Inhibition of ROCK ameliorates pulmonary fibrosis by suppressing M2 macrophage polarisation through phosphorylation of STAT3

Abstract

Background: Emerging evidence provides mechanistic insights into the pathogenesis of pulmonary fibrosis (PF), and rare anti-PF therapeutic method has promising effect in its treatment. Rho-associated coiled-coil kinases (ROCK) inhibition significantly ameliorates bleomycin-induced PF and decreases macrophage infiltration, but the mechanism remains unclear. We established bleomycin and radiation-induced PF to identify the activity of WXWH0265, a newly designed unselective ROCK inhibitor in regulating macrophages.

Methods: Bleomycin-induced PF was induced by intratracheal instillation and radiation-induced PF was induced by bilateral thoracic irradiation. Histopathological techniques (haematoxylin and eosin, Masson's trichrome and immunohistochemistry) and hydroxyproline were used to evaluate PF severity. Western blot, quantitative real-time reverse transcription-polymerase chain reaction and flow cytometry were performed to explore the underlying mechanisms. Bone marrow-derived macrophages (BMDMs) were used to verify their therapeutic effect. Clodronate liposomes were applied to deplete macrophages and to identify the therapeutic effect of WXWH0265.

Results: Therapeutic administration of ROCK inhibitor ameliorates bleomycin-induced PF by inhibiting M2 macrophages polarisation. ROCK inhibitor showed no significant anti-fibrotic effect in macrophages-depleted mice. Treatment with WXWH0265 demonstrated superior protection effect in bleomycin-induced PF compared with positive drugs. In radiation-induced PF, ROCK inhibitor effectively ameliorated PF. Fibroblasts co-cultured with supernatant from various M2 macrophages phenotypes revealed that M2 macrophages stimulated by interleukin-4 promoted extracellular matrix production. Polarisation of M2 macrophages was inhibited by ROCK inhibitor treatment in vitro. The p-signal transducer and activator of transcription 3 (STAT3) in lung tissue and BMDMs was significantly decreased in PF in vivo and vitro after treated with ROCK inhibitors.

Conclusion: Inhibiting ROCK could significantly attenuate bleomycin- and radiation-induced PF by regulating the macrophages polarisation via phosphorylation of STAT3. WXWH0265 is a kind of efficient unselective ROCK inhibitor in ameliorating PF. Furthermore, the results provide empirical evidence that ROCK inhibitor, WXWH0265 is a potential drug to prevent the development of PF.

Keywords: idiopathic pulmonary fibrosis; macrophage; polarisation; radiation-induced pulmonary fibrosis.

FIGURE 1

Inhibition of ROCK ameliorated bleomycin‐induced lung fibrosis. (A) The key structural features of the ROCK inhibitor (WXWH0265). (B) Schematic representation of the experimental protocol for bleomycin exposure, treatment and endpoints in bleomycin‐induced lung fibrosis mice. For bleomycin‐induced mice, 4 mg/kg bleomycin was administered by intratracheal instillation. At the day after bleomycin injection, the ROCK inhibitor WXWH0265 at 10 and 25 mg/kg was administered intragastrically every day. (C–E) After intratracheal instillation at 4 mg/kg bleomycin or saline, the mice were treated with WXWH0265 or saline. The wet weight of lung (C), lung index (D) and hydroxyproline assay in lung (E) on days 7, 14 and 28 are shown. Lung index was referred to lung/body weight ratio. (F) H&E staining of representative mice lung tissues on days 7 and 28. Representative pictures were shown, scale bar = 50 μm. (G) The inflammation changes in the lungs were quantified by a numerical inflammation score (Szapiel score) histopathological. (H) The fibrotic changes in the lungs were quantified by a numerical fibrotic score (Ashcroft score) histopathological. (I–L) The mice were sacrificed on days 7, 14 and 28 and the lung specimens were harvested for immunohistochemical analysis and stained with ROCK1, ROCK2, α‐SMA and collagen‐Ⅰ. The mean optical density of ROCK1 (I), ROCK2 (J), α‐SMA (K) and collagen‐Ⅰ (L), expression in the lung tissue on the days 7, 14 and 28. Data are shown as mean ± SEM. ^* p < .05, ^** p < .01, ^*** p < .001

