Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Type III IFNs are produced by and stimulate human plasmacytoid dendritic cells 1

doi: 10.4049/jimmunol.1102038

Figure Lengend Snippet: PBMCs were stimulated with Influenza virus (panel A) or HSV (panel B) for different time periods, and then stained for pDC markers and intracellular expression of IFN-α and IFN-λ expression. Data are representative of 2 separate experiments. C) PBMC were stimulated with HSV-1 for varying times, with BFA added two hours before each time point. Data are representative of two separate donors. D) Kinetics of expression of IFN-α (left panel) and IFN-λ (right panel). Enriched pDC were stimulated with HSV-1 (without the addition at BFA) and supernatants were collected at the indicated time points and tested by ELISA for IFN-α and IFN-λ. Data for two separate donors are shown. E) Summary of IFN-α and –λ in supernatants of purified pDC stimulated with HSV for 18 hr. Data are mean ± 1 SD for 4 separate donors. F) ELISA-determined concentration of IFN-λ in the supernatants of purified pDC after treatment with CpGA or CpGB for 18 hr. Data are mean ± 1 SD for 4 separate donors.

Article Snippet: For some experiments, PE- or FITC-labeled anti-IFN-α (Miltenyi Biotec) was used.

Techniques: Virus, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Purification, Concentration Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Type III IFNs are produced by and stimulate human plasmacytoid dendritic cells 1

doi: 10.4049/jimmunol.1102038

Figure Lengend Snippet: A, B) Co-expression of IFN-α and –λ: After stimulation of PBMC with HSV for 7hr, pDC were stained and the cells were permeabilized and stained for the co-expression of IFN-α and IFN-λ1/3 (Panel A), and for IFN-α and IFN-λ2 (panel B) and for IFN-λ1/3 and IFN-λ2 (panel C). Data are representative of three separate experiments for each panel.

Article Snippet: For some experiments, PE- or FITC-labeled anti-IFN-α (Miltenyi Biotec) was used.

Techniques: Expressing, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Type III IFNs are produced by and stimulate human plasmacytoid dendritic cells 1

doi: 10.4049/jimmunol.1102038

Figure Lengend Snippet: A) PBMC were stimulated with purified HSV-GFP with final stimulation conditions containing <5 pg/ml of IFN-λ, then stained for IFN-λ expression in pDC as described in Figure 2. Data re representative of two similar experiments. B) HSV-1 was pelleted in an ultracentrifuge then PBMC were stimulated with pelleted virus (MOI of 1) or supernatant from the centrifuged virus for 7 hr and intracellular expression of IFN-α and IFN-λ1/3 were determined as described in Figure 3. C) Purified pDC were incubated with HSV for 14 hr with or without a 30 min pre-treatment with IFN-α receptor neutralizating antibody (αRAb, 12.5µg/ml). The amount of IFN-λ (i) and IFN-α (ii) in supernatants was measured by ELISA, with mock and IFN-α receptor neutralization antibody (12.5µg/ml) treatment alone as controls. Data are mean ± 1 SD (n=3). D) Purified pDC (98–99% pure) were treated with 10,000 IU rIFN-α for the times shown; as a control, pDC were stimulated for 24 with HSV-1 or without HSV (MOCK). Supernatants were collected and the levels of IFN-λ were determined by ELISA. Data are representative of two similar experiments.

Article Snippet: For some experiments, PE- or FITC-labeled anti-IFN-α (Miltenyi Biotec) was used.

Techniques: Purification, Staining, Expressing, Virus, Incubation, Enzyme-linked Immunosorbent Assay, Neutralization

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Type III IFNs are produced by and stimulate human plasmacytoid dendritic cells 1

doi: 10.4049/jimmunol.1102038

Figure Lengend Snippet: A) Cross-linking of CD4 or BDCA-2 on pDC inhibits IFN-λ and IFN-α production of PDC: CD4 or BDCA-2 on pDC within PBMC was cross-linked with anti-CD4 or anti-BDCA-2 microbeads, and then PBMC were simulated for 7 hr with HSV. The expression of IFN-α (i) or IFN-λ1 (ii) by pDC was measured by flow cytometry as described above. Data represent mean ± 1 SD for 6 separate experiments. * P<0.05 vs. no crosslinking, ** P<0.01 vs. no crosslinking as determined by ANOVA. B) A TLR9 inhibitor inhibits HSV- but not Flu-induced IFN-λ production of pDC. IFN-λ1 production by pDC in PBMC was measured by flow cytometry after stimulation with HSV (i) and Flu (ii), with or without a TLR9 inhibitory ODN (5.42 µg/ml). Data represent mean ± 1 SD for 6 separate donors. ** P<0.01 vs. sample without CpG inhibitor as determined by ANOVA. C) HSV replication is not required for the induction of IFN-λ in pDC. PBMC were simulated with HSV or UV-HSV. The expression of IFN-λ1 of PDC in PBMC was measured by flow cytometry as described above. Data represent mean ± 1 SD for 3 separate donors.

