IL-6/gp130 signaling in CD4+ T cells drives the pathogenesis of pulmonary hypertension

Abstract

Pulmonary arterial hypertension (PAH) is characterized by stenosis and occlusions of small pulmonary arteries, leading to elevated pulmonary arterial pressure and right heart failure. Although accumulating evidence shows the importance of interleukin (IL)-6 in the pathogenesis of PAH, the target cells of IL-6 are poorly understood. Using mice harboring the floxed allele of gp130, a subunit of the IL-6 receptor, we found substantial Cre recombination in all hematopoietic cell lineages from the primitive hematopoietic stem cell level in SM22α-Cre mice. We also revealed that a CD4⁺ cell-specific gp130 deletion ameliorated the phenotype of hypoxia-induced pulmonary hypertension in mice. Disruption of IL-6 signaling via deletion of gp130 in CD4⁺ T cells inhibited phosphorylation of signal transducer and activator of transcription 3 (STAT3) and suppressed the hypoxia-induced increase in T helper 17 cells. To further examine the role of IL-6/gp130 signaling in more severe PH models, we developed Il6 knockout (KO) rats using the CRISPR/Cas9 system and showed that IL-6 deficiency could improve the pathophysiology in hypoxia-, monocrotaline-, and Sugen5416/hypoxia (SuHx)-induced rat PH models. Phosphorylation of STAT3 in CD4⁺ cells was also observed around the vascular lesions in the lungs of the SuHx rat model, but not in Il6 KO rats. Blockade of IL-6 signaling had an additive effect on conventional PAH therapeutics, such as endothelin receptor antagonist (macitentan) and soluble guanylyl cyclase stimulator (BAY41-2272). These findings suggest that IL-6/gp130 signaling in CD4⁺ cells plays a critical role in the pathogenesis of PAH.

Keywords: CD4+ T cell; SM22α-Cre; inflammation; interleukin-6; pulmonary arterial hypertension.

Fig. 1.

Gp130 deletion using SM22α-Cre showed a tendency to improve pulmonary hypertension in the HPH mouse model. (A) Experimental protocols and the groups are shown. Male 8-wk-old mice were exposed to a 10% hypoxic chamber for 4 wk. To induce CreERT2-mediated Cre recombination, 1 mg of tamoxifen was injected intraperitoneally on 5 consecutive days. (B) Right ventricular systolic pressure (RVSP) of each group is shown. (C) Right ventricle/left ventricle + septum (RV/LV+S) weight ratio (Fulton’s index) of each group is shown.

Fig. 2.

Unintended Cre recombination in all hematopoietic lineage cells in SM22α-Cre mice. (A) Representative image of immunofluorescence staining of the bone marrow from SMMHC-CreERT2 reporter mice. The arrows indicate smooth muscle cells around arteries in the bone marrow. SMMHC-CreERT2 mice were crossed with Rosa26-loxP-stop-loxP-tdTomato mice. (B) Flow cytometry analysis of hematopoietic tissues from SMMHC-CreERT2 reporter mice. FITC channels were used to remove cells that emit nonspecific autofluorescence. Gates indicate tdTomato-positive cells, and the percentages of cells in each gate are shown in each panel. (C) Representative image of immunofluorescence staining of the bone marrow from SM22α-Cre reporter mice. The arrows indicate smooth muscle cells around arteries in the bone marrow. SM22α-Cre mice were crossed with Rosa26-loxP-stop-loxP-tdTomato mice. (D) Flow cytometry analysis of hematopoietic tissues from SM22α-Cre reporter mice. FITC channels were used to remove cells that emit nonspecific autofluorescence. Gates indicate tdTomato-positive cells, and the percentages of cells in each gate are shown in each panel.

Fig. 3.

Gp130 expression in CD4⁺ T cells was partially abolished in gp130flox/flox; SM22α-Cre mice. (A) Mouse gp130 (IL6ST) expression pattern in hematopoietic lineage cells was retrieved using the mouse hematopoiesis model on Gene Expression Commons (23). (B) Representative flow cytometry analyses of gp130 expression patterns on CD3ε⁺ CD4⁺ T cells from the spleen of Cre(−) control, gp130flox/flox; SM22α-Cre, and gp130flox/flox; CD4-Cre mice are shown. Solid red lines and dashed black lines represent gp130 and background levels, respectively. (C) The percentages of gp130-negative cells in CD3ε⁺ CD4⁺ cells from the spleen of each mouse are shown. (D) Representative flow cytometry analyses of gp130 and IL-6Rα expression patterns on CD3ε⁺ CD4⁺ T cells from the spleen of Cre(−) control, gp130flox/flox; SM22α-Cre, and gp130flox/flox; CD4-Cre mice are shown. (E) Representative flow cytometry analyses of gp130 and IL-6Rα expression patterns on CD3ε⁺ CD4⁺, CD3ε⁺ CD8⁺, and CD3ε⁻ cells from the spleen of Cre(−) control, gp130flox/flox; SM22α-Cre, and gp130flox/flox; CD4-Cre mice are shown. (F) The percentages of gp130⁺ IL-6Rα⁺ cells in CD3ε⁺ CD4⁺ cells from the spleen of each mouse are shown.

