点燃素-3调控中性粒细胞功能的新型分子机制研究

Release of neutrophil extracellular traps (NETs) is a newly recognized defensive mechanism in innate immunity. NETs are chromatin network lined with granular components released from neutrophils into the extracellular milieu upon inflammatory challenges. In addition to microbicidal activity, NETs are also involved in the progression of multiple noninfectious pathological conditions, such as autoimmunity, deep vein thrombosis and cancer. Multiple lines of evidence show that reactive oxygen species (ROS) generated by NADPH oxidase in neutrophils play an essential role in promoting NET release. However, the detailed mechanisms are largely unknown. Kindlin-3, a hematopoietic cell-specific protein, is an important integrin activator by directly binding to the β-integrin cytoplasmic tails. Surprisingly, our recently collected preliminary data indicate that kindlin-3 can negatively regulate ROS/NETs signaling in neutrophils in an integrin-independent manner, and such a function may be due to a direct association of kindlin-3 with one of the key components of NADPH oxidase. Based on these observations, here we propose to explore the molecular and mechanistic basis of kindlin-3 in suppressing ROS/NET signaling in neutrophils. We will first map the key region in kindlin-3 that is responsible for the negative regulation; subsequently, we will interpret how the association of kindlin-3 with NADPH oxidase influences ROS production and NET release in neutrophils; and finally, we will evaluate the pathological significance of such a mechanism in different mouse models. Taken together, we believe that this study will further our mechanistic understanding of NET release, and may help to develop new NETs-targeted strategies for preventing and treating relevant diseases.

中性粒细胞在应激条件下向胞外释放网状核物质（NETs）是一种新发现的免疫防御机制。NETs对入侵病源体具有直接的杀伤作用。研究发现NETs也与自身免疫性疾病、深静脉血栓症、恶性肿瘤等多种非感染性疾病正相关联。目前已知NETs释放是由NADPH氧化酶产成的活性氧自由基 （ROS）所诱发的，但具体的分子调控机理尚不清除。点燃素-3是血细胞内整合素结合和激活因子，并促进整合素所介导的中性粒细胞的粘附和迁移。重要的是，我们近来的多项预试验结果表明中性粒细胞中点燃素-3还具有以非整合素结合依赖的方式负调控ROS/NETs信号的功能。鉴于此，该申请项目将重点解析点燃素-3该项新功能的分子机制和病理重要性。具体内容包括：（1）鉴定点燃素-3中的核心功能域；（2）阐明点燃素-3调控NADPH氧化酶活性的分子机制；（3）病理重要性评价。我们相信该项目研究成果将为NETs相关疾病的针对性治疗提供新的理论依据。

中性粒细胞激活时可释放胞外网状核物质（NETs），从而促使深静脉血栓形成。NETs的形成主要是由NADPH oxidase产生的活性氧分子介导的。本项目主要内容是研究中性粒细胞中Kindlin-3对深静脉血栓形成的影响和分子调控机制。Kindlin-3分子表达在血细胞中，是重要的胞内整合素激活因子。首先，我们制作了Kindlin-3的条件性基因剔除小鼠（Kindlin-3fl/fl）并获得了在髓系粒细胞中特异敲除Kindlin-3的Kindlin-3fl/fLysM-Cre小鼠。通过功能验证，我们发现Kindlin-3分子在粒细胞表达缺失后对深静脉血栓的形成有明显的促进作用；同时我们阐明了中性粒细胞中Kindlin-3的表达缺失可促进NETs的释放，进而诱发深静脉血栓的形成。通过蛋白结晶学手段，我们成功解析了Kindlin-3蛋白的3D分子结构，证明了其可形成类似“三叶草”形状的典型接头分子。在此基础上，我们发现Kindlin-3可以直接通过结合参与负向调控NADPH oxidase的活性，从而揭示了其调控深静脉血栓形成的分子机理。这些重要发现为将来开发更安全高效的抗深静脉血栓形成药物提供了新的靶点和理论基础。