Exogenous Dendritic Cells Aggravates Atherosclerosis Via P-selectin/PSGL-1 Pathway

Objective:To investigate the role of exogenous dendritic cells（DCs）in atherosclerosis?associated immunity mediated by P-selectin/PSGL-1 pathway and their effect on the development of atherosclerosis.Methods:ApoE^-/-P^-/-and ApoE^-/-/PSGL^-/-double knockout mice were set up.The study included 6 experimental groups of animals:（1）AS（atherosclerosis）model group(ApoE^-/-mice+high-fat diet+NS);（2）ApoE^-/-P^-/-group(ApoE^-/-/P^-/-mice+high-fat diet+NS);（3）ApoE^-/-P^-/-+P-sel group(ApoE^-/-/P^-/-mice+high-fat diet+P-selectin);（4）ApoE^-/-/P^-/-+DCs group(ApoE^-/-/P^-/-mice+high-fat diet+DCs)（5）ApoE^-/-/PSGL^-/-+DCs group(ApoE^-/-/PSGL^-/-mice+high-fat diet+DCs)（6）ApoE^-/-/PSGL^-/-+NS group(ApoE^-/-/PSGL^-/-mice+high-fat diet+NS).Bone marrow mononuclear cells were collected from the wild type mice.Then purified mature dendritic cells（DCs）were obtained by immunomagnetic bead sorting.All experimental groups mice were treated at the beginning of ninth weeks according to the following scheme:AS model group and ApoE^-/-P^-/-group were injected with 200ul NS into the tail vein of mice in the 9th week,and then 200ul NS was subcutaneously injected into the medial thigh of the mouse every week until sixteenth weeks;The mice in ApoE^-/-P^-/-+P-sel group were injected with P-selectin through the tail vein once in the9th week,then subcutaneously injected with 200ul NS in the medial thigh of the mouse every week until sixteenth weeks;The mice of the ApoE^-/-P^-/-+DCs group were injected with wild type exogenous mature DCs via the tail vein once in the 9th week,then subcutaneously injected with wild type exogenous mature DCs into the thigh of the mouse per week until sixteenth weeks;In ApoE^-/-/PSGL^-/-+DCs group,wild type exogenous mature DCs was injected into the tail vein of mice in ninth weeks,then subcutaneously injection of wild type exogenous mature DCs to the thigh of the mice per week until sixteenth weeks;And the same amount of NS was subcutaneously injected into the mice in the ApoE^-/-/PSGL^-/-+NS group in the same route,and observed for a total of 16 weeks.Then all the groups mice were killed and the corresponding specimens were tested.The expression profile of the phenotypes of mature DCs including CD40,CD80,CD83,CD86,and MHC II,T lymphocyte subsets in the spleen,the percentages of Treg marker（CD4⁺CD25⁺Foxp3⁺）in CD4⁺cells were detected by Flow cytometry（FCM）.The levels of IL-6 and TNF-αin the peripheral blood serum of mice were determined by Enzyme linked immunosorbent assay（ELISA）.The severity of atherosclerosis in aortic root was observed by HE staining.Oil red staining was applied to observe the pathological changes of aortic atherosclerosis in mice.Masson’s trichrome staining was used to observe the severity of vascular fibrosis in the aortic root of mice.The expressions of IL-6 and TNF-a mRNA in the aorta of mice were detected by Real-time PCR method.The expressions of CD11c,TLR4,MyD88 and NF-κb in the aortic root atherosclerotic lesions were detected by Immunofluorescence method.Results:As determined by FCM,the expression of mature DCs related phenotypes in the peripheral blood of mice.The results showed that:the expression level of AS model group was the highest,and the expression level of ApoE^-/-/PSGL^-/-+DCs group and ApoE^-/-P^-/-+DCs group was the second,the expression level of ApoE^-/-P^-/-+P-sel group was the third,ApoE^-/-/PSGL^-/-+NS group and