The Role Of The PD-1 Pathway Regulated Immune Responses In Atherosclerosis In Mice

Objective : The prevalence and morbidity of atherosclerosis in our country were significantly increased, and had become one of the main causes of mortality in elderly people in our country. Recent studies have shown that inflammation and immune response play an important role in the genesis and development of atherosclerosis. Immune cells, in particular the infiltration, activation, proliferation of T cell play an vital role in the formation and progression of vulnerable atherosclerotic plaque. Programmed cell death factor 1(PD-1) is a costimulatory receptor which mediates negative regulatory signals and mainly expressed on activated T cells, B cells and myeloid cells. Recently, some researches have shown that PD-1/PD-Ls pathway is associated with autoimmune diseases, immune tolerance, tumour, acute and chronic inflammation. Because the formation and development of atherosclerosis are closely correlated with inflammation and immune reactions, we suppose that PD-1 pathway might play an important role in atherosclerosis. Therefore, by using PD-1-/-Apo E-/-mice, we studied the role of the PD-1 pathway in the progression of atherosclerosis.Method: PD-1-/- mice were mated with ApoE-/- mice to obtain PD-1-/-ApoE-/- mice. PD-1+/+Apo E-/- mice were used as control. We assessed body weight and fasting serum total cholesterol, triglyceride, high-density lipoprotein of 6-week old mice in both groups as the baseline level. We fed both groups of mice with high-fed diet for 12 weeks. Then, serum total cholesterol, triglyceride, high-density lipoprotein of mice in both groups were analyzed again. After anesthesia, chest of mice was opened. Then we flushed the residual blood in left ventricle and aortic endovascular with cold phosphate buffer(PBS). Aorta and heart were separated from ascending aorta, and the lower half part of heart was resected. Then we washed the residual blood in the upper part of heart with cold PBS and embedded it by using OCT. Samples of aortic arch and descending aortas as well as aortic sinuses were taken for oil-red staining. We also analyzed the CD3+T cell, CD4+T cell,CD8+ T cell, macrophages and smooth muscle cell in plaque by using immunohistochemistry. Spleen cell total numbers, CD4+T cell, CD8+T cell and the expression level of CD26 L and CD25 on the T cells surface were detected by using FACS. The purified CD4+ and CD8+ T cells were collected by using cell purification column and then labeled with CFSE. DC and macrophages were separated from normal mice and treated with LDL. The T cells were mixed with DC or macrophages in different proportion and then the proliferation of CD4+ and CD8+ T cells were determined by FACE. The purified CD4+ and CD8+ T cells were stimulated by anti-CD3 antibody and the levels of IL-2、IFN-γ、TNF-α in the supernatant were detected by using ELISA. After feeding with high-fed diet for 12 weeks, the serum levels of IL-2、IFN-γ、TNF-α of both groups were determined by ELISA.Result:6 weeks of baseline and after 12 weeks of high-fat diet, no significant changes were observed between the experimental group and the control group of mice in the levels of body weight, fasting serum cholesterol, triglyceride and high-density lipoprotein. After 12 weeks of high fat feeding, atherosclerotic plaque in aortic arch and descending aorta intima increased significantly in PD-1-/-Apo E-/- mice compared with the control group, as well as severe lumen stenosis in the experimental group. Aortic root sections showed atherosclerotic plaque area is about 2 times bigger than that of the control group [(0.51±0.08)mm2 vs(0.29±0.06)mm2,P<0.001]. Immunohistochemical analysis in plaque showed that after 12 weeks of high fat feeding, the number of CD3+T lymphocyte content(154.88 + 36.64)/mm2 in the experimental group increased significantly than those in the control group(59.38 + 25.28)/mm2(t =2.14, P < 0.01). Further analysis of the composition of the cells in plaque showed that the number of CD4+ T cell in PD-1-/-Apo E-/- group is three times than control. More CD8+ T cells and macrophages in the lesion of PD-1-/-Apo E-/- mice were also observed. However, there is no difference between the number of smooth muscle cells in both groups. The total cell numbers, the CD4+ and CD8+T cells in the spleen of PD-1-/-Apo E-/- group significantly increased, as well as the proportion of CD3+CD62L-, CD3+CD25+T cells. Moreover, The capability of secreting IL-2、IFN-γ、TNF-α of T cells were enhanced after activation of CD3 antibody in the experimental group. After co-cultured with macrophages or DCs, the proliferation of T cells were also enhanced than that of control group. After 12 weeks of high-fed diet, the serum levels of TNF-α in PD-1-/-Apo E-/- group was higher than that of control group. However, the serum level of IL-2 and IFN-γ were no significant difference between two groups.Conclusion : In this study, we observed the atherosclerosis accelerate progress,analysed the cell composition in plaque, and T cell function in PD-1-/-Apo E-/- double gene knockout mice. Our results suggest that PD-1/PD-Ls pathway has an important role in limiting atherosclerosis by downregulating proatherogenic T-cell responses.