新型凝集素分子CLEC18A通过线粒体自噬调控NLRP3炎性体信号在脓毒症中的作用和机制研究

Dysfunction of monocyte-macrophage contributes to sepsis-induced immunosupression, which is the main cause of sepsis-related mortality. However, the underlying mechanism is still elusive. During sepsis, monocytes/macrophages secrete mature IL-1β，IL-18 through activation of inflammasomes ,including NLRP3, AIM2 and NLRC4 inflammasomes. We found that CLEC18A was upregulated in PBMC in immunosupression phase sepstic patients. Mechanistically, we found NF-κb involved in LPS-induced upregulation of CLEC18A expression. We also found that NLRP3 driven proinflamatory cytokines increased significantly in CLEC18A knockdowned mice peritoneal macrophages compared with control macrophages, indicated that CLEC18A might involved in negative regulation of NLRP3 signaling pathway. Furthermore, CLEC18A knockdown resulted in decreased LPS-induced autophagy and activation of autophagy by rapamycin could reverse the inhibitory effect of CLEC18A on regulation of NLRP3 inflammasome. We assumed that sepsis induce CLEC18A mRNA upregulation through NF-κb signaling pathway and CLEC18A contribute to the negative regulation of NLRP3 inflammasome through mitophagy. Our project aims to investigate the role of CLEC18A in sepsis and to reveal the potential mechanism underlying the regulation of NLRP3 inflammasome signaling pathway.

单核巨噬细胞功能障碍是脓毒症免疫抑制的主要原因。脓毒症中，单核细胞通过激活NLRP3等炎性体释放成熟的IL-1β，IL-18和诱导细胞焦亡。我们发现CLEC18A在脓毒症病人免疫抑制期高表达。体外，用RNA干扰技术发现该分子特异参与NLRP3炎性体的负性调控。机制探讨发现，NF-κb通路可能参与CLEC18A表达上调；敲减CLEC18A后造成LPS诱导的自噬降低，用自噬激活剂可逆转其对炎性体的作用，表明该分子可能通过自噬途径调控炎性体激活。我们假设：炎症通过NF-κb诱导CLEC18A的表达上调，通过线粒体自噬途径调控NLRP3炎性体激活，参与脓毒症免疫抑制。目前，关于CLEC18A调控脓毒症及炎性体的研究未见报道。本项目将从临床、细胞及转基因动物水平，探讨CLEC18A在脓毒症免疫抑制中的作用，阐明炎症诱导其上调及负向调控炎性体信号通路的机制，为靶向CLEC18A治疗脓毒症提供实验依据。

炎性体激活造成的IL-1β上调与多种疾病相关。作为C-型凝集素家族唯一的外分泌分子，CLEC18A在炎性体相关疾病中的作用和机制以及受体未知。我们在LPS/GalN诱导的急性肝损伤模型和LPS诱导的急性肺损伤2种模型明确了CLEC18A的负向调控作用。机制探讨发现了炎症通过JNK/ERK/c-jun通路诱导CLEC18A的表达上调；在细胞水平和在体水平明确了该分子的受体是mincle，阻断mincle可逆转外源性CLEC18A的抑制作用。在C57b/l6小鼠和CLEC18A条件敲除小鼠，我们发现CLEC18A可能通过调控自噬抑制炎性体的表达，在此过程中，mincle下游syk分子是必不可缺的。