ApoE^-/-P^-/-group had the lowest expression level.Comparison between groups,the related phenotypes expression of ApoE^-/-/PSGL^-/-+DCs group was significantly higher than that of ApoE^-/-/PSGL^-/-+NS group（P<0.05）;the related phenotypes expression of ApoE^-/-/P^-/-+P-sel group was apparently higher than that of ApoE^-/-/P^-/-group（P<0.05）;ApoE^-/-/PSGL^-/-+NS group and ApoE^-/-/P^-/-group were lower than AS model group（P<0.05）.The proportion of mature DCs related phenotypes expression were similar among ApoE^-/-P^-/-+P-sel group,ApoE^-/-P^-/-+DCs group,ApoE^-/-/PSGL^-/-+DCs group and AS model group in peripheral blood.There were no significant difference.Treg（its marker:CD4⁺CD25⁺Foxp3⁺cells）accounted for the proportion of CD4⁺T cells in the spleen of mouse,we also observed that,CD4⁺CD25⁺Foxp3⁺proportion in the ApoE^-/-/PSGL^-/-+NS group and ApoE^-/-/P^-/-group were the highest,while the ApoE^-/-/PSGL^-/-+DCs group and ApoE^-/-/P^-/-+DCs group were the second,ApoE^-/-/P^-/-+P-sel group was the third,the proportion of CD4⁺CD25⁺Foxp3⁺in the AS model group was the lowest.Comparison among these groups,the proportion of CD4⁺CD25⁺Foxp3⁺cells in ApoE^-/-/PSGL^-/-+DCs group was significantly lower than that of ApoE^-/-/PSGL^-/-+NS group,（P<0.05）;the proportion of CD4⁺CD25⁺Foxp3⁺cells in ApoE^-/-/P^-/-+P-sel group was lower than that of ApoE^-/-/P^-/-group,（P<0.05）;ApoE^-/-/PSGL^-/-+NS group and ApoE^-/-/P^-/-group were higher than that of AS model group（P<0.05）;There was no significant difference among ApoE^-/-P^-/-+P-sel group,ApoE^-/-P^-/-+DCs group,ApoE^-/-/PSGL^-/-+DCs group and AS model group.Results of HE staining in the mice aortic root showed the degree of atherosclerotic lesion of ApoE^-/-/P^-/-group and ApoE^-/-/PSGL^-/-+NS group was the least,followed by ApoE^-/-/P^-/-+P-sel group,and among the AS model group,ApoE^-/-/P^-/-+DCs group and ApoE^-/-/PSGL^-/-+DCs group had the most seriously degree of atherosclerosis.We found that the severity degree of atherosclerotic lesion of mice aortic root in the ApoE^-/-/P^-/-group were significantly decreased than that of the ApoE^-/-/P^-/-+P-sel group;compared with the Apo E^-/-/PSGL^-/-+NS group and ApoE^-/-/PSGL^-/-+DCs group,the change was also similar,the degree of atherosclerosis was significantly reduced,the difference was significant（p<0.05）.Compared with ApoE^-/-/PSGL^-/-+NS group and ApoE^-/-/P^-/-group,local lesion of plaque in ApoE^-/-/P^-/-group was smaller,but there was no significant difference between them.And among the ApoE^-/-/P^-/-+P-sel group,ApoE^-/-/P^-/-+DCs group,ApoE^-/-/PSGL^-/-+DCs group and AS model group,the aortic root severity were similar;compared with the AS model group,ApoE^-/-/P^-/-+P-sel group and ApoE^-/-/PSGL^-/-+DCs group in aortric lesions,plaque area,lipid core size was slightly increased,however,there were no statistically difference（p>0.05）;Similarly,aortic plaque deposition was also seen in the ApoE^-/-/P^-/-+DCs group,accompanied by a small amount of necrotic lipid pool formed in some white needle like crystals of cholesterol,but the extent of lesion,the plaque area were lower than that of AS model group,the difference was statistically significant（p<0.05）.Result of Masson’s trichrome staining also showed that the severity of vascular fibrosis was most seriously in AS model group,followed by the ApoE^-/-/PSGL^-/-+DCs group and ApoE^-/-/P^-/-+DCs group,ApoE^-/-/P^-/-+P-sel group had a higher degree of vascular fibrosis,ApoE^-/-/PSGL^-/-+NS group and ApoE^-/-/P^-/-group