The Role And Significance Of Th9/IL-9 In The Progress Of Experimental Atherosclerosis

Background: Atherosclerosis(AS)is the main pathological basis of many cardio-cerebrovascular diseases and peripheral vascular diseases,and is also a leading cause of death in some western developed countries.In recent years,with the improvement of people’s living standard,the disease has gradually become one of the main causes of death in the middle-aged and elderly in China.At present,the pathogenesis of AS has not been fully elucidated,however,lots of clinical and basic studies have shown that AS is a chronic inflammatory disease,which variety of immune cells and cytokines are involved,including CD4+ T lymphocytes.T-helper 9(Th9)cells are newly discovered CD4+T lymphocytes subset.Naive CD4+T cells can differentiate into Th9 cells and secrete a large number of interleukin-9(IL-9)after antigen stimulation in the presence of transforming growth factor-β(TGF-β)and IL-4.The production and function of Th9 cells are regulated by transcription factor PU.1.Studies have shown that Th9 cells play an important role in various inflammatory diseases,such as asthma and inflammatory bowel disease.However,it is unclear about the role of Th9 cells in atherogenesis currently.Th17 cells are also new CD4+T lymphocytes subset which were found in recent years,and Th17 cells have a strong role in promoting inflammation and involve in the occurrence and development of a variety of autoimmune diseases.Previous studies have demonstrated that Th17 cells play a role in promoting AS.Foam cells formation is an important pathological basis of atherogenesis,and oxidized-low density lipoprotein(ox-LDL)can promote the formation of foam cells which is closely related to occurrence of AS.In our study,we established atherosclerosis model to analyze Th9/IL-9 expression and its correlation with Th17/IL-17 and ox-LDL,furthermore,the role of Th9/IL-9 in the process of atherosclerosis was confirmed by intervention experiment in vivo and cellular experiment in vitro,and the mechanism of Th9 cells in the progress of atherosclerosis were explore preliminarily.We hope to provide a new target for the prevention and treatment of AS.Objective: To clarify the expression and significance ofTh9 and IL-9 in atherosclerotic model of Apo E-/-mice and to explore the mechanism of Th9 cells in the progress of AS.Methods :(1)The atherosclerotic mice model was established by feeding high fat diet and the role and significance of Th9/IL-9 in the process of experimental atherosclerosis was analyzed by detecting various markers.(1)48 male Apo E-/-mice aged 8 weeks were fed with high fat diet for 0,4,12 and 24 weeks respectively and their age-matched C57BL/6 control mice were fed with normal diet;(2)Plasma lipid levels of different aged mice were measured;(3)Aorta of different aged mice were separated and paraffin sections were prepared,then the formation of aortic plaques were detected with HE staining;(4)The frequencies of Th9 cells and Th17 cells in spleen of different aged mice were measured by flow cytometry;(5)Enzyme linked immunosorbent assay(ELISA)was used to detect the concentrations of IL-9,IL-17 and ox-LDL in serum of different aged mice;(6)Reverse transcription polymerase chain reaction(RT-PCR)was used to detect the expression of PU.1m RNA and RORγt m RNA in spleen and aorta of different aged mice.(2)The effects of recombinant IL-9(r IL-9)protein and IL-9 monoclonal antibody(IL-9m Ab)on the occurrence and development of AS were detected by intervention experiment of Apo E-/-mice in vivo.(1)15 Apo E-/-mice were randomly divided into three groups: PBS control group,recombinant IL-9(r IL-9)treatment group,and anti-IL-9 monoclonal antibody(IL-9m Ab)treatment group.From 8 weeks old they were injected via tail vein injection once a week for a period of 12 weeks;(2)The plagues of aorta with were detected by HE staining and serum IL-9 concentrations were detected by ELISA.(3)The mechanism of Th9 cells in the progress of atherosclerosis was explored by the experiment of foam cells formation.(1)Peritoneal macrophages of C57BL/6 mice were collected and cultured;(2)Peritoneal macrophages were divided into three groups at random: control group,ox-LDL induction group and r IL-9 treatment group,then the formation of foam cells were observed with Oil red O after 3 days culture.Results:(1)The total cholesterol and triglyceride level of Apo E-/-mice increased and the high density lipoprotein level decreased with the extension of feeding time,while there were no significant changes in age-matched C57BL/6 mice.The aorta of Apo E-/-mice with high fat diet only thicken at week 4,the aortic plaques appeared at week 12,plaque area at week 24 increased significantly compared with that at the week 12(P<0.01),while there was no obvious thickening or plaque formation in age-matched C57BL/6 mice.(2)The frequencies of Th9 cells and Th17 cells were elevated markedly in Apo E-/-mice with high fat diet than in age-matched C57BL/6 mice since week 4(P<0.01),and the frequencies of Th9 cells and Th17 cells were increased with the extension of feeding time.The frequencies of Th9 cells at week 24 were significantly higher than those at week 12(P<0.01),the frequencies of Th17 cells were increased at week 24 but there was no significant difference in those between week 12 and week 24.(3)The concentrations of IL-9,IL-17 and ox-LDL in Apo E-/-mice with high fat diet were significantly higher compared with the age-matched C57BL/6J mice since week 4,and the concentrations of IL-9,IL-17 and ox-LDL were increased with the extension of feeding time.IL-9 concentrations achieved the maximal value at week 24 while IL-17 concentrations achieved the maximal value at week 12.(4)The concentrations of IL-9 in serum and the frequencies of Th9 cells in spleen of Apo E-/-mice were significantly positively correlated with ox-LDL,IL-17 concentrations in serum and the frequencies of Th17 in spleen(P<0.01).(5)The levels of PU.1 and RORγt expression in spleen and aortic plague of Apo E-/-mice with high fat diet were significantly higher than those in age-matched C57BL/6 mice since week 4(P<0.01),and the levels of PU.1 and RORγt expression were increased with the extension of feeding time.(6)Administration of r IL-9 led to a significant elevation of aortic plaque area and serum IL-9 concentrations,while administration of IL-9m Ab led to an obvious reduction of aortic plaque area and serum IL-9 concentrations compared with PBS control group.(7)The foam cells of r IL-9 treatment group were significantly higher than those of ox-LDL induction group.Conclusions:(1)With the extension of feeding time,the aortic plaques of Apo E-/-mice formed and expanded,and the expression of Th9/IL-9 and Th17/IL-17 were significantly increased,in addition,Th9/IL-9 was significantly increased in the whole process of atherosclerotic plaque formation,while Th17/IL-17 increased mainly in the early stage of atherosclerotic plaque formation.(2)Th9/IL-9 levels were significantly positively correlated with the concentration of ox-LDL,an important promoting atherosclerosis factor,and Th17/IL-17 levels.(3)The intervention experiment in vivo showed that r IL-9 could significantly promote the formation of atherosclerosis,while IL-9m Ab could inhibit the occurrence and development of AS.(4)IL-9 can promote the formation of foam cells induced by ox-LDL.In conclusion,the role of Th9/IL-9 in the process of AS is related to influencing the expression of Th17/IL-17 and promoting foam cells formation.