FIGURE 2

Blockade of ROCK inhibited the M2 macrophages infiltration in bleomycin‐induced fibrotic mice. Mice with intratracheal administration of bleomycin (4 mg/kg) or saline were treated with WXWH0265 (10 or 25 mg/kg) or saline. The mice were sacrificed on days 7, 14 and 28 and the lung specimens were harvested for immunohistochemical analysis and stained with F4/80, Arg‐1 and iNOS. (A) Representative pictures of IHC analysis on day 28 were shown. Scale bar = 50 μm. (B–D) The mean optical density of F4/80 (B), Arg‐1 (C) and iNOS (D) expression in the lung tissue on the days 7, 14 and 28. After the establishment of bleomycin‐induced PF, mice were treated with WXWH0265. On days 7, 14 and 28, the lung tissues and blood were collected and digested for further analyses. (E–H) The infiltration of M2 macrophages (CD45⁺ F4/80⁺ CD206⁺ CD11c^–) in lung tissue and monocytes (CD45⁺ CD11b⁺ Ly6G^– Ly6C^high) in blood was detected by flow cytometry. Flow cytometric cytometry analysis of the percentage of infiltrated M2 macrophages (E and F) and monocytes (G and H) were shown. Flow cytometry dot plots of macrophage, M1/M2 macrophage and alveolar macrophage in lung tissue of the saline‐treated mice on day 7. Data are shown as mean ± SEM. ^* p < .05, ^** p < .01, ^*** p < .001

FIGURE 3

Clodronate liposomes depletes macrophages and WXWH0265 attenuates lung fibrosis via macrophages. C57Bl/6 mice were intratracheally administrated by 4 mg/kg bleomycin on Day 0 ± clodronate liposomes (200 μl) via tail vein on days 1, 7, 14 and 21. Control liposomes was given as a control. (A and B) The infiltration of M2 macrophages (CD45⁺ F4/80⁺ CD206⁺ CD11c^–) and M1 macrophages (CD45⁺ F4/80⁺ CD206^– CD11c^–) in lung on day 28 was detected by flow cytometry. Flow cytometry analysis of the percentage of infiltrated M2 and M1 macrophages. The wet weight (C) and lung index (D) was shown on day 28. (E) H&E staining of representative mice lung tissues on day 28. (F) The inflammation changes in the lungs were quantified by a numerical inflammation score (Szapiel score) histopathological. (G) Masson's staining of representative mice lung tissues on day 28. (H) The fibrotic changes in the lungs were quantified by a numerical fibrotic score (Ashcroft score) histopathological. Representative pictures were shown, scale bar = 50 μm. Data are shown as mean ± SEM. ^* p < .05, ^** p < .01, ^*** p < .001