of vascular fibrosis were the lightest extent.Comparison between ApoE^-/-/P^-/-group and ApoE^-/-/P^-/-+P-sel group,the degree of the aortic root fibrosis was significantly reduced,the difference was statistically significance（p<0.05）;And compared with the ApoE^-/-/PSGL^-/-+NS group and ApoE^-/-/PSGL^-/-+DCs group,the change also seen similar,the degree of fibrosis of the intima lumen significantly reduced,the difference was statistically significant（p<0.05）;and the degree of fibrosis of ApoE^-/-/PSGL^-/-+NS group was lighter than that of ApoE^-/-/P^-/-group,but the difference was not significant（p>0.05）;and ApoE^-/-/P^-/-+P-sel group;And compared with the AS model group,ApoE^-/-/P^-/-+P-sel group,ApoE^-/-/P^-/-+DCs group and ApoE^-/-/PSGL^-/-+DCs group,the severity of aortic root fibrosis were similar among these groups（p>0.05）.Results of oil red staining of the aorta showed the similar results with HE staining.We found that the AS model group,ApoE^-/-/P^-/-+DCs group and ApoE^-/-/PSGL^-/-+DCs group had the highest degree of oil red staining,ApoE^-/-/P^-/-+P-sel group had moderate degree of oil red staining,the ApoE^-/-/PSGL^-/-+NS group and the ApoE^-/-/P^-/-group had the lowest degree of oil red staining.Comparison between groups,the degree of aortic oil red staining in ApoE^-/-/P^-/-group was significantly decreased than that of ApoE^-/-/P^-/-+P-sel group;Compared with ApoE^-/-/PSGL^-/-+NS group and Apo E^-/-/PSGL^-/-+DCs group,the change was also similar,the arteries oil red staining reduced obviously;Among the AS model group,ApoE^-/-/P^-/-+DCs group and ApoE^-/-/PSGL^-/-+DCs group,oil red staining degree of ApoE^-/-/PSGL^-/-+DCs group was slightly increased than AS model group,oil red staining degree of ApoE^-/-/P^-/-+P-sel group was slightly decreased.There were no significantly difference（p>0.05）.Results of ELISA test showed that serum IL-6 and TNF-αlevels in peripheral blood.We found that the level of IL-6 was as follows:the level of IL-6 in ApoE^-/-/PSGL^-/-+DCs group was the highest,while the level of AS model group and ApoE^-/-/P^-/-+DCs were lower than that of ApoE^-/-/PSGL^-/-+DCs group,and the IL-6 level of ApoE^-/-/P^-/-+P-sel group was alleviated than that of ApoE^-/-/P^-/-+DCs group and ApoE^-/-/PSGL^-/-+DCs group,however,there was no significant difference（p>0.05）.The concentration level of IL-6 level among the ApoE^-/-/P^-/-group and ApoE^-/-/PSGL^-/-+NS group were the lowest.The levels of TNF-a were as follows:peripheral plasma concentration level of TNF-a in the AS model group was the highest,while ApoE^-/-/P^-/-+DCs group and ApoE^-/-/P^-/-+P-sel group were less than that of AS model group,and that the concentration level of TNF-a were least between the ApoE^-/-/P^-/-group and ApoE^-/-/PSGL^-/-+NS group.We found that the comparison plasma levels of IL-6 and TNF-a were significantly reduced between the ApoE^-/-/P^-/-group and ApoE^-/-/P^-/-+P-sel,there was statistically significance（p<0.05）;Compared with the ApoE^-/-/PSGL^-/-+NS group and ApoE^-/-/PSGL^-/-+DCs group,plasma levels of IL-6 and TNF-a had the similarity reduce.The difference was statistically significant（p<0.05）;Compared with ApoE^-/-/PSGL^-/-+NS group and ApoE^-/-/P^-/-group,although the concentration level of IL-6 and TNF-a in ApoE^-/-/PSGL^-/-+NS group was lower,there was no significant difference between them.Among the ApoE^-/-/P^-/-+P-sel group,ApoE^-/-/P^-/-+DCs group,ApoE^-/-/PSGL^-/-+DCs group and AS model group,these groups had the