Article Snippet: For some experiments, PE- or FITC-labeled anti-IFN-α (Miltenyi Biotec) was used.

Techniques: Expressing, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Type III IFNs are produced by and stimulate human plasmacytoid dendritic cells 1

doi: 10.4049/jimmunol.1102038

Figure Lengend Snippet: A) PBMC were treated with either rIFN-α (1000 IU/ml) or IFN-λ1 (10 ng/ml) for 40 minutes; the cells were collected and immediately surface stained with CD123-PE and BDCA2-APC to identify the pDC population, then permeabilized for intracellular staining for the detection of phosphorylated Stat1. Histogram overlays comparing mock (filled) and stimulated pDC (lines). Data are representative of two independent experiments. B) Functional activation of pDC by IFN-λ1 priming: PBMC were primed with IFN-λ1 (10 ng/ml) for 3hr, and then cells were stimulated by HSV for another 4 hr. The percentage and mean fluorescence intensity (MFI) of IFN-α positive (left panels) and IFN-λ positive (right panels) pDC were detected by flow cytometry. Data are mean ± 1 SD for 5 separate donors. C) PBMC were treated with IFN-λ1 (25ng/ml) for 6 hr, then PDC, monocytes, B cells, NK cells, CD4+ T cells, and CD8+ T cells were labeled with specific markers. The expression of HLA-ABC in these subpopulations was measured by flow cytometry with anti-HLA-ABC antibody. (Filled: isotype control; Open: anti-HLA-ABC antibody.) Data are representative of 3 different donors. D) PBMC were treated with IFN-α (1000 U/ml) or IFN-λ1 (25 ng/ml) for 7 hr, and stained with anti-CD83-FITC or anti-HLA-ABC-FITC vs. isotype controls. The mean fluorescence intensities (MFI) of HLA-ABC (i) and CD83 (ii) of PDC after IFN-α or IFN-λ treatment was compared with that of mock for n=3 donors. *p<0.05.

Article Snippet: For some experiments, PE- or FITC-labeled anti-IFN-α (Miltenyi Biotec) was used.

Techniques: Staining, Functional Assay, Activation Assay, Fluorescence, Flow Cytometry, Labeling, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Type III IFNs are produced by and stimulate human plasmacytoid dendritic cells 1

doi: 10.4049/jimmunol.1102038

Figure Lengend Snippet: A) Enriched pDC were treated with 1 µM dexamethasone (Dex) for 1 hr prior to stimulation with HSV-1, HIV-1 or highly purified GFP-HSV for another 18 hr. IFN-λ in supernatants was determined by ELISA. Data represent mean and range for two separate donors. B) Exogenous IFN-λ inhibits apoptosis of pDC. PBMC were treated with Dex, with or without IFN-α (10,000 U/ml) or IFN-λ1 (10ng/ml) for 6 hr. Active caspase-3 in pDC was stained intracellularly in pDC. Data are representative of 3 different donors. C) Annexin V/PI staining was carried out for pDC within PBMC treated with 1 µM Dex in the presence or absence of rIFN-α (100 ng/ml) or –λ1 (10 or 100 ng/ml). Data are representative of three experiments.

Article Snippet: For some experiments, PE- or FITC-labeled anti-IFN-α (Miltenyi Biotec) was used.

Techniques: Purification, Enzyme-linked Immunosorbent Assay, Staining

Journal: Cancer gene therapy

Article Title: Type I IFN gene delivery suppresses regulatory T cells within tumors.

doi: 10.1038/cgt.2014.60

Figure Lengend Snippet: Figure 1. Antitumor effect of intratumoral IFN-α gene transfer. (a) Growth of tumors injected with Ad-mIFN. Tumor volumes were measured at indicated days following the intratumoral injection of Ad-mIFN (n = 6) or Ad-AP (n = 8). Relative tumor volumes compared with those at day10 were presented. Data are shown as means ± standard deviation (s.d.). (b) ELISpot assay of IFN-γ-producing cells in response to stimulation of CT26 cells. Twenty-two days after tumor inoculation, splenocytes were isolated from mice injected with Ad-mIFN (n = 4) or Ad-AP (n = 3), and were cultured with CT26 or syngeneic lymphocytes. Data are presented as means ± s.d. (c) Intracellular cytokine staining of IFN-γ-producing cells in response to CT26 cells. The splenocytes from mice injected with Ad-mIFN (n = 4) or Ad-AP (n = 3) at day 22 were incubated with CT26 cells and stained by anti-mouse IFN-γ antibody. The activated cell fractions were analyzed by staining with anti-mouse CD8 antibody. Representative FACS plots (right panel) are shown.

Article Snippet: The amounts of cytokines in cell culture supernatants and tumors were assayed with antibodies for IL-6 and mouse IFN-α (Quantikine; R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s recommendations.

