| Part 1 MiR-155 expression and Th17/Treg cell immune response in experimental autoimmune myocarditisObjective:To identify a positive role of miR-155 and an imbalance of Th17/Treg in autoimmune myocarditis, we detected the expression level of miR-155 and Th17/Treg cell immune response in EAM mice.Methods:Experimental autoimmune myocarditis was elicited in BALB/c mice by immunization of self-peptides derived from murine α-cardiac myosin heavy chain sequence. The miR-155 expression levels in heart tissue, peripheral blood mononuclear cells (PBMCs), splenic lymphocytes and splenic CD4+T cells were detected by RT-PCR. Th17-related factors (RORγt, IL-6, IL-17A) were detected in cardiac tissue by western blot and RT-PCR. Th17 and Treg cells percentages in splenic CD4+T cells and CD11c+DC percentages in splenocytes were investigated by flow cytometry. Expression of Th17-related factors (RORγt, IL-17A and IL-6) and Treg related factors (foxp3, TGF-β, IL-10, IL-35) in splenic CD4+T cells, and Thl7-polarzing cytokines in splenic CD11c+DC were detected by RT-PCR. T cell proliferation and suppression assay was conducted to evaluate the suppression function of Treg cells in EAM.Results:Compared with control mice, the expression levels of miR-155 in heart tissue, PBMCs and splenic CD4+T cells were significantly elevated. Heart tissue from EAM mice expressed higher levels of Thl7-related factors including RORyt, IL-17A and IL-6. Flow cytometric analysis indicated a higher frequency of Th17 cells in splenic CD4+T cells of EAM group. However, there was no significant difference in the frequencies of Treg cells between these two groups. The expression of Th17-related proinflammatory factors (RORyt, IL-17A, IL-21, IL-22) and Treg related anti-inflammatory factors (foxp3, TGF-β, IL-10, IL-35) were all increased in splenic CD4+T cell of EAM mice. Furthermore, EAM mice presented a higher percentage of splenic CD11c+DCs, and splenic CDllc+DCs from EAM mice expressed elevated mRNA levels of Thl7-polarizing cytokines (IL-6, IL-23, IL-1β, TNF-a). Our CFSE-based in vitro proliferation assay indicated that CD4+effector T cells of EAM exhibited low susceptibility to Treg-mediated suppression.Conclusions:MiRNA-155 expression is highly elevated in experimental autoimmune myocarditis; EAM presented a proliferative and functional imbalance of Th17/Treg cells.Part 2 MiR-155 promotes Th17 cell development and function in experimental autoimmune myocarditisObjective:To detect the effect and mechanism of miR-155 on the development and function of Th17 and Treg cells in experimental autoimmune myocarditis (EAM), we performed’loss of function’ experiments through miR-155 inhibition in vivo.Methods:EAM was induced in BALB/c mice. Animal groups included control groups injected with PBS or antagomir-155 and EAM groups injected with PBS or antagomir-155. The body weight change, the heart weight/body weight (HW/BW) ratio, the heart weight/tibia length (HW/TL) ratio and total splenic lymphocytes counts in each group were measured. The miR-155 expression levels in heart tissue, peripheral blood mononuclear cells (PBMCs), splenic lymphocytes and splenic CD4+T cells were detected by RT-PCR. Histopathologic analysis and echocardiography were conducted to detect the extent of myocarditis and cardiac function of each group. Th17-related factors (RORyt, IL-6, IL-17A) were detected in cardiac tissue by western blot and RT-PCR. Immunochemistry was performed to detect IL-17A in myocarditis hearts. Th17 cells in isolated cardiac-infiltrating cells were detected by flow cytometry.Th17 and Treg cells percentages in splenic CD4+T cells and CD11c+DC percentages in splenocytes were investigated by flow cytometry. Expression of Th17-related factors (RORyt, IL-17A and IL-6) and Treg related factors (foxp3, TGF-β, IL-10, IL-35) in splenic CD4+T cells, and Th17-polarzing cytokines in splenic CDllc+DC were detected by RT-PCR. Splenic lymphocytes from EAM groups were isolated and subjected to MyHC-α peptide-specific proliferation assay in vitro. The proliferation and IL-17A secretion of CD4+T cells were detected by flow cytometry.Results:compared with PBS-treated EAM mice, antagomir-155-treated EAM mice presented reduced total splenocytes and body weight loss, elevated ratios of HW/BW at baseline and HW/TL, diminished inflammation severity