FIGURE 4

Conditioned medium (CM) collected from BMDMs‐treated lung fibroblast. BMDMs were extracted from the femurs of untreated wild‐type mice treated by M‐CSF and various cytokines in the culture medium. M2 macrophages could be further classified into M2a, M2b and M2c subtypes based on their stimulated response and BMDMs treated with M‐CSF acted as control. BMDMs were stimulated with IL‐4, LPS plus IgG, and IL‐10, respectively. The relative mRNA expressions of CCL24, CCL1 and CXCL13 were quantified by qRT‐PCR. (A) After the macrophages were stimulated for 48 h, the CM from M2a, M2b and M2c was collected. To identified which phenotypes of macrophages could stimulate the fibroblast activation, the CM from M0, M2a, M2b and M2c were used to co‐cultured with the fibroblasts, fibroblasts cultured with CM from M0 macrophages were considered as control. Relative mRNA expressions of α‐SMA, collagen‐1A1 and TGF‐β1 in the lung fibroblasts were quantified by qRT‐PCR (B). (C and D) BMDMs were extracted from the femurs of untreated wild‐type mice treated by M‐CSF and IL‐4 was added at 20 ng/ml in the culture medium. After cultured for 2 h, 10 and 1 μM of WXWH0265 was added into the medium for another 48 h and then the BMDMs were harvested for further analyses. The relative mRNA expressions of CCL24, CD206, Arg‐1, IL‐4, IL‐10 and TGF‐β1 (C) in the BMDM were quantified by qRT‐PCR. The proportion of M2 macrophage (CD45⁺ CD11b⁺ F4/80⁺ CD206⁺) was analysed by flow cytometry (D). WX: WXWH0265; the CM from M2a macrophages treated or untreated with WXWH0265 were co‐cultured with fibroblasts. The relative mRNA expressions of α‐SMA, collagen‐1A1 and TGF‐β1 in the lung fibroblasts were quantified by qRT‐PCR (E). BMDMs treated by M‐CSF and LPS together with IgG was added to the culture medium. After cultured for 2 h, 10 and 1 μM of WXWH0265 was added into the medium for another 48 h and then the BMDMs were harvested for further analyses. The relative mRNA expressions of CCL1 (F) in the BMDM were quantified by qRT‐PCR. BMDMs treated by M‐CSF and IL‐10 was added to the culture medium. After cultured for 2 h, 10 and 1 μM of WXWH0265 was added into the medium for another 48 h and then the BMDMs were harvested for further analyses. The relative mRNA expressions of CXCL13 (G) in the BMDM were quantified by qRT‐PCR. The CM from M2b or M2c macrophages treated or untreated with WXWH0265 were co‐cultured with fibroblasts. The relative mRNA expressions of TGF‐β1 in the lung fibroblasts were quantified by qRT‐PCR (H and I). Data were presented as mean ± SEM of three separated experiments. ^* p < .05, ^** p < .01, ^*** p < .001

FIGURE 5

Inhibition of ROCK ameliorated radiation‐induced lung fibrosis. (A) Schematic representation of the experimental protocol for radiation‐induced lung fibrosis, treatment and endpoints in radiation‐induced lung fibrosis mice. For radiation‐induced pulmonary fibrosis, mice were exposed to a single dose of 18 Gy irradiation on the bilateral thorax. At the day after radiation, the ROCK inhibitor WXWH0265 was given at 10 and 25 mg/kg via administration intragastrical every day. At the endpoint, the lung tissue and blood were collected for further experiments. (B–D) The wet weight of lung (B), lung index (C) and hydroxyproline assay in lung (D) on day 7, week 12, week 16 are shown. Lung index was referred to lung/body weight ratio. (E) Masson's staining of representative mice lung tissues on day 7 and week 16. Representative pictures were shown, scale bar = 50 μm. (F) The inflammation changes in the lungs were quantified with a numerical inflammation score (Szapiel score) histopathological. (G) The fibrotic changes in the lungs were quantified with a numerical fibrotic score (Ashcroft score) histopathological. (H–K) The mice were sacrificed on day 7, week 12 and week 16 and the lung specimens were harvested for immunohistochemical analysis and stained with ROCK1, ROCK2, α‐SMA and collagen‐Ⅰ. The mean optical density of ROCK1 (H), ROCK2 (I), α‐SMA (J) and collagen‐Ⅰ (K), expression in the lung tissue on the day 7, week 12 and week 16. Data are shown as mean ± SEM. ^* p < .05, ^** p < .01, ^*** p < .001

FIGURE 6

Phosphorylation of STAT3 acted as a regulator of M2 macrophage polarisation in vitro. BMDMs were extracted from the femurs of untreated wild‐type mice treated by M‐CSF and IL‐4 was added at 20 ng/ml in the culture medium. After cultured for 2 h, 5 and 1 μM of BB1608 (STAT3 inhibitor) was added into the medium for another 48 h and then the BMDMs were harvested for further analyses (A). The relative mRNA expressions of CD206, Arg‐1, IL‐4, IL‐10 and TGF‐β1 (B) in the BMDMs were quantified by qRT‐PCR. (C) The expression of total‐STAT3, the phosphorylation of STAT3, CD206, Arg‐1 and TGF‐β1 were determined by Western blot analysis on BMDM cells with indicated treatment. β‐Tubulin were used as a loading control. (D) Histograms showing the densitometry analysis of Western protein bands changes in the expression of phosphorylation of STAT3, CD206, Arg‐1 and TGF‐β1. (E) The BMDM cells were stained for p‐STAT3 (red) and CD206 (green) foci. DAPI staining (blue fluorescence), scale bar = 20 μm (magnification, 1000×). (F) Quantitative analysis of immunofluorescence staining for p‐STAT3 (red) and CD206 (green). (G) The proportion of M2 macrophage (CD45⁺ CD11b⁺ F4/80⁺ CD206⁺) was analysed by flow cytometry. Data were presented as mean ± SEM of three separated experiments, ^* p < .05, ^** p < .01, ^*** p < .001

