Interleukin-5 alleviates cardiac remodelling via the STAT3 pathway in angiotensin II-infused mice

Abstract

Interleukin-5 (IL-5) has been reported to be involved in cardiovascular diseases, such as atherosclerosis and cardiac injury. This study aimed to investigate the effects of IL-5 on cardiac remodelling. Mice were infused with angiotensin II (Ang II), and the expression and source of cardiac IL-5 were analysed. The results showed that cardiac IL-5 expression was time- and dose-dependently decreased after Ang II infusion, and was mainly derived from cardiac macrophages. Additionally, IL-5-knockout (IL-5-/-) mice were used to observe the effects of IL-5 knockout on Ang II-induced cardiac remodelling. We found knockout of IL-5 significantly increased the expression of cardiac hypertrophy markers, elevated myocardial cell cross-sectional areas and worsened cardiac dysfunction in Ang II-infused mice. IL-5 deletion also promoted M2 macrophage differentiation and exacerbated cardiac fibrosis. Furthermore, the effects of IL-5 deletion on cardiac remodelling was detected after the STAT3 pathway was inhibited by S31-201. The effects of IL-5 on cardiac remodelling and M2 macrophage differentiation were reversed by S31-201. Finally, the effects of IL-5 on macrophage differentiation and macrophage-related cardiac hypertrophy and fibrosis were analysed in vitro. IL-5 knockout significantly increased the Ang II-induced mRNA expression of cardiac hypertrophy markers in myocardial cells that were co-cultured with macrophages, and this effect was reversed by S31-201. Similar trends in the mRNA levels of fibrosis markers were observed when cardiac fibroblasts and macrophages were co-cultured. In conclusions, IL-5 deficiency promote the differentiation of M2 macrophages by activating the STAT3 pathway, thereby exacerbating cardiac remodelling in Ang II-infused mice. IL-5 may be a potential target for the clinical prevention of cardiac remodelling.

Keywords: STAT3 pathway; angiotensin II; cardiac remodelling; interleukin‐5; macrophage differentiation.

FIGURE 1

Effects of Ang II infusion on IL‐5 expression. (A, B) Cardiac IL‐5 mRNA expression and serum IL‐5 levels were determined after Ang II infusion for different times. (C, D) The expression of cardiac IL‐5 and serum IL‐5 was measures after treatment with different doses of Ang II. (E) Cardiac IL‐5 levels (green signal) in the control and Ang II groups were analysed by immunofluorescence staining. (F) Double immunofluorescence staining of anti‐F4/80 (red signal) and anti‐IL‐5 (green signal) in Ang II‐infused mice, and the target signal was yellow. Scale bar = 50 μm, N = 5 in each group. *p < 0.05 versus the control group. ^# p < 0.05 versus the previous time or previous dose group.

FIGURE 2

Effects of IL‐5 knockout on Ang II‐induced cardiac hypertrophy and cardiac dysfunction. (A). The HW/BW ratios were analysed; N = 10 in each group. (B–D). mRNA expression of ANP, BNP and β‐MHC in the LV in the four groups; N = 5 in each group. (E). WGA staining and the myocyte CSA in each group; more than 200 cells were analysed. (F, G). The LVEDD, LVESD, LVEF and EF were determined by echocardiography; N = 10 in each group. Scale bar = 50 μm, *p < 0.05 versus the saline WT group. ^# p < 0.05 versus the Ang II WT group. ANA, atrial natriuretic peptide; BNP, B‐type natriuretic peptide; BW, body weight; CSA, cross‐sectional areas; EF, ejection fraction; FS, fractional shortening; HW, heart weight LVEDD, end‐diastolic diameter; LVESD, LV end‐systolic diameter. WGA, wheat germ agglutinin; WT, Wild‐type.

FIGURE 3

Effects of IL‐5 deletion on Ang II‐induced cardiac fibrosis. (A) The degree of fibrotic area in LV in each group was measured by PSR staining and analysed (400×). (B, C) Protein levels and mRNA expression of α‐SMA, collagen I, collagen III and TGF‐β in the LV in the four groups were measured by western blotting and RT‐qPCR, respectively. Scale bar = 100 μm, N = 5 in each group. *p < 0.05 versus the saline WT group. ^# p < 0.05 versus the Ang II WT group. LV, left ventricle, PSR, picrosirius red; WT, wild‐type.

FIGURE 4

Effects of IL‐5 deficiency on M2 macrophage differentiation in Ang II‐treated mice. (A) The expression of p‐STAT3, t‐STAT3 and Arg‐1 in the LV in the four groups was measured by western blotting. (B) p‐STAT3 protein (green signal) and CD206 protein (green signal) expression in cardiac Møs (red signal), of Ang II WT mice and Ang II IL‐5−/− mice was measured by double immunofluorescence staining, and the target signal was yellow. (C) The mRNA levels of Arg‐1, CD163, CD206, TGF‐β, IL‐4, IL‐10 and IL‐13 in the LV were measured in the four groups. Scale bar = 50 μm, N = 5 in each group. *p < 0.05 versus the saline WT group. ^# p < 0.05 versus the Ang II WT group. LV, left ventricle; WT, wild‐type.

FIGURE 5

Effects of S31‐201 on cardiac remodelling in Ang II IL‐5−/− mice. (A) The HW/BW ratios were measured in the four groups. (B–D). The mRNA expression of ANP, BNP and β‐MHC for each group were measured. (E) Myocyte CSA and cardiac fibrosis in the LV were measured. (F, G) The LVEDD, LVESD, LVEF and FS in each group were determined by echocardiography. Scale bar = 50 μm, N = 5–10 in each group. *p < 0.05 versus the Ang II IL‐5−/− + DMSO group. ANA, atrial natriuretic peptide; BNP, B‐type natriuretic peptide; BW, body weight; CSA, cross‐sectional areas; FS, fractional shortening; HW, heart weight LVEDD, end‐diastolic diameter; LVESD, LV end‐systolic diameter.

FIGURE 6

Effects of S31‐201 on M2 macrophage differentiation in Ang II IL‐5−/− mice. (A) Cardiac Arg‐1, p‐STAT3 and t‐STAT3 levels were measured and analysed in each group. (B, C) The mRNA expression levels of α‐SMA, collagen I, collagen III, TGF‐β, Arg‐1, CD163, CD206, TGF‐β, IL‐4, IL‐10 and IL‐13 in the LV were measured in the four groups. N = 5 in each group. *p < 0.05 versus the Ang II IL‐5−/− + DMSO group. LV, left ventricle.

FIGURE 7

Effects of macrophage IL‐5 knockout on Ang II‐induced myocardial cell hypertrophy and collagen synthesis in cardiac fibroblasts. (A, B) ANP and BNP mRNA levels in MCs and (C–E) α‐SMA, collagen I and collagen III mRNA levels in CFs were measured. N = 5 in each group. *p < 0.05 versus the MCs/CFs + WT Mø group. ^# p < 0.05 versus the MCs/CFs + WT Mø + Ang II group. ^& p < 0.05 versus the MCs/CFs + IL‐5−/− Mø + Ang II group. ANA, atrial natriuretic peptide; BNP, B‐type natriuretic peptide; CFs, cardiac fibroblasts; MCs, myocardial cells; WT, wild‐type.