Techniques: Injection, Standard Deviation, Enzyme-linked Immunospot, Isolation, Cell Culture, Staining, Incubation

Journal: Cancer gene therapy

Article Title: Type I IFN gene delivery suppresses regulatory T cells within tumors.

doi: 10.1038/cgt.2014.60

Figure Lengend Snippet: Figure 2. Intratumoral IFN-α gene transfer reduced the frequency of Tregs in tumors. (a) Frequency of CD4+Foxp3+ Tregs per CD4+ T cells in tumors. Tumors injected with viruses were harvested at days 16, 22 and 28, and processed into single-cell suspension. The percentage of CD4+

Article Snippet: The amounts of cytokines in cell culture supernatants and tumors were assayed with antibodies for IL-6 and mouse IFN-α (Quantikine; R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s recommendations.

Techniques: Injection, Suspension

Journal: Cancer gene therapy

Article Title: Type I IFN gene delivery suppresses regulatory T cells within tumors.

doi: 10.1038/cgt.2014.60

Figure Lengend Snippet: Figure 3. Intratumoral IL-6 concentration was significantly increased by IFN-α gene transfer. (a) IL-6 concentration in tumors. Tumors injected with Ad-mIFN (n = 5) or Ad-AP (n = 5) were harvested at days 16, 22 and 28, and IL-6 concentration was measured by ELISA. (b) Relationship between IL-6 and IFN-α concentration in tumors. Tumors injected with Ad-mIFN (n = 5) or Ad-AP (n = 5) were harvested at day 16, and the concentrations of IL-6 and IFN-α were compared by ELISA. (c) IL-6 production from tumor CD11c+ cells. The CD11c+ and CD11c −cells were isolated from tumors injected with Ad-mIFN (n = 2) or Ad-AP (n = 2) at day 16, and 5 × 104 cells were plated in 96-well plates. After 48 h, supernatants were assayed for the measurement of IL-6 concentration by ELISA. (d) IL-6 production from splenic CD11c+ cells in response to a recombinant IFN-α protein. The CD11c+ and CD11c−cells isolated from naïve splenocytes, and 5 × 104 of CT26 cells were cultured in 96-well plates with depicted concentration of recombinant mouse IFN-α (Miltenyi Biotech). After 48 h, supernatants were assayed for the measurement of IL-6 concentration by ELISA (n = 2 for each group).

Article Snippet: The amounts of cytokines in cell culture supernatants and tumors were assayed with antibodies for IL-6 and mouse IFN-α (Quantikine; R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s recommendations.

Techniques: Concentration Assay, Injection, Enzyme-linked Immunosorbent Assay, Isolation, Recombinant, Cell Culture

Journal: Cancer gene therapy

Article Title: Type I IFN gene delivery suppresses regulatory T cells within tumors.

doi: 10.1038/cgt.2014.60

Figure Lengend Snippet: Figure 4. IL-6 receptor blockade suppressed IFN-α-mediated Treg reduction in tumors. (a) Schema of experiment. The 1000 μg of the monoclonal anti-IL-6 receptor antibody was intraperitoneally injected into the mice at days 7, 14 and 21 after tumor inoculation. Ad-mIFN or Ad-AP was injected once at day 10 after inoculation. (b) Frequency of CD4+Foxp3+ cells per CD4+ T cells in tumors treated with IL-6R ab. Tumors were harvested at day 22, and CD4+ T cells and CD4+Foxp3+ Tregs were analyzed by flow cytometry (n = 5 for the group of Ad-AP i.t. +IL-6R ab i.p., n = 4 for the other groups). (c) Ratio of CD8+ T cells to CD4+Foxp3+ Tregs in tumors. Frequency of CD8+ T cells within whole tumor cells (left panel). Frequency of CD4+Foxp3+ Tregs within whole tumor cells (middle panel). The number of CD8+ T cells was compared with that of CD4+Foxp3+ Tregs in tumors at day 16 (right panel) (n = 4 for the group of Ad-mIFN i.t.+IL-6R ab i.p., n = 6 for the other groups). IL-6R ab, anti-IL-6 receptor antibody; i.t., intratumoral injection; i.p., intraperitoneal administration; TDLNs, tumor-draining lymph nodes.

Article Snippet: The amounts of cytokines in cell culture supernatants and tumors were assayed with antibodies for IL-6 and mouse IFN-α (Quantikine; R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s recommendations.

Techniques: Injection, Cytometry

Journal: Cancer gene therapy

Article Title: Type I IFN gene delivery suppresses regulatory T cells within tumors.

doi: 10.1038/cgt.2014.60

Figure Lengend Snippet: Figure 5. IL-6 receptor blockade partially attenuated IFN-α-mediated tumor growth suppression. (a) Growth of tumors treated with IL-6R ab. Tumor volumes in mice treated with the viruses and/or IL-6R ab were measured at the indicated days (n = 5 for the group of Ad-AP i.t.+IL-6R ab i.p., n = 6 for the other groups). Relative tumor volumes compared with those at day 10 were presented. (b) ELISpot assay of IFN-γ- producing cells in mice treated with IL-6R ab. The splenocytes were isolated from mice as shown in Figure 4a at day 28, and the cells were cultured with CT26 or syngeneic splenocytes (n = 5 for the group of Ad-AP i.t.+IL-6R ab i.p., n = 6 for the other groups).