FIGURE 7

Reduced ROCK directs the M2 macrophages via downregulated STAT3 phosphorylation. BMDMs were extracted from the femurs of untreated wild‐type mice treated by M‐CSF and IL‐4 was added at 20 ng/ml in the culture medium. After cultured for 2 h, 10 and 1 μM of WXWH0265 was added into the medium for another 48 h and then the BMDMs were harvested for further analyses. Western blot analysis of the expression of ROCK1, ROCK2, p‐STAT3, STAT3 and TGF‐β1 in BMDMs treated with 20 ng/ml IL‐4 or the ROCK inhibitor WXWH0265 10 or 1 μM for 48 h (A). (B) Histograms showing the densitometry analysis of Western protein bands changes in the expression of ROCK1, ROCK2, p‐STAT3 and TGF‐β1. BMDMs were stimulated with IL‐4, LPS plus IgG and IL‐10, respectively. After cultured for 2 h, 10 and 1 μM of WXWH0265 was added into the medium for another 48 h and then the BMDMs were harvested for further analyses. After intratracheal instillation of bleomycin, mice were treated with WXWH0265 and scarified on day 28. The lung tissues were collected and digested for further analyses. (C) The relative mRNA expression of IL‐4, IL‐10 and TGF‐β1 in the lung on day 28. (D) Western blot analysis of the expression of ROCK1, ROCK2, p‐STAT3, STAT3 in lung tissue of bleomycin‐induced mice on day 28. β‐Tubulin were used as a loading control. BLM: bleomycin + saline; Sal: saline; WH: bleomycin + 25 mg/kg WXWH0265; WL: bleomycin + 10 mg/kg WXWH0265. (E) Histograms showing the densitometry analysis of Western protein bands changes in the expression of ROCK1, ROCK2, p‐STAT3 and TGF‐β1. (F) The relative mRNA expression of IL‐4, IL‐10 and TGF‐β1 in the lung on week 16. (G) Western blot analysis of the expression of ROCK1, ROCK2, p‐STAT3, STAT3 in lung tissue of radiation‐induced mice on week 16. β‐Tubulin were used as a loading control. Con: control; RA: radiation; WH: radiation + 25 mg/kg WXWH0265; WL: radiation + 10 mg/kg WXWH0265. (H) Histograms showing the densitometry analysis of Western protein bands changes in the expression of ROCK1, ROCK2, p‐STAT3 and TGF‐β1.Data were presented as mean ± SEM of three separated experiments *p < .05, ^** p < .01, ^*** p < .001

FIGURE 8

Elementary diagram of the proposed model of macrophages in lung contribution to radiation or bleomycin‐induced pulmonary fibrosis. After radiation or bleomycin stimulation, an increase of monocytes was observed in blood and lung. Several days later, the number of interstitial macrophages (pro‐inflammatory tissue‐infiltrating macrophages) was increased. In the interstitial space (parenchyma), secretion of cytokines such as interleukin‐4 (IL‐4) and IL‐10 is related to the number and the proportion of M2 macrophages derived from interstitial macrophages. The interplay between interstitial macrophages and fibroblasts contributes to pathogenesis of pulmonary fibrosis. WXWH0265, an unselective inhibitor of Rho‐associated coiled‐coil kinases (ROCK), could significantly inhibit the polarisation of M2 macrophages in parenchyma. Inhibiting the polarisation of M2 macrophage derived from interstitial macrophages, the fibrosis score and related markers are reduced