CD38调控TLR4介导的B细胞发育机制研究

Developmental disorders of B-cells may result in humoral immune deficiency, TLR4 is the important signal molecule for promoting B cell development and maturation. We found in TLR4mut and CD38-/- mouse models that in addition to TLR4 and CD38 gene defects may result in B-cell developmental disorders, CD38-/- mice show an increased cell apoptosis of B-cells, accompanied by increased expression of Sirt1. And in the CD38-/-TLR4mut mouse model, we observed that B cell apoptosis induced by CD38 gene deletion could be reversed by TLR4mut, these preliminary data suggested that CD38 can activating TLR4, and promote the development of B-cells by inhibiting the apoptosis of B-cells, and Sirt1 may be involved in CD38 gene deletion induced B-cell developmental disorders. The aim of this study is to investigate: 1) the promoting effects of CD38 on TLR4 and its regulatory mechanisms, the role of Sirt1 in the deletion of CD38 gene, 2) the role of CD38/TLR4/NFκB and CD38/TLR4/BAFF pathway in B cell maturation, 3) the key targets of CD38/TLR4 pathway in regulation of B-cell apoptosis. All the results will be validated in CD38-/-TLR4-/- mouse model. We hope the results of this study will reveal the role of CD38 gene in TLR4 mediated B-cell development and its mechanisms.

B细胞发育障碍可引起体液免疫缺陷，TLR4是促进B细胞发育成熟的重要信号分子。我们在TLR4mut和CD38-/-小鼠模型上发现，除引起B细胞发育障碍外，CD38-/-小鼠B细胞凋亡增加伴Sirt1表达升高，且在CD38-/-TLR4mut小鼠模型上观察到CD38基因缺失引起的B细胞凋亡增加可被TLR4mut逆转，提示CD38可通过激活TLR4抑制B细胞凋亡以促进B细胞发育，Sirt1可参与CD38基因缺失引起的B细胞发育障碍。本项目拟研究：1）CD38对TLR4的促进作用、调控机制及CD38基因缺失时Sirt1的作用，2）CD38/TLR4/NFκB和CD38/TLR4/BAFF通路在促进B细胞成熟中的作用，3）CD38/TLR4通路在抑制B细胞凋亡中的关键靶点；并借助制备CD38-/-TLR4-/-小鼠模型进行结果验证。研究期望揭示CD38在TLR4介导的B细胞发育中的调控作用及机制。

B细胞发育障碍可引起体液免疫缺陷，TLR4信号在B细胞发育和败血症肾损伤中起着重要的促进作用。我们的前期研究提示CD38可通过激活TLR4抑制B细胞凋亡以促进B细胞发育，但CD38对TLR4的调控机制及其在B细胞发育及败血症肾损伤中的作用靶点尚不清楚。在此项目中，我们从分子、细胞和动物三个层面分析了CD38对TLR4的促进作用、调控机制及CD38基因缺失时Sirt1的作用，探讨了CD38/TLR4/NFκB和CD38/TLR4/BAFF通路在促进B细胞成熟中的作用，揭示了CD38/TLR4/NFκB通路在促进B细胞发育及败血症肾损伤中的关键靶点。结果表明：1）CD38基因敲除后，小鼠脾脏B细胞中TLR4、NF-κB和Sirt1蛋白表达均显著上升。2）CD38与TLR4之间存在相互作用。3）TLR4mut会引起CD38-/-小鼠脾脏中Sirt1-NF-κB通路的活化。4）与WT小鼠比较，CD38-/-小鼠脾脏B淋巴细胞中TLR4及Phospho-NF-κB蛋白显著升高（P<0.05）；与CD38-/-小鼠比较，CD38-/-TLR4mut小鼠脾脏B淋巴细胞中TLR4及 Phospho-NF-κB蛋白显著下降(P<0.05)。5）与WT小鼠比较，CD38-/-小鼠脾脏B淋巴细胞中炎性因子TNF-α、IL-1β和IL-4表达水平显著下降，脾脏中炎性细胞减少；与CD38-/-小鼠比较，CD38-/-TLR4mut小鼠中炎性因子TNF-α和IL-1β表达升高，脾脏中炎性细胞增加。6）CD38缺失会引起小鼠脾脏B细胞成熟障碍（T1-T2转型障碍），但TLR4非主要影响因素。7）CD38阻断可显著增加B细胞中TLR4、BAFF及BAFFR的表达。8）CD38缺失小鼠肾损伤及炎性反应显著增强伴TLR4/NF-κB通路的激活。综上所述，我们的研究结果表明：CD38主要通过激活TLR4/NFκB通路介导B细胞成熟，TNF-a和IL-1β是CD38/TLR4通路调控B细胞发育成熟的关键靶点，CD38缺失对TLR4介导的B细胞发育的影响可能与Sirt1的活性升高相关，CD38缺失通过激活TLR4/NF-κB通路加重LPS诱导的败血症急性肾损伤。研究结果揭示了CD38在TLR4介导的B细胞发育及败血症肾损伤中的调控作用及机制，为B细胞发育障碍性疾病及LPS诱导的败血症肾损伤防治提供了新策略。