The Effects And Its Mechanisms Underlying TLR2 Deficiency On Allograft Rejection In The Murine Cardiac Transplantation

Background:The emergence of IL-17+ alloreactive T cells that mediate allograft rejection has provided an impetus to understand the factors affecting the generation of these IL-17+T cells in allograft transplantation.Toll-like receptor 2(TLR2)plays a different role in the differentiation of Th17 cells in various conditions and disease models.How TLR2 signalling regulates the generation of IL-17-producing alloreactive T cells upon alloantigen stimuli remains unclearObjective:In this study,we used a mouse model of cardiac allograft transplantation to investigate whether TLR2 signalling influences the development of IL-17-producing alloreactive T cellsMethods:1.Establishment of the murine cardiac transplantation model:C57BL/6J wild type mice(WT)and C57BL/6J TLR2 knockout mice(TLR2-/-;B6.129 tmIkir/JNju)mice were used as recipients,and BALB/c mice were used as donors in the allogeneic graft transplantation models.In the syngeneic graft transplantation models,C57BL/6J WT and C57BL/6J TLR2-/-mice were used as recipients,and C57BL/6J mice were used as donors2.Detection contents and methods in vivo experiments(1)Survival of cardiac graft of each group was recorded.H&E staining was used to detect the inflammatory cell infiltration and myocardial damage in cardiac graft,and the severity of cardiac rejection was measured by the International Society for Heart and Lung(ISHLT)score;(2)Immunohistochemical staining was performed to detect the neutrophils and macrophages infiltration in the grafts;(3)IL-17+CD3+ T cells,IL-17+CD4+ T cells(Th17),IL-17+CD8＋T cells,IL-17＋γδT cells,IFN-γ+ cells and regulatory T cells(Tregs)in spleens and allografts were analysed by flow cytometry;(4)IL-6+CD11C+ cells and IL-6+F4/80+ cells in spleens after cardiac allograft transplantation were analysed by flow cytometry;(5)The expression of IL-17A,IL-6,IL-1β,TNF-α,CCL20,CCR6,IL-10,IL-4,IFN-γand other cytokines in allografts were analysed by using quantitative real-time PCR;(6)Western blots were used to investigate the expression of STAT3 and p-STAT3 in spleens and allografts3.Detection contents and methods in vitro experiments(1)T cells from recipient mice(TLR2-/-and WT)were co-cultured with BMDCs from donor mice with or without TGF-β and IL-6,then the percentages of Th17 and IL-17+γδ T cells were analysed by flow cytometry;(2)Spleen cells from recipient mice(TLR2-/-and WT)were co-cultured with BMDCs from donor mice,and the percentages of Th17 and IL-17+ γδ T cells were analysed by flow cvtometrv.And the concentration of IL-6 in the suDernatant was detected bv ELISA;(3)Anti-IL-6 antibody was used in the mixed lymphocyte culture,and the percentages of Th17 and IL-17+ γδ T cells were analysed by flow cytometryResults:1.This study successfully established an acute rejection model of murine cardiac transplantation2.The effects of TLR2 gene knockout on the allograft rejection(1)TLR2 deficiency led to more severe infiltration of inflammatory leukocytes in the cardiac allograft in the early period of acute allograft rejection;(2)Neutrophil and macrophage infiltration in allografts was enhanced in TLR2-deficient mice;(3)TLR2 deficiency led to an increase in Th17 cells and IL-17+ γδ T cells;(4)No significant difference in Thl cells was observed between recipient TLR2-/-and WT mice;(5)mRNA expression of IL-17,IL-6,IL-1β,TNF-a,CCL20 and CCR6 within the allografts markedly increased in recipient TLR2-/-mice compared to WT mice3.The increase in IL-17-producing T cells of TLR2-/-mice was related to the increased production of IL-6 from DCs(1)TLR2 deficency of T cells themselves did not affect the reactivity of T cells to alloantigen stimulation;(2)IL-6 enhanced the production of Th17 and IL-17+ γδ T cells in both the TLR2-/-and WT groups,and blockade of IL-6 suppressed Th17 and IL-17+ γδ T cell production in TLR2-/-group;(3)Production of IL-6 from DCs was significantly increased in TLR2-/-mice after allograft transplantation4.Lack of TLR2 signalling resulted in a remarkable increase of p-STAT3 both in spleens and allografts5.TLR2 deficiency led to a significant decrease in the generation of TregsConclusion:1.TLR2 deficiency enhances cardiac transplantation rejection in mice;2.TLR2-deficient recipient mice exhibit high Th17 and IL-17+ alloreactive T cells;3.IL-6 secreted by dendritic cells of TLR2-deficient mice contributes to driving Th17 skewing.And TLR2 deficiency leads to upregulation of STAT3 phosphorylation;4.Modulation of TLR2 signaling is a potential therapeutic target for transplantation patients.