TLR4活化树突状细胞调节中性粒细胞参与脓毒症发生发展的机制研究

It has been accepted that there is close relationship among TLR4 signaling, Dendritic cells and sepsis, however, the underlying mechanisms is still elusive. We found that, after CLP procedure, bacterial clearance in DC TLR4 knockout mice improved significantly, simultaneously the percentages of neutrophils in the peritoneal cavity also incresed; whereas the IL-10 production by isolated DCs from these mice decreased significantly. The above data strongly indicate that, during sepsis, Dendritic cells activated by TLR4 may regulate the recruitment of neutrophils into inflammatory sites via the production of IL-10, then influence the pathiophysiology of sepsis.Therefore, in the present study, we will first investigate the numbers, function,apoptosis of Dendritic cells, and the effects on bacterial clearance, inflammatory response and survival by using DC TLR4 KO, Flox, TLR4 KO and WT mice; Secondly，we will analysis the numbers, chemotasis, and CXCR2 expression of neutrophils in peritoneal cavity, in combination with adoptive transfer and antibody depletion experiment; finally, we plan to demonstrate whether DCs regulate neutrophils recruitment via production of IL-10. The accomplishment of the present proposal will not only help us to make a better understanding of the pathophysiology of sepsis, but also provide possible targets for early intervention of sepsis.

TLR4与树突状细胞（DC）变化和脓毒症发生发展密切相关，但机制不详。我们发现，DC条件性TLR4敲除（DC TLR4 KO）小鼠盲肠结扎穿孔（CLP）术后，腹腔内中性粒细胞比例增加，细菌清除改善，而DC IL-10生成减少，提示，脓毒症中TLR4可能通过活化DC，生成IL-10，调节中性粒细胞趋化，影响细菌清除参与脓毒症病生理过程。为此，拟采用DC TLR4 KO、Flox、TLR4 KO和WT小鼠，检测CLP术后，DC数量、凋亡和功能等，及细菌清除、炎症反应和生存率等，揭示TLR4在DCs变化中的作用及病生理意义；检测腹腔内中性粒细胞数量、趋化和CXCR2表达等，结合转移输注和抗体清除，证实DC TLR4对中性粒细胞趋化的影响和效应；通过体内外实验，证实DC TLR4以IL-10依赖机制影响中性粒细胞趋化参与脓毒症发病；以深化对脓毒症病生理机制的认识，并为脓毒症治疗提供潜在作用靶点。

脓毒症（sepsis）是机体对感染反应失调而导致的危及生命的器官功能障碍，在其病生理过程中，调节性T细胞（Regulatory T cell，Treg)发挥重要作用。本项目中，我们分别分析了TLR4信号途径、IL-3和内质网应激对Treg细胞功能的影响及其在脓毒症病生理机制中的作用。结果显示：⑴与WT小鼠相比，TLR4-/-小鼠生存率明显改善，相应的，Treg比例和数量、Foxp3表达和IL-10生成均明显上调，提示TLR4信号途径可能通过抑制Treg的数量和功能活性，导致脓毒症免疫紊乱;⑵Treg表达IL-3R，活化时能够产生IL-3，并以自分泌机制负反馈调节Treg细胞功能；脓毒症中IL-3既是导致促炎细胞因子大量释放的重要原因，也通过下调Treg细胞的功能活性参与脓毒症免疫紊乱的发生发展；⑶通过体外实验，我们发现TG能够诱导Treg发生明显内质网应激反应，并通过PERK信号途径影响Treg细胞Foxp3表达和IL-10、TGF-β的生成，对Treg细胞的功能活性有重要的调节作用。本项目培养博士生1名、硕士生1名。已发表学术论文5篇，其中SCI收录2篇（IF分别为5.345和3.056）。