Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Interleukin-6/interleukin-21 signaling axis is critical in the pathogenesis of pulmonary arterial hypertension

doi: 10.1073/pnas.1424774112

Figure Lengend Snippet: IL-6 blockade inhibits accumulation of Th17 cells in the murine lung after hypoxia exposure. (A) qRT-PCR analysis of Il-17A mRNA expression in the lungs of C57BL/6 WT mice after hypoxia exposure. The results are pooled data from at least three independent experiments with 5–10 mice per group. (B–E) qRT-PCR analysis of IL-17A (B), Rorc (C), Cxcl1 (D), and Cxcl5 (E) mRNA expression in the lungs of mice treated with control antibody or MR16-1 after exposure to hypoxia or normoxia for 2 d (n = 8). (F and G) Western blot analysis of IL-17A in the lung homogenates from mice treated with control antibody or MR16-1 after exposure to hypoxia or normoxia for 2 d (n = 6). Relative levels of IL-17A protein (normalized to β-tubulin) compared with the normoxic control group are shown. (H) Flow cytometric analysis of IL-17A–expressing cells in the CD4+ gated T-cell population isolated from the lungs of mice treated with control antibody or MR16-1 after exposure to hypoxia or normoxia for 3 d (n = 6). (I) Percentage of IL-17A+ cells within the CD4+ population. Values shown are the mean ± SEM; *P < 0.05, **P < 0.01 calculated using ANOVA. (J) Experimental protocol for examining the effect of anti–IL-17A antibody treatment on HPH. An anti–IL-17A neutralizing monoclonal antibody or control antibody (IgG) was administered to C57BL/6 mice under hypoxic conditions. (K and L) Assessment of antibody-treated mice. RVSP (n = 6) (K) and Fulton's index (n = 6) (L) are shown. (M–O) IL-17A blockade significantly attenuates the hypoxia-induced IL-21 up-regulation in the murine lungs. (M) qRT-PCR analysis of IL-21 mRNA expression in the lungs of mice treated with control antibody or an anti–IL-17A neutralizing antibody after exposure to hypoxia or normoxia for 2 d (n = 3). (N and O) Western blot analysis of IL-21 in the lung homogenates from mice treated with control antibody or an anti–IL-17A neutralizing antibody after exposure to hypoxia or normoxia for 2 d (n = 3). Values shown are the mean ± SEM; *P < 0.05, **P < 0.01 calculated using ANOVA. NS, not significant.

Article Snippet: To determine the effect of the MR16-1–mediated blockade of IL-6, mice were i.v. injected with 2 mg of MR16-1 or purified rat nonimmune isotype control IgG (MP Biomedicals) just before exposure to hypoxia or normoxia and subsequently were injected i.p. with 0.5 mg of MR16-1 or control IgG, respectively, once a week.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Isolation