The Role Of Peripheral Th17/Treg Imbalance In Atherosclerosis And The Effect Of Atra On Th17/Treg Imbalance As Well As Its Mechanism

Background Atherosclerosis (AS) is the major pathologic basis of cardiovascular and cerebrovascular disease, which is threaten serviously to people's health and life. However, the pathogenesis of AS has not been fully investigated. More evidences incidate that AS is a chronic inflammatory disease, and inflammation is the main pathogenesis of AS. The inherent and acquired immune inflammatory mechanisms involved in the development of AS. Acute coronary syndrome (ACS) including acute myocardial infarction (AMI) and unstable angina (UA), is caused by atherosclerotic plaque instability under a variety of factors, and lead to plaque rupture and secondary thrombosis. The immune inflammatory response is one important mechanism of plaque rupture and the onset of ACS. Accordingly, it has important significance for medical science to lucubrate the immune mechanism of AS and ACS and to look for new medicine to treat AS and ACS.CD4~+CD25~+ regulatory T (Treg) cells and T-helper 17 (Th17) cells are 2 original subsets distinguished from Th1 and Th2 cells. Treg cells can control autoimmune diseases and maintain immune tolerance effectively. The alterations of Treg number and function are closely related to tumor, infection, autoimmune diseases and other diseases. Th17 cells, a new CD4+ T cell subtype, have strong pro-inflammatory effects, and involve in the occurrence and development of many autoimmune diseases. There is complex relationship between Treg and Th17 cells, and the two cells are mutually antagonistic in functions. The balance of Treg and Th17 cells plays important roles in tissue inflammation and autoimmune diseases. However, Th17/Treg balance in AS, especially ACS patients has not been fully investigated.Oxidized low density lipoprotein (ox-LDL) is closely correlated with the development of AS. Ox-LDL can damage endothelial, promote the proliferation of smooth muscle cells, and favor foam cell formation though various ways, which lead to the formation of lipid streaks and atherosclerotic plaque. Ox-LDL may also affect plaque stability and contribute to the occurrence of ACS, but it is unclear whether ox-LDL also affects the occurrence of AS and ACS by disrupting the balance of the peripheral Tregs and Th17 cells.All-trans retinoic acid (ATRA) is the main derivative of vitamin A. As an important bioactive substance, ATRA has hormone-like effect, and produce wide biological effects. It has been reported that ATRA take an important effect on the differentiation of Treg and Th17 cells.In our study, we examined the frequencies of Th17 and Treg cells, key transcription factors and relevant cytokines in patients with AMI, UA, stable angina (SA) and controls, and investigated the alteration of Th17/Treg balance in ACS patients. Then by cellular experiments in vitro, we analyze the effect of ATRA and ox-LDL on the number and function of Treg and Th17, and elucidate the roles of ox-LDL and ATRA in the onset of ACS from the perspective of cell differentiation. We also explored the mechanism of Th17/Treg imbalance in ACS patients from the angel of cellular apoptosis. In addition, we duplicated early AS rat model, and verified the effect of ATRA on Th17/Treg balance and the role in AS. We hope to find new method and basis for prevention and control of ACS, and provide a new approach for development of drugs for AS treatment. Aim To clarify the effect and significance of Th17/Treg balance in AS, to analyze the effect of ATRA and ox-LDL on Th17/Treg balance as well as atherogenesis, and to explore the mechanism of Th17/Treg imbalance and ATRA on the balance.Methods (1)We chose the subjects including AMI, UA, stable angina (SA) and control with normal coronary arteries (NCA). Surface and cytoplasma antigens (such as CD4, CD25, CD127, FoxP3, IL-17 et al.) were detected by Multicolor Immunofluorescence labeling method, and the frequency of Treg and Th17 cells was analyzed by multiparameter flow cytometry. CD4~+CD25~+CD127low and CD4+CD25- cells were sorted by flow cytometry. Treg cells from 4 groups were assayed for their suppressive activity in the allogeneic mixed lymphocyte response (MLR) assay. RORγt and Foxp3 expression levels were detected by RT-PCR. We analyzed the correlations of serum ox-LDL to Th17/Treg frequency, and the effects of ox-LDL on Th17/Treg cells in vitro. The levels of serum Interleukin 10 (IL-10), IL-17 and ox-LDL were measured by ELISA. The effects of ox-LDL on Th17/Treg cells were analyzed in vitro. (2) The mechanisms of Th17/Treg imbalance were explored by experiment in vitro and apoptosis analysis. The effects of ATRA on Th17/Treg imbalance were analyzed by ATRA intervention test in vitro, and