Fig. 4.

Gp130 deletion in CD4⁺ T cells ameliorated the PH phenotype of HPH mice. (A) Right ventricular systolic pressure of each group. (B) Right ventricle/left ventricle + septum (RV/LV+S) weight ratio (Fulton’s index) of each group. (C) Representative images of pulmonary arteries with a diameter ~50 µm stained with Elastica van Gieson. (Scale bar: 20 µm.) (D) Phosphorylation of STAT3 (pSTAT3) in CD4⁺ cells from the lungs of each mouse at hypoxia day 3 is shown. (E) Median fluorescence intensity (MFI) of pSTAT3 in CD4⁺ cells from the lungs of each mouse at hypoxia day 3 is shown. (F) Phosphorylation of STAT3 (pSTAT3) in CD4⁺ cells from the spleen of each mouse at hypoxia day 3 is shown. (G) mRNA expression levels of IL-6 family cytokines in the lungs of wild-type C57BL/6 mice after hypoxic exposure are shown. (H) Representative flow cytometry analysis of IL-17A and IL-21 expression in CD4⁺ cells in the lungs at hypoxia day 21. (I and J) Percentages of IL-21⁺ (I) and IL-17A⁺ (J) cells among the CD4⁺ cells at hypoxia day 21.

Fig. 5.

Il6 KO rats are resistant to PH in the SuHx rat model. (A) Experimental protocol for examining the effect of Il6 deletion on SuHx rats. (B and C) Assessment of the effect of Il6 deletion on the PH phenotype of SuHx rats in terms of RVSP (B) and Fulton’s index (C) (n = 8 in each group). (D) Representative images of the vascular remodeling of distal acinar arterioles in lung sections subjected to EVG staining. (Scale bar: 20 μm.) (E) Medial wall thickness index of rats (normoxia WT: n = 6, normoxia Il6 KO: n = 5, SuHx WT: n = 6, SuHx Il6 KO: n = 5). (F) Pulmonary arterial occlusions were graded as open (no luminal occlusion; white), partial (<50% occlusion; blue), or closed (≥50% occlusion; red). Percentages of open, partial, and closed pulmonary arteries of outer diameter (OD) <100 μm in SuHx rats) (n = 4 in each group). (G) qRT-PCR analysis of Il6 mRNA expression in the lungs of WT rats after SuHx treatment. (H and I) Western blot analysis of STAT3 phosphorylation (Tyr705) and total STAT3 in lung homogenates from WT and Il6 KO rats 8 wk after normoxia or SuHx treatment (n = 8 in each group). (J) Representative immunofluorescence images of pulmonary arteries stained for pSTAT3 (red) and CD4 (green), and pSTAT3 (red) and Ahr (green) in the lung tissues of WT and Il6 KO rats 8 wk after SU5416 administration. Arrowheads indicate copositive cells. (K) Z-score of respiration-related genes in CD4⁺ cells. RHC: right heart catheterization, WB: western blotting, IHC: immunohistochemistry. Values are the means ± SEM. ***P < 0.001, **P < 0.01, and *P < 0.05.

Fig. 6.

Il6 deficiency and standard of care vasodilator therapy combination in rats with SuHx-induced PH. (A) Experimental protocol for disease initiation and the macitentan treatment time course. A macitentan-containing diet (30 mg/kg/d) or a control diet was fed to rats every day. (B and C) Assessment of the effect of Il6 deletion combined with macitentan treatment on the PH phenotype of SuHx rats in terms of RVSP (B) and Fulton’s index (C) (normoxia WT: n = 6, normoxia Il6 KO: n = 5, SuHx WT: n = 6, SuHx Il6 KO: n = 6, SuHx+macitentan WT: n = 6, SuHx+macitentan Il6 KO: n = 5). (D) Representative images of the vascular remodeling of distal acinar arterioles in lung sections subjected to EVG staining. (Scale bar: 20 μm.) (E) Vascular occlusion rate of rats (normoxia WT: n = 4, normoxia Il6 KO: n = 4, SuHx WT: n = 5, SuHx Il6 KO: n = 5, SuHx+macitentan WT: n = 5, SuHx+macitentan Il6 KO: n = 5). (F) Experimental protocol for disease initiation and the macitentan treatment time course. A BAY41-2272-containing diet (10 mg/kg/d) or a control diet was fed to rats every day. (G and H) Assessment of the effect of Il6 deletion combined with BAY41-2272 treatment on the PH phenotype of SuHx rats in terms of RVSP (G) and Fulton’s index (H) (n = 6 in each group). (I) Representative images of the vascular remodeling of distal acinar arterioles in lung sections subjected to EVG staining. (Scale bar: 20 μm.) (J) Vascular occlusion rate of rats (normoxia WT: n = 4, normoxia Il6 KO: n = 4, SuHx WT: n = 5, SuHx Il6 KO: n = 5, SuHx+BAY41-2272 WT: n = 5, SuHx+BAY41-2272 Il6 KO: n = 5). Values are the means ± SEM. ***P < 0.001, **P < 0.01, and *P < 0.05.