Article Snippet: The amounts of cytokines in cell culture supernatants and tumors were assayed with antibodies for IL-6 and mouse IFN-α (Quantikine; R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s recommendations.

Techniques: Enzyme-linked Immunospot, Isolation, Cell Culture

Journal: Cancer gene therapy

Article Title: Type I IFN gene delivery suppresses regulatory T cells within tumors.

doi: 10.1038/cgt.2014.60

Figure Lengend Snippet: Figure 6. Intratumoral IFN-α expression increased the number of Th17 cells in tumors. (a) Expression of RORγt and Foxp3 genes in tumors. The tumors injected with viruses were harvested at day 16 and were subjected to real-time PCR analysis (Ad-mIFN: n = 4, Ad-AP: n = 3). (b) Expression of IL-17 in tumors. The tumors were harvested at day 28, and subjected to RT-PCR for IL-17 expression (n = 3 for the group of Ad-AP i.t.+PBS i.p., n = 2 for the group of Ad-mIFN i.t.+PBS i.p., n = 3 for the group of Ad-mIFN i.t.+IL-6R ab i.p.). (c) Intracellular cytokine staining of IL-17A in CD4+ T cells. The tumors and tumor-draining lymph nodes were harvested at day 22, and IL-17A expressions were analyzed by flow cytometry (n = 3 for the group of Ad-AP i.t.+PBS i.p., n = 4 for the group of Ad-mIFN i.t.+PBS i.p., n = 4 for the group of Ad-AmIFN i.t.+IL-6R ab i.p.) (upper panel). Representative FACS plots of tumors (lower left panel) and tumor-draining lymph nodes (lower right panel) were shown.

Article Snippet: The amounts of cytokines in cell culture supernatants and tumors were assayed with antibodies for IL-6 and mouse IFN-α (Quantikine; R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s recommendations.

Techniques: Expressing, Injection, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Staining, Cytometry

Journal: The Journal of Clinical Investigation

Article Title: Human pDCs preferentially sense enveloped hepatitis A virions

doi: 10.1172/JCI77527

Figure Lengend Snippet: (A) pDCs (4 × 105/ml) were exposed to nonenveloped HAV (MOI = 20), either with or without anti-HAV antibody, or cocultured with HM175/p16 virus-infected cells (1 × 106/ml). Supernatant IFN-α was measured at 20 hours by ELISA. CpG DNA (ODN2216, 1 μM) was used as a positive control. LOD, limit of detection. (B) pDCs were exposed to influenza A virus (IAV, 6 HA units/ml), R848 (1 μM), or CpG in the presence or absence of HAV. (C) pDCs from multiple donors were exposed to CpG or HAV or cocultured with mock or HAV-infected Huh-7.5 cells or FRhK-4 cells for 20 hours. Red bars indicate median. (D) pDCs and HAV-infected cells were cocultured in the presence of monoclonal anti-HAV antibody or control IgG, following treatment with either bafilomycin A (BAF, 50 nM) or chloroquine (CLQ, 50 μM) or 1 μM TLR7 antagonist IRS-661 or control IRS. Results are representative of 2 independent experiments. (E) Total or pDC-depleted PBMCs (-pDC) were exposed to 1 μM CpG A or cocultured with mock or HAV-infected Huh-7.5 cells. (F) pDCs were cocultured with cells infected for increasing numbers of days. Supernatant IFN-α (bars) and intracellular HAV RNA (red line) are shown. GE, genome equivalents. (G) pDCs and HAV-infected cells were cocultured with or without separation by a permeable membrane (pore size = 1 μm). Data represent mean ± SEM in A, B, and D–G.

Article Snippet: Supernatant IFN-α levels were determined with the human IFN-α ELISA Kit (PBL Interferon Source) following the manufacturer’s suggested procedure.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Positive Control

Journal: The Journal of Clinical Investigation

Article Title: Human pDCs preferentially sense enveloped hepatitis A virions

doi: 10.1172/JCI77527

Figure Lengend Snippet: (A) Serial dilutions of concentrated supernatant (100,000-g pellet) from HAV-infected cells were mixed with pDCs (1 × 106/ml) and incubated for 20 hours. IFN-α levels (mean ± range in replicate assays) are plotted against HAV RNA content. Unconcentrated supernatant from infected (red arrow and triangle) and mock-infected (white square) cells were tested in parallel. (B) Concentrated supernatant fluids from HAV-infected cells were subjected to isopycnic gradient centrifugation, and individual gradient fractions were incubated with pDCs (1 × 106/ml). HAV RNA content of fractions was determined by RT-qPCR. IFN-α production shown represents results from 3 donors (mean ± SEM). AChE activity was measured by enzyme assay. (C) Correlation (Spearman’s test) between eHAV content of isopycnic gradient fractions, shown as genome equivalent per pDC, and IFN-α produced (mean ± range in replicate assays). (D) Northern blot of HAV RNA: full-length in vitro–transcribed HM175/18f HAV RNA, RNA extracted from peak isopycnic gradient fractions containing eHAV, or nonenveloped HAV (mean ± range in replicate assays). (E) Consecutive gradient fractions containing eHAV from a gradient similar to that in B were pooled, concentrated, and subjected to rate-zonal ultracentrifugation. Fractions were collected from the top and assessed for HAV RNA content, AChE activity, and pDC stimulating activity (P105 and P106 indicate individual donors) (mean ± range in replicate assays). Representative results from 1 of 3 independent experiments are shown. Western blots of ALIX and flotillin-1 in the rate-zonal gradient fractions are shown.