it's mechanism about Treg and Th17 expression was demonstrated by cytokine-induced experiment. Treg apoptosis and expression of Fas, FasL in ACS patients were detected by FCM. (3) A rat model about early AS was established by immune injury and cholesterol diet. Expression of Treg and Th17 was measured by FCM. The effect of ATRA on Th17/Treg balance was further validated in vivo.Results (1) ACS patients have shown a significant decline of Treg frequency, Foxp3 expression, suppressive function, and serum IL-10, and a obvious increase of Th17 frequency, RORγt expression and serum IL-17. The ratio of Treg/Th17 cells was markedly lower in AMI and UA patients than in SA and NCA groups (P < 0.01). The ratio of CD4~+CD25~+CD127low /Th17 cells was mostly > 5 in NCA subjects and 2-4 in SA patients, but 1-1.5 in UA patients and <0.7 in AMI patients. The concentration of ox-LDL increased more significantly in the AMI and UA patients than in SA and NCA groups (P < 0.01). In addition, ox-LDL concentrations in serum were negatively correlated with the frequency of Treg cells (P < 0.01), and positively correlated with the frequency of Th17 cells (P < 0.01). We found that, with increased ox-LDL concentrations and prolonged incubation times, the frequency of Treg cells was decreased while the frequency of Th17 cells was elevated. Treg and Th17 cells from AMI and UA patients were more susceptible to the influence of ox-LDL when compared to those in NCA and SA subjects. (2) Frequency of Treg cells and Treg suppressive function was higher significantly while Frequency of Treg cells was lower obviously in group treated with ox-LDL and ATRA than in group only treated with ox-LDL (all P < 0.01). When PBMCs from NCA and SA groups were co-cultured with ox-LDL and ATRA for 48h, Treg and Th17 expression and Treg function had been close to cellular control. In addition, recovery of Treg and Th17 expression and Treg function were also significant in PBMCs from UA and AMI patients. Induced by TGF-β1 and IL-2 for 72h, expression of Treg cells were increased significantly, while Induced by IL1-β, IL-6 and IL-23 for 72h, expression of Th17 cells were also elevated obviously. Expression of Treg cells in ATRA treatment group was higher significantly than those in cytokine-induced group (P < 0.01), but expression of Th17 cells in ATRA treatment group was lower obviously than those in cytokine-induced group (P < 0.01). There was no difference in expression of Treg and Th17 cells between ox-LDL treatment group and cytokine-induced group (all P> 0.05). Apoptotic Treg cells were higher significantly in AMI and UA groups than in NCA and SA groups(P < 0.01).Compared with NCA and SA groups, Fas and FasL expression in Treg from AMI and UA groups was higher significantly(all P < 0.05). (3) About rat model, the results of HE staining shown that there are edematous thickening of the intima with deposition of a large number of foam cells and a mild atrophy in the membrane in the group treated with cholesterol diet and immune injury, there was a less extent on pathological changes in immune injurey groups and ATRA treatment groups.Total cholesterol (TC) in serum of the cholesterol diet group, the cholesterol diet plus immune injury group increased significantly compared with normal group and immune injurey group (P < 0.05), but the levels of TC did not differ significantly between ATRA treatment group and other groups. Treg expression in immune injurey group and cholesterol diet plus immune injury group was lower significantly while Th17 expression was higher obviously than that in normal and cholesterol diet group (P < 0.05). Treg levels in ATRA treatment group were increased significantly while Th17 levels in ATRA treatment group were decreased significantly than those in cholesterol diet plus immune injury group.Conclusions (1) A early AS rat model was established successfully by immune injury and cholesterol diet. (2) Th17/Treg imbalance exists in ACS patients and early AS rat model. The Th17/Treg imbalance is not only closely related to the onset of ACS, but also possibly used as a sensitive indicator to early diagnosis and dynamic monitoring of ACS. (3) Ox-LDL has a direct effect on Th17/Treg imbalance, and differential sensitivity in ACS patients to ox-LDL results in the aggravation of the Th17/Treg imbalance, which may contribute to plaque destabilization and pathogenesis of ACS. However, this action was not correlated with effect on Treg and Th17 cell induced expression. (4) It is shown in experiment in vitro and rat model that ATRA can up-regulated Treg expression, down-regulated Th17 expression, and altert the state of Th17/Treg imbalance effectively, which can suppress atherogenesis. The possible mechanism is that ATRA could promote Treg cell differentiation and inhibit