C型凝集素家族分子ClEC18A负向调控TLR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. We found that CLEC18A was upregulated in PBMC in immunosupression phase sepstic patients and its upregulation was negative related to the function of human CD14+ monocytesevidenced by HLA-DR. TLR driven proinflamatory cytokines and activation of NF-κb/MAPKs increased significantly in CLEC18A knockdowned mice peritoneal macrophages compared with control macrophages, indicating that CLEC18A might involved in negative regulation of TLR signaling pathway. Mechanistically，we found a 195KD molecular integrated to CLEC18A, which might be the target of CLEC18A. Deferent from other well known C-type lectin family molecules, CLEC18A might function as a secretary protein rather than as a cell membrane expressed molecular, as we detected CLEC18A in the serum of septic patients. We assumed that sepsis induce CLEC18A mRNA upregulation which contribute to the high concentration in serum. After ligation with its cognate receptor, CLEC18A activate its downstream signaling molecular and interact with TLR induced signaling molecular which contribute to the negative regulation of TLR pathway. Our project aims to investigate the role of CLEC18A in sepsis and to reveal the potential mechanism underlying the regulation of TLR 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，但调控机制未明。我们发现CLEC18A在免疫抑制期脓毒症病人外周血单个核细胞高表达，且与单核细胞功能呈负相关。用RNA干扰敲减小鼠巨噬细胞CLEC18A后发现，TLR诱导产生的炎症因子和转录因子均明显增高，提示CLEC18A可能负向调控TLR信号通路。机制上，我们发现一个195KD的蛋白分子诱导性地与CLEC18A结合，疑似CLEC18A的靶分子。这些结果有待于在CLEC18A基因敲除小鼠（已获得）进一步证实。我们假设：炎症诱导CLEC18A表达上调并分泌到胞外，再与相应受体结合后激活其下游信号，从而负向调控TLR激活。CLEC18A与脓毒症及调控TLR信号通路的研究均未见报导。本项目将从临床、细胞及转基因动物水平，探讨CLEC18A在脓毒症中的作用，阐明CLEC18A负向调控TLR信号通路的机制，为靶向CLEC18A治疗脓毒症提供实验依据。

本研究明确了一种新型 TLR 负向调控因子 CLEC18A 在脓毒症巨噬细胞炎症反应中的作用及相关作用机制。研究发现CLEC18A可负向调控TLR4信号，降低LPS刺激下巨噬细胞IL-6及TNF-α的分泌，并对脓毒症小鼠具有保护作用。敲除CLEC18A导致巨噬细胞炎症反应增强，脓毒症小鼠生存率下降。机制研究中，研究发现CLEC18A的特异性受体 Mincle 及下游信号的转导机制，发现了 CLEC18A 通过 Mincle-Syk 途径介导 MyD88 的泛素化降解调控炎症反应的作用。本研究提示了CLEC18A在脓毒症中的保护作用，更加深入的探索了脓毒症的发生发展机理，为靶向 CLEC18A 治疗脓毒症提供了理论和实验依据。