1 μg/ml Pam3CSK4 (Invivogen, San Diego, CA, USA), 10 ng/ml TNFα (Peprotech, Rocky Hill, NJ, USA), 0.1–10 μg/ml ultrapure LPS from E. coli 0111:B4 strain (Alexis, Enzo Life Sciences, Farmingdale, NY, USA), 1,000 U/ml pegylated IFNα-2a (Pegasys, Roche, Basel, Switzerland), 100 U/ml IFNγ (R & D Systems, Minneapolis, MN, USA), 10 ng/ml GM-CSF (Miltenyi Biotec), 1,000 U/ml G-CSF (Myelostim, Italfarmaco Spa, Milano, Italy), 10–100 ng/ml IL-18 (MBL International, Nagoya, Japan), 10–100 ng/ml IL-33 (Peprotech), 10 nM fMLF (Sigma, Saint Louis, MO, USA), 500 μg/ml particulate β-glucan (InvivoGen), or 500 μg/ml curdlan (Sigma).

    Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

    Expression of CCR1-binding chemokine mRNAs in human neutrophils incubated with R848 . Human neutrophils were incubated with or without 5 μM R848 for 24 h, and then total RNA was extracted and processed for RNA-seq experiments. Data are presented as FPKM (fragment per kilobase of exons per million fragments mapped) from two independent experiments (mean ± SEM).

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Human Neutrophils Produce CCL23 in Response to Various TLR-Agonists and TNFα

    doi: 10.3389/fcimb.2017.00176

    Figure Lengend Snippet: Expression of CCR1-binding chemokine mRNAs in human neutrophils incubated with R848 . Human neutrophils were incubated with or without 5 μM R848 for 24 h, and then total RNA was extracted and processed for RNA-seq experiments. Data are presented as FPKM (fragment per kilobase of exons per million fragments mapped) from two independent experiments (mean ± SEM).

    Article Snippet: Cells were then suspended at 5 × 106 /ml in RPMI 1640 medium supplemented with 10% low endotoxin FBS ( < 0.5 EU/ml; from BioWhittaker-Lonza, Basel, Switzerland) and then plated either in 6/24-well tissue culture plates or in polystyrene flasks (from Greiner Bio-One, Kremsmünster, Austria) for culture at 37°C, 5% CO2 atmosphere, in the presence or the absence of: 0.2–50 μM R848, 5–50 μM R837, 1 μg/ml Pam3CSK4 (Invivogen, San Diego, CA, USA), 10 ng/ml TNFα (Peprotech, Rocky Hill, NJ, USA), 0.1–10 μg/ml ultrapure LPS from E. coli 0111:B4 strain (Alexis, Enzo Life Sciences, Farmingdale, NY, USA), 1,000 U/ml pegylated IFNα-2a (Pegasys, Roche, Basel, Switzerland), 100 U/ml IFNγ (R & D Systems, Minneapolis, MN, USA), 10 ng/ml GM-CSF (Miltenyi Biotec), 1,000 U/ml G-CSF (Myelostim, Italfarmaco Spa, Milano, Italy), 10–100 ng/ml IL-18 (MBL International, Nagoya, Japan), 10–100 ng/ml IL-33 (Peprotech), 10 nM fMLF (Sigma, Saint Louis, MO, USA), 500 μg/ml particulate β-glucan (InvivoGen), or 500 μg/ml curdlan (Sigma).

    Techniques: Expressing, Binding Assay, Incubation, RNA Sequencing Assay

    Both IFN-α and IFN-γ have direct priming effect on purified pDCs. (A) Representative flow cytometry plots showing TLR7/9-mediated IFN-α production by purified pDCs after pre-treatment with IFN-α and IFN-γ. (B) TLR7/9-mediated IFN-α production by purified pDCs after pre-treatment with IFN-α and IFN-γ. Data are mean ± S.E.M. of 3 independent experiments.

    Journal: Frontiers in Immunology

    Article Title: Up-Regulation of TLR7-Mediated IFN-α Production by Plasmacytoid Dendritic Cells in Patients With Systemic Lupus Erythematosus

    doi: 10.3389/fimmu.2018.01957

    Figure Lengend Snippet: Both IFN-α and IFN-γ have direct priming effect on purified pDCs. (A) Representative flow cytometry plots showing TLR7/9-mediated IFN-α production by purified pDCs after pre-treatment with IFN-α and IFN-γ. (B) TLR7/9-mediated IFN-α production by purified pDCs after pre-treatment with IFN-α and IFN-γ. Data are mean ± S.E.M. of 3 independent experiments.

    Article Snippet: PBMCs (1 × 106 /well) from patients with SLE or RA, or healthy subjects in 48-well plates or pDCs (2.5 × 104 /well) from healthy subjects in 96-well flat-bottom plates were stimulated with TLR7 agonist, loxoribine (1 mmol/L) or R837 (5 μg/mL), or TLR9 agonist, CpG2216 (2 μmol/L) or CpG2006 (2 μmol/L, all from InvivoGen) for 5 h, and brefeldin A (2.5 μg/mL: SIGMA-Aldrich) was added during the final 3 h of stimulation to block cytokine secretion.

    Techniques: Purification, Flow Cytometry, Cytometry

    TLR7-mediated IFN-α production correlates with disease activity in SLE patients. Correlation between TLR7/9-mediated IFN-α production and SLEDAI or anti-dsDNA Ab titer. Statistical analysis by the Spearman's correlation coefficient.

    Journal: Frontiers in Immunology

    Article Title: Up-Regulation of TLR7-Mediated IFN-α Production by Plasmacytoid Dendritic Cells in Patients With Systemic Lupus Erythematosus

    doi: 10.3389/fimmu.2018.01957

    Figure Lengend Snippet: TLR7-mediated IFN-α production correlates with disease activity in SLE patients. Correlation between TLR7/9-mediated IFN-α production and SLEDAI or anti-dsDNA Ab titer. Statistical analysis by the Spearman's correlation coefficient.

    Article Snippet: PBMCs (1 × 106 /well) from patients with SLE or RA, or healthy subjects in 48-well plates or pDCs (2.5 × 104 /well) from healthy subjects in 96-well flat-bottom plates were stimulated with TLR7 agonist, loxoribine (1 mmol/L) or R837 (5 μg/mL), or TLR9 agonist, CpG2216 (2 μmol/L) or CpG2006 (2 μmol/L, all from InvivoGen) for 5 h, and brefeldin A (2.5 μg/mL: SIGMA-Aldrich) was added during the final 3 h of stimulation to block cytokine secretion.

    Techniques: Activity Assay

    TLR7/9 response of pDCs is regulated by the priming effects of types I and II IFNs in PBMCs of healthy subjects. (A) Representative flow cytometry plots showing TLR7/9-mediated IFN-α production in pDCs after pre-treatment with each cytokine. (B) TLR7/9-mediated IFN-α production in pDCs after pre-treatment with each cytokine. (C) TLR7/9 expression in pDCs after pre-treatment with each cytokine. (D) TLR7/9-mediated IFN-α production in pDCs after pre-treatment with different doses of IFN-α and IFN-γ. Data are mean ± S.E.M. of 4 independent experiments. * p

    Journal: Frontiers in Immunology

    Article Title: Up-Regulation of TLR7-Mediated IFN-α Production by Plasmacytoid Dendritic Cells in Patients With Systemic Lupus Erythematosus

    doi: 10.3389/fimmu.2018.01957

    Figure Lengend Snippet: TLR7/9 response of pDCs is regulated by the priming effects of types I and II IFNs in PBMCs of healthy subjects. (A) Representative flow cytometry plots showing TLR7/9-mediated IFN-α production in pDCs after pre-treatment with each cytokine. (B) TLR7/9-mediated IFN-α production in pDCs after pre-treatment with each cytokine. (C) TLR7/9 expression in pDCs after pre-treatment with each cytokine. (D) TLR7/9-mediated IFN-α production in pDCs after pre-treatment with different doses of IFN-α and IFN-γ. Data are mean ± S.E.M. of 4 independent experiments. * p

    Article Snippet: PBMCs (1 × 106 /well) from patients with SLE or RA, or healthy subjects in 48-well plates or pDCs (2.5 × 104 /well) from healthy subjects in 96-well flat-bottom plates were stimulated with TLR7 agonist, loxoribine (1 mmol/L) or R837 (5 μg/mL), or TLR9 agonist, CpG2216 (2 μmol/L) or CpG2006 (2 μmol/L, all from InvivoGen) for 5 h, and brefeldin A (2.5 μg/mL: SIGMA-Aldrich) was added during the final 3 h of stimulation to block cytokine secretion.