Article Snippet: Supernatant IFN-α levels were determined with the human IFN-α ELISA Kit (PBL Interferon Source) following the manufacturer’s suggested procedure.

Techniques: Infection, Incubation, Gradient Centrifugation, Quantitative RT-PCR, Activity Assay, Enzymatic Assay, Produced, Northern Blot, In Vitro, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Human pDCs preferentially sense enveloped hepatitis A virions

doi: 10.1172/JCI77527

Figure Lengend Snippet: (A) pDCs were exposed to equal quantities of gradient-purified eHAV or nonenveloped HAV, and cell-associated viral RNA and supernatant IFN-α levels were determined at intervals. Circles and squares represent cells from 2 individual donors, respectively. (B) pDCs were incubated with eHAV in the presence or absence of annexin V (2 or 10 μg/ml), and cell-associated viral RNA was determined at intervals by RT-qPCR. (C) IFN-α produced by pDCs exposed to concentrated supernatant fluids from mock- or HM175/p16-infected cell cultures in the presence or absence of annexin V (2 μg/ml). (D) IFN-α produced by pDCs exposed to gradient-purified eHAV following inactivation by UV light or cultured with eHAV in the presence of 100 μM HAV 3Cpro inhibitor (3C-inh) (EC90 = 62 μM, CC50 = 3 mM). Efficiency of UV inactivation and 3C inhibitor treatment. Huh-7.5 cells were infected with UV-inactivated eHAV or nontreated eHAV in the presence of 3C-inh, and cell-associated HAV RNA was measured by RT-qPCR and compared with that in cells infected with untreated HAV at 48 hours. (E) pDCs were transfected with HM175/18f genomic RNA in the presence and absence of IRS-661 or subgenomic HAV-luc RNA or a replication incompetent variant HAV-Luc-Δ3D. Supernatant IFN-α levels were measured by ELISA. Results represent the mean ± SEM (n = 2 or 3 cultures) obtained with pDCs from single donors.

Article Snippet: Supernatant IFN-α levels were determined with the human IFN-α ELISA Kit (PBL Interferon Source) following the manufacturer’s suggested procedure.

Techniques: Purification, Incubation, Quantitative RT-PCR, Produced, Infection, Cell Culture, Transfection, Variant Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Clinical Investigation

Article Title: Human pDCs preferentially sense enveloped hepatitis A virions

doi: 10.1172/JCI77527

Figure Lengend Snippet: (A) Serum IFN-α and changes in intrahepatic ISG15 and IFIT1 mRNA abundance are shown together with levels of intrahepatic HAV RNA in a chimpanzee, 4x0293, experimentally infected with HAV. The ISG15 response and virologic events have been described previously (12). Sections of archived liver specimens collected from chimpanzee 4x0293 at indicated times prior to and after intravenous challenge with HAV were stained with rabbit polyclonal antibody to human BDCA2. (B) Serum IFN-α and ISG15 and IFIT1 mRNA and ALT activities and intrahepatic HAV RNA in chimpanzee 4x0395 that was similarly infected with HAV (12). Sections of archived liver biopsies from 4x0395 were stained with murine monoclonal antibody 15B to human BDCA2. Scale bars: 200 μm.

Article Snippet: Supernatant IFN-α levels were determined with the human IFN-α ELISA Kit (PBL Interferon Source) following the manufacturer’s suggested procedure.

Techniques: Infection, Staining

Journal: The Journal of Experimental Medicine

Article Title: NFATC3 promotes IRF7 transcriptional activity in plasmacy­­toid dendritic cells

doi: 10.1084/jem.20160438

Figure Lengend Snippet: Identifying IRF7 binding proteins and investigating their roles in IFN-α production. (A) Gen2.2 cells were left untreated or treated with 1 µM CpG A for 6 h, and then the cells were pooled together, lysed, and immunoprecipitated with anti-IRF7 antibody or isotype control. IRF7 binding proteins were analyzed by mass spectrometry. (B) Selected IRF7 binding proteins and the peptides hit in mass spectrometry are listed. (C) IRF7-associated genes were knocked down by using siRNA targeting specific molecules or nonspecific siRNA control (siNS), and the knockdown cells were stimulated with 1 µM CpG A for 20 h. IFN-α production in the supernatants was analyzed by ELISA. Data are representative of two independent experiments and are presented as the mean ± SEM of triplicate wells. *, P < 0.05; **, P < 0.01 by Student’s t test.