and reduced collagen deposition. In situ hybridization of miR-155 on heart sections indicated an apparent decline of miR-155 expression in cardiac tissue of EAM mice treated with antagomir-155. Furthermore, RT-PCR analysis confirmed significant repression of miR-155 in hearts, PBMC and spleens by antagomir-155. Cardiac dysfunction, as shown by obviously decreased Ejection fraction (EF) and Fractional shorting (FS), was observed in PBS-treated EAM group, whereas miR-155 inhibition significantly increased the EF and FS, indicating improved cardiac function in antagomir-155-treated EAM group. Meanwhile, anti-miR-155 treatment largely reduced the heart size of EAM mice. Immunochemistry of IL-17A and flow cytometric analysis of CD4+IL-17+T cells indicated that miR-155 inhibition significantly decreased the Th17 cells infiltration in myocarditis heart. The expression levels of Th17-related factors (RORyt, IL-17A, IL-6) were all decreased in myocarditis heart after antagomir-155 treatment. In addition, antagomir-155 treatment in EAM mice reduced the percentages of Th17 cells and Treg cells among total splenic CD4+T cells, and the frequency of splenic CDllc+DCs. The expression of Th17-related factors (RORyt, IL-17A and IL-6) and Treg-related factors (foxp3, TGF-β,IL-10, IL-35) in splenic CD4+T cells, and Th17-polarzing cytokines in splenic CD11c+DCs were all downregulated in antagomir-155-treated EAM group, compared with PBS-treated EAM group. MyHC-α peptide-specific proliferation assay indicated that CD4+T cells from EAM mice treated with antagomir-155 exhibited diminished proliferation and IL-17A production in comparison with CD4+T cells from PBS-treated EAM mice.Conclusions:MiRNA-155 inhibition significantly ameliorated myocardial damage through suppressing Th17 cell development and function in experimental autoimmune myocarditis.Part 3 In vitro analysis of the effect of miR-155 on the differentiation and function of Thl7 cells and dendritic cellsObjective:To further investigate the impact of miR-155 on the differentiation and function of Th 17 cells and bone marrow derived dendritic cells (BMDCs), we performed in vitro studies.Methods:Splenic CD4+T cells sorted from normal BALB/c mice were transfected with agomiR-155 or antagomir-155 for miRNA-155 overexpression or inhibition, and then cultured and activated for Th17-polarizing. The IL-17A secretion in each group were detected by ELISA. The mRNA expression of IL-17A, essential transcriptional factor RORyt, single transducer factor STAT3 and miR-155 target SOCS1 were detected by RT-PCR. BMDCs were generated in vitro. Cells were transfected with antagomir-155 or antagomir-NC for miRNA-155 inhibition before LPS stimulation for maturation. Surface markers MHCⅡ, CD86 and CDllc in each cell group were detected by Flow cytometry. RT-PCR was conducted to detect the mRNA expression of miR-155 direct tragets (SOCS1, SHIP1, PU.1) and proinflammatory cytokines (IL-6, IL-23, IL-1β, TNF-a). Secretion of the proinflammatory cytokines in the culture supernatants were measured by ELISA.Results:MiRNA-155 expression was highly elevated in agomiR-155-treated cells while apparently suppressed in cells transfected with antagomir-155 in comparison with other groups. Flow cytometric analysis revealed that miR-155-overexpressed CD4+T cells differentiated into Th17 cells most remarkably while miR-155-inhibited CD4+T cells had a defective ability to produce Th17 cells. The IL-17A secretion and the mRNA expression of IL-17A, RORyt and STAT3 were significantly increased in agomiR-155-treated group whereas obviously decreased in antagomir-155-treated group. Additionally, the mRNA expression of miR-155 target SOCS1 was decreased in agomiR-155-treated cells, while increased in antagomir-155-treated cells. Derepressed expression of SOCS1, SHIP1 and PU.1 were observed in miR-155-inhibited BMDCs. Although miR-155 inhibition in BMDCs had no significant influence on the expression of surface markers MHC Ⅱ, CD86 and CD11c after LPS stimulation, miR-155-inhibited BMDCs had obviously impaired ability of producing Th17-polarizing cytokine (IL-6, TNF-α, IL-1β, IL-23).Conclusions:MiRNA-155 is necessary for proper Th17 differentiation and dendritic cell function of secreting Th17-polarizing cytokines in vitro. |
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