    Techniques: Flow Cytometry, Cytometry, Expressing

    TLR7 response was quickly up-regulated by IFN-α, but down-regulation of TLR9 response by IFN-γ was required long time. PBMCs were pre-treated with IFN-α and IFN-γ for 2, 12, and 24 h, followed by stimulation with TLR7/9 agonist for 5 h. TLR7/9-mediated IFN-α production in pDCs after pre-treatment with each condition. Data are mean ± S.E.M. of 3 independent experiments. * p

    Journal: Frontiers in Immunology

    Article Title: Up-Regulation of TLR7-Mediated IFN-α Production by Plasmacytoid Dendritic Cells in Patients With Systemic Lupus Erythematosus

    doi: 10.3389/fimmu.2018.01957

    Figure Lengend Snippet: TLR7 response was quickly up-regulated by IFN-α, but down-regulation of TLR9 response by IFN-γ was required long time. PBMCs were pre-treated with IFN-α and IFN-γ for 2, 12, and 24 h, followed by stimulation with TLR7/9 agonist for 5 h. TLR7/9-mediated IFN-α production in pDCs after pre-treatment with each condition. Data are mean ± S.E.M. of 3 independent experiments. * p

    Article Snippet: PBMCs (1 × 106 /well) from patients with SLE or RA, or healthy subjects in 48-well plates or pDCs (2.5 × 104 /well) from healthy subjects in 96-well flat-bottom plates were stimulated with TLR7 agonist, loxoribine (1 mmol/L) or R837 (5 μg/mL), or TLR9 agonist, CpG2216 (2 μmol/L) or CpG2006 (2 μmol/L, all from InvivoGen) for 5 h, and brefeldin A (2.5 μg/mL: SIGMA-Aldrich) was added during the final 3 h of stimulation to block cytokine secretion.

    Techniques:

    Increased localization of TLR7 in late endosome and lysosome by IFN-α. (A) Representative images showing TLR7 (red) and indicated endosomal maturation markers (green) in pDCs pre-treated with or without IFN-α. White arrows indicate robust co-localization of TLR7 with Rab7 and LAMP1. (B) Quantification of co-localization between TLR7 and EEA1, Rab7, and LAMP-1. Data shows 10 cells per each condition in one of three independent experiments.

    Journal: Frontiers in Immunology

    Article Title: Up-Regulation of TLR7-Mediated IFN-α Production by Plasmacytoid Dendritic Cells in Patients With Systemic Lupus Erythematosus

    doi: 10.3389/fimmu.2018.01957

    Figure Lengend Snippet: Increased localization of TLR7 in late endosome and lysosome by IFN-α. (A) Representative images showing TLR7 (red) and indicated endosomal maturation markers (green) in pDCs pre-treated with or without IFN-α. White arrows indicate robust co-localization of TLR7 with Rab7 and LAMP1. (B) Quantification of co-localization between TLR7 and EEA1, Rab7, and LAMP-1. Data shows 10 cells per each condition in one of three independent experiments.

    Article Snippet: PBMCs (1 × 106 /well) from patients with SLE or RA, or healthy subjects in 48-well plates or pDCs (2.5 × 104 /well) from healthy subjects in 96-well flat-bottom plates were stimulated with TLR7 agonist, loxoribine (1 mmol/L) or R837 (5 μg/mL), or TLR9 agonist, CpG2216 (2 μmol/L) or CpG2006 (2 μmol/L, all from InvivoGen) for 5 h, and brefeldin A (2.5 μg/mL: SIGMA-Aldrich) was added during the final 3 h of stimulation to block cytokine secretion.

    Techniques:

    Up-regulation of TLR7-mediated IFN-α production by pDCs in SLE patients. (A) Representative flow cytometry plots showing TLR7/9-mediated IFN-α production in Lin − HLA-DR + CD11c − CD123 + pDCs from healthy control subjects and SLE patients. (B) TLR7/9-mediated IFN-α production in pDCs of each group. (C) Representative flow cytometry plots showing TLR7/9 expression in pDCs of healthy control subjects and SLE patients. (D) TLR7/9 expression in pDCs of each group. Horizontal lines represent the mean value of each group. * p

    Journal: Frontiers in Immunology

    Article Title: Up-Regulation of TLR7-Mediated IFN-α Production by Plasmacytoid Dendritic Cells in Patients With Systemic Lupus Erythematosus

    doi: 10.3389/fimmu.2018.01957

    Figure Lengend Snippet: Up-regulation of TLR7-mediated IFN-α production by pDCs in SLE patients. (A) Representative flow cytometry plots showing TLR7/9-mediated IFN-α production in Lin − HLA-DR + CD11c − CD123 + pDCs from healthy control subjects and SLE patients. (B) TLR7/9-mediated IFN-α production in pDCs of each group. (C) Representative flow cytometry plots showing TLR7/9 expression in pDCs of healthy control subjects and SLE patients. (D) TLR7/9 expression in pDCs of each group. Horizontal lines represent the mean value of each group. * p

    Article Snippet: PBMCs (1 × 106 /well) from patients with SLE or RA, or healthy subjects in 48-well plates or pDCs (2.5 × 104 /well) from healthy subjects in 96-well flat-bottom plates were stimulated with TLR7 agonist, loxoribine (1 mmol/L) or R837 (5 μg/mL), or TLR9 agonist, CpG2216 (2 μmol/L) or CpG2006 (2 μmol/L, all from InvivoGen) for 5 h, and brefeldin A (2.5 μg/mL: SIGMA-Aldrich) was added during the final 3 h of stimulation to block cytokine secretion.

    Techniques: Flow Cytometry, Cytometry, Expressing

    Specificity of phloretin with various TLR-specific agonists that selectively activate different TLRs determined by monitoring the inhibition activity of TNF-α production in Raw264.7 cells. Pam 3 CSK 4 (200 ng/mL), Pam 2 CSK 4 (200 ng/mL), poly(I:C) (1 µg/mL), LPS (20 ng/mL), imiquimod (1 µg/mL), and ODN1826 (10 µg/mL) were used to selectively activate respective TLRs. TNF-α secreted into the supernatant was measured by ELISA. Each sample was measured in triplicate. The error bars represent ± SEM. (* p

    Journal: Nutrients

    Article Title: Phloretin as a Potent Natural TLR2/1 Inhibitor Suppresses TLR2-Induced Inflammation

    doi: 10.3390/nu10070868

    Figure Lengend Snippet: Specificity of phloretin with various TLR-specific agonists that selectively activate different TLRs determined by monitoring the inhibition activity of TNF-α production in Raw264.7 cells. Pam 3 CSK 4 (200 ng/mL), Pam 2 CSK 4 (200 ng/mL), poly(I:C) (1 µg/mL), LPS (20 ng/mL), imiquimod (1 µg/mL), and ODN1826 (10 µg/mL) were used to selectively activate respective TLRs. TNF-α secreted into the supernatant was measured by ELISA. Each sample was measured in triplicate. The error bars represent ± SEM. (* p

    Article Snippet: Therefore, we tested the effects of phloretin (Sigma-Aldrich, St. Louis, MO, USA) on the TNF-α production induced by various TLRs, including TLR2/1, TLR2/6, TLR3, TLR4, TLR7, and TLR9, using known TLR-specific ligands such as Pam3 CSK4 (200 ng/mL; Invivogen, San Diego, CA, USA), Pam2 CSK4 (200 ng/mL; Invivogen, San Diego, CA, USA), Polyinosinic:polycytidylic acid (Poly(I:C)) (1 µg/mL; Invivogen, San Diego, CA, USA), lipopolysaccharide (LPS; 20 ng/mL; Invivogen, San Diego, CA, USA), imiquimod (1 µg/mL; Sigma-Aldrich, St. Louis, MO, USA), and ODN1826 (10 µg/mL; Invivogen, San Diego, CA, USA), respectively, to selectively activate a particular TLR-mediated TNF-α response.

