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

Abstract

IL-6 is a multifunctional proinflammatory cytokine that is elevated in the serum of patients with pulmonary arterial hypertension (PAH) and can predict the survival of patients with idiopathic PAH (IPAH). Previous animal experiments and clinical human studies indicate that IL-6 is important in PAH; however, the molecular mechanisms of IL-6-mediated pathogenesis of PAH have been elusive. Here we identified IL-21 as a downstream target of IL-6 signaling in PAH. First, we found that IL-6 blockade by the monoclonal anti-IL-6 receptor antibody, MR16-1, ameliorated hypoxia-induced pulmonary hypertension (HPH) and prevented the hypoxia-induced accumulation of Th17 cells and M2 macrophages in the lungs. Consistently, the expression levels of IL-17 and IL-21 genes, one of the signature genes for Th17 cells, were significantly up-regulated after hypoxia exposure in the lungs of mice treated with control antibody but not in the lungs of mice treated with MR16-1. Although IL-17 blockade with an anti-IL-17A neutralizing antibody had no effect on HPH, IL-21 receptor-deficient mice were resistant to HPH and exhibited no significant accumulation of M2 macrophages in the lungs. In accordance with these findings, IL-21 promoted the polarization of primary alveolar macrophages toward the M2 phenotype. Of note, significantly enhanced expressions of IL-21 and M2 macrophage markers were detected in the lungs of IPAH patients who underwent lung transplantation. Collectively, these findings suggest that IL-21 promotes PAH in association with M2 macrophage polarization, downstream of IL-6-signaling. The IL-6/IL-21-signaling axis may be a potential target for treating PAH.

Keywords: M2 macrophage; Th17 cells; interleukin-21; interleukin-6; pulmonary arterial hypertension.

Fig. 1.

Blockade of IL-6 signaling prevents HPH. (A) qRT-PCR analysis of Il-6 mRNA expression in the lungs of C57BL/6 WT mice after hypoxia exposure. Each data point represents the analysis of 5–10 mice. (B) Representative IL-6 immunostaining of lung sections from C57BL6 mice after exposure to hypoxia or normoxia for 2 d (n = 5). (C) Western blot analysis of STAT3 phosphorylation (Tyr705) and total STAT3 in lung homogenates from mice treated with control antibody or MR16-1 after exposure to normoxia or hypoxia for 2 d (n = 3). (D) Experimental protocol for examining the effect of antibody treatment on HPH. MR16-1 or control antibody was administered to C57BL/6 mice under normoxic or hypoxic conditions. (E–G) Assessment of antibody-treated mice. (E) RVSP (n = 8–12). (F) Fulton’s index (n = 8–12). (G) Medial wall thickness index (percent wall thickness) (n = 5–6). Distal acinar arterioles (50–100 μm in diameter) were examined. (H) Representative images of the vascular remodeling of distal acinar arterioles in lung sections subjected to elastic Van Gieson (EVG) staining. (Scale bars: 25 μm.) Br, bronchus; PA, pulmonary artery. Values shown are the mean ± SEM; *P < 0.05, **P < 0.01 calculated using ANOVA.

Fig. 2.

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.

Fig. 3.

IL-21, a downstream target of IL-6, mediates the development of HPH in mice. (A) qRT-PCR analysis of Il-21 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) qRT-PCR analysis of IL-21 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). (C and D) Relative levels of IL-21 protein (normalized to β-tubulin) compared with the normoxic control group. A.U., arbitrary units. (E) Flow cytometric analysis of IL-17A⁺/IL-21⁺ 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). (F and G) Percentage of IL-21⁺ (F) and IL-17A⁺/IL-21⁺ cells (G) within the CD4⁺ population. (H) Experimental protocol for examining the effect of IL-21R deficiency on HPH. (I–K) Assessment of WT and IL-21RKO (KO) mice. RVSP (n = 5–10) (I), Fulton's index (n = 5–10) (J), and percent wall thickness (n = 5) (K) are shown. Distal acinar arterioles (50–100 μm in diameter) were examined. (L) Representative images of vascular remodeling of the distal acinar arterioles, subjected to elastic Van Gieson staining. (Scale bar: 40 μm.) Values shown are the mean ± SEM; *P < 0.05, **P < 0.01 calculated using ANOVA.

Fig. 4.

