脓毒症中MMP8(+)树突状细胞功能转化及其介导T细胞耗竭和二次感染的作用和机制研究

It is well known that, after a transient hyper-inflammatory phase, septic patients rapidly enter a prolonged immunosuppressive state which is characterized by T-cell exhaustion and increased levels of immunosuppressive cells. This unique immunosuppressive state has been shown to be directly related to sepsis-related mortality because of the inability to fight secondary infections.. Previous studies illustrated that sepsis-induced impairments in immunobiology of dendritic cells (DCs) contribute to T-cell exhaustion and the expansion of regulatory T cells (Tregs). Nevertheless, the functional reprogramming of DCs during sepsis are not fully understood. In a septic mouse model, we found that a new DC subset, MMP8 positive dendritic cells (MMP8+DCs), was increased significantly post-sepsis. This finding was confirmed by microarray-based gene expression profiling, real-time PCR, and flow cytometric analysis. Additionally, MMP8 inhibition significantly decreased the levels of IL-10 and increased the levels of IL-12 in septic DCs. As IL-10 plays a crucial role in sepsis-induced T-cell exhaustion and Tregs expansion, we speculate that the increased number of MMP8+DCs may be responsible for T-cell exhaustion and secondary infection post-sepsis. To verify this hypothesis, the percentage of MMP8+DCs will be determined in both septic mice and septic patients. Additionally, in the proposed study, we will investigate the effect of MMP8+DCs on T-cell exhaustion and secondary infection post-sepsis. The exact role of MMP8+DCs in T-cell exhaustion and secondary infection post-sepsis will be determined using DC-specific MMP8 knockout mice. To evaluate the function of MMP8+DCs in vitro, human monocyte-derived DCs and splenic DCs from mice will be transfected with lentivirus vectors (Lv)-encoding MMP8 or MMP8-siRNA. Furthermore, using different cell co-culture systems and IL-10 knock-down cells, the mechanism of MMP8+DCs in T-exhaustion will be investigated in the proposed study.. It has been reported that β-catenin activation programs DCs to a regulatory state. In human umbilical vein endothelial cells (HUVECs), knock-down of MMP8 significantly decreased β-catenin nuclear levels. Additionally, β-catenin activation is regulated by Akt through both GSK-3β dependent and independent pathways. Our preliminary study found that β-catenin nuclear translocation and phosphorylated Akt were decreased after MMP8 inhibition in septic DCs. So, the Akt/β-catenin pathway may contribute to the unique function of MMP8+DCs. In the proposed study, the role of the Akt/β-catenin pathway in regulating MMP8+DC function will be investigated both in vivo and in vitro. Our research may be helpful in developing novel strategies to modulate the immune response and to decrease the incidence of secondary infection post-sepsis.

在脓毒症致病过程中，免疫功能麻痹导致二次感染的发生率显著增加，已经成为脓毒症治疗的新难题。前期研究发现，T淋巴细胞耗竭不仅是脓毒症免疫功能紊乱的重要表现，也是二次感染发生的原因。鉴于树突状细胞（DCs）在T细胞功能维持和调节中的关键性作用，我们通过对DCs差异性基因筛查、RT-PCR和流式细胞术发现，脓毒症中MMP8+DCs明显升高，且具有免疫抑制和免疫调节特征。在上述研究基础上，项目拟通过临床和基础研究相结合，从整体、细胞和分子水平三个层面，采用DC细胞特异性MMP8敲除动物、基因转染技术等，论证MMP8+DCs在脓毒症T细胞耗竭中的作用和地位，探讨其在二次感染临床预测和防治中的应用价值；同时基于预实验结果，以Akt/β-catenin为切入点，分析MMP8+DCs功能转化的机制。该项目从MMP8+DCs这一全新细胞亚型的角度，为脓毒症免疫监测和调理治疗提供新线索。

树突状细胞（Dendritic cells, DCs）免疫功能向免疫抑制型转化与脓毒症的不良预后相关。基质金属蛋白酶（Matrix metalloproteinases, MMPs) 已被证明具有免疫调节作用。然而，MMPs是否参与DCs的功能转化尚不清楚。本研究使用晚期脓毒症模型，分析了DCs中MMPs的表达规律发现，MMP8、MMP9、MMP14、MMP19、MMP25和MMP27的表达水平升高，其中MMP8表达最高。临床研究发现，CD11c+MMP8+细胞在脓毒症中明显升高，且与脓毒症严重程度有关。通过MMP8敲除小鼠和体外基因转染发现，MMP8缺乏在体内和体外均显著减轻脓毒症诱导的DC免疫耐受。脓毒症晚期过继输注来源脓毒症小鼠的MMP8-/-BMDCs能够抑制调节性T细胞扩增并增强的Th1应答，减低小鼠的细菌载量，最终减低脓毒症小鼠死亡率。对其机制研究发现，MMP8能够与TLR4直接结合，并下调其表达。同时，MMP8缺失可促进脓毒症DCs核因子κB p65活化，并下调β-catenin磷酸化水平，且MMP8缺失介导的β-catenin磷酸化水平改变可以被核因子κB p65抑制剂所抑制。这些发现表明，脓毒症晚期其DCs中MMP8水平的增加可能作为负反馈回路，从而抑制促炎反应并诱导DC耐受。CD11c+MMP8+细胞可以作为新型免疫标志物用于辅助脓毒症诊断和预后判断。