similar results about the concentration levels of IL-6 and TNF-a,there were no statistical significance（p>0.05）;compared with the AS model group,the concentration level of IL-6 and TNF-a have significantly reduced between the ApoE^-/-/P^-/-group and ApoE^-/-/PSGL^-/-+NS group,there was statistical difference（p<0.05）.The expression of IL-6 and TLR-4 mRNA in the aorta was quantified by Real-time PCR.Determination of IL-6 mRNA level,the results showed that:compared with AS model group,IL-6 mRNA levels were similar among the ApoE^-/-/PSGL^-/-+DCs group,ApoE^-/-/P^-/-+DCs group and ApoE^-/-/P^-/-+P-sel group,and that IL-6 mRNA level of the ApoE^-/-/PSGL^-/-+DCs group was more than other groups relatively,while the IL-6mRNA levels were relatively less between the ApoE^-/-/P^-/-+DCs group and ApoE^-/-/P^-/-+P-sel group,but there were no statistical difference（p>0.05）.However,compared with ApoE^-/-/P^-/-group and ApoE^-/-/P^-/-+P-sel group,the expression of IL-6mRNA decreased significantly,and the expression of IL-6 was also significantly reduced between the ApoE^-/-/PSGL^-/-+NS group and ApoE^-/-/PSGL^-/-+DCs group,there was statistical significance（p<0.05）.Determination of TNF-a mRNA level,the results showed that the TNF-a expression were the highest level in the AS model group and ApoE^-/-/PSGL^-/-+DCs group,and that the level of TNF-a mRNA was relatively smaller among the Apo E^-/-/P^-/-+DCs group and ApoE^-/-/P^-/-+P-sel group,there was no statistical significance（p>0.05）.However,compared with ApoE^-/-/P^-/-group and ApoE^-/-/P^-/-+P-sel group,the level of TNF-a mRNA decreased significantly,and the expression of TNF-a m RNA in the ApoE^-/-/PSGL^-/-+NS group decreased significantly than that of the ApoE^-/-/PSGL^-/-+DCs group.Immunofluorescence results showed that expression of TLR4,MyD88 and NF-kb in the local plaque was the highest in AS model group;followed by the ApoE^-/-/PSGL^-/-+DCs group and ApoE^-/-/P^-/-+DCs group;ApoE^-/-/P^-/-+P-sel group was the third;ApoE^-/-/PSGL^-/-+NS group and ApoE^-/-/P^-/-group were the lowest.Comparison between groups,expression of TLR4,MyD88 and NF-kb levels in the ApoE^-/-/P^-/-group and ApoE^-/-/PSGL^-/-+NS group were significantly decreased than that of AS model group,there was significant difference（p<0.05）;Compare with ApoE^-/-/P^-/-group and ApoE^-/-/P^-/-+P-sel group,the expression of TLR4,MyD88 and NF-kb levels were significantly reduced,there was statistical significance（p<0.05）;the expression level of TLR4,MyD88 and NF-kb in the ApoE^-/-/PSGL^-/-+NS group was significantly reduced than that of ApoE^-/-/PSGL^-/-+DCs group,the difference was significant（p<0.05）.Compared with AS model group,the expression levels of TLR4,MyD88 and NF-kb were similar to those of ApoE^-/-/P^-/-+P-sel group,Apo E^-/-/P^-/-+DCs group and ApoE^-/-/PSGL^-/-+DCs group（P>0.05）.Conclusion:From these results of mature DCs costimulatory molecule expression,local lesion of aortic root and gross pathological artery,vascular fibrosis,the level of concentration inflammatory factors,the expression level of mRNA and immunofluorescence means that exogenous DCs could have mature the stimulation of immature DCs’s maturation through P-selectin/PSGL-1 pathway in vivo,by up-regulating the expression of TLR-4 receptors on the surface of immature DCs by the activation of the TLR-4-MyD88-NF-κB signaling pathway,promoting the immune inflammatory reaction intensified,thereby affecting the aortic atherosclerosis occurrence and development in vivo.