Article Snippet: The mouse IFN-α ELISA kit was from eBioscience.

Techniques: Binding Assay, Immunoprecipitation, Control, Mass Spectrometry, Knockdown, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: NFATC3 promotes IRF7 transcriptional activity in plasmacy­­toid dendritic cells

doi: 10.1084/jem.20160438

Figure Lengend Snippet: Knockdown of NFATC3 reduces CpG DNA–induced IFN-α in Gen2.2 cells. (A) Gen2.2 cells were knocked down by using nonspecific siRNA (si-NS) or siRNA targeting MyD88, NFATC1–4, and NFAT5. The knockdown efficiency was analyzed by immunoblotting. (B) siRNA knockdown Gen2.2 cells were stimulated with 1 µM CpG A for 24 h. IFN-α, TNF-α, and IL-6 in supernatants were analyzed by ELISA. Data are representative of four independent experiments and are presented as the mean ± SEM of triplicate wells. *, P < 0.05; **, P < 0.01 by Student’s t test.

Article Snippet: The mouse IFN-α ELISA kit was from eBioscience.

Techniques: Knockdown, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: NFATC3 promotes IRF7 transcriptional activity in plasmacy­­toid dendritic cells

doi: 10.1084/jem.20160438

Figure Lengend Snippet: Inhibition of NFAT reduces CpG DNA–induced IFN-α in human primary pDCs. (A) Human primary pDCs stimulated with 1 µM CpG A for 24 h together with FK506 or vehicle (DMSO). Production of IFN-α was measured by ELISA. (B and C) Human primary pDCs stimulated with 1 µM CpG B for 24 h together with FK506 or vehicle (DMSO). Production of TNF-α and IL-6 were measured by ELISA. (D) Human primary pDCs stimulated with 1 µM CpG A for 24 h together with Cs A or vehicle (DMSO). Production of IFN-α was measured by ELISA. (E and F) Human primary pDCs stimulated with 1 µM CpG A for 24 h together with Cs A or vehicle (DMSO). Production of TNF-α and IL-6 was measured by ELISA. Data are representative of four independent experiments.

Article Snippet: The mouse IFN-α ELISA kit was from eBioscience.

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: NFATC3 promotes IRF7 transcriptional activity in plasmacy­­toid dendritic cells

doi: 10.1084/jem.20160438

Figure Lengend Snippet: Knockout of NFATC3 reduces CpG DNA–induced IFN-α and inhibits IRF7 nuclear translocation, and NFATC3 restoration rescues the responses. (A–C) CRISPR knockout Gen2.2 cells were stimulated with different doses of CpG A. NT guide RNA–transduced cells were used as controls. Production of IFN-α, TNF-α, and IL-6 were measured by ELISA. P-values are NT compared with NFATC3 knockout Gen2.2 cells. (D) CRISPR knockout Gen2.2 cells were stimulated with CpG A for 3 h. IRF7 and p50 nuclear translocation were analyzed by immunoblotting. Anti-transferrin receptor (Tfr) antibody and anti-HDAC1 antibody were used to show the cytoplasmic fraction and nuclear fraction. (E) NFATC3 knockout Gen2.2 cells that were stably transduced with mutated NFATC3 expression lentiviral particles that contained the mutated guide RNA–targeting sequence (NFATC3a) to restore NFATC3 expression. The lentiviral particles that contained empty lentiviral vector were used as a control. NFATC3 expression level was measured by immunoblotting. (F) NFATC3 knockout and NFATC3-restored Gen2.2 cells were stimulated with CpG A for 24 h. Production of IFN-α was measured by ELISA. Data are presented as the mean ± SEM of triplicate wells and are representative of three independent experiments. *, P < 0.05; **, P < 0.01 by Student’s t test.

Article Snippet: The mouse IFN-α ELISA kit was from eBioscience.

Techniques: Knock-Out, Translocation Assay, CRISPR, Enzyme-linked Immunosorbent Assay, Western Blot, Stable Transfection, Transduction, Expressing, Sequencing, Plasmid Preparation, Control

Journal: The Journal of Experimental Medicine

Article Title: NFATC3 promotes IRF7 transcriptional activity in plasmacy­­toid dendritic cells