    Techniques: Inhibition, Activity Assay, Enzyme-linked Immunosorbent Assay

    Cell-Based TLR Activation Assays. Various CEP adducts and TLR agonists were tested in HEK293 or THP-1 cells. Readouts were NFkB reporter signal or IL-8 secretion (columns), as indicated. In addition, the same wells were analyzed for viability with the CellTiter-Glo kit (axis on right; square symbols) to ensure that any lack of activation was not due to cell toxicity. HEK293 cells were treated with the following reagents: HSA-CTL1, HSA-CTL2, or HSA-CEP: 0, 3.9, 7.8, 15.6, 32.6, 62.5, and 125 and 250 µg/ml; Pam3CSK4∶ 0, 1.5, 3.2, 6.3, 12.5, 25, 50, 100 ng/mL. THP-1 cells were treated with the following reagents: HSA-CTL1, HSA-CTL2, or HSA-CEP: 62.5, 125, and 250 µg/mL; Pam3CSK4∶ 4, 20, and 100 ng/mL; FSL-1∶0.4, 2, and 10 ng/mL; LPS: 4, 20, and 100 ng/mL; R837 or R848∶0.4, 2, and 10 µM; ODN2006G5∶0.2, 1, and 5 µM.

    Journal: PLoS ONE

    Article Title: Lack of Involvement of CEP Adducts in TLR Activation and in Angiogenesis

    doi: 10.1371/journal.pone.0111472

    Figure Lengend Snippet: Cell-Based TLR Activation Assays. Various CEP adducts and TLR agonists were tested in HEK293 or THP-1 cells. Readouts were NFkB reporter signal or IL-8 secretion (columns), as indicated. In addition, the same wells were analyzed for viability with the CellTiter-Glo kit (axis on right; square symbols) to ensure that any lack of activation was not due to cell toxicity. HEK293 cells were treated with the following reagents: HSA-CTL1, HSA-CTL2, or HSA-CEP: 0, 3.9, 7.8, 15.6, 32.6, 62.5, and 125 and 250 µg/ml; Pam3CSK4∶ 0, 1.5, 3.2, 6.3, 12.5, 25, 50, 100 ng/mL. THP-1 cells were treated with the following reagents: HSA-CTL1, HSA-CTL2, or HSA-CEP: 62.5, 125, and 250 µg/mL; Pam3CSK4∶ 4, 20, and 100 ng/mL; FSL-1∶0.4, 2, and 10 ng/mL; LPS: 4, 20, and 100 ng/mL; R837 or R848∶0.4, 2, and 10 µM; ODN2006G5∶0.2, 1, and 5 µM.

    Article Snippet: Thp1 (ATCC, cat# TIB-202) was primed with 0.5% DMSO for overnight, at 100,000/well, then incubate with HSA-CEP for 24 hr, with TLR ligands (Pam3CSK4, FSL1, R837 and R848 were all from Invivogen; LPS was purchased from Sigma) as controls.

    Techniques: Activation Assay

    TLR ligands induced allergic rhinitis (AR)-derived monocyte-derived DCs (mo-DCs) to express IL-17RB, ST2, and thymic stromal lymphopoietin (TSLPR). (A,B) Representative flow plots of IL-17RB, ST2, and TSLPR expression on CD1c + mo-DCs derived from AR ( n = 9) and healthy control (HC) ( n = 6) subjects before and 48 h after stimulation with Pam3CSK4, PGN-BS, Poly I:C, Der.p1, FLA-ST, FSL-1, Imiquimod-R837, ssRNA, and ODN2395. Gray histograms were negative isotypes. (C,D) Quantification of percentages of IL-17RB + , ST2 + , and TSLPR + cells in CD1c + mo-DCs derived from AR and HC subjects. (E) Quantification of percentages of IL-17RB + ST2 + TSLPR + cells in CD1c + mo-DCs derived from AR subjects. Data are expressed as means ± standard deviation (SD). Significant differences were tested using the paired t -test between control and TLR groups, and using Student’s t -test between TLR stimulation groups. * P

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Combinatorial IL-17RB, ST2, and TSLPR Signaling in Dendritic Cells of Patients With Allergic Rhinitis

    doi: 10.3389/fcell.2020.00207

    Figure Lengend Snippet: TLR ligands induced allergic rhinitis (AR)-derived monocyte-derived DCs (mo-DCs) to express IL-17RB, ST2, and thymic stromal lymphopoietin (TSLPR). (A,B) Representative flow plots of IL-17RB, ST2, and TSLPR expression on CD1c + mo-DCs derived from AR ( n = 9) and healthy control (HC) ( n = 6) subjects before and 48 h after stimulation with Pam3CSK4, PGN-BS, Poly I:C, Der.p1, FLA-ST, FSL-1, Imiquimod-R837, ssRNA, and ODN2395. Gray histograms were negative isotypes. (C,D) Quantification of percentages of IL-17RB + , ST2 + , and TSLPR + cells in CD1c + mo-DCs derived from AR and HC subjects. (E) Quantification of percentages of IL-17RB + ST2 + TSLPR + cells in CD1c + mo-DCs derived from AR subjects. Data are expressed as means ± standard deviation (SD). Significant differences were tested using the paired t -test between control and TLR groups, and using Student’s t -test between TLR stimulation groups. * P

    Article Snippet: To investigate the effects of Toll-like receptor (TLR) ligands on DCs, mo-DCs were cultured for 48 h for flow cytometry in the presence of 300 ng/mL Pam3CSK4, 1 μg/mL PGN-BS, 10 μg/mL Poly I:C, 100 ng/mL FLA-ST, 100 ng/mL FSL-1, 2 μg/mL Imiquimod-R837, 500 ng/mL ssRNA40/LyoVecTM, and 7 μg/mL ODN2395 (all from InvivoGen, San Diego, CA, United States), or 1 μg/mL natural Dermatophagoides pteronyssinus allergen 1 (Der.p1) (Indoor Biotechnologies, Charlottesville, VA, United States).

    Techniques: Derivative Assay, Expressing, Standard Deviation

    Expression dynamics of genes involved in innate immune signaling in response to PMA (A) Heatmap showing the proteome fold change of the subset of genes involved in innate immune signaling. Scale indicates the level of expression (Log2-expression values, z-transformed, scaled). (B) Dynamic expression pattern to assess the expression levels of pro-inflammatory cytokine mRNA induced by TLR agonists: CL075-TLR7/8, CpG2006-TLR9, Flagellin-TLR5, FSL1- TLR2/6, LPS 0111:B4, and LPS K12 – TLR4, Pam3CSK4-TLR2/1, Poly I:C-TLR3, R837-TLR7, and R848-TLR7/8. Heatmap depicts Log10 fold change of normalized gene expression for pairwise comparisons of mRNA levels.

    Journal: bioRxiv

    Article Title: Dose-dependent phorbol 12-myristate-13-acetate-mediated monocyte-to-macrophage differentiation induces unique proteomic signatures in THP-1 cells

    doi: 10.1101/2020.02.27.968016

    Figure Lengend Snippet: Expression dynamics of genes involved in innate immune signaling in response to PMA (A) Heatmap showing the proteome fold change of the subset of genes involved in innate immune signaling. Scale indicates the level of expression (Log2-expression values, z-transformed, scaled). (B) Dynamic expression pattern to assess the expression levels of pro-inflammatory cytokine mRNA induced by TLR agonists: CL075-TLR7/8, CpG2006-TLR9, Flagellin-TLR5, FSL1- TLR2/6, LPS 0111:B4, and LPS K12 – TLR4, Pam3CSK4-TLR2/1, Poly I:C-TLR3, R837-TLR7, and R848-TLR7/8. Heatmap depicts Log10 fold change of normalized gene expression for pairwise comparisons of mRNA levels.

    Article Snippet: RNA isolation and quantitative real-time PCR (qPCR) analysis Undifferentiated THP-1 and PMA-differentiated cells (2 × 105 cells) in biological duplicates per condition were stimulated with TLR agonists- CL075 (TLR8; tlrl-c75; Invivogen, 5 μg/ml), CpG2006 (TLR9; tlrl-2006; Invivogen, 10 μM), Flagellin (TLR5; tlrl-stfla; Invivogen, 100 ng/ml), FSL1 (TLR2/6; tlrl-fsl; Invivogen, 100 ng/ml), LPS 0111:B4 (TLR4; tlrl-3pelps; Invivogen, 200 ng/ml), LPS K12 (TLR4; tlrl-eklps; Invivogen, 200 ng/ml), Pam3CSK4 (TLR1/2; tlrl-pms; Invivogen, 200 ng/ml), Poly (I:C) (TLR3; vac-pic; Invivogen, 10 μg /ml), R837 (TLR7; tlrl-imqs; Invivogen, 10 μg/ml), and R848 (TLR7/8; tlrl-r848; Invivogen, 100 ng/ml) for 4 hours.

