IL-27 promotes cardiac fibroblast activation and aggravates cardiac remodeling post myocardial infarction

Figures

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  Fig. 1. IL-27 expression in the injured heart after MI. a) Flow chart of the experimental treatments. b) ELISA of IL-27 protein expression in serum and heart from C57BL/6 mice at various time points after MI. c) p28 and EBI3 mRNA levels in the LV at 7 and 14 days after MI (n = 8). For qPCR, all data were analyzed using the 2−ΔΔct formula. d) The expression of WSX-1 and gp130 mRNA in the LV at 7 and 14 days after MI (n = 8). All sham samples were collected at day 1 post sham operation. All data are shown as the mean ± SEM. Each experiment was repeated at least three times. *P < 0.05; **P < 0.01; ***P < 0.001 vs. sham.

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  Fig. 2. Elevation of IL-27 in macrophages after MI. a) Representative immunofluorescence images of the expression of IL-27 (red) and CD45+ (green) cells in the heart at 7 days after MI. The nuclei were stained with DAPI. Scale bar = 20 μm. b) Quantified data of immunofluorescence images in (a). c) Representative immunofluorescence images of the expression of IL-27 (red) and F4/80+ (green) cells in the heart at 7 days after MI. The nuclei were stained with DAPI. Scale bar = 20 μm. d) Quantified data of IL-27 + F4/80+ cell percentages in (c). e) Representative immunofluorescence images of the expression of IL-27 (red) and F4/80+ (green) cells or vimentin+ (green) cells in the heart at 14 days after MI. The nuclei were stained with DAPI. Scale bar = 20 μm. For quantitative analysis of the immunofluorescence staining results, we counted 5 random fields in each slide. f) p28 and EBI3 mRNA levels in CD45+ F4/80+ macrophages that were sorted by flow cytometry at 7 days after MI. Data are shown as the mean ± SEM. Each experiment was repeated at least three times. *P < 0.05; **P < 0.01; ***P < 0.001 vs. control. (For interpretation of the references to colour in this figure legend, the reader is…

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  Fig. 3. The expression of IL-27 in serum is associated with cardiac function and cardiac fibrosis. a) Serum IL-27 was significantly associated with LVEF after MI. b) Serum IL-27 was significantly correlated with heart Col l mRNA expression after MI. c) Serum IL-27 was significantly associated with the area of cardiac fibrosis by Masson staining after MI. For quantitative analysis of the Masson staining, we counted six random fields of each slide for each mouse. The percentage of the fibrosis area (blue) in each section was calculated by ImageJ software. Data are expressed as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

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  Fig. 4. Effect of IL-27 on the proliferation and migration of cardiac fibroblasts induced by Ang II. a) The mRNA expression of p28 and EBI3 in cardiac fibroblasts treated with Ang II (1 μm) for 0, 6, 12, 18, 24, and 48 h. b) The mRNA expression of WSX-1 and gp130 in cardiac fibroblasts treated with Ang II (1 μm) for 0, 6, 12, 18, 24, and 48 h. c) Cell viability of cardiac fibroblasts was measured in different groups by CCK-8 assay. d) Representative immunofluorescence images of the expression of Ki67 (green) protein in different groups. The nuclei were stained with DAPI. Scale bar = 200 μm. e) Statistical results of Ki67+ cell percentages in (d). f) Cell migration distance in different groups at 12 or 24 h after scratching. g) Quantification of scratch widths in separate experiments at 12 h. Wound closure distance (%) = [(the initialized width of the scratch) - (the final width of the scratch)]/(the initialized width of the scratch). h) Quantification of scratch widths in separate experiments at 24 h. The images used for the statistics are the six random fields of the scratch widths captured for statistics. Values are the mean ± SEM from at least three experiments. *P < 0.05; **P < 0.01;…

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  Fig. 5. Effect of IL-27 on the differentiation of CFs to myofibroblasts and ECM production induced by Ang II. a) α-SMA mRNA expression in cardiac fibroblasts after treatment with IL-27 (100 ng/ml) for 0, 2, 6, 12, 18, and 24 h. b) α-SMA mRNA levels in cardiac fibroblasts treated with different conditions for 24 h. c) Western blotting was used to analyze the protein level of α-SMA in cardiac fibroblasts in different groups. d) The results were quantified using ImageJ software. e) Col I mRNA expression in cardiac fibroblasts in different groups. f) Col III mRNA expression in cardiac fibroblasts in different groups. g) Western blotting was used to analyze the protein levels of Col I and Col III in cardiac fibroblasts in different groups. h-i) Col I and Col III mRNA expression in cardiac fibroblasts in different groups. All data are shown as the mean ± SEM. Each experiment was repeated at least three times. *P < 0.05; **P < 0.01; ***P < 0.001 vs. control.

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  Fig. 6. IL-27 promotes the activation of the JAK/STAT3 signaling pathway in Ang II-induced CFs. a) Western blotting was used to analyze the protein levels of P-JAK2, JAK2, P-STAT3, and STAT3 in cardiac fibroblasts in different groups. b-g) Quantified data of immunoblotting band intensity in A. All data are shown as the mean ± SEM from at least three experiments. *P < 0.05; **P < 0.01; ***P < 0.001 vs. control.