MDL-1调控脓毒症时单核巨噬细胞功能异常的作用与机制研究

Sepsis is the leading cause of death globally each year. Dysfunction of monocytes/macrophages (Mφ) is the major cause of immunosuppression during sepsis. However, its underlying mechanism is still not clear. Recent literature indicates that MDL-1, also known as CLEC5A, a kind of C-Type lectin receptor, plays a critical role in regulation of Mφ in several diseases. However, the exact role of MDL-1 during sepsis is poorly understood. Our preliminary experiments showed that the expression of MDL-1 on CD14+ monocytes in septic patients is significantly increased. Our later research found that LPS can trigger the MDL-1 expression on BMDMs from mice, and the concentration of TNF-α in culture supernatant was significantly increased. We then propose that MDL-1 might be a critical molecule involved in Mφ dysfunction during sepsis. Our current program was designed to illuminate the role of MDL-1 in regulation of Mφ function in sepsis from bedside to bench, including septic patients, in vitro Mφ study, and MDL-1 gene knockout mice. The following scientific questions raised in the proposal will be answered. First, the relationship between MDL-1 expression on Mφ during sepsis will be determined. Second, the role of MDL-1 in regulation of Mφ will be explored. Third, the potential pathways involved in MDL-1 mediated Mφ dysfunction will be investigated. Collectively, through the research into the role of MDL-1 in Mφ during sepsis, our program will provide valuable insights into the mechanisms of sepsis and might raise a new potential therapeutic area in near future.

单核巨噬细胞功能异常是脓毒症时免疫紊乱重要原因，机制尚不清楚。新近发现C型凝集素受体MDL-1调控巨噬细胞促炎反应。我们前期研究发现，脓毒症患者外周血单核细胞MDL-1表达水平显著升高，其病理生理机制值得深入研究。我们进一步实验表明，内毒素可诱导小鼠骨髓来源巨噬细胞MDL-1表达上调，培养液上清TNF-α等浓度显著升高。我们推测：MDL-1是调控脓毒症时单核巨噬细胞功能异常的关键分子。本项目将从临床患者、离体细胞及基因敲除动物，探索脓毒症时单核巨噬细胞功能异常与MDL-1的关系；MDL-1调控单核巨噬细胞功能异常的方式；基于ChIP-seq、荧光素酶报告基因、激酶通路等，探索MDL-1/DAP12调控单核巨噬细胞功能的新机制。本研究以单核巨噬细胞功能异常为着眼点，以备受关注的C型凝集素受体MDL-1为切入点，将加深对脓毒症病理生理机制认识，为通过MDL-1防治脓毒症提供新的实验依据。

单核巨噬细胞功能异常是脓毒症时免疫功能紊乱重要原因，具体作用机制目前尚不完全清楚。新型C型凝集素受体MDL-1可有效调控巨噬细胞炎症反应过程。通过本研究初步证实MDL-1与脓毒症患者严重程度存在密切相关性，并通过小鼠在体进一步验证MDL-1表达与脓毒症的相关性；采用MDL-1-/-基因敲除小鼠，进一步明确MDL-1分子与脓毒症小鼠单核巨噬细胞的功能存在相关性；最后探索MDL-1在调控脓毒症小鼠单核巨噬细胞功能异常方面潜在的机制，并通过小鼠在体验证调控MDL-1对脓毒症病理生理的影响。通过本研究，我们发现，脓毒症患者外周血单核细胞MDL-1表达水平较健康志愿者显著升高；脓毒症小鼠外周血中单核细胞MDL-1表达显著高于正常对照组，进一步研究发现，脓毒症小鼠肺组织中巨噬细胞、内皮细胞及上皮细胞MDL-1表达均显著高于正常组。MDL-1基因敲除小鼠骨髓来源的巨噬细胞培养液上清液细胞因子IFN-γ、IL-6及TNF-α水平均显著升高；采用RT-PCR结果显示，脓毒症MDL-1-/-小鼠细胞因子IL-6、TNF-α和IL-0及趋化因子KC和MDL-1 分子mRNA表达水平均显著高于野生型小鼠。RT-PCR结果进一步显示，MDL-1 mRNA与PD-1及其配体PD-L1 mRNA表达趋势一致。脓毒症MDL-1-/-小鼠肺组织中，调节因子Syk表达显著高于野生型小鼠。据此我们推测：MDL-1是参与调控脓毒症时单核巨噬细胞功能异常的重要分子。本项目从临床患者、离体细胞及基因敲除动物，探索脓毒症时单核巨噬细胞功能异常与MDL-1的关系；MDL-1调控单核巨噬细胞功能异常的方式；基于ChIP-seq、激酶通路等，探索MDL-1调控单核巨噬细胞功能的潜在机制。本研究以单核巨噬细胞功能异常为着眼点，以备受关注的C型凝集素受体MDL-1为切入点，进一步加深了对脓毒症病理生理机制认识，为通过MDL-1有效防治脓毒症提供新的实验依据。