Blockade of IL-6 Trans signaling attenuates pulmonary fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease with progressive fibrosis and death within 2-3 y of diagnosis. IPF incidence and prevalence rates are increasing annually with few effective treatments available. Inhibition of IL-6 results in the attenuation of pulmonary fibrosis in mice. It is unclear whether this is due to blockade of classical signaling, mediated by membrane-bound IL-6Rα, or trans signaling, mediated by soluble IL-6Rα (sIL-6Rα). Our study assessed the role of sIL-6Rα in IPF. We demonstrated elevations of sIL-6Rα in IPF patients and in mice during the onset and progression of fibrosis. We demonstrated that protease-mediated cleavage from lung macrophages was important in production of sIL-6Rα. In vivo neutralization of sIL-6Rα attenuated pulmonary fibrosis in mice as seen by reductions in myofibroblasts, fibronectin, and collagen in the lung. In vitro activation of IL-6 trans signaling enhanced fibroblast proliferation and extracellular matrix protein production, effects relevant in the progression of pulmonary fibrosis. Taken together, these findings demonstrate that the production of sIL-6Rα from macrophages in the diseased lung contributes to IL-6 trans signaling that in turn influences events crucial in pulmonary fibrosis.

FIGURE 1.

sIL-6Rα in IPF and a chronic bleomycin mouse model of pulmonary fibrosis. sIL-6Rα expression was assessed in humans and mice with pulmonary fibrosis. (A and B) ELISA measurement and Western blot analysis of sIL-6Rα in lung lysates from patients with COPD and IPF. (C) Western blot analysis of sIL-6Rα in lung lysates and (D) ELISA measurement of sIL-6Rα in BAL fluid from wild-type C57BL/6 mice given saline or bleomycin, day 33. (E) Sircol analysis of soluble collagen in BAL fluid during development and progression of pulmonary fibrosis in a chronic bleomycin mouse model. Western blot analysis (F) and ELISA quantification (G) of sIL-6Rα in BAL fluid throughout the model. All data are presented as means ± SEM, n ≥ 4. *p < 0.05, **0.001 < p < 0.01, ***p < 0.001 for difference from COPD or PBS-treated cohort.

FIGURE 2.

ADAM17 expression in a mouse model of chronic bleomycin-induced pulmonary fibrosis. Expression of the protease ADAM17 was evaluated in mice with pulmonary fibrosis. Western blot analysis of (A) ADAM17 and (B) ADAM10 in lung lysates from wild-type C57BL/6 mice given saline or bleomycin, day 33. (C) Western blot analysis of ADAM17 in BAL fluid from day 33 mice. (D) Immunostaining for ADAM17 (brown) in the lungs of day 33 mice and (E) immunofluorescence staining for mIL-6R α (green) and DAPI (blue) on BAL fluid cells, day 33. Arrows denote positive cells. Images are representative of n ≥ 4 animals from each group. Scale bars, 50 μm (×100 oil immersion). (F) Western blot analysis of mIL-6Rα in protein lysates made from BAL fluid cell pellets, day 33. (G) Western blot analysis of ADAM17 in BAL fluid as pulmonary fibrosis develops and progresses in a chronic bleomycin-induced mouse model. (H) BAL macrophages throughout the model. Data are presented as means ± SEM, n ≥ 4. *p < 0.05 for difference from PBS-treated cohort.

FIGURE 3.

Pharmacologic neutralization and siRNA-mediated silencing of ADAM17 activity in macrophages. (A) Western blot analysis of mIL-6Rα and arginase 1 expression in bone marrow–derived macrophages stimulated with IL-4 and IL-13. (B) ELISA measurement of sIL-6Rα in culture media of bone marrow macrophages stimulated with PMA, with and without TAPI-1. (C) Western blot analysis of ADAM17 in protein lysates of bone marrow macrophages transfected with control or ADAM17 siRNA. (D) ELISA measurement of sIL-6Rα in culture media of bone marrow macrophages transfected with control or ADAM17 siRNA and then stimulated with PMA. (E) ELISA measurement of sIL-6Rα in culture media of primary lung macrophages stimulated with PMA, with and without TAPI-1. All data are presented as means ± SEM, n ≥ 6 for (B) and (D), n = 1 or 3 for PBS or bleomycin cohorts in (E). ***p < 0.001 for difference from media only cohort, ^###p < 0.001 for difference from PMA-stimulated cohort.

FIGURE 4.

