Exogenous interleukin 37 ameliorates atherosclerosis via inducing the Treg response in ApoE-deficient mice

Abstract

Our previous study indicated that interleukin (IL)-37 is involved in atherosclerosis. In the present study, Anterior tibial arteries were collected from diabetes patients and controls. A histopathological analysis showed that IL-37 was over-expressed in human atherosclerotic plaques. Many types of cells including macrophages, vascular smooth muscle cells (VSMCs), endothelial cells and T lymphocyte expressed IL-37 in human atherosclerotic plaques. ApoE-/- mice were divided into a control group and a recombinant human IL-37-treated group. The IL-37 treatment resulted in a significant decrease in macrophages and CD4+ T lymphocytes and a substantial increase in VSMCs and collagen in atherosclerotic plaques, resulting in a reduction in atherosclerotic plaque size. Furthermore, the IL-37 treatment modulated the CD4+ T lymphocyte activity, including a decrease in T helper cell type 1 (Th1) and Th17 cells and an increase in regulatory T (Treg) cells, and inhibited the maturity of dendritic cells both in vivo and in vitro. In addition, treatment with anti-IL-10 receptor monoclonal antibody abrogated the anti-atherosclerotic effects of IL-37. These data suggest that exogenous IL-37 ameliorates atherosclerosis via inducing the Treg response. IL-37 may be a novel therapeutic to prevent and treat atherosclerotic disease.

Figure 1

Over-expression of IL-37 in human atherosclerotic plaques. (A) Normal human anterior tibial artery stained with hematoxylin-eosin. (B) Human atherosclerotic plaque. (C) Little IL-37 expression in ECs and VSMCs in normal artery (indicated by arrowheads). (D) Abundant IL-37 expression in foam cells in atherosclerotic plaque. D through F, IL-37 expression (D) in macrophages identified by anti-CD68 antibody (E) and VSMCs identified by anti-α-SMA antibody (F). IL-37 expression (G) in T lymphocytes identified by anti-CD3 antibody (H). (I) Results of statistical analysis of IL-37 expression in normal arteries and atherosclerotic plaques. Values are presented as the means ± SEM, n = 8. Black bar = 200μm. **p < 0.01.

Figure 2

IL-37 ameliorates the development of atherosclerosis. (A) Representative photographs of Oil Red O staining in the surface lesion area of the entire aorta, and quantitative analysis of the percent lesion area of the entire vessel area (*p < 0.05). (B) Representative sections of aortic sinus stained with Oil Red O (Black bar = 200 μm), antibodies to MOMA-2 (Black bar = 200 μm), α-SMA (Black bar = 200 μm), collagen (Black bar = 200 μm) and CD4 (Black bar = 200 μm) staining in the two groups (Black bar = 200 μm), and quantitative analysis of the lesion size of aortic sinus, MOMA-2, α-SMA, collagen and CD4 staining in the two groups. MOMA indicates monocytes/macrophages; SMA, smooth muscle actin. Values are presented as the means ± SEM, n = 12. **p < 0.01.

Figure 3

Effect of IL-37 administration on DCs and CD4⁺T cells in spleen of ApoE−/− mice. (A) Flow cytometry results of DCs (CD86+MHC-II+CD11c+) are shown. The numbers in upper right quadrants indicate positive percentages of these cells. Results of statistical analysis of DCs. (B) Flow cytometry results of Th1 (CD4+IFN-γ+), Th2 (CD4+IL-4+), Th17 (CD4+IL-17+), and Tregs (CD4+CD25+FOXP3+) are shown. The numbers in upper right quadrants indicate positive percentages of these cells. Results of statistical analysis of Th1 cells, Th2 cells, Th17 cells, and Tregs. Values are presented as the means ± SEM, n = 8. *p < 0.05, and **p < 0.01.

Figure 4

Effect of IL-37 on the maturation of BMDCs in vitro. (A) Flow cytometry results of CD86 and MHC-II. (B) Representative MFI of CD86 and MHC-II in the cultured BMDCs. (C) Results of statistical analysis of the cytokine levels in the cultured supernatant. The data represent 3 experiments in each group. Values are presented as the means ± SEM. *p < 0.05, and **p < 0.01.

Figure 5

Effect of IL-37 on the Th1/Th2/Th17/Treg paradigm in vitro. (A) Flow cytometry results of (CD4+IFN-γ+), Th2 (CD4+IL-4+), Th17 (CD4+IL-17+), and Tregs (CD4+CD25+FOXP3+) are shown. Results of statistical analysis of Th1 cells, Th2 cells, Th17 cells, and Tregs. (B) Results of statistical analysis of the cytokine levels in the cultured supernatant. The data represent 3 experiments in each group. Values are presented as the means ± SEM. *p < 0.05, and **p < 0.01.

Figure 6

The levels of plasma cytokines in the two groups and the mRNA expression in the aorta. (A) The levels of plasma G-CSF, GM-CSF, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-17, IL-21,IL-23, IFN-γ, TNF-α and TGF-β1 in the two groups. (B) Expression of IL-1β, IL-4, IL-6, IL-10, IL-12p70, IFN-γ, TBX21, RORγT, IL-17, IL-23, GATA-3, TGF-β1 and FOXP3 mRNA in aorta in the two groups. Values are presented as the means ± SEM, n = 5 to 7. *p < 0.05, and **p < 0.01.

Figure 7

Anti-IL-10 treatment abrogates beneficial effects of IL-37 on atherosclerosis. (A) Representative sections of aortic sinus stained with Oil Red O staining in ApoE−/− mice at 18 weeks. Black bar = 200 μm. n = 12. (B) Through (D) Quantitative analysis of lesion size, MOMA-2, and α-SMA staining. (E) Levels of plasma neutralizing antibodies of anti-IL-10R IgG. Values are presented as the mean ± SEM, n = 6. MOMA indicates monocyte/macrophage; SMA, smooth muscle actin. *p < 0.05, and **p < 0.01.