Journal: Science Advances

Article Title: Innate immune sensing of dietary alcohol ignites inflammation to drive alcohol-related disease

doi: 10.1126/sciadv.aea3979

Figure Lengend Snippet: ( A and B ) Real-time analysis of cell death in WT (A) pBMDMs and (B) iBMDMs treated with the indicated doses of ethanol (EtOH). The representative images of cell death (red color) at 9 hours are shown (below). Scale bars, 100 μm. ( C ) Heatmap depicting differentially regulated pathways in WT iBMDMs compared with pBMDMs. NES, normalized enrichment scores. ( D ) Real-time analysis of cell death in WT pBMDMs following stimulation with 0.6 M EtOH in the presence of PBS, IFN-γ (50 ng/ml), or IFN-β (20 ng/ml) added 12 hours before EtOH. The representative images of cell death (red color) at 12 hours are shown (right). Scale bar, 100 μm. ( E ) Immunoblot analysis of LDHA and HMGB1 in WT pBMDMs stimulated with EtOH plus IFN-β. Densitometric quantification of the representative blot is shown, relative to the untreated (UT) group. ( F and G ) Real-time analysis of cell death in WT pBMDMs infected with (F) IAV or (G) HSV-1 in the presence or absence of EtOH. ( H ) Real-time analysis of cell death in WT and Ifnar1 −/− pBMDMs treated with EtOH plus IFN-β. Data are representative of at least two (C) or three [(A), (B), (D), (E), (F), (G), and (H)] independent experiments. * P < 0.05, ** P < 0.01, and **** P < 0.0001. Analysis was performed using one-way analysis of variance (ANOVA) [(D), (F), and (G)] and t test (H). Data are shown as mean ± SEM [(A), (B), (D), (F), (G), and (H)].

Article Snippet: Ifnar1 −/− and Zbp1 −/− mice on a C57BL/6J background were purchased from the Jackson Laboratory (stock number 028288) and Cyagen , respectively.

Techniques: Western Blot, Infection

Journal: Science Advances

Article Title: Innate immune sensing of dietary alcohol ignites inflammation to drive alcohol-related disease

doi: 10.1126/sciadv.aea3979

Figure Lengend Snippet: ( A to C ) Immunoblot analysis of (A) pro- (P45) and cleaved (P20) CASP1, pro- (P54) and activated (P34) GSDME, and pro- (P53) and activated (P30) GSDMD; (B) pro- (P55) and cleaved (P18) CASP8, pro- (P35) and cleaved (P19 and P17) CASP3, pro- (P35) and cleaved (P20) CASP7, pro- (P35) and cleaved (P18) CASP6, and pro- (P47) and cleaved (P37 and P35) CASP9; and (C) phosphorylated MLKL (pMLKL), total MLKL (tMLKL), phosphorylated RIPK3 (pRIPK3), and total RIPK3 (tRIPK3) in WT pBMDMs treated with 0.6 M EtOH in the presence of PBS or IFN-β (20 ng/ml). Densitometric quantification of the representative blot is shown, relative to the untreated (UT) group. ( D to F ) Immunoblot analysis of (D) GSDME; (E) CASP8, CASP3, CASP7, and CASP9; and (F) pMLKL, tMLKL, pRIPK3, and tRIPK3 in WT and Ifnar1 −/− pBMDMs stimulated with EtOH plus IFN-β. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as an internal control. Data are representative of at least three independent experiments.

Article Snippet: Ifnar1 −/− and Zbp1 −/− mice on a C57BL/6J background were purchased from the Jackson Laboratory (stock number 028288) and Cyagen , respectively.

Techniques: Western Blot, Control

Journal: Science Advances

Article Title: Innate immune sensing of dietary alcohol ignites inflammation to drive alcohol-related disease

doi: 10.1126/sciadv.aea3979

Figure Lengend Snippet: ( A ) Histopathological analysis, H&E, Oil red O, and TUNEL stain of liver tissues from WT and Ifnar1 −/− mice fed with EtOH diet and injected with IFN-β. Black arrows indicate TUNEL + cells. Scale bars, 400 μm (H&E) and 600 μm (Oil red O and TUNEL). Quantifications of histopathological analyses are shown (right). ( B ) Analysis of serum ALT in WT ( n = 14) and Ifnar1 −/− ( n = 5) mice and IL-1β in WT ( n = 18) and Ifnar1 −/− ( n = 15) mice fed with EtOH diet and injected with IFN-β. ( C ) Heatmap depicting differentially regulated pathways in liver transcriptomes of patients with alcoholic liver disease (ALD) compared with healthy people (control). Data are pooled from two independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001. Analysis was performed using t test and data are shown as mean ± SEM [(A) and (B)].

Article Snippet: Ifnar1 −/− and Zbp1 −/− mice on a C57BL/6J background were purchased from the Jackson Laboratory (stock number 028288) and Cyagen , respectively.

Techniques: TUNEL Assay, Staining, Injection, Control

Journal: Science Advances

Article Title: Inflammatory arthritis irAE may represent a unique autoimmune disease primarily driven by T cells but likely not autoantibodies

doi: 10.1126/sciadv.aea4262

Figure Lengend Snippet: ( A to G ) PBMCs from the patients with irAE were cultured in the plate coated with anti-CD3 and anti-CD28 (10 μg/ml) in the presence of IgG1 isotype control or anti–human IL-6R (50 μg/ml), anti–human IL-12p40 (50 μg/ml), and anti–human IFNAR1 (50 μg/ml) for 3 days; n = 9. (A) Expression of CD38 and CD127 on CD8 + T cells. Right: Percentage of CD38 + CD127 − CD8 + T cells. (B and C) CD8 + T cells activated for 5 days were restimulated with PMA, ionomycin, and monensin for 5 hours. (B) Expression of granzyme B and IFN-γ. Right: Percentage of granzyme B + IFN-γ + CD8 + T cells. (C) Expression of perforin and IFN-γ. Right: Percentage of perforin + IFN-γ + CD8 + T cells. [(D) and (E)] MFIs of GluCy5 (D), TMRM, and MTDR (E) in CD8 + T cells were presented. [(F) and (G)] CD4 + T cells activated for 5 days were restimulated with PMA, ionomycin, and monensin for 5 hours. (F) Expression of IL-21 and IL-2. Right: Percentage of IL-21 + IL-2 + CD4 + T cells. (G) Expression of CD38 and CXCR5 on CD4 + T cells. Right: Percentage of CD38 + CXCR5 − CD4 + T cells. Data in graphs represent mean ± SEM. Significance was tested paired Student’s t test [(A) to (G)].

Article Snippet: PBMCs from patients with irAE were thawed and rested in a complete medium for 1 hour and then activated with plate-coated anti–human CD3 and anti–human CD28 (10 μg/ml) with IgG1 isotype control (Bio X Cell, catalog no. CP174) or a combination of anti–human IL-6R 50 μg/ml; Bio X Cell, catalog no. SIM0014), anti–human IL-12p40 (50 μg/ml; Bio X Cell, catalog no. SIM0020), and anti–human IFNAR1 (50 μg/ml; Bio X Cell, catalog no. SIM0022) for 3 days.

Techniques: Cell Culture, Control, Expressing