Th17 differentiation. (5) Expression of Fas and FasL in ACS patients was elevated, and Treg apoptosis was induced by Fas/FasL pathway, which may lead to Treg reduction and Th17/Treg imbalance. The Effect and Mechanism of ATRA on Peripheral Th17/Treg Balance in Patients with Acute Coronary SyndromeAim To analyze the effect of ATRA on Peripheral Th17/Treg balance and atherogenesis, and to explore the mechanism of Th17/Treg imbalance and ATRA on the balance.Methods The effects of ATRA on Th17/Treg imbalance were analyzed by ATRA intervention test in vitro, and it's mechanism about Treg and Th17 differentiation was demonstrated by cytokine-induced experiment. Treg apoptosis and expression of Fas, FasL in ACS patients were detected by FCM.Results Frequency of Treg cells and Treg suppressive function was higher significantly while Frequency of Treg cells was lower obviously in group treated with ox-LDL and ATRA than in group only treated with ox-LDL (all P < 0.01). When PBMCs from NCA and SA group were co-cultured with ox-LDL and ATRA for 48h, Treg and Th17 expression and Treg function had been close to cellular control. In addition, recovery of Treg and Th17 expression and Treg function were also significant in PBMCs from UA and AMI patients. Induced by TGF-β1 and IL-2 for 72h, expression of Treg cells were increased significantly, while Induced by IL1-β, IL-6 and IL-23 for 72h, expression of Th17 cells were also increased obviously. Expression of Treg cells in ATRA treatment group was higher significantly than those in cytokine-induced group (P < 0.01), but expression of Th17 cells in ATRA treatment group was lower obviously than those in cytokine-induced group (P < 0.01). There was no difference in expression of Treg and Th17 cells between ox-LDL treatment group and cytokine-induced group (all P> 0.05). Apoptotic Treg cells were higher significantly in AMI and UA group than in NCA and SA group(P < 0.01).Compared with NCA and SA group, Fas and FasL expression in Treg from AMI and UA group was also higher significantly(all P< 0.01)Conclusins ATRA can up-regulated Treg expression, down-regulated Th17 expression, and change the state of Th17/Treg imbalance effectively. The possible mechanism is that ATRA could promote Treg cell differentiation and inhibition of Th17 differentiation. Ox-LDL has a direct effect on Th17/Treg imbalance, but this effect was not correlated with Treg and Th17 cell differentiation. Expression of Fas and FasL in ACS patients was elevated, and Treg apoptosis was possibly induced by Fas/FasL pathway, which may lead to Treg reduction and Th17/Treg imbalance. Effects of ATRA on Peripheral Th17/Treg ImBalance in early experimental arteriosclerosis rat modelAim To establish early experimental AS rat model, and to investigate Peripheral Th17/Treg ImBalance and the effects of ATRA on the balance in the model.Methods Fourty 8-week-old Sprague Dawley rats were randomly divided into 5 groups: the normal group (Group 1), cholesterol diet group (Group 2), immune injury group (Group 3), cholesterol diet plus immune injury group (Group 4), and cholesterol diet+ immune injury + ATRA group (Group 5). Rat in Groups 1 and 3 were fed with normal diet, and other group rats fed with cholesterol diet. Rat in all the groups were fed for 16 weeks. Rat in Groups 3 and 4 were immunized with ovalbumin. Rat in Group 5 were immunized with ovalbumin and treated with ATRA.For all the 5 groups, blood and aorta samples were obtained after 16 weeks. TC and TG in serum were measured by ELISA, histological changes in aorta were analyzed by HE staining, and the frequencies of Th17 and Treg cells in peripheral blood were detected by flow cytometry.Results A early AS rat model was established successfully. The results of HE staining shown that there are edematous thickening of the intima with deposition of a large number of foam cells and a mild atrophy in the membrane in Groups 4, there was a less extent on pathological changes in Group 3 and Group 5. TC in serum of Groups 2 and 4 increased significantly compared with Groups 1 and 3 (P <0.05), but the levels of TC in Group 5 have no significant difference compared with those of other groups. Expression of Treg was lower significantly while expression of Th17 was higher obviously in group 3 and group 4 than that in group1 and group 2 (P < 0.05, P < 0.01 respectively). Treg levels in Group 5 were increased significantly while Th17 levels in Group 5 were decreased significantly than those in Group 4. Conclusions A early AS rat model was established successfully by immune injury and cholesterol diet. The shift of the Th17/Treg cell balance from Treg cells towards Th17 cells exists in rat model, and ATRA may play an important role in inhibiting AS development by affecting the peripheral Th17/Treg balance.