Inhibition of netosis with PAD inhibitor attenuates endotoxin shock induced systemic inflammation. Inhibition of netosis with PAD inhibitor attenuates endotoxin shock induced systemic inflammation

Neutrophils play a pivotal role in innate immunity and participate in a range of immunological disorders. Excessive neutrophil extracellular traps formed at infection and tissue damage sites are detrimental to the local tissue and further exacerbate inflammation. Protein arginine deiminases mediate histone citrullination and NET formation thereby regulating endotoxin shock response. However, the molecular mechanisms are less known. Here we report that inhibition of netosis attenuates inflammation in a LPS induced lethal endotoxic shock model in mice. Albeit a higher number of neutrophils are accumulated in peritoneal cavity the primary inflammatory site, the level of inflammatory signals is greatly reduced after netosis inhibition with a PAD inhibitor. In lungs under endotoxic stress, gene expression analyses indicate that LPS induced a drastic increase in inflammatory gene expression. In contrast, lung tissue damage and inflammation were much reduced after PAD inhibition to reduce netosis. In summary, we found NET formation inhibition as a new avenue to manage inflammatory damages under endotoxic stress. Overall design: We performed gene expression profiling analysis using data obtained from RNA-seq of 3 different treatment groups of mice lung tissue: control group with 3 samples, LPS group with 3 samples, and dual treatment group with PAD4 inhibitor pretreated and LPS with 2 samples.

中性粒细胞（Neutrophils）在固有免疫（innate immunity）中发挥关键作用，并参与多种免疫紊乱性疾病。在感染与组织损伤部位形成的过量中性粒细胞胞外陷阱（neutrophil extracellular traps）会对局部组织造成损害，进而加重炎症反应。蛋白精氨酸脱亚胺酶（Protein arginine deiminases, PADs）可介导组蛋白瓜氨酸化与中性粒细胞胞外陷阱形成，从而调控内毒素休克应答。然而，其具体分子机制尚未明确。本研究发现，抑制中性粒细胞胞外陷阱形成（NETosis）可减轻脂多糖（lipopolysaccharide, LPS）诱导的小鼠致死性内毒素休克模型中的炎症反应。尽管作为主要炎症部位的腹腔（peritoneal cavity）内中性粒细胞浸润数量有所增加，但使用PAD抑制剂抑制中性粒细胞胞外陷阱形成后，炎症信号水平显著降低。在内毒素应激状态下的肺组织中，基因表达分析显示脂多糖可诱导炎症相关基因表达大幅上调；与之相反，通过PAD抑制剂抑制中性粒细胞胞外陷阱形成后，肺组织损伤与炎症程度均显著减轻。综上，本研究证实抑制中性粒细胞胞外陷阱形成可作为内毒素应激状态下炎症损伤的新型干预策略。

实验设计概述：本研究通过分析小鼠肺组织的RNA测序（RNA-seq）数据开展基因表达谱分析，共设置3组不同处理方案：对照组（3个样本）、脂多糖处理组（3个样本），以及PAD4抑制剂预处理联合脂多糖处理组（2个样本）。