    Techniques: Expressing, Transformation Assay

    Inhibitory effect of TAC5-a on the TLR signaling pathways. ( A ) The chemical structure of TAC5 and its derivatives represented by different R groups. ( B ) RAW 264.7 cells were treated with various concentrations of compounds for 24 h and the cell viability was measured using an MTT assay. ( C ) The inhibitory effects of TAC5-a, TAC5-c, TAC5-d, and TAC5-e on the TNF-α secretion level in THP1 derived macrophage cells under the influence of different TLR7/8 ligands. R848 and CL075 were used to activate TLR7/8, imiquimod (IMQ) to activate TLR7, and TL8-506 to activate TLR8, specifically. ( D – F ) The inhibitory effect of TAC5-a on the TNF-α secretion level in THP1-derived macrophage cells was evaluated following the treatment of cells with different TLR ligands ( D ) Lipopolysaccharide was used to activate TLR4, Pam 3 CSK 4 to activate TLR1/2, and FSL-1 to activate TLR2/6). ( E ) poly(I:C) was used to activate TLR3 and CpG ODN to activate TLR9. ( F ) R848 and CL075 were used to activate TLR7/8, IMQ to activate TLR7, and TL8-506 to activate TLR8. ( G ) The inhibitory effects of TAC5-a on the IL-6 secretion in RAW264.7 cells stimulated with agonists of TLR7/8 (R848 and CL075), TLR7 (IMQ), or TLR8 (TL8-506). All data shown represent the results of five independently conducted experiments; the mean ± SEM of the independent experiments were calculated and used in the two-tailed paired Student’s t -test (* p

    Journal: Cells

    Article Title: A Novel Small-Molecule Inhibitor of Endosomal TLRs Reduces Inflammation and Alleviates Autoimmune Disease Symptoms in Murine Models

    doi: 10.3390/cells9071648

    Figure Lengend Snippet: Inhibitory effect of TAC5-a on the TLR signaling pathways. ( A ) The chemical structure of TAC5 and its derivatives represented by different R groups. ( B ) RAW 264.7 cells were treated with various concentrations of compounds for 24 h and the cell viability was measured using an MTT assay. ( C ) The inhibitory effects of TAC5-a, TAC5-c, TAC5-d, and TAC5-e on the TNF-α secretion level in THP1 derived macrophage cells under the influence of different TLR7/8 ligands. R848 and CL075 were used to activate TLR7/8, imiquimod (IMQ) to activate TLR7, and TL8-506 to activate TLR8, specifically. ( D – F ) The inhibitory effect of TAC5-a on the TNF-α secretion level in THP1-derived macrophage cells was evaluated following the treatment of cells with different TLR ligands ( D ) Lipopolysaccharide was used to activate TLR4, Pam 3 CSK 4 to activate TLR1/2, and FSL-1 to activate TLR2/6). ( E ) poly(I:C) was used to activate TLR3 and CpG ODN to activate TLR9. ( F ) R848 and CL075 were used to activate TLR7/8, IMQ to activate TLR7, and TL8-506 to activate TLR8. ( G ) The inhibitory effects of TAC5-a on the IL-6 secretion in RAW264.7 cells stimulated with agonists of TLR7/8 (R848 and CL075), TLR7 (IMQ), or TLR8 (TL8-506). All data shown represent the results of five independently conducted experiments; the mean ± SEM of the independent experiments were calculated and used in the two-tailed paired Student’s t -test (* p

    Article Snippet: LPS (Escherichia coli 0111:B4) was purchased from Sigma-Aldrich Co. Pam3 CSK4, FSL-1, poly(I:C), R848, CL075, imiquimod (IMQ; R837), TL8-506, and CpG ODN were all purchased from InvivoGen, San Diego, CA, USA.

    Techniques: MTT Assay, Derivative Assay, Two Tailed Test

    TAC5 inhibits the CL075-induced proinflammatory response. RAW 264.7 cells were treated with 50 µM of TAC5 for 1 h, followed by treatment with CL075 (1 µg/mL) for 15 and 30 min, respectively. ( A ) Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation (NF-κB translocation into the nucleus and degradation of IκBα in the cytosol) was measured by western blotting. Histone deacetylase-1 was used as a nucleus loading control. β-actin was used as a cytosol loading control. ( B ) The expression levels of mitogen-activated protein kinases (MAPKs) were evaluated by western blot analysis using whole protein extracts, with inactive MAPKs used as controls (β-actin was used as a loading control). ( C ) Inhibition of TNF-α secretion by TAC5 in the presence of CL075 and a combination of CL075 and poly(dT). ( D ) Confocal microscopy image of the phosphorylated NF-κB (p-p65) expression level. The red staining corresponds to phosphorylated p65 subunit of NF-κB (p-p65) and blue staining (Hoechst33258) indicates nuclei (the scale bar is 20 µm in width). ( E , F ) The expression level of TNF-α was measured by ELISA when the TAC5 was co-treated with ( E ) imiquimod (TLR7), TL8-506 (TLR8), CpG ODN (TLR9) (F) Pam 3 CSK 4 (TLR1/2), poly(I:C) (TLR3), LPS (TLR4), or CL075 (TLR7/8) agonists in RAW 264.7 cells. ( G ) Surface plasmon resonance (SPR) sensorgram illustrating the competi tive binding of TAC5 to TLR7 in the presence of imiquimod. The data shown represent five independent experiments, and the bars represent means ± SEM (* p

    Journal: Cells

    Article Title: A Novel Small-Molecule Inhibitor of Endosomal TLRs Reduces Inflammation and Alleviates Autoimmune Disease Symptoms in Murine Models

    doi: 10.3390/cells9071648

    Figure Lengend Snippet: TAC5 inhibits the CL075-induced proinflammatory response. RAW 264.7 cells were treated with 50 µM of TAC5 for 1 h, followed by treatment with CL075 (1 µg/mL) for 15 and 30 min, respectively. ( A ) Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation (NF-κB translocation into the nucleus and degradation of IκBα in the cytosol) was measured by western blotting. Histone deacetylase-1 was used as a nucleus loading control. β-actin was used as a cytosol loading control. ( B ) The expression levels of mitogen-activated protein kinases (MAPKs) were evaluated by western blot analysis using whole protein extracts, with inactive MAPKs used as controls (β-actin was used as a loading control). ( C ) Inhibition of TNF-α secretion by TAC5 in the presence of CL075 and a combination of CL075 and poly(dT). ( D ) Confocal microscopy image of the phosphorylated NF-κB (p-p65) expression level. The red staining corresponds to phosphorylated p65 subunit of NF-κB (p-p65) and blue staining (Hoechst33258) indicates nuclei (the scale bar is 20 µm in width). ( E , F ) The expression level of TNF-α was measured by ELISA when the TAC5 was co-treated with ( E ) imiquimod (TLR7), TL8-506 (TLR8), CpG ODN (TLR9) (F) Pam 3 CSK 4 (TLR1/2), poly(I:C) (TLR3), LPS (TLR4), or CL075 (TLR7/8) agonists in RAW 264.7 cells. ( G ) Surface plasmon resonance (SPR) sensorgram illustrating the competi tive binding of TAC5 to TLR7 in the presence of imiquimod. The data shown represent five independent experiments, and the bars represent means ± SEM (* p

    Article Snippet: LPS (Escherichia coli 0111:B4) was purchased from Sigma-Aldrich Co. Pam3 CSK4, FSL-1, poly(I:C), R848, CL075, imiquimod (IMQ; R837), TL8-506, and CpG ODN were all purchased from InvivoGen, San Diego, CA, USA.

    Techniques: Activation Assay, Translocation Assay, Western Blot, Histone Deacetylase Assay, Expressing, Inhibition, Confocal Microscopy, Staining, Enzyme-linked Immunosorbent Assay, SPR Assay, Binding Assay

    TCDD decreases BMDC cytokine production following TLR stimulation: Vehicle- and TCDD-BMDCs were harvested on day 10 and stimulated for 24 h with (A) LPS (1 μg/ml) or (B) CpG (0.5μM) or (C) Imiquimod (30 μg/ml) or unstimulated.

    Journal: Toxicological Sciences

    Article Title: Consequences of AhR Activation in Steady-State Dendritic Cells

    doi: 10.1093/toxsci/kfq354

    Figure Lengend Snippet: TCDD decreases BMDC cytokine production following TLR stimulation: Vehicle- and TCDD-BMDCs were harvested on day 10 and stimulated for 24 h with (A) LPS (1 μg/ml) or (B) CpG (0.5μM) or (C) Imiquimod (30 μg/ml) or unstimulated.

    Article Snippet: LPS ( Escherichia coli ; 055:B5) was obtained from Sigma-Aldrich, and CpG (ODN 1826; type B) and Imiquimod (R837) were purchased from Invivogen (San Diego, CA).

