Batf3-dependent Dendritic Cells Promote Atherosclerosis Through The Induction Of Th1Differentiation

Objective:Atherosclerosis is a immune-related chronic inflammatory disease,both innateand adaptive immunity play a vital role in that process.As well, Different T cellsubsets also play an important role in the development of atherosclerosis. dendriticcells(DCs) also participate in regulating T cell responses in this process. Baft3is a keytranscription factor that regulate the differentiation of DCs. This topic will study thatwhat is the effects of Baft3–dependent DCs subsets on atherosclerotic disease andexplore its regulation to innate immunity and adaptive immunity deeply to providetheoretical basis for the prevention and treatment of atherosclerosis in the future.Method：1. Build Batf3-/-Apoe-/-double genetic defect mice by hybridization. Mainmethods is that Batf3-/-mice hybrids with Apoe-/-mice to produce F1generation, thencontinue to close cage to F2generation. after several rounds of hybridization, throughPCR amplification and identify genes in mice by agarose gel electrophoresis.thenanalyse the DC subsets of spleen and aorta by flow cytometry.2. Using Apoe-/-mice as a control, we induce the model of Batf3-/-Apoe-/-doublegenetic defect atherosclerotic disease in mice by high-fat diet.3. Feed Batf3-/-Apoe-/-double genetic defect mice on a high-fat diet for nineweeks,then cardiac perfusion, embedding the heare and freezing microtome sections.4. Evaluate the formation of plaque in the section of the aortic root through theoil red O staining, then calculate the area of plaque and the ratio of lumen bysoftware. Using HE staining to observe the infiltration of inflammatory cells in aortaplaque.5. Test the changes of the weight between the experimental group and the control group, test total plasma cholesterol and triglyceride concentrations by biochemicalanalyzer.6. Take the spleen and aortic to prepare the single cell suspension. Use mul-ticolor fluorescence labeling and flow cytometry testing, we analyze the changes ofspleen DC subsets and the expression of functional molecules as well as the ratio ofthe neutrophils, macrophages, mononuclear cells, T cells, B cells and effects about theactivation and proliferation.7. Intracellular cytokines staining to detect the changes of Th1and Th17cells.8. Intracellular cytokine staining to detect the changes of regulatory T cells.9. Use C57mice as a control, feeding Apoe-/-on a high-fat diet for9weeks,separate CD8+DC and CD8-DC subsets of spleen, then detect IL-12p35geneexpression through the Real-time PCR.Results:1. Batf3-/-Apoe-/-mice displays reduction of spleen CD8a+DC subsets (P <0.001)and absence of aortic CD103+DC subsets by flow cytometry.2. After9weeks of high-fat diets, oil red O staining shows that compared withthe control group, Batf3-/-Apoe-/-double genetic defect mice displays the significantdecreased plaque areas, As well, HE staining shows the significant reducedflammatory cells infiltration.3. Through biochemical analyzer test,we find that Batf3-/-Apoe-/-mice has nosignificant difference in total cholesterol and triglycerides in comparison with thecontrol group as well as the weight.4. Through the flow cytometry, the spleen CD8a+DC subsets of Batf3-/-Apoe-/-mice significantly reduce (P <0.001) and aortic CD103+DC subgroup is absent.Therates of the spleen cDC increases significantly, while the expression of its IA/IEreduces remarkablely.5. By flow cytometry, we find that compared with Apoe-/-mice, Batf3-/-Apo-e-/-mice has no significant difference about the ratio of neutrophils, macrophages,monocytes, CD8+T cells, B cells, in the contrary, the ratio of the CD4+T cellsincreased remarkablely.6. The results of flow cytometry shows that the activation and proliferation of theCD8+T cell are no significant difference in spleen as well as B cells. We also findthat the absence of Batf3do not affect the activation of CD4+T cell but itsproliferation which is significantly increased. 7. By intracellular cytokine staining and flow cytometry analysis, we find thatthere are no significant difference in Th17and Tregs between the two groups, but theproportion of Th1cells significantly reduce (P <0.001).8. Through the Real-time PCR to detect the expression of IL-12p35genes, wefind that in comparison with the C57mice, Apoe-/-mice increases the expression of IL-12p35on CD8+DCs,while CD8–DCs has no difference.Conclusions:1. In the process of atherosclerosis disease, the absence of Batf3causes thesignificant decrease of plaque area and inflammatory cells infiltration withoutaffecting the plasma concentrations of total cholesterol and triglyceride.2. The absence of Batf3causes the significant reduction of spleen CD8a+DCsubgroup (P <0.001), aortic CD103+DC subgroup is also missing, in turn, it mayinduces the differentiation of Th1by high expression of IL-12p35to promote theformation of atherosclerotic plaque.