doi: 10.1084/jem.20160438

Figure Lengend Snippet: pDCs from Nfatc3 −/− mice show impaired IFN-α production in response to CpG A. (A) Mouse primary pDCs were isolated from wild-type or Nfatc3 −/− mice. Expression of NFATC3 and IRF7 were analyzed by immunoblotting. (B and C) pDCs from wild-type or Nfatc3 −/− mice stimulated with different concentrations of CpG A (D19) for 24 h. IFN-α and IL-12p40 in supernatants were measured by ELISA. (D–K) Bone marrow–derived pDCs from wild-type or Nfatc3 −/− mice were left unstimulated or stimulated with 1 µM CpG A for 20 h, and Irf7 , Ifna1 , Ifna2 , Ifna4 , Ifna5 , Ifna6 , Ifnb1 , and IL12a gene expression were detected by RT-PCR. (L and M) Bone marrow–derived pDCs from wild-type or Nfatc3 −/− mice were left unstimulated or stimulated with 10 µg/ml R848 for 20 h, and IFN-α and IL-12p40 were measured by ELISA. (N and O) Wild-type or Nfatc3 −/− mice were injected with CpG A for 6 h. IFN-α and IL-12p40 in serum were measured by ELISA. Data are representative of three independent experiments and are presented as the mean ± SEM of triplicate wells. *, P < 0.05; **, P < 0.01 by Student’s t test.

Article Snippet: The mouse IFN-α ELISA kit was from eBioscience.

Techniques: Isolation, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Derivative Assay, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Injection

Journal: Nature immunology

Article Title: High density lipoprotein mediates anti-inflammatory transcriptional reprogramming of macrophages via the transcriptional repressor ATF3

doi: 10.1038/ni.2784

Figure Lengend Snippet: HDL inhibits TLR-induced cytokine production from macrophages in vivo and in vitro. ( a – d ) Mice were injected intraperitoneally (i.p.) with 2 mg HDL for 6 h before i.p. injection of TLR ligand (CpG: 20 μg, P3C 2 μg) and D-galactosamine (10 mg). Serum cytokines were measured after 1 h and serum alanine aminotransferase (ALT) and liver histology after 10 h . a C3H/HeJ mice injected with native HDL and CpG: ALT release ( n =12) and serum cytokines (CpG n= 28, native HDL+CpG n = 27) were measured. b , c C57BL/6 mice injected with HDL and CpG: ALT release ( n =9); serum cytokines (CpG n =18, HDL+CpG n =19) ( b ), and liver histology by hemotoxylin and eosin staining shows hepatic cell death. ( c ). d , C57BL/6 injected with HDL and P3C: ALT release ( n =15) and serum cytokines ( n =15) were measured. e , ELISA of BMDMs treated with 2 mg/ml HDL for 6 h followed by overnight stimulation with the TLR4 ligand, LPS (100 ng/ml); TLR9 ligand, CpG (100 nM); TLR7/8 ligand, R848 (5 ng/ml); or TLR2/1 ligand P3C (50 ng/ml). f , Cell viability was measured with CellTitre- Blue® reagent. g , BMDMs were pre-treated with recombinant or native HDL (2 mg/ml) for 6 h and stimulated overnight with CpG (100 nM) or P3C (50 ng/ml) and IL-6 cytokine secretion measured by ELISA. a , b , d Data are presented as mean values ±S.E.M, CpG versus HDLs+CpG *p<0.05, **p<0.01, ***p<0.005, c , images are representative of mice with median ALT concentration; scale bars, 100 μm, e , f , g data is shown as mean ±S.D. and is representative of at least three independent experiments.

Article Snippet: Murine IL-6, IL-12p40 and TNF, and human IL-6 and TNF ELISA kits were from R & D Systems; the human IFN-α ELISA kit was from eBiosciences.

Techniques: In Vivo, In Vitro, Injection, Staining, Enzyme-linked Immunosorbent Assay, Recombinant, Concentration Assay

Journal: Nature immunology

Article Title: High density lipoprotein mediates anti-inflammatory transcriptional reprogramming of macrophages via the transcriptional repressor ATF3

doi: 10.1038/ni.2784

Figure Lengend Snippet: HDL inhibits TLR-induced pro-inflammatory cytokine transcription. a , Bodipy-labeled LPS, Alexa 647-labeled CpG 1826 or HDL were run over an S200 size exclusion column separately ( a , upper panel) or together ( a , lower panel) and absorbance profiles examined. b , LPS (200 ng/ml), CpG (100 nM) or P3C (50 ng/ml) were incubated with HDL (2 mg/ml), and BMDMs stimulated for 30 min. Whole cell lysates were analyzed for p38 phosphorylation (p-p38) relative to total β-actin. c , d BMDMs were pre-treated with HDL (2 mg/ml) for 6 h before stimulation with CpG (100 nM) for indicated times: total cellular cholesterol ( c ) and phosphorylation of p38, JNK, NF-κB p65 and IκBα degradation ( d ). e , f BMDMs were pre-treated with HDL as before, and stimulated with CpG (100 nM) for 30 min: subcellular localization of NF-κB p65 (β-tubulin: cytoplasmic loading control; poly ADP ribose polymerase (PARP): nuclear loading control) ( e ) and NF-κB binding to a target probe as analysed by EMSA ( f ). g , h BMDMs were pre-treated with HDL as before: CpG-induced phospho-NF-κB p65 and intracellular IL-6 and IL-1β were measured in whole cell extracts, ( g ); secreted IL-6 measured by ELISA ( h ). mRNA expression of BMDMs pre-treated with HDL as before, and stimulated with 100 nM CpG for 4 h ( i ). j , Liver mRNA profile 1 h after C57BL/6 mice were injected with CpG following 6 h HDL pre-treatment ( n =8). a , Data is representative of at least three independent experiments. b , Representative immunoblot and densitometric analysis combined from three independent experiments (mean ±S.E.M, each ligand; no HDL versus HDL incubation ***p<0.005). c – i , Representative data from at least three independent experiments (mean ±S.D). j , Mean values ±S.E.M, CpG versus HDL+CpG *p<0.05.

