| Background: Early studies proved that the inflammation and Oxidative Stresstriggered the initiation of atherosclerosis and also contributed to its progression. Up todate, more and more researchers believe atherosclerosis is an autoimmune disease due tothe disorders of immune responses. And diabetes mellitus, as the equivalence of AS,always aggravates atherosclerosis. Dendritic cells, as the typical antigen presenting cells,are considered to play an vital role in AS, which induces the adaptive immune responses.During the prevention of atherosclerosis, studies show that the vaccination against the ASrelevant antigen could attenuate the progression of atherosclerosis, and our previous studyperformed the same results that the vaccination against AGE-LDL could haveatheroprotective effects in diabetic LDLr-/-mice fed with high fat diets. Although the ASvaccines are promising towards AS prevention, both protein and cell vaccines have somelimits during application, and researcher are focusing on a new particles called exosomeswhich derived from many cell types and harbor the similar functions with the cells, andtrying to overcome the limits of previous vaccines by using exosome vaccins.Objective: To investigate whether the vaccination of DC derived exosomes couldattenuate atherosclerosis, and partially explore its mechanisms.Methods:(1) Six-week-old LDL-/-mice was obtained and randomly divided into twogroups. All the mice started high-fat-diet after two weeks of adaptive normal feeding. onthe9thweek, The mice was vaccinated against AGE-LDL plus Al(OH)3subcutaneouslywith a dose of25ug and the equal PBS as control. The vaccination was strengthened everytwo weeks and had a total of3repeats. All the mice was executed on17thweek withspleen and aorta obtained and splenocytes were on the procession of DC isolation usingMACS under germ-free conditions. The DCs was cultured with exo-depleted media andthe supernatant was utilized to isolate exosomes using Exo-quick TC，and the proteinquantification was measured by BCA, the exosomes were also identified by flowcytometry.(2) Another batch of6-week-old LDLr-/-mice was obtained and randomlydivided into three groups (the PBS group,n=8; the exo-control group, n=10; theexo-immune group, n=11). After the same diet procedure previously,the mice wereimmunized with1ug exosomes subcutaneously (DC derived exosomes either fromAGE-LDL immunized or PBS injected mice DC) with PBS as control and the vaccinationwas strengthened2times every2weeks. On the15thweek, all the mice were injected STZ(50mg/kg) intraperitoneally for6days continuously to develop diabetes. All the micewere executed on the25thweek, serum was collected and the lipid measurement was doneby the auto-analyzer technique, and lipid distribution was measured by Fast protein liquidchromatography (FPLC). The aorta samples were isolated and en face oil red staining wasperformed to calculate the plaque area of different groups. The cryo-section of aortic rootwere prepared and morphological and cytological changes were observed by oil redstaining, Masson staining, MOMA-2staining to evaluate the stability of AS plaque. Thenwe investigated the percentage of T helper lymphocytes(Th1/2/17) and regulatory T cells(Treg) to identify the cellular immune alterations by flow cytometry, and serum totalantibodies and AGE-LDL specific antibodies were detected by Elisa. At last, weperformed the qPCR to find whether any transcriptional factors expressed differentially.Statistical approach: All the data were analyzed using SPSS18.0statistical software,P<0.05was considered statistically significant.Results:(1) After MACS isolation and the maturation stimulated by AGE-LDL, DCsexpressed high surface markers with CD11c95.34%positive, CD8678.62%positive andMHC-II83.05%positive, CD4082.31%positive, and the exosomes were also having aDC phenotype with CD86positive for25.79%, CD11c positive for9.41%, MHC-IIpositive for7.03%and CD63positive for18.73%.(2) The immunization of DC derivedexosomes from AGE-LDL vaccinated LDLr-/-mice could attenuate the progression ofatherosclerosis: The FPLC and lipid measurement showed that the HDL concentration inthe exo-immune group was higher than the other two groups while the non-HDL was lowerin the two exo group than PBS control. The en face aorta oil red staining showed that theplaque area of exo-immune group was significantly smaller than those of both theexo-control and PBS group, about44%and54%reduction respectively, while there’s nostatistic difference between the two control groups. Meanwhile the cryo-section oil-redstaining also revealed the same results of plaque area that the exo-immune group had a25%and a34%less than the exo-control and PBS-control group respectively.(2) Theimmunization of DC derived exosomes from AGE-LDL vaccinated LDLr-/-mice couldmodulate the immune response that alleviated atherosclerosis. In our study, theexo-immune group had less Th1lymphocyte compared with the two controls (p<0.05),and there were more Th2T cells in the exo-immune group and exo-control groupcompared with the PBS control though there were no difference between the two exo group. Total serum IgG antibodies was equivalent among the three groups while theAGE-LDL specific antibodies was higher in the exo-immune group (p<0.05). The qPCRunveiling that IFN-γ mRNA had a lower expression while IDO-1mRNA had a higherpattern in exo-immune group.Conclusion: Our study has identified that the vaccination against AGE-LDL couldattenuate atherosclerosis, while the dendritic cell derived exosomes from AGE-LDLimmunized LDLr-/-mice could harbor the same effects, briefly by inhibiting the Th1immune response and increasing serum HDL concentrations and alleviating inflammationthrough IDO expression, which represents that exosomes may play a role on theconnection between APCs and immune response that throughout the initiation andprogression of atherosclerosis, and possibly the vaccination of exosomes may shed lighton the immune therapy of atherosclerosis. |
PDF Full Text Request