    Techniques:

    TCDD alters NF-kB activity: Vehicle- and TCDD-treated BMDCs were stimulated with LPS (1 μg/ml) or CpG (0.5μM) for 45 min. Following stimulation, BMDCs were harvested, nuclear lysates isolated, and RelB-, p65-, p52-, and p50-binding activity

    Journal: Toxicological Sciences

    Article Title: Consequences of AhR Activation in Steady-State Dendritic Cells

    doi: 10.1093/toxsci/kfq354

    Figure Lengend Snippet: TCDD alters NF-kB activity: Vehicle- and TCDD-treated BMDCs were stimulated with LPS (1 μg/ml) or CpG (0.5μM) for 45 min. Following stimulation, BMDCs were harvested, nuclear lysates isolated, and RelB-, p65-, p52-, and p50-binding activity

    Article Snippet: LPS ( Escherichia coli ; 055:B5) was obtained from Sigma-Aldrich, and CpG (ODN 1826; type B) and Imiquimod (R837) were purchased from Invivogen (San Diego, CA).

    Techniques: Activity Assay, Isolation, Binding Assay

    Reduced uptake of soluble antigen by BMDC in the presence of TLR agonists. Granulocyte macrophage colony-stimulating factor (GM-CSF) BMDC were cultured for 4 h with OVA–Alexa555 (5 μg ml −1 ) in the presence of polyI:C (50 μg ml −1 ), CpG (0.5 μg ml −1 ), R837 (1 μg ml −1 ), LPS (1 μg ml −1 ) or left untreated. Cells were stained with anti-CD11c antibody and samples were acquired by flow cytometry. (A) The mean fluorescent intensity of cells for Alexa555 was determined after gating on the CD11c + cell population. The data of two independent representative experiments were pooled ( n = 4) and the standard deviation is indicated. (B) The frequency of OVA–Alexa555 + cells was determined after gating on the CD11c + cell population. The relative percentage of OVA–Alexa555 + BMDC in samples stimulated with TLR agonists is depicted in relation to the frequency of OVA–Alexa555 + BMDC in the absence of TLR stimulation (unst), which was set to 100%. The results of eight independent experiments were compiled and the standard deviation is indicated. (C) Mice were vaccinated intravenously with GM-CSF BMDC that had been pulsed overnight with OVA in the presence or absence of various TLR ligands. Seven days after vaccination, CFSE-labelled target cells were injected and antigen-specific lysis of peptide-pulsed targets cells versus unpulsed target cells was determined by flow cytometry the following day. The graph compiles data from two independent experiments ( n = 10). For statistical analysis, one-way analysis of variance in combination with Dunnett's (A and B) or Tukey's (C) multiple comparison test was used (** P

    Journal: International Immunology

    Article Title: PolyI:C-induced reduction in uptake of soluble antigen is independent of dendritic cell activation

    doi: 10.1093/intimm/dxp053

    Figure Lengend Snippet: Reduced uptake of soluble antigen by BMDC in the presence of TLR agonists. Granulocyte macrophage colony-stimulating factor (GM-CSF) BMDC were cultured for 4 h with OVA–Alexa555 (5 μg ml −1 ) in the presence of polyI:C (50 μg ml −1 ), CpG (0.5 μg ml −1 ), R837 (1 μg ml −1 ), LPS (1 μg ml −1 ) or left untreated. Cells were stained with anti-CD11c antibody and samples were acquired by flow cytometry. (A) The mean fluorescent intensity of cells for Alexa555 was determined after gating on the CD11c + cell population. The data of two independent representative experiments were pooled ( n = 4) and the standard deviation is indicated. (B) The frequency of OVA–Alexa555 + cells was determined after gating on the CD11c + cell population. The relative percentage of OVA–Alexa555 + BMDC in samples stimulated with TLR agonists is depicted in relation to the frequency of OVA–Alexa555 + BMDC in the absence of TLR stimulation (unst), which was set to 100%. The results of eight independent experiments were compiled and the standard deviation is indicated. (C) Mice were vaccinated intravenously with GM-CSF BMDC that had been pulsed overnight with OVA in the presence or absence of various TLR ligands. Seven days after vaccination, CFSE-labelled target cells were injected and antigen-specific lysis of peptide-pulsed targets cells versus unpulsed target cells was determined by flow cytometry the following day. The graph compiles data from two independent experiments ( n = 10). For statistical analysis, one-way analysis of variance in combination with Dunnett's (A and B) or Tukey's (C) multiple comparison test was used (** P

    Article Snippet: PolyI:C was from GE Healthcare (Chalfont St Giles, UK), R837 and LPS (from Escherichia coli K12) were from Invivogen (Toulouse, France) and CpG-containing oligonucleotide (ODN) 1668 was purchased from MWG (Edersberg, Germany).

    Techniques: Cell Culture, Staining, Flow Cytometry, Cytometry, Standard Deviation, Mouse Assay, Injection, Lysis

    Specific RNA homopolymers partially inhibit antigen uptake by BMDC independently of DC activation. Uptake of OVA–Alexa555 by wild-type (A), TRIF −/− (B) and TLR4 −/− (C) BMDC was assessed after 4 h of incubation in the presence of the indicated stimuli. Cells were stained with anti-CD11c antibody and the frequency of OVA–Alexa555 + CD11c + cells was determined by flow cytometry. The relative percentage of OVA–Alexa555 + BMDC is depicted in relation to BMDC in the absence of TLR agonists. The following concentrations of TLR agonists were used: 0.4, 2, 10 and 50 μg ml −1 for polyI:C, Ampligen, polyI, polyG and polyU and 0.01, 0.1 and 1 μg ml −1 for LPS. The standard deviation is shown and data are representative of at least three independent experiments. Data were analysed by one-way analysis of variance in combination with Dunnett's multiple comparison test (* P

    Journal: International Immunology

    Article Title: PolyI:C-induced reduction in uptake of soluble antigen is independent of dendritic cell activation

    doi: 10.1093/intimm/dxp053

    Figure Lengend Snippet: Specific RNA homopolymers partially inhibit antigen uptake by BMDC independently of DC activation. Uptake of OVA–Alexa555 by wild-type (A), TRIF −/− (B) and TLR4 −/− (C) BMDC was assessed after 4 h of incubation in the presence of the indicated stimuli. Cells were stained with anti-CD11c antibody and the frequency of OVA–Alexa555 + CD11c + cells was determined by flow cytometry. The relative percentage of OVA–Alexa555 + BMDC is depicted in relation to BMDC in the absence of TLR agonists. The following concentrations of TLR agonists were used: 0.4, 2, 10 and 50 μg ml −1 for polyI:C, Ampligen, polyI, polyG and polyU and 0.01, 0.1 and 1 μg ml −1 for LPS. The standard deviation is shown and data are representative of at least three independent experiments. Data were analysed by one-way analysis of variance in combination with Dunnett's multiple comparison test (* P

    Article Snippet: PolyI:C was from GE Healthcare (Chalfont St Giles, UK), R837 and LPS (from Escherichia coli K12) were from Invivogen (Toulouse, France) and CpG-containing oligonucleotide (ODN) 1668 was purchased from MWG (Edersberg, Germany).

    Techniques: Activation Assay, Incubation, Staining, Flow Cytometry, Cytometry, Standard Deviation

    CD40 ligation on DCs boosts PRR-induced cytokine production. ( A–C ) Spleen-derived DCs were incubated with the indicated doses of CpG, curdlan, or zymosan in the absence or presence of 2 or 10 μg/ml of anti-CD40 antibody. After 20 h, surface

    Journal:

    Article Title: CD40-CD40L cross-talk integrates strong antigenic signals and microbial stimuli to induce development of IL-17-producing CD4+ T cells

    doi: 10.1073/pnas.0810769106

    Figure Lengend Snippet: CD40 ligation on DCs boosts PRR-induced cytokine production. ( A–C ) Spleen-derived DCs were incubated with the indicated doses of CpG, curdlan, or zymosan in the absence or presence of 2 or 10 μg/ml of anti-CD40 antibody. After 20 h, surface

    Article Snippet: LPS from Escherichia coli (0111:B4), zymosan A from Saccharomyces cerevisiae , and curdlan from Alcaligenes faecalis were purchased from Sigma-Aldrich.

