新细胞因子FAM19A4促进中性粒细胞活化在脓毒症急性肺损伤中的作用和机制研究

Sepsis is a syndrome characterized by a dysregulated response of the host to invading pathogens which involves hemodynamic alterations that lead to multiple life-threatening organ dysfunctions and imposes a substantial global burden in terms of morbidity and mortality. Among the injured organs, the lung is the first and most frequent organ to fail. Accompanying acute respiratory distress syndrome (ARDS), which is the clinical term for acute lung injury (ALI), is one of the most critical prognostic factors for mortality in patients with sepsis. The recruitment of neutrophils to the lung makes them a key factor in the pathogenesis of ALI. Unregulated recruitment and activation of neutrophils via formyl peptide receptor 1 (FPR1) could induce lung injury through the release of inflammatory mediators, including cytokines and reactive oxygen species (ROS). FAM19A4 is a new novel cytokine identified in 2015. It is a novel ligand of FPR1. In our previous study, it showed that FAM19A4 can chemoattract and activate neutrophils in vivo. We also indicated that the expression of FAM19A4 is upregulated in septic patients and in the murine model of sepsis. This cytokine can aggravate the lung injury and increase the mortality of mice. Thus, we supposed that FAM19A4 may recruit and activate neutrophils in lung via FPR1 in sepsis, and then prompt the development of sepsis-related ALI. In this study, we will identify the expression of FAM19A4 in septic patients and the model of sepsis mice. We will also clarify the influence of FAM19A4 on the sepsis-related ALI and the neutrophil function, and then explore the downstream signaling events. Our results will provide a new basis for the study of the mechanism of sepsis-related ALI and provides new targets for clinical treatment.

脓毒症是机体应对感染时宿主反应失调导致的致命性器官功能障碍综合征，急性肺损伤是最早且最常见的并发症，发病率及死亡率极高。中性粒细胞通过膜表面受体FPR1被激活，募集至肺部并释放细胞毒性物质、加重炎症反应，造成弥漫性肺泡损伤，是推动疾病发展最核心的因素。我们前期研究发现新细胞因子FAM19A4可结合膜受体FPR1促进中性粒细胞活化；它在脓毒症中表达升高，可加重小鼠器官损伤并增加死亡率。本项目拟利用成功构建的Fam19a4基因敲除鼠和Fpr1基因敲除鼠，从分子、细胞、动物模型和患者四个层面，研究FAM19A4在脓毒症急性肺损伤中的表达特征、生物学功能及分子机制。以论证脓毒症中FAM19A4表达升高，结合FPR1受体促进中性粒细胞活化，可加重肺损伤；p38 MAPK等信号通路，可能是作用的主要分子机制。为深入理解脓毒症急性肺损伤发病机制提供新的理论依据，也可为疾病治疗提供新的干预靶点。

急性肺损伤是脓毒症（sepsis）病程中最早且最常见的并发症，发病率及死亡率极高。疾病早期中性粒细胞大量募集肺部，活化释放蛋白酶、氧活性物质和炎性因子等，造成弥漫性肺泡损伤，是推动疾病发生发展最核心的因素。我们前期研究发现细胞因子FAM19A4可促进中性粒细胞吞噬细菌并释放活性氧物质（ROS）；其在脓毒症中表达明显升高，可加重小鼠器官损伤并增加死亡率。本项目中，我们验证了小鼠脓毒症模型中，肺组织Fam19a4基因表达水平增高，外周血和肺泡灌洗液中FAM19A4蛋白水平表达显著增高。另外，我们纳入脓毒症患者进行检测，发现FAM19A4蛋白在患者外周血和肺泡灌洗液中表达较显著升高，升高程度与白细胞介素-6和C反应蛋白的升高趋势类似，且存活患者的FAM19A4表达水平较死亡患者低。为了解FAM19A4对急性肺损伤的影响，我们利用fam19a4 KO小鼠盲肠结扎穿孔法（CLP）制备脓毒症模型，证实缺失FAM19A4蛋白的脓毒症小鼠，肺组织损伤程度较轻，血氧合情况较好，肺水肿较轻，肺毛细血管渗漏较轻，外周血促炎细胞因子水平较低，7天生存率较高。对FAM19A4-null小鼠腹腔注射FAM19A4重组蛋白，小鼠肺损伤严重程度与对照组类似，不过在对WT小鼠CLP制备模型后腹腔注射FAM19A4重组蛋白，小鼠的疾病程度并不会进一步加重。在对FAM19A4作用机制研究的部分，体外研究证实FAM19A4蛋白可促进脂多糖活化的中性粒细胞吞噬大肠杆菌、释放ROS；而利用fam19a4 KO小鼠的体内实验发现，FAM19A4-null小鼠肺泡灌洗液和肺组织中，中性粒细胞数较WT小鼠显著减少，小动物成像技术显示FAM19A4-null小鼠体内ROS水平显著低于WT小鼠。为了进一步探索FAM19A4作用于中性粒细胞的分子机制，我们用LPS预处理HL-60细胞系分化的中性粒细胞细胞，之后用FAM19A4重组蛋白进行刺激。利用转录组测序技术，筛选了免疫相关的基因，利用qPCR法进行了验证，最后证实FAM19A4蛋白可增加中性粒细胞内p38 MAPK信号分子的转录；并通过阻断该信号分子，发现其与FAM19A4促进中性粒细胞释放ROS相关。本研究的结果，为深入理解脓毒症急性肺损伤发病机制提供新的理论依据，也可为疾病治疗提供了新的干预靶点。