IL-6 blockade ameliorates the hypoxia-induced generation of M2 macrophages in the murine lung. (A) qRT-PCR analysis of Fizz1 mRNA expression in the alveolar macrophages isolated from the BALF of C57BL/6 WT mice after hypoxia exposure. The results are pooled data from three independent experiments with 6 mice per group. (B–F) qRT-PCR analysis of M2 macrophage marker genes, including Fizz1 (B), Arg1 (C), Chi3l3 (D), Mrc1 (E), and Cxcl12 (F) in the alveolar macrophages isolated from mice treated with control antibody or MR16-1 after exposure to hypoxia or normoxia for 4 d (n = 6). (G) Western blot analysis of Fizz1 in lung homogenates from mice treated with control antibody or MR16-1 after exposure to hypoxia or normoxia for 1 wk (n = 6). (H) Relative Fizz1 protein levels (normalized to β-tubulin level) compared with normoxic mice treated with control antibody. (I) Representative Fizz-1 immunostaining of the alveolar areas to detect the accumulation of M2 macrophages (arrowheads) in mice treated with control antibody or MR16-1 after exposure to hypoxia or normoxia for 1 wk (n = 5). (Scale bars: 25 μm.) (J) Quantification of Fizz1⁺ cells in the alveolar areas. Data shown are the mean number of positive cells per high-power field ± SEM; *P < 0.05, **P < 0.01 calculated using ANOVA.

Fig. 5.

IL-21–induced M2 macrophage polarization is associated with hypoxia-induced pulmonary vascular remodeling. (A–C) qRT-PCR analysis of M2 macrophage markers in IL-21–treated primary mouse alveolar macrophages. (D–F) qRT-PCR analysis of M2 macrophage markers in alveolar macrophages isolated from WT and IL-21RKO mice after exposure to hypoxia for 4 d (n = 6). (G and H) Fizz1 Western blot analysis of lung homogenates from mice exposed to hypoxia for 1 wk (n = 6). (I) Fizz1 immunostaining of lung sections from mice treated with control or an anti–IL-21 neutralizing antibody after exposure to hypoxia for 1 wk (n = 5). (J) Quantification of Fizz1⁺ cells in the alveolar areas. Values are the mean number of Fizz1⁺ cells per high-power field ± SEM. (K) Effects of the conditioned medium (CM) obtained from primary mouse alveolar macrophages cultivated with IL-21 on the proliferation of HPASMCs. (L) Inhibitory effect of AMD3100 on the proliferation of HPASMCs cultured with the conditioned medium of IL-21–treated primary mouse alveolar macrophages. IL-21-CM, conditioned medium of IL-21–treated alveolar macrophages; Med-CM, conditioned medium of control alveolar macrophages. (M) p-HH3 (green) and α-smooth muscle actin (α-SMA) (red) immunostaining of lung sections from mice treated with an anti–mouse IL-21 neutralizing antibody after exposure to hypoxia for 1 wk (n = 5). (N) Percentage of p-HH3⁺ nuclei (arrowheads) in the PASMCs. (Scale bars: 40 μm.) Br, bronchus; PA, pulmonary artery. Values shown are the mean ± SEM; *P < 0.05, **P < 0.01 calculated using ANOVA.

Fig. 6.

Significantly enhanced expression of IL-21 and M2 macrophage markers is detected in the lungs of IPAH patients who underwent lung transplantation. (A, D, and G) Representative IL-21, Arg1, and MRC-1 immunostaining of the pulmonary artery, plexiform lesion, and alveolar area from the lungs of a control patient and patients with IPAH. Arrowheads indicate IL-21⁺ (A), Arg1⁺ (D), and MRC-1⁺ (G) cells. (Scale bars: 100 μm.) (B, E, and H) The numbers of IL-21⁺ (B), Arg1⁺ (E), and MRC-1⁺ (H) cells around one pulmonary artery of a control patient and IPAH patients. The horizontal line in the graph indicates the mean number in each group. (C, F, and I) The numbers of IL-21⁺ (C), Arg1⁺ (F), and MRC-1⁺ (I) cells per high-power field in the alveolar areas of the control patient and IPAH patients. The horizontal line in the graph indicates the mean number in each group.

Fig. 7.

Schematic illustration of the development of PAH through the IL-6/Th17/IL-21–signaling axis. Hypoxia induces the up-regulation of IL-6 in the PAECs (orange) and PASMCs (yellow). IL-6 promotes the differentiation of Th17 cells from naive T cells, presumably in cooperation with TGF-β in the lung. IL-17, derived mainly from Th17 cells, is partly responsible for the production of IL-21 in these cells. IL-21, secreted from CD4⁺ T cells, including Th17 cells, promotes macrophage skewing toward the M2 phenotype in the alveolar macrophages. M2 macrophages positively regulate the hypoxia-induced proliferation of PASMCs by releasing soluble factors such as CXCL12. Taken together, these data show that IL-21 plays a critical role in the pathogenesis of PAH in concert with M2 macrophage polarization downstream of the IL-6/IL-21–signaling axis and may be a potential therapeutic target for treating patients with PAH.