共抑制分子TIGIT负性调控ECMO辅助患者炎性单核细胞亚群功能的机制研究

Extracorporeal membrane oxygenation (ECMO) has become a life-saving procedure in patients with severe acute heart and/or lung failure. The application of ECMO has greatly improved the short and long survival rate of patients with severe heart and/or lung failure, but the immune imbalance induced by ECMO still leads to many complications, such as infection, multi-organ failure, SIRS, et al. These complications have been proved to contribute to the mortality rate of patients supported with ECMO. Human monocytes are phenotypically and functionally heterogeneous, and they are subdivided into three subsets: classic monocytes (Mo1), intermediated monocyte (Mo2) and non-classic monocytes (Mo3) subsets, which have different immune functions. With the support of the National Natural Science Foundation of China, we found that the intermediate Mo2 monocyte subsets is one of the major sources of inflammatory cytokines TNF-a, which not only involves in the development of acute lung injury induced by cardiopulmonary bypass, and also was observed to increase in the patients receiving ECMO assistance. However, the mechanism of negative regulation on the immune function of the inflammatory monocyte subsets is not yet clear. Recent studies have shown that co-inhibitory molecular pathways not only inhibit adaptive immunity, but also regulate innate immune responses. In our previous studies, we found that the expression of co-inhibitory molecular TIGIT was up-regulated in the monocytes of ECMO-supported patients, and these results suggested that TIGIT might negatively regulate the function of the inflammatory monocytes in the patients supported with ECMO. We propose hypothesis: TIGIT regulates the immune function of inflammatory monocytes. We propose to carry out prospective studies and monitor the expression of TIGIT on monocytes; to explore the regulatory role of TIGIT on monocytes by functional study and TIGIT knockdown experiments; and to clarify the mechanism of TIGIT up-regulation and to reveal the potential cellular and molecular mechanism on TIGIT regulating the monocyte by cell signal pathway. Our study might provide novel ideas and theoretical evidence for the prevention and intervention of ECMO-related inflammatory responses.

体外膜氧合（ECMO）的应用提高了严重心肺衰竭患者的生存率，但免疫失衡导致的器官功能障碍是影响患者预后的重要原因。单核细胞是参与天然免疫反应的主要群体，其亚群表型和功能各不相同。在国自然资助下，我们发现中间型Mo2炎性单核细胞亚群是TNF-a的主要来源之一，不仅参与体外循环相关急性肺损伤，而且在ECMO辅助患者外周血中持续存在。但是负性调控炎性单核细胞亚群免疫功能的机制尚不明确。最新研究发现：共抑制分子通路不仅抑制适应性免疫，而且调控天然免疫炎性反应。我们发现ECMO辅助患者外周血单核细胞共抑制分子TIGIT表达上调，提出：共抑制分子TIGIT可能负性调控ECMO辅助患者炎性单核细胞功能。本研究拟采用TIGIT knockdown方法，证实TIGIT调控炎性单核细胞功能；阐明TIGIT上调的机制以及TIGIT抑制炎性反应的信号通路，为预防和干预ECMO相关炎性反应提供理论证据。

体外膜氧合（ECMO）极大提高了重症救治水平，ECMO引发的免疫系统失衡是导致患者多种并发症的重要因素。单核细胞作为天然免疫的重要组成部分，在ECMO辅助中的作用引起了广泛关注，前期国自然研究发现，中间型Mo2炎性单核细胞亚群是TNF-的主要来源之一，不仅参与体外循环相关急性肺损伤，而且在ECMO辅助患者外周血中持续存在，但具体功能及调控机制尚未研究。通过本次的研究，我们发现接受ECMO辅助患者单核细胞亚群Mo2、Mo3及Mo0比例增高，与正常人相比，接受ECMO辅助的患者在辅助期间以及脱机后单核细胞表面共抑制分子TIGIT、CD200R、LAG-3、TIM3和BTLA表达均增加。通过体外刺激单核细胞，单核细胞分泌细胞因子TNF-、IL-6、IL-1β、IL-10及GM-CSF的能力降低，进一步分析TIGIT+单核细胞与TIGIT-单核细胞，发现与TIGIT-单核细胞相比，TIGIT+单核细胞分泌TNF-，IL-6，IL-1β的能力显著下降。与正常对照相比，接受ECMO辅助患者单核细胞吞噬能力降低。单核细胞表面TIGIT配体CD122表达增高，可能是单核细胞通过CD122与TIGIT相互作用，负性调控自身免疫功能的机制。根据这部分结果，我们发现ECMO辅助患者单核细胞整体处于免疫抑制状态，单核细胞表面共抑制分子TIGIT表达增高，通过CD122与TIGIT相互作用调控免疫功能，该研究为减少ECMO并发症，提高生存率提供理论证据。