Changes in CD4+CD25+ Tregs in the pathogenesis of atherosclerosis in ApoE-/- mice

Abstract

The goal of this study was to observe the pathological characteristics of atherosclerotic plaques in the aortic walls of ApoE^-/- and C57BL/6J mice and the changes of CD4⁺CD25⁺ regulatory T cells (Tregs) in atherosclerotic mice. Twenty ApoE^-/- mice were split into high-fat diet (AH) and normal diet (AN) groups and 10 C57BL/6J male mice were designated as the control group (BN). The serum concentrations of IL-10 and TGF-β1 were detected by enzyme-linked immunosorbent assay; paraffin sections of the aorta were stained with hematoxylin & eosin, and morphometric parameters were measured using the Image Pro Plus 6.0 system. Verhoeff stain was used to observe the distribution of elastic fibers, and immunohistochemical staining was performed to verify the phenotype of the forkhead box protein 3 (Foxp3⁺) CD25⁺ cells in the atherosclerotic tissue. The proportion of CD4⁺CD25⁺ Tregs in the spleen was calculated by flow cytometry. The thickness of the intima, the intima/media ratio, the plaque area, and the plaque/lumen ratio of mice in AN group were significantly larger than those of mice in BN group. The thickness of the intima, the plaque area, and the plaque/lumen ratio of the mice in AH group were significantly increased compared with those of the AN group mice. The serum concentrations of IL-10 and TGF-β1 and the percentage of splenic CD4⁺CD25⁺ Tregs in AN group mice were significantly decreased compared with the control group. The serum concentrations of IL-10 and TGF-β1 and the percentage of splenic CD4⁺CD25⁺ Tregs in the mice in AH group were significantly decreased compared with those in AN group. The proportions of Foxp3⁺ and CD25⁺ cells within the total lymphocyte population were significantly decreased in AH group mice compared with those in AN group mice. Atherosclerosis in an experimental mouse model was correlated with Treg depletion in the lymphoid tissues and plaques, indicating the important antiatherosclerotic role of CD4⁺CD25⁺ Tregs. Impact statement In this article, we conclude that Tregs decreased with atherosclerosis (AS) as determined in ApoE knockout mice fed a high fat diet. It is an important matter for understanding the AS pathology.

Keywords: ApoE-KO mouse (ApoE−/− mice); Atherosclerosis; CD4+CD25+ Tregs; forkhead box protein 3.

Figure 1

The pathological morphology of the aortic artery in different groups revealed by H&E and Masson staining. AS plaques were significantly increased in the aortas of mice in AH group (H&E (a)). Early plaque formation was observed in the aortic walls of mice in AN group (H&E (a)); the aortic walls of mice in BN group were normal, with no AS plaque formation (H&E (a)). The elastic fibers in AH group were arranged loosely, disordered, and partially ruptured (Masson b, red arrow), which was a more severe phenotype than AN group (Masson b), while the elastic fibers in BN group were arranged neatly (Masson b), bar represented 50 µm. AH: ApoE^−/− mice + high-fat diet; AN: ApoE^−/− mice + normal diet; AS: atherosclerosis; BN: control group; H&E: hematoxylin & eosin. (A color version of this figure is available in the online journal.)

Figure 2

The proportion of CD4⁺CD25⁺ Tregs in a splenic cell suspension in different treatment groups. The proportion of CD4⁺CD25⁺ Tregs within splenic cell suspensions from the mice in AH group was lower than that in AN group (P = 0.017), and the proportion of CD4⁺CD25⁺ Tregs was lower in AN group than in BN group (P = 0.023). AH: ApoE^−/− mice + high-fat diet; AN: ApoE^−/− mice + normal diet; BN: control group; Treg: regulatory T cells. (A color version of this figure is available in the online journal.)

Figure 3

The numbers of Foxp3⁺ and CD4⁺CD25⁺ cells in different treatment groups. Foxp3⁺ and CD4⁺CD25⁺ cells were present in atherosclerotic plaques. However, none of these cells were observed in the intima of the mice in BN group. The proportion of Foxp3⁺, CD4⁺CD25⁺ cells within the total lymphocyte population was significantly decreased (1.28 ± 1.20 versus 3.04 ± 1.92, P = 0.024; 2.00 ± 1.39 versus 3.98 ± 1.67, P = 0.010 in aortic root; 1.55 ± 0.98 versus 3.20 ± 1.46, P = 0.032; 2.13 ± 0.99 versus 3.86 ± 1.24, P = 0.022 in aortic arch; and 1.08 ± 0.76 versus 3.00 ± 1.31, P = 0.012; 1.45 ± 0.78 versus 3.45 ± 1.26, P = 0.008 in aortic, respectively) ((a) and (b)), bar represented 50 µm. BN: control group; Foxp3⁺: forkhead box protein 3. (A color version of this figure is available in the online journal.)