Overexpression of OSM and IL-6 impacts the polarization of pro-fibrotic macrophages and the development of bleomycin-induced lung fibrosis - PubMed (original) (raw)
Overexpression of OSM and IL-6 impacts the polarization of pro-fibrotic macrophages and the development of bleomycin-induced lung fibrosis
Ehab A Ayaub et al. Sci Rep. 2017.
Abstract
Although recent evidence indicates that gp130 cytokines, Oncostatin M (OSM) and IL-6 are involved in alternative programming of macrophages, their role in lung fibrogenesis is poorly understood. Here, we investigated the effect of transient adenoviral overexpression of OSM or IL-6 in mice during bleomycin-induced lung fibrosis. Lung fibrosis and M2-like macrophage accumulation were assessed by immunohistochemistry, western blotting, gene expression and flow cytometry. Ex-vivo isolated alveolar and bone marrow-derived macrophages were examined for M2-like programming and signalling. Airway physiology measurements at day 21 demonstrated that overexpression of OSM or IL-6 exacerbated bleomycin-induced lung elastance, consistent with histopathological assessment of extracellular matrix and myofibroblast accumulation. Flow cytometry analysis at day 7 showed increased numbers of M2-like macrophages in lungs of mice exposed to bleomycin and OSM or IL-6. These macrophages expressed the IL-6Rα, but were deficient for OSMRβ, suggesting that IL-6, but not OSM, may directly induce alternative macrophage activation. In conclusion, the gp130 cytokines IL-6 and OSM contribute to the accumulation of profibrotic macrophages and enhancement of bleomycin-induced lung fibrosis. This study suggests that therapeutic strategies targeting these cytokines or their receptors may be beneficial to prevent the accumulation of M2-like macrophages and the progression of fibrotic lung disease.
Conflict of interest statement
The authors declare that they have no competing interests.
Figures
Figure 1
OSM and IL-6 worsen bleomycin-induced increase in lung elastance and bleomycin-induced fibrotic changes. Mice were intubated with AdDL70, AdOSM and AdIL-6, alone, or in combination with bleomycin (0.03 U/mouse). Additional control groups were included, receiving bleomycin plus saline with no adenovectors. Mice were sacrificed following 7 and 21 days of exposure, with the fibrotic outcome was assessed at day 21. (A,B) IL-6 and OSM were assessed in the BALF by ELISA (day 7). (C) Elastance derived from pressure-driven pressure volume loops are shown and graphed as an average value of all the animals per group. (D) Lung collagen content was assessed by Sircol collagen assay. (E) Ashcroft score demonstrating the grade of fibrosis and (F) representative images from Masson’s trichrome and αSMA stained lung sections. Bar graphs represent mean ± SEM from 5-8 mice per group. All samples were derived at the same time and processed in parallel. This figure shows one of two representative experiments. *P < 0.05; **P < 0.01; ***P < 0.001; #P < 0.05; ##P < 0.01; ###P < 0.001; * represent a difference between bleomycin-exposed groups and their respective controls (Saline-Bleo vs. Saline, AdDL70- Bleo vs. AdDL70, AdOSM-Bleo vs. AdOSM, AdIL-6-Bleo vs. AdIL-6); #represent a difference between the indicated groups. Significance was established using GraphPad, Prism 7.0 with one-way ANOVA using Newman-Keuls Multiple Comparison test.
Figure 2
Enhanced production of several mediators in the lungs of bleomycin-exposed mice during IL-6 or OSM overexpression. Following intratracheal intubation of AdDL70, AdOSM and AdIL-6 alone, or in combination with bleomycin (0.03 U/mouse), mice were sacrificed after 7 days. Different cytokine/chemokine mediators were assessed in the BALF, either by ELISA or multiplex assay. The differentially regulated factors include (A) TGFβ1 (B) G-CSF (C) MIG (D) IP-10 (E) MCP-1 and (F) LIF. All other mediators were below the level of detection, and are therefore not included in this figure. Bar graphs represent mean ± SEM from 3-5 mice per group, *P < 0.05; **P < 0.01; ***P < 0.001; #P < 0.05; ##P < 0.01; ###P < 0.001; *represent a difference between bleomycin-exposed groups and their respective controls (AdDL70- Bleo vs. AdDL70, AdOSM-Bleo vs. AdOSM, AdIL-6-Bleo vs. AdIL-6); #represent a difference between the indicated groups. Significance was established using GraphPad, Prism 7.0 with one-way ANOVA using Newman-Keuls Multiple Comparison test.
Figure 3
Increased induction of arginase-1 in the lungs of bleomycin-exposed groups during IL-6 or OSM overexpression. Following intratracheal intubation of AdDL70, AdOSM and AdIL-6, alone, or in combination with bleomycin (0.03 U/mouse), mice were sacrificed after 7 and 21 days. (A,B) Immunoblot images and densitometry analyses of STAT3, pSTAT3 and arginase-1 of lung homogenates following 7 days of exposure. (C–E) Representative arginase-1 immunostaining and quantification as well as arginase-1 mRNA signal in lung tissues following 7 days of exposure. Bar graphs represent mean ± SEM from 3–6 mice per group. All samples were derived at the same time and processed in parallel. The appropriate Western blot area depicting the antibody band was cropped and enclosed by black boxes, as indicated above. *P < 0.05; **P < 0.01; ***P < 0.001; #P < 0.05; ##P < 0.01; ###P < 0.001; *represent a difference between bleomycin-exposed groups and their respective controls (AdDL70- Bleo vs. AdDL70, AdOSM-Bleo vs. AdOSM, AdIL-6-Bleo vs. AdIL-6); #Represent a difference between the indicated groups. Significance was established using GraphPad, Prism 7.0 with one-way ANOVA using Newman-Keuls Multiple Comparison test.
