Interleukin‐38 alleviates cardiac remodelling after myocardial infarction

Figure 1. IL‐38 was increased in mice after MI. IL‐38 mRNA and protein expression levels were assessed by real‐time PCR and Western blot, respectively. Relative mRNA expression of IL‐38 in the border zone (A) and remote zone (B) of infarcted hearts in C57BL/6 at different time points post‐MI. Representative image of western blot for IL‐38 protein expression levels in the border zone (C) and remote zone (D) of infarcted hearts in C57BL/6 at different time points post‐MI. E, IL‐38 expression was reflected by immunohistochemical staining with IL‐38 antibody in infarcted heart at different time points post‐MI. (sham n = 6, 24 h n = 8, 48 h n = 8, 7 d n = 8).*P < .05 vs sham; **P < .01 vs sham

Figure 2. Cardiomyocytes are responsible for producing IL‐38 in infarcted heart. A, Representative images of infarcted heart sections stained with antibodies against cardiomyocyte marker α‐actinin (green), macrophage marker CD68 (green), and the cytokine IL‐38(red), and a nuclear stain (blue). Scar bar: 50 μm (each time point n = 8). B, IL‐38 mRNA expression levels in cultured cardiomyocytes with H2O2 stimulation for 6h and 12 h. C, IL‐38 protein expression levels in cultured cardiomyocytes with H2O2 stimulation for 6 and 12 h (control n = 6, 6 h n = 8, 12 h n = 8). D, Real‐time analysis of Bcl‐2 and Bax mRNA levels after incubation with rIL‐38 (50 ng/mL) for the indicated times. The results are expressed as the Bax/Bcl‐2 ratio (control n = 6, 6 h n = 8, 12 h n = 8). *P < .05 vs sham; **P < .01 vs sham

Figure 3. IL‐38 improves left ventricular function post‐MI. A, Survival analysis in PBS‐treated and IL‐38‐treated mice after MI within 28 days (sham n= 15, MI+PBS n=40 and MI+IL‐38 n = 40). B, Representative M‐mode echocardiography images of the left ventricle on day 28 post‐MI in different groups. C‐F, Ejection fraction (EF), ventricular end‐diastolic diameter (LVEDD), left ventricular end‐systolic diameter (LVESD) and fractional shortening (FS) on day 7 and day 28 post‐MI (sham n = 6, MI+PBS n = 8 and MI+IL‐38 n = 8). *P < .05 vs sham; **P < .01 vs sham; # P < .05 vs MI+PBS; ## P < .01 vs MI+PBS

Figure 4. IL‐38 inhibited cardiomyocyte apoptosis and cardiac fibrosis in vivo. A, Representative images of terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL)‐stained heart sections from different experimental groups 1 and 28 d post‐MI. TUNEL (green) and 4, 6‐diamidino‐2‐phenylindole (blue) staining of nuclei in apoptotic cardiomyocytes (red) in the peri‐infarct zone. Scar bar: 100 μm. B, Quantitative analysis of the percentages of TUNEL‐positive nuclei (each group n = 6). C, Real‐time PCR determined mRNA expression levels of Bax and Bcl‐2 in the in infarcted heart on day 1 after MI. The results were also expressed as ratio of Bax/Bcl‐2 (n = 4). D, Representative Masson’s trichrome staining images of collagen deposition (blue) in both infarct and remote areas on day7 post‐MI. E, The extent of fibrosis, as assessed by the fibrotic area/left ventricle and interstitial fibrosis, was compared among the different groups (each group n = 6). **P < .01 vs sham; # P < .05 vs MI+PBS; ## P < .01 vs MI+PBS

Figure 5. IL‐38 inhibits inflammatory response in the infarcted heart. A, Representative images of haematoxylin and eosin (HE) staining, infiltration of myeloperoxidase (MPO+) neutrophils and mouse CD68+ macrophages in the border area of infarcted hearts. Images for HE staining and neutrophils are from day 3 after MI, and images for macrophages are from day7 post‐MI. B, Infiltration of neutrophils and macrophages were compared between the different groups at set time points (each group n = 6). C–F, Analysis of mRNA levels of IL‐6, IL‐1β, TNF‐α and IL‐17A on day 3 and 7 after MI. Data are depicted as fold changes vs sham and shown as the mean ± SEM of 3‐6 independent experiments. **P < .01 vs sham; # P < 0.05.vs MI+PBS; ## P < .01 vs MI+PBS

Figure 6. The role of IL‐38 on DCs. Bone marrow‐derived DCs were cultured in the absence of stimulus (imDCs) or in the presence of 100 ng/mL LPS (mDCs) or/and 100 ng/mL LPS plus 50 ng/mL IL‐38. DCs were stained with specific antibodies against major histocompatibility complex class II (MHC‐II), CD40 and CD86 and analyzed by flow cytometry. A, Comparison of the CD45+CD11b+CD11c+ cells in infarcted heart. B, Comparison of the CD11c+MHC‐II+, CD11c+CD40+ and CD11c+CD86+cells in different cultured DCs. C, TNF‐α, IL‐23 and IFN‐γ expression levels were assessed by ELISA in cultured supernatants of different DCs. D, IDO and IL‐10 mRNA expressions in cultured DCs.( each group n = 4 and repeat at least three times).*P < .05, **P < .01 vs sham

Figure 7. IL‐38 plus TNI treated DCs augment the percentage of regulatory T cells in vitro. We used small doses of LPS (10 ng/mL) and TNI (1 μg/mL) to induce the tolerance of DCs. A‐B, IL‐10 and IDO mRNA expression levels in cultured DCs. C‐E, IL‐23, TNF‐α, and IFN‐γ expression levels were assessed by ELISA in cultured supernatants of different DCs. F, Splenic CD4+ T cells (1 × 106 cells/mL) were cultured for 3 d in the presence of medium alone or combined with imDCs, mDCs, or tDCs (2 × 105 cells/mL). The cells were stained with anti‐CD4, anti‐CD25, and anti‐Foxp3 and analyzed by flow cytometry. G‐J, Analysis of the expression levels of IFN‐γ, IL‐17A, IL‐22 and IL‐10 in culture supernatants of CD4+ T cells using ELISA in different groups. (each group n=4 and repeat at least three times). *P < .05,**P < .01