Article Snippet: Murine IL-6, IL-12p40 and TNF, and human IL-6 and TNF ELISA kits were from R & D Systems; the human IFN-α ELISA kit was from eBiosciences.

Techniques: Labeling, Incubation, Phospho-proteomics, Control, Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, Injection, Western Blot

Journal: Nature immunology

Article Title: High density lipoprotein mediates anti-inflammatory transcriptional reprogramming of macrophages via the transcriptional repressor ATF3

doi: 10.1038/ni.2784

Figure Lengend Snippet: Microarray analysis identifies ATF3 as a candidate gene for the anti-inflammatory function of HDL. a , Immortalized-BMDMs were pre-treated with HDL (2 mg/ml) for indicated times, then either washed twice in serum-free DMEM, or left unwashed, before overnight stimulation with CpG (100 nM). TNF secretion was measured by ELISA and normalized to CpG treatment alone. b , BMDMs were pre-treated with HDL (2 mg/ml) for 12 h, then either washed twice in serum-free DMEM, or left unwashed, before stimulation with CpG (100 nM) for indicated times. IL-6 was measured in culture supernatants and normalized to CpG treatment alone. c – f , Microarray analysis of BMDMs pre-treated for 6 h with HDL (2 mg/ml) then stimulated with CpG (100 nM) for 4 h. c , Venn diagram shows genes with differential expression (vs control) and the overlap in these genes (Fold Change limit 1.8, False discovery rate p<0.05). d , A fold change/fold change plot shows directional gene expression resulting from CpG treatment (red), CpG and HDL treatment (blue) or genes that are co-regulated in both treatments (purple). e , Expression of transcription factors following HDL, CpG or combined treatment as described in the methods and presented as a heat map. TFs are ranked according change in expression across treatments, while color intensity shows the mean expression value per condition. a,b , Representative data from at least three independent experiments (mean ±S.D) c – e , At least three biological replicates per condition were generated.

Article Snippet: Murine IL-6, IL-12p40 and TNF, and human IL-6 and TNF ELISA kits were from R & D Systems; the human IFN-α ELISA kit was from eBiosciences.

Techniques: Microarray, Enzyme-linked Immunosorbent Assay, Quantitative Proteomics, Control, Gene Expression, Expressing, Generated

Journal: Nature immunology

Article Title: High density lipoprotein mediates anti-inflammatory transcriptional reprogramming of macrophages via the transcriptional repressor ATF3

doi: 10.1038/ni.2784

Figure Lengend Snippet: ATF3 is required for the anti-inflammatory effect of HDL in vitro and in vivo . a , ELISA from WT or Atf3 -deficient BMDMs pre-treated with HDL (2 mg/ml) for 6 h prior to overnight stimulation with CpG (indicated doses). b , WT or Atf3 -deficient mice injected i.p with HDL (2 mg) 6 h before subsequent injection with CpG (30 μg) and D-gal (10 mg) for a further 10 h. Serum ALT, serum TNF and hepatic TNF (WT n= 10, Atf3 −/− n= 8,), and serum IL-12p40 ( n= 6) were measured. c , d , HDL increased re-endothelialization after carotid artery injury in WT, but not Atf3- deficient mice. Carotid injury was performed on WT or Atf3- deficient mice, 3 h later PBS or HDL (20 mg/kg) was injected i.v. Re-endothelialization was evaluated 3 days following carotid injury (WT PBS n= 8, WT HDL n= 7, Atf3 −/− PBS n= 7, Atf3 −/− HDL n= 9). e Gene set enrichment analysis using the macrophage/carotid injury overlapping gene set applied to the carotid injury dataset assessing gene enrichment in HDL treated samples derived from WT versus Atf3 −/− mice. a , Combined data from three independent experiments are shown as the mean ±S.E.M (WT versus Atf3 −/− *p<0.05, **p<0.01). b , Data are presented as mean values ±S.E.M, CpG versus HDL+CpG (per genotype) *p<0.05, ***p<0.001. c , Data are presented as mean values ±S.E.M, PBS vs HDL (per genotype) **p<0.01. d , Images are representative of each group in each genotype. e , At least three biological replicates per condition were generated.

Article Snippet: Murine IL-6, IL-12p40 and TNF, and human IL-6 and TNF ELISA kits were from R & D Systems; the human IFN-α ELISA kit was from eBiosciences.

Techniques: In Vitro, In Vivo, Enzyme-linked Immunosorbent Assay, Injection, Derivative Assay, Generated