    Techniques: Ligation, Derivative Assay, Incubation

    Activation and apoptosis were not affected in Blimp-1-deficient FLpDCs. (A) Flow cytometric analysis showing the frequency of Annexin V positive cells in 4-OHT treated Ctrl-ER or CKO-ER FLpDCs stimulated with 1 µM CpG-A or medium alone for 6 and 16 h. (B) Flow cytometric analysis of CD86, CD69, and MHCII expression on 4-OHT treated Ctrl-ER or CKO-ER FLpDCs with or without 16 h of 1 µM CpG-A stimulation. The positive frequency of each marker is indicated. Results represent the mean ± SEM and were analyzed by two-tailed unpaired Student’s t -test [ n = 3 in (A,B) ].

    Journal: Frontiers in Immunology

    Article Title: Blimp-1-Mediated Pathway Promotes Type I IFN Production in Plasmacytoid Dendritic Cells by Targeting to Interleukin-1 Receptor-Associated Kinase M

    doi: 10.3389/fimmu.2018.01828

    Figure Lengend Snippet: Activation and apoptosis were not affected in Blimp-1-deficient FLpDCs. (A) Flow cytometric analysis showing the frequency of Annexin V positive cells in 4-OHT treated Ctrl-ER or CKO-ER FLpDCs stimulated with 1 µM CpG-A or medium alone for 6 and 16 h. (B) Flow cytometric analysis of CD86, CD69, and MHCII expression on 4-OHT treated Ctrl-ER or CKO-ER FLpDCs with or without 16 h of 1 µM CpG-A stimulation. The positive frequency of each marker is indicated. Results represent the mean ± SEM and were analyzed by two-tailed unpaired Student’s t -test [ n = 3 in (A,B) ].

    Article Snippet: The purified pDCs were stimulated with 1 µM CpG-A (InvivoGen), CpG-C (InvivoGen), or 2 µg/ml R837 (InvivoGen). cDCs were treated with 50 ng/ml poly(I:C) (InvivoGen) or 10 ng/ml LPS (Sigma-Aldrich) at a density of 1 × 106 cells/ml for the indicated time points.

    Techniques: Activation Assay, Flow Cytometry, Expressing, Marker, Two Tailed Test

    Increased IRAK-M expression in pDCs lacking Blimp-1 contributes to impaired IFN-I production. (A,B) RT-qPCR showing Irak3 mRNA levels (A) , IRAK-M and Blimp-1 protein levels (B) in Ctrl-ER and CKO-ER FLpDCs treated with 4-OHT and then stimulated with 1 µM CpG-A. The quantitation of Blimp-1 in (B) was presented by the ratios of Blimp-1 band intensity vs. Lamin-B band intensity at each time point. The quantitation of IRAK-M in (B) was presented by the ratios of IRAK-M band intensity vs. actin band intensity at each time point. (C) Five putative Blimp-1 consensus binding sites were identified within 5 kb upstream and downstream of the Irak3 transcriptional start site (TSS, indicated by an arrow). (D) ChIP assay using chromatin isolated from FLpDCs following 4 h stimulation with 1 µM CpG-A showing the levels of binding of Blimp-1 at various putative sites. Gapdh was used as the negative control locus. (E) IFN-α production by Blimp-1-deficient and control FLpDCs transfected with control siRNA (siCtrl) or siRNA-pools with three different siRNAs against Irak3 (siIrak3) and stimulated with 1 µM CpG-A for 16 h. (F) Model of the action of Blimp-1 in the regulation of induction of IFN-I signaling in pDCs. Abbreviations: Rac, Ras-related C3 botulinum toxin substrate; IRAK-M, interleukin-1 receptor-associated kinase M; OPN, osteopontin; pDCs, plasmacytoid dendritic cells; IFN-I, type I IFN; siRNA, small-interfering RNA; Irak3, interleukin-1 receptor-associated kinase 3 ; ChIP, chromatin immunoprecipitation; RT-qPCR, RT-quantitative PCR. Data represent the mean ± SEM and were analyzed by two-tailed unpaired Student’s t -test [ n = 4 in (A) and 3 in (D,E) ]. * p

    Journal: Frontiers in Immunology

    Article Title: Blimp-1-Mediated Pathway Promotes Type I IFN Production in Plasmacytoid Dendritic Cells by Targeting to Interleukin-1 Receptor-Associated Kinase M

    doi: 10.3389/fimmu.2018.01828

    Figure Lengend Snippet: Increased IRAK-M expression in pDCs lacking Blimp-1 contributes to impaired IFN-I production. (A,B) RT-qPCR showing Irak3 mRNA levels (A) , IRAK-M and Blimp-1 protein levels (B) in Ctrl-ER and CKO-ER FLpDCs treated with 4-OHT and then stimulated with 1 µM CpG-A. The quantitation of Blimp-1 in (B) was presented by the ratios of Blimp-1 band intensity vs. Lamin-B band intensity at each time point. The quantitation of IRAK-M in (B) was presented by the ratios of IRAK-M band intensity vs. actin band intensity at each time point. (C) Five putative Blimp-1 consensus binding sites were identified within 5 kb upstream and downstream of the Irak3 transcriptional start site (TSS, indicated by an arrow). (D) ChIP assay using chromatin isolated from FLpDCs following 4 h stimulation with 1 µM CpG-A showing the levels of binding of Blimp-1 at various putative sites. Gapdh was used as the negative control locus. (E) IFN-α production by Blimp-1-deficient and control FLpDCs transfected with control siRNA (siCtrl) or siRNA-pools with three different siRNAs against Irak3 (siIrak3) and stimulated with 1 µM CpG-A for 16 h. (F) Model of the action of Blimp-1 in the regulation of induction of IFN-I signaling in pDCs. Abbreviations: Rac, Ras-related C3 botulinum toxin substrate; IRAK-M, interleukin-1 receptor-associated kinase M; OPN, osteopontin; pDCs, plasmacytoid dendritic cells; IFN-I, type I IFN; siRNA, small-interfering RNA; Irak3, interleukin-1 receptor-associated kinase 3 ; ChIP, chromatin immunoprecipitation; RT-qPCR, RT-quantitative PCR. Data represent the mean ± SEM and were analyzed by two-tailed unpaired Student’s t -test [ n = 4 in (A) and 3 in (D,E) ]. * p

    Article Snippet: The purified pDCs were stimulated with 1 µM CpG-A (InvivoGen), CpG-C (InvivoGen), or 2 µg/ml R837 (InvivoGen). cDCs were treated with 50 ng/ml poly(I:C) (InvivoGen) or 10 ng/ml LPS (Sigma-Aldrich) at a density of 1 × 106 cells/ml for the indicated time points.

    Techniques: Expressing, Quantitative RT-PCR, Quantitation Assay, Binding Assay, Chromatin Immunoprecipitation, Isolation, Negative Control, Transfection, Small Interfering RNA, Real-time Polymerase Chain Reaction, Two Tailed Test

    Impaired IKKα and IRF7 activation in plasmacytoid dendritic cells (pDCs) with reduced Blimp-1 expression. (A) Immunoblot analysis of nuclear extracts showing the levels of IRF7 in 4-OHT treated Ctrl-ER and CKO-ER FLpDCs following stimulation with 1 µM CpG-A at indicated time points. (B) Immunoblot analysis of total cell lysates showing the levels of IRF7 phosphorylation at Ser471/472 in 1 µM CpG-A stimulated Ctrl-ER and CKO-ER FLpDCs. (C) Immunoblot analysis showing the levels of phospho-IKKα at Ser176/180, OPN and phospho-AKT at Ser473, in 4-OHT treated and CpG-A stimulated Ctrl-ER and CKO-ER FLpDCs. The quantification of pIKKα was presented by the ratios of pIKKα band intensity vs. IKKα band intensity at each time point. (D) Immunoblot analysis using nuclear extracts of 4-OHT treated Ctrl-ER and CKO-ER FLpDCs showing the levels of p50 and p65 translocation after 1 µM CpG-A treatment at indicated time points. (E) Immunoblot analysis showing the levels of phosphorylated STAT1 at Tyr701 at the indicated time points in Ctrl-ER and CKO-ER FLpDCs treated with 4-OHT and then stimulated with 1 µM CpG-A at the indicated time points. (F) Immunoblot analysis showing levels of phosphorylated STAT1 at Tyr701 in FLpDCs in the presence of 500 U/ml mIFN-α.