Figure 4
Increased accumulation of CD206+Arg1+ macrophages in the lungs of bleomycin-exposed mice during IL-6 or OSM overexpression. Following intratracheal intubation of AdDL70, AdOSM and AdIL-6, alone, or in combination with bleomycin (0.03 U/mouse) mice were sacrificed after 7 days. Lungs were processed for flow cytometry and AMs derived from BALF cells were isolated by adhesion and subsequently subjected to RNA analysis and Nanostring gene expression. (A) A graph showing absolute numbers of CD45+ CD206+ CD11b+Arg1+ F4/80+ cells. (B,C) graphs showing Arg1 mRNA expression of adhered AMs and arginase-1/DAPI immunofluorescence staining of BALF cells. (D) Il-4rα mRNA expression of adhered AMs. All samples were derived at the same time and processed in parallel. Bar graphs represent mean ± SEM from 3–5 mice per group, *P < 0.05; **P < 0.01; ***P < 0.001; #P < 0.05; ##P < 0.01; ###P < 0.001; *represent a difference between bleomycin-exposed groups and their respective controls (AdDL70- Bleo vs. AdDL70, AdOSM-Bleo vs. AdOSM, AdIL-6-Bleo vs. AdIL-6); #represent a difference between the indicated groups. Significance was established using GraphPad, Prism 7.0 with one-way ANOVA using Newman-Keuls Multiple Comparison test.
Figure 5
IL-6, but not OSM, directly acts on macrophages to potentiate their alternatively activated phenotype. Bone-marrow derived macrophages were cultured from naïve C57BL/6 for either 18 or 30 hours with recombinant OSM, IL-6, IL-4/IL-13, alone or in combination as indicated. AMs were isolated from naïve mice by adherence. BMDMs were either lysed and processed for (A) arginase activity assay, or analysed by flow cytometry to show (B) percentage of arg1+CD206+ macrophages, (C–D) fold change of percentage of IL-4Rα+ cells from the arg1+CD206+ population relative to controls. At least 100,000 events were captured per condition, and repeated twice. (E) Western blot analysis of cell lysates probed for arginase-1, pSTAT6, pSTAT3, and Actin. (F) Densitometry of pSTAT6 (corrected to STAT6), (G) arginase-1 (corrected to actin) and (H) pSTAT3 (corrected to STAT3) represented as a fold-change relative to control. (I) Normalized mRNA counts of Osmrβ and Il6rα from control (unstimulated) BMDMs and AMs. Flow cytometry and western blot results are from experiments completed in duplicates. The appropriate Western blot area depicting the antibody band was cropped and enclosed by black boxes, as indicated above. All samples were derived at the same time and processed in parallel. For Nanostring gene expression, lower than 5 counts was considered not detected “ND”. Bar graphs represent mean ± SEM from 2-3 replicates per group (showing one of two representative experiments), *P < 0.05; **P < 0.01; ***P < 0.001; #P < 0.05; ##P < 0.01; ###P < 0.001; *represent a difference between any sample relative to the control (control vs. IL-4/IL-13, IL-6 vs. IL-4/IL-13 + IL-6); #represent a difference between the indicated groups. Significance was established using GraphPad, Prism 7.0 with One-way ANOVA and non-parametric independent Student’s t-test.
Figure 6
IL-6 increases the expression of pro-fibrotic factors in IL-4/IL-13 stimulated BMDMs. BMDMs were cultured from naïve C57BL/6 for 30 hours with recombinant IL-6 plus IL-4/IL-13. The harvested RNA and cellular supernatant were later examined for gene expression and cytokine/chemokine mediators, respectively. Anti-fibronectin antibody was used to stained lung tissues (Day 7). Significantly regulated pro-fibrotic related genes include (A) Fn1 (B) Mrc1 (C) Timp-1 and (D) Mcp-1. Analysis of mediators showed (E) MCP-1 protein level to be differentially regulated. (F) Representative images showing fibronectin positive staining. Bar graphs represent mean ± SEM from 3–5 samples per group, *P < 0.05; **P < 0.01; ***P < 0.001; #P < 0.05; ##P < 0.01; ###P < 0.001; *represent a difference between any sample relative to the control (control vs. IL-4/IL-13 or IL-4/IL-13/IL-6); #represent a difference between the indicated groups. Significance was established using GraphPad, Prism 7.0 with One-way ANOVA using Newman-Keuls Multiple Comparison test.
Figure 7
An illustration suggesting OSM or IL-6 presence in the lung acts as stimuli to further potentiate fibrotic disease development. Bleomycin exposure initiate epithelial injury, leading to the recruitment of immune cells and pro-fibrotic alternative macrophage polarization. Our data suggest that both gp130 cytokines, OSM or IL-6, have the capacity to enhance the fibrotic response to bleomycin, associated with an increased number of alternatively activated macrophages. We show further that IL-6 have the capacity to act directly on pulmonary macrophages as opposed to OSM, as macrophages do not express the OSM receptor.
References
- Gifford AH, Matsuoka M, Ghoda LY, Homer RJ, Enelow RI. Chronic inflammation and lung fibrosis: pleotropic syndromes but limited distinct phenotypes. Mucosal Immunol. 2012;5:480–484. -PubMed
- Ask, K., Martin, G. E., Kolb, M. & Gauldie, J. Targeting genes for treatment in idiopathic pulmonary fibrosis: challenges and opportunities, promises and pitfalls. Proceedings of the American Thoracic Society3, 389–393, 10.1513/pats.200602-021TK (2006). -PubMed
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