Effect Of Tolerogenic DC Loaded With HSP60 On The Progression Of The Atherosclerotic Plaque In Mice

Part 1 The effect of aspirin on maturation and function of murine myeloid dendritic cells in vitroObjective To study effect of aspirin on the function of murine myeloid dendritic cell in vitro. Methods Bone marrow-derived dendritic cell progenitors from C57BL/6J mice were treated with aspirin or without. Flow cytometric analysis was used to detect the expression of co-stimulating factor CD80, CD86 on DC. The stimulating capacity of DC was determined in mixed lymphocyte reaction (MLR). ELISA was used to analyze the level of cytokines in the suspension of MLR. Result Compared with untreated group, CD80, CD86 on aspirin-treated DC expressed significantly less (P<0.01); the stimulating T-cell capacity of aspirin-treated DC in MLR was lower(P<0.01); T lymphocytes in MLR secreted lower levels of pro-inflammation cytokines(TNF-α)(P<0.01); Above effects were evident on higher concentration. Conclusion Aspirin has suppressive influences on the expression of co-stimulating factor and stimulating capacity of DC,and inhibitory effect of aspirin on inflammation dose-dependently. Part 2 Effect of tolerogenic DC loaded with HSP60 on the progression of the atherosclerotic plaque in mouseObjective To evaluate whether tolerogenic dendritic cell(DC) loaded with heat shock protein60(HSP60) could inhibit the progression of aortic atherosclerotic plaque in apolipoprotein(apo) E-/- mice. Methods Bone marrow derived DC of the mice were incubated with PBS,HSP60 or co-cultured with aspirin and HSP60 respectively, and gained DCHSP60,asp-DCHSP60 and DCPBS. Then the treated DCs were observed the morphological changes and detected the variation of CD80 and CD86 phenotype with flow cytometric analysis (FCA) in vitro. mDCs were marked by fluorescent CFSE and injected into the homogenous ApoE-/- mice subcutaneously for three times at a one-week interval and fed high-cholesterol diet 8 weeks to form plaque. Then pathological analysis of atherosclerotic lesion were performed and serum cytokines (IL-10, IFN-γ) were analyzed by ELISA assay. Then aortas were harvested for Haematoxylin-Eosin stained to mearure plaque area and plaque/lumia area ratio and anti-CD4+T cell immunostained to observe CD4+T cell and DC infiltration in plaques. Result Compared with DCPBS, DCHSP60 up-regulated CD80,CD86 levels and stimulated T lymphocyte proliferation significantly; while asp-DCHSP60 expressed lower levels of CD80,CD86. After treated mice, DCHSP60 made inflammatory cells infiltration in atherosclerotic plaques greatly, and plaques tended to vulnerability. Then asp-DCHSP60 reduced inflammatory cells infiltration in atherosclerotic plaques and plaques tended to stabilization(P<0.01). And asp-DCHSP60 and DCHSP60 migated to atherosclerotic plaques more greatly than DCPBS group(P<0.01), but no significant deviation between asp-DCHSP60 and DCHSP60 groups. Compared with DCPBS group, serum IFN-γwas lower in asp-DCHSP60 group (P<0.01), but DCHSP60 group was reverse. While serum IL-10 was no significant difference among the three groups.Conclusion Our data shows that tolerogenic asp-DCHSP60 reduces the HSP60 specific immuno-inflammatory process and inhibits atherogensis.