Pulmonary inflammation following chronic bleomycin exposure in mice treated with recombinant gp130Fc. Wild-type C57BL/6 male mice were injected i.p. with saline or bleomycin twice weekly for 4 wk. Beginning on day 19, when pulmonary fibrosis has been established, daily treatment with vehicle (saline) or recombinant gp130Fc was performed. Mice were sacrificed and samples collected on day 33. (A–C) Total cell count and cell differential from BAL fluid of wild-type C57BL/6 mice given saline or bleomycin, with and without gp130Fc. (D) Expression of MCP-1 transcript in whole-lung RNA. Data are presented as means ± SEM, n ≥ 6. *p < 0.05, **0.001 < p < 0.01, ***p < 0.001 for difference from PBS-treated cohort. ^#p < 0.05, ^###p < 0.001 for difference from bleomycin-exposed mice.

FIGURE 5.

Changes in pulmonary fibrosis following chronic bleomycin exposure in mice treated with recombinant gp130Fc. Wild-type C57BL/6 male mice were injected i.p. with saline or bleomycin twice weekly for 4 wk. Beginning on day 19, when pulmonary fibrosis had been established, daily treatment with vehicle (saline) or recombinant gp130Fc was performed. Mice were sacrificed and samples collected on day 33. Lung sections from day 33 mice were stained with (A) Masson’s trichrome for visualization of collagen deposition (blue). Sections are representative of n ≥ 6 mice from each group. Scale bars, 200 μm. (B) Sircol measurement of soluble collagen protein levels in BAL fluid from day 33 mice. (C) Collagen 1A2 transcripts were measured in whole-lung RNA. (D) Pulmonary fibrosis was quantified by the Ashcroft method. All data are presented as mean ± SEM, n ≥ 6. **0.001 < p < 0.01, ***p < 0.001 for difference from PBS-treated cohort. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 for difference from bleomycin-exposed mice.

FIGURE 6.

Changes in pulmonary fibrosis following chronic bleomycin exposure in mice treated with recombinant gp130Fc. Wild-type C57BL/6 male mice were injected i.p. with saline or bleomycin twice weekly for 4 wk. Beginning on day 19, when pulmonary fibrosis has been established, daily treatment with vehicle (saline) or recombinant gp130Fc was performed. Mice were sacrificed and samples collected on day 33. Lung sections from day 33 mice were stained with (A) an Ab against α-SMA for detection of myofibroblast accumulation (red). Sections are representative of n ≥ 6 mice from each group. Scale bars, 200 μm. (B) Western blot analysis of fibronectin expression in whole-lung lysates. (C) Arterial oxygen saturation was measured using a neck collar. All data are presented as means ± SEM, n ≥ 6. ***p < 0.001 for difference from PBS-treated cohort, ^###p < 0.001 for difference from bleomycin-exposed mice.

FIGURE 7.

STAT3 activation following chronic bleomycin exposure in mice treated with mouse recombinant gp130Fc and in IPF lungs. Wild-type C57BL/6 male mice were injected i.p. with saline or bleomycin twice weekly for 4 wk. Beginning on day 19, when pulmonary fibrosis had been established, daily treatment with vehicle (saline) or recombinant gp130Fc was performed. Mice were sacrificed and samples collected on day 33. (A) Expression of p-STAT3 was examined by Western blot analysis of lung lysates and (B) quantified by ImageJ analysis of Western blot images. Data are presented as mean ± SEM. (C) p-STAT3 immunofluorescence (red) in myofibroblasts (green) in lung sections. (D) p-STAT3 immunopositivity (brown) in myofibroblasts (red) in control and IPF lungs. Arrows denote positive cells. Images are representative of n ≥ 4 from each group. Scale bars, 50 μm (×100 oil immersion).

FIGURE 8.

Effect of IL-6 trans signaling on proliferation rates and extracellular matrix protein production in control and IPF fibroblasts. Control and IPF fibroblasts were serum-starved for 24 h and then stimulated for 48 h with IL-6 alone or IL-6 and sIL-6Rα. Proliferation rates and collagen and fibronectin production were assessed. (A) Changes to proliferation rate in response to IL-6 and sIL-6Rα. (B) Western blot analysis of mIL-6Rα in control and IPF fibroblasts at baseline. (C) Western blot analysis of collagen and fibronectin production in normal and IPF fibroblasts in response to IL-6 and sIL-6Rα. (D and E) Collagen and fibronectin production as quantified by ImageJ analysis of Western blot images. All data are presented as means ± SEM, n ≥ 6 for (A), n = 1 or 2 for (D) and (E). **0.001 < p < 0.01, ***p < 0.001 for difference from media only cohort. ^###p < 0.001 for difference from IL-6 only group.

FIGURE 9.

Model of IL-6 trans signaling in pulmonary fibrosis. In fibrotic lungs, elevated ADAM17 expression in M₂ macrophages leads to cleavage of mIL-6Rα to produce sIL-6Rα. sIL-6Rα binds IL-6. The IL-6/sIL-6Rα complex can then activate various cells in the lung in a paracrine manner. (A and B) Stimulation of IL-6 trans signaling in fibroblasts results in 1) increased extracellular matrix protein production and 2) increased proliferation.