IL-33介导脓毒症状态下树突状细胞焦亡及其免疫学效应研究

Impaired immune function and associated immunosuppression are hallmarks of sepsis and multiple organ dysfunction syndrome (MODS), the leading cause of mortality in intensive care units. Deactivation and profoundly depletion of dendritic cell (DC) has recently been highlighted in the pathogenesis of sepsis. However, fundamental questions regarding the mechanisms of DC dysfuntion remain unresolved. In our preliminary experiments, we have found that there is significant pyroptosis in splenic DC after CLP, and level of IL-33 was increased in spleen and up-regulated in splenic DC in these mice. However, IL-33 KO could reduce the pyroptosis of splenic DC after CLP, and treatment with recombination IL-33 (rIL-33) could induce pyroptosis in mice splenic DCs in vitro. We suspect whether IL-33-induced pyroptosis is related to DCs dysfunction in sepsis. The current study was conducted to clarify the effects of continued stimulation with rIL-33 on DCs pyroptosis and also the intrinsic relation with dysfunction of DC in sepsis. Firstly, we plan to access the relationship between IL-33 stimulation and pyroptosis of DCs, as well as DC function and the ablility of regulating T cell immunity. And we will explore the involvement of signaling pathway of ST2/MyD88/TRAF6 activated NLRP3inflammasome in DCs pyroptosis in sepsis. Our findings will help to clarify the underlying mechanisms of pyroptosis of DC induced by IL-33, thus shed a light to the development of novel therapeutic strategies for improving the clinical outcome of patients with severe injury and subsequent sepsis.

免疫障碍是创烧伤感染并发脓毒症的重要机制，树突状细胞(DC)死亡、功能下降是关键所在，但DC免疫功能下降及细胞死亡的具体分子机制尚未阐明，临床缺乏有效调控策略。我们预实验研究发现，CLP模型小鼠脾脏DC发生明显焦亡(Casp-1依赖的炎性程序性细胞死亡模式)，且CLP小鼠脾脏及DC细胞内IL-33表达显著上调；而IL-33 KO小鼠CLP术后DC焦亡减轻，重组IL-33体外刺激可直接诱导DC焦亡，提示IL-33在介导DC焦亡中具有重要意义，可能是脓毒症状态下DC功能障碍、数量减少的重要机制。本项目拟利用细胞及动物模型研究脓毒症状态下IL-33通过ST2/MyD88/TRAF6激活NLRP3/ASC炎性小体这一信号通路介导DC细胞焦亡，经由“自分泌”正反馈放大效应对DC免疫功能状态发挥重要调控效应，深入探索脓毒症状态下DC死亡的分子机制及调控新策略。

白细胞介素(IL)-33作为T辅助型2(Th2)细胞的激活剂被发现，在维持组织稳态和修复、2型免疫应答、过敏性和非过敏性触发引发的炎症以及病毒感染和恶性肿瘤中发挥重要作用。证据表明，IL-33是一种多效细胞因子，它不仅激活Th2细胞，还诱导Th1细胞、固有淋巴细胞(ILC2s)和T调节细胞(Tregs)。除了适应性免疫外，在感染、过敏反应及组织损伤时，IL-33大量释放，参与多种炎症性疾病的发生发展。细胞焦亡(Pyroptosis)又称为细胞炎性坏死，是近年来新发现并证实的一种程序性细胞死亡方式。细胞焦亡依赖于炎症性胱天蛋白酶（caspases，主要是caspase-1、4、5、11）并伴有大量促炎因子的释放，其形态学特征、发生和调节机制不同于凋亡、坏死等细胞死亡方式，表现为细胞的持续扩张，直到细胞膜破裂，导致细胞内容物的释放和强烈炎症反应的激活。在前期科研成果的基础上，本课题重点研究了脓毒症过程中重要抗原提呈细胞树突状细胞(Dendritic cell, DC)焦亡情况以及IL-33的大量合成、释放对DC焦亡的促进作用和相关信号机制。首先检测CLP脓毒症小鼠模型脾脏DC的焦亡水平，发现CLP术后小鼠脾脏DC焦亡明显增加，且与小鼠免疫紊乱和不良预后密切相关。CLP小鼠脾脏及各重要脏器IL-33水平升高，是诱导DC细胞焦亡的重要因素。我们进而通过细胞实验分析 IL-33 对DC 焦亡的直接诱导效应，及其对DC免疫功能状态的确切影响，并利用IL-33和它的特异性受体ST2的基因敲除小鼠复制CLP模型，从不同层次、多个角度检测并论证 IL-33 对DC免疫功能的重要调控作用及其在脓毒症免疫功能障碍发病机制中的潜在意义。发现IL-33/ST2信号轴可能对应激状态下DC内质网选择性自噬具有重要影响，这为寻求脓毒症病理过程中调理 DC 免疫效应的可能干预靶点提供新的线索。项目总体目标已完成，但临床验证及转化应用有待加强，需进一步重点澄清IL-33参与调控DC免疫功能与临床脓毒症患者病情进展的本质联系，以及患者预后评估中的价值及临床意义，为制定脓毒症免疫障碍早期防治新策略提供实验数据支撑。