    Journal: Frontiers in Immunology

    Article Title: Blimp-1-Mediated Pathway Promotes Type I IFN Production in Plasmacytoid Dendritic Cells by Targeting to Interleukin-1 Receptor-Associated Kinase M

    doi: 10.3389/fimmu.2018.01828

    Figure Lengend Snippet: Impaired IKKα and IRF7 activation in plasmacytoid dendritic cells (pDCs) with reduced Blimp-1 expression. (A) Immunoblot analysis of nuclear extracts showing the levels of IRF7 in 4-OHT treated Ctrl-ER and CKO-ER FLpDCs following stimulation with 1 µM CpG-A at indicated time points. (B) Immunoblot analysis of total cell lysates showing the levels of IRF7 phosphorylation at Ser471/472 in 1 µM CpG-A stimulated Ctrl-ER and CKO-ER FLpDCs. (C) Immunoblot analysis showing the levels of phospho-IKKα at Ser176/180, OPN and phospho-AKT at Ser473, in 4-OHT treated and CpG-A stimulated Ctrl-ER and CKO-ER FLpDCs. The quantification of pIKKα was presented by the ratios of pIKKα band intensity vs. IKKα band intensity at each time point. (D) Immunoblot analysis using nuclear extracts of 4-OHT treated Ctrl-ER and CKO-ER FLpDCs showing the levels of p50 and p65 translocation after 1 µM CpG-A treatment at indicated time points. (E) Immunoblot analysis showing the levels of phosphorylated STAT1 at Tyr701 at the indicated time points in Ctrl-ER and CKO-ER FLpDCs treated with 4-OHT and then stimulated with 1 µM CpG-A at the indicated time points. (F) Immunoblot analysis showing levels of phosphorylated STAT1 at Tyr701 in FLpDCs in the presence of 500 U/ml mIFN-α.

    Article Snippet: The purified pDCs were stimulated with 1 µM CpG-A (InvivoGen), CpG-C (InvivoGen), or 2 µg/ml R837 (InvivoGen). cDCs were treated with 50 ng/ml poly(I:C) (InvivoGen) or 10 ng/ml LPS (Sigma-Aldrich) at a density of 1 × 106 cells/ml for the indicated time points.

    Techniques: Activation Assay, Expressing, Translocation Assay

    Type I IFN (IFN-I) production was impaired in CKO-11c mice and in plasmacytoid dendritic cells (pDCs) from CKO-11c mice. (A) ELISA showing the levels of IFN-α production in the serum of Ctrl-11c and CKO-11c mice 6 h after intravenous injection with 5 µg CpG-A + DOTAP or DOTAP alone. (B,C) ELISA determining the levels of IL-6 (B) and TNF-α (C) in serum from Ctrl-11c and CKO-11c mice from panel (A) . (D,E) ELISA determining the levels of IFN-α production at 24 h in medium alone treated, 1 µM CpG-A-stimulated or virus-infected splenic pDCs isolated from Ctrl-11c and CKO-11c mice. (F) ELISA measurement of the levels of IL-6 produced by Ctrl-11c and CKO-11c splenic pDCs at 24 h after treatment with medium alone, 1 µM CpG-A and Japanese encephalitis virus (JEV) at MOI of 10. Data represent the mean ± SEM and were analyzed by two-tailed unpaired Student’s t -test [ n = 3 in DOTAP, 5−7 in CpG-A + DOTAP in (A) , 3−4 in (B,C) , 3 in medium, 6 in CpG-A treated group in (D) , 3 in (E) , and 3−4 in (F) ]. ** p

    Journal: Frontiers in Immunology

    Article Title: Blimp-1-Mediated Pathway Promotes Type I IFN Production in Plasmacytoid Dendritic Cells by Targeting to Interleukin-1 Receptor-Associated Kinase M

    doi: 10.3389/fimmu.2018.01828

    Figure Lengend Snippet: Type I IFN (IFN-I) production was impaired in CKO-11c mice and in plasmacytoid dendritic cells (pDCs) from CKO-11c mice. (A) ELISA showing the levels of IFN-α production in the serum of Ctrl-11c and CKO-11c mice 6 h after intravenous injection with 5 µg CpG-A + DOTAP or DOTAP alone. (B,C) ELISA determining the levels of IL-6 (B) and TNF-α (C) in serum from Ctrl-11c and CKO-11c mice from panel (A) . (D,E) ELISA determining the levels of IFN-α production at 24 h in medium alone treated, 1 µM CpG-A-stimulated or virus-infected splenic pDCs isolated from Ctrl-11c and CKO-11c mice. (F) ELISA measurement of the levels of IL-6 produced by Ctrl-11c and CKO-11c splenic pDCs at 24 h after treatment with medium alone, 1 µM CpG-A and Japanese encephalitis virus (JEV) at MOI of 10. Data represent the mean ± SEM and were analyzed by two-tailed unpaired Student’s t -test [ n = 3 in DOTAP, 5−7 in CpG-A + DOTAP in (A) , 3−4 in (B,C) , 3 in medium, 6 in CpG-A treated group in (D) , 3 in (E) , and 3−4 in (F) ]. ** p

    Article Snippet: The purified pDCs were stimulated with 1 µM CpG-A (InvivoGen), CpG-C (InvivoGen), or 2 µg/ml R837 (InvivoGen). cDCs were treated with 50 ng/ml poly(I:C) (InvivoGen) or 10 ng/ml LPS (Sigma-Aldrich) at a density of 1 × 106 cells/ml for the indicated time points.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay, Injection, Infection, Isolation, Produced, Two Tailed Test

    Inducible deletion of Prdm1 confirmed the important role of Blimp-1 in type I IFN (IFN-I) production in plasmacytoid dendritic cells (pDCs). (A,B) RT-quantitative PCR (RT-qPCR) (A) and immunoblotting (B) showing Blimp-1 mRNA and protein levels in FLpDCs from Ctrl-ER and CKO-ER mice treated with 500 nM 4-OHT and then stimulated with 1 µM CpG-A at indicated time points. The quantitation of Blimp-1 in (B) was presented by the ratios of Blimp-1 band intensity vs. Lamin-B band intensity at each time point. (C) 4-OHT treated FLpDCs cultured from Ctrl-ER and CKO-ER mice were stimulated with 1 µM CpG-A or medium alone for 16 h, followed by ELISA to measure the levels of IFN-α, IL-6, and TNF-α production. (D,E) RT-qPCR showing mRNA levels of IFN-I (D) and proinflammatory cytokines (E) in 4-OHT treated FLpDCs from Ctrl-ER and CKO-ER mice after stimulation with 1 µM CpG-A at indicated time points. Data represent the mean ± SEM and were analyzed by two-tailed unpaired Student’s t -test [ n = 6 in (A) , 3 in medium, 9−14 in CpG-A treatment in (C) , 7 in (D) , and 3−4 in (E) ]. * p

    Journal: Frontiers in Immunology

    Article Title: Blimp-1-Mediated Pathway Promotes Type I IFN Production in Plasmacytoid Dendritic Cells by Targeting to Interleukin-1 Receptor-Associated Kinase M

    doi: 10.3389/fimmu.2018.01828

    Figure Lengend Snippet: Inducible deletion of Prdm1 confirmed the important role of Blimp-1 in type I IFN (IFN-I) production in plasmacytoid dendritic cells (pDCs). (A,B) RT-quantitative PCR (RT-qPCR) (A) and immunoblotting (B) showing Blimp-1 mRNA and protein levels in FLpDCs from Ctrl-ER and CKO-ER mice treated with 500 nM 4-OHT and then stimulated with 1 µM CpG-A at indicated time points. The quantitation of Blimp-1 in (B) was presented by the ratios of Blimp-1 band intensity vs. Lamin-B band intensity at each time point. (C) 4-OHT treated FLpDCs cultured from Ctrl-ER and CKO-ER mice were stimulated with 1 µM CpG-A or medium alone for 16 h, followed by ELISA to measure the levels of IFN-α, IL-6, and TNF-α production. (D,E) RT-qPCR showing mRNA levels of IFN-I (D) and proinflammatory cytokines (E) in 4-OHT treated FLpDCs from Ctrl-ER and CKO-ER mice after stimulation with 1 µM CpG-A at indicated time points. Data represent the mean ± SEM and were analyzed by two-tailed unpaired Student’s t -test [ n = 6 in (A) , 3 in medium, 9−14 in CpG-A treatment in (C) , 7 in (D) , and 3−4 in (E) ]. * p

    Article Snippet: The purified pDCs were stimulated with 1 µM CpG-A (InvivoGen), CpG-C (InvivoGen), or 2 µg/ml R837 (InvivoGen). cDCs were treated with 50 ng/ml poly(I:C) (InvivoGen) or 10 ng/ml LPS (Sigma-Aldrich) at a density of 1 × 106 cells/ml for the indicated time points.

    Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Mouse Assay, Quantitation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    Blimp-1 was induced via Rac activation after TLR7/9 ligand treatment in plasmacytoid dendritic cells (pDCs). (A) RT-quantitative PCR (RT-qPCR) showing Blimp-1 mRNA in human pDCs 24 h after treatment with medium alone, 1 µM CpG-A and H1N1 at a titer of 10 4 TCID50/ml. (B) Blimp-1 protein levels were determined by immunoblotting in human pDCs 24 h after treatment with medium alone or 1 µM CpG-A. (C) ELISA showing the levels of IFN-α produced by human pDCs as described in (A) . (D) Blimp-1-yellow fluorescent protein reporter mice were intravenously injected with DOTAP alone or DOTAP + CpG-A. The frequency of Blimp-1 + pDCs in splenic CD11c int B220 + Siglec-H + Bst2 + gate was examined at indicated time after infection. The frequency of Blimp-1 + pDCs from untreated group (naïve) was shown for comparison. (E) Nuclear Blimp-1 protein levels were detected by immunoblotting in mouse FLpDCs stimulated with medium alone or 1 µM CpG-A at indicated time points. Freshly isolated FLpDCs at 0 h, before addition of medium alone or CpG-A, were also used as the control. (F) RT-qPCR showing the Blimp-1 mRNA levels in Tlr9 knockout (KO) FLpDCs treated with 1 µM CpG-A. (G) RT-qPCR showing Blimp-1 mRNA levels in FLpDCs after 1 h pre-treatment with EHop-016 and further treatment with 1 µM CpG-A for 1 h. (H) RT-qPCR showing the Blimp-1 mRNA levels in Tlr7 KO FLpDCs treated with 2 µg/ml R837 for 1 h. (I) Rac1 activation determined by PAK1 PBD agarose beads pulled down and immunoblotting with antibody against Rac1 in FLpDCs from WT and TLR7 KO mice after stimulation with 2 µg/ml R837. (J) RT-qPCR showing Blimp-1 mRNA expression in FLpDCs after 1 h pre-treatment with EHop-016 and further treatment with 2 µg/ml R837 for 1 h. Data represent the mean ± SEM and were analyzed by two-tailed unpaired Student’s t -test [ n = 3−6 in (A) , 4−7 in (C) , 3 in (F) , 5−6 in (G) , 3 in (H) , and 4−5 in (J) ]. * p

    Journal: Frontiers in Immunology

    Article Title: Blimp-1-Mediated Pathway Promotes Type I IFN Production in Plasmacytoid Dendritic Cells by Targeting to Interleukin-1 Receptor-Associated Kinase M

    doi: 10.3389/fimmu.2018.01828

    Figure Lengend Snippet: Blimp-1 was induced via Rac activation after TLR7/9 ligand treatment in plasmacytoid dendritic cells (pDCs). (A) RT-quantitative PCR (RT-qPCR) showing Blimp-1 mRNA in human pDCs 24 h after treatment with medium alone, 1 µM CpG-A and H1N1 at a titer of 10 4 TCID50/ml. (B) Blimp-1 protein levels were determined by immunoblotting in human pDCs 24 h after treatment with medium alone or 1 µM CpG-A. (C) ELISA showing the levels of IFN-α produced by human pDCs as described in (A) . (D) Blimp-1-yellow fluorescent protein reporter mice were intravenously injected with DOTAP alone or DOTAP + CpG-A. The frequency of Blimp-1 + pDCs in splenic CD11c int B220 + Siglec-H + Bst2 + gate was examined at indicated time after infection. The frequency of Blimp-1 + pDCs from untreated group (naïve) was shown for comparison. (E) Nuclear Blimp-1 protein levels were detected by immunoblotting in mouse FLpDCs stimulated with medium alone or 1 µM CpG-A at indicated time points. Freshly isolated FLpDCs at 0 h, before addition of medium alone or CpG-A, were also used as the control. (F) RT-qPCR showing the Blimp-1 mRNA levels in Tlr9 knockout (KO) FLpDCs treated with 1 µM CpG-A. (G) RT-qPCR showing Blimp-1 mRNA levels in FLpDCs after 1 h pre-treatment with EHop-016 and further treatment with 1 µM CpG-A for 1 h. (H) RT-qPCR showing the Blimp-1 mRNA levels in Tlr7 KO FLpDCs treated with 2 µg/ml R837 for 1 h. (I) Rac1 activation determined by PAK1 PBD agarose beads pulled down and immunoblotting with antibody against Rac1 in FLpDCs from WT and TLR7 KO mice after stimulation with 2 µg/ml R837. (J) RT-qPCR showing Blimp-1 mRNA expression in FLpDCs after 1 h pre-treatment with EHop-016 and further treatment with 2 µg/ml R837 for 1 h. Data represent the mean ± SEM and were analyzed by two-tailed unpaired Student’s t -test [ n = 3−6 in (A) , 4−7 in (C) , 3 in (F) , 5−6 in (G) , 3 in (H) , and 4−5 in (J) ]. * p

    Article Snippet: The purified pDCs were stimulated with 1 µM CpG-A (InvivoGen), CpG-C (InvivoGen), or 2 µg/ml R837 (InvivoGen). cDCs were treated with 50 ng/ml poly(I:C) (InvivoGen) or 10 ng/ml LPS (Sigma-Aldrich) at a density of 1 × 106 cells/ml for the indicated time points.

    Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Produced, Mouse Assay, Injection, Infection, Isolation, Knock-Out, Expressing, Two Tailed Test

    Plexin-A4 is required for TNF and IL-6 production in macrophages. (A) Expression of Plxna4 mRNA in different immune subpopulations was normalized to Actb mRNA level. T cells, B cells, NK cells, BM pDCs, and splenic mDCs were isolated by FACS sorting. BMMs and BMDCs were, respectively, cultured from BM cells in the presence of L929 cell supernatant and GM-CSF plus IL-4. Macrophages were isolated by adherence for 2 h at 37°C. Results are expressed as mean ± SD. (B) Immunofluorescence staining of plexin-A4 and F4/80 in WT and Plxna4 −/− peritoneal macrophages. (C) WT and Plxna4 −/− peritoneal macrophages were stained with IgG control (gray fill) or antibody against mouse plexin-A4 (black lines). Histograms show gated CD11b + cells. The data shown in A–C are representative of two independent experiments. (D and E) Peritoneal macrophages were left unstimulated (−) or were stimulated with 5 µg/ml Pam3Cys, 10 µg/ml poly(I:C), 1 µg/ml ultrapure LPS, 10 µg/ml R837, or 4 µg/ml CpG-B for 4 h. TNF and IL-6 mRNA (D) and protein (E) were measured by RT-PCR and ELISA, respectively. The results shown in D and E are representative of five independent experiments and are expressed as mean ± SD. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Plexin-A4-semaphorin 3A signaling is required for Toll-like receptor- and sepsis-induced cytokine storm

    doi: 10.1084/jem.20101138

    Figure Lengend Snippet: Plexin-A4 is required for TNF and IL-6 production in macrophages. (A) Expression of Plxna4 mRNA in different immune subpopulations was normalized to Actb mRNA level. T cells, B cells, NK cells, BM pDCs, and splenic mDCs were isolated by FACS sorting. BMMs and BMDCs were, respectively, cultured from BM cells in the presence of L929 cell supernatant and GM-CSF plus IL-4. Macrophages were isolated by adherence for 2 h at 37°C. Results are expressed as mean ± SD. (B) Immunofluorescence staining of plexin-A4 and F4/80 in WT and Plxna4 −/− peritoneal macrophages. (C) WT and Plxna4 −/− peritoneal macrophages were stained with IgG control (gray fill) or antibody against mouse plexin-A4 (black lines). Histograms show gated CD11b + cells. The data shown in A–C are representative of two independent experiments. (D and E) Peritoneal macrophages were left unstimulated (−) or were stimulated with 5 µg/ml Pam3Cys, 10 µg/ml poly(I:C), 1 µg/ml ultrapure LPS, 10 µg/ml R837, or 4 µg/ml CpG-B for 4 h. TNF and IL-6 mRNA (D) and protein (E) were measured by RT-PCR and ELISA, respectively. The results shown in D and E are representative of five independent experiments and are expressed as mean ± SD. *, P

    Article Snippet: Pam3Cys, poly(I:C), ultrapure LPS, imiquimod (R837), and mouse CpG-B (ODN 1826) were purchased from InvivoGen.

    Techniques: Expressing, Isolation, FACS, Cell Culture, Immunofluorescence, Staining, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay