TLR7激动剂Imiquimod负向调控Tfh细胞在系统性红斑狼疮中的作用机制研究

Follicular helper T cells (Tfh cells), a novel subset of CD4+T cells are specialized in helping B cells. It leads to the autoimmune diseases such as Systemic Lupus Erythematosus (SLE) when its numbers and functions are been mis-conducted. Recently, a new study has been found that Toll Like Receptor 7 (TLR7) can negatively regulate the function of CD4 + T cells. So far, there is not clear whether TLR7 can negatively regulate the function of Tfh cells in SLE and the relative molecular mechanisms. Therefore, this project plans to investigate the proliferation and differentiation of Tfh cells and its effect on helping B cells to produce antibodies from Mice in vivo and in vitro study by Flow Cytometry, Immunofluorescence technique, Real-time PCR，Western blot and so on, to identify the function of the regulating Tfh cells immune response through Ligand, Imiquimod(IMQ) activation. Furthermore, in order to explore the molecular mechanism of regulating Tfh cells by TLR7, it plans to evaluate the expression of mRNA and protein of the transcriptional factor of Bcl-6、STAT3、NFAT、IRF4 and so on during the differentiation of Tfh cells to IMQ activating response. Finally, we will establish the SLE mice model for the study, to explore the mechanism of negatively regulating Tfh cells by TLR7 in SLE mice model, providing the new ideas and new targets for treating SLE.

滤泡辅助性T（Tfh）细胞是一群专职辅助B细胞免疫的新型CD4+T细胞亚群，其数量和功能异常可导致系统性红斑狼疮（SLE）的发生。最新研究发现，Toll样受体7（TLR7）能负向调控CD4+T细胞的功能，但TLR7能否负向调控Tfh细胞在SLE中的作用及其机制尚不清楚。为此，本研究拟通过流式细胞术、免疫荧光显微术、Real-time PCR、Western blot等方法，在小鼠体内外观察TLR7激动剂Imiquimod（IMQ）对Tfh细胞增殖分化及其辅助B细胞免疫的影响，明确TLR7对Tfh细胞的负向调控作用；通过观察IMQ对Tfh细胞分化相关的转录因子Bcl-6、STAT3、NFAT、IRF4等mRNA及蛋白表达的影响，探讨TLR7调节Tfh细胞的分子机制；最后，以SLE模型小鼠为研究对象，阐明TLR7激动剂负向调控Tfh细胞在SLE中的作用及机制，为SLE的治疗提供新思路和新靶点。

本课题通过体内外实验明确TLR7激动剂咪喹莫特对Tfh细胞分化及功能的影响，并探讨其作用机理，进一步体内研究咪喹莫特对系统性红斑狼疮（Systemic lupus erythematosus，SLE）模型小鼠（MRL/lpr小鼠）Tfh细胞的影响，为TLR7在SLE中的作用机理提供新的实验数据。体外实验结果显示：培养3天后，与阴性组相比，咪喹莫特能明显下调 Tfh细胞（CD4+CXCR5+）的数量，抑制Tfh细胞表面分子ICOS、PD-1和转录因子BCL-6细胞的表达，呈剂量依赖性；咪喹莫特能明显降低细胞因子IL-21和IL-4的分泌，呈剂量依赖性；咪喹莫特能明显抑制Tfh细胞相关转录因子BCL-6、C-MAF、NFATc2、BATF3、STAT3和LEF-1mRNA的表达水平，明显上调Blimp-1、STAT5a和STAT5b mRNA的表达水平。体内结果显示，连续腹腔注射4周后，咪喹莫特能明显下调小鼠Tfh细胞表面分子ICOS和转录因子BCL-6细胞的表达，咪喹莫特能明显降低细胞因子IL-21的分泌。咪喹莫特能明显下调GC B细胞和IgG1型浆细胞的数量，咪喹莫特能降低血清中IgG、IgG2a、IgG3的含量。咪喹莫特能明显抑制BCL-6、C-MAF 、NFATc2、STAT3 mRNA的表达水平；明显上调Blimp-1和STAT5b mRNA的表达水平。TLR7激动剂咪喹莫特对MRL/lpr小鼠的影响：连续腹腔注射4周后，与生理盐水组相比，咪喹莫特能明显下调MRL/lpr小鼠Tfh细胞表面分子ICOS、PD-1和转录因子BCL-6的表达，咪喹莫特能明显降低细胞因子IL-21和IL-4的分泌。咪喹莫特能明显下调IgG1型浆细胞的数量，咪喹莫特能降低血清中IgG、IgG1、IgG2a、IgG3、IgA、IgM的含量，咪喹莫特能明显抑制BCL-6、C-MAF、NFATc2、STAT3、BATF3、LEF-1mRNA的表达水平，明显上调Blimp-1和STAT5a mRNA的表达水平。HE染色和免疫荧光结果显示：咪喹莫特能明显减轻MRL/lpr小鼠肾脏肾小球及肾小球淋巴细胞浸润和肾小球基底膜IgG的沉积。结论：TLR7通过调控Tfh细胞相关转录因子，抑制Tfh细胞的分化和功能；抑制B细胞，减弱体液免疫应，减缓狼疮肾损害，为TLR7在SLE中的作用机理提供新的实验数据。