Data Sheet 1_Inhibition of EETosis with an anti-citrullinated histone antibody: a novel therapeutic approach for eosinophilic inflammatory disorders.pdf

Eosinophils are a subset of granulocytes that protect the host against fungal and parasitic infection through secretion of their granular contents. In response to specific stimuli, eosinophils also undergo a type of lytic cell death, referred to as eosinophil extracellular trap (EET)-associated cell death (EETosis), where histone citrullination facilitates chromatin decondensation, cell rupture and release of pro-inflammatory, decondensed chromatin into the extracellular environment as EETs. In this study, we show the abundant presence of eosinophils and citrullinated histones in nasal polyp tissue of patients with eosinophilic chronic rhinosinusitis (ECRS). Using live imaging microscopy on purified human eosinophils, we demonstrate that physiologically relevant stimuli induce release of citrullinated EETs and the marker of eosinophil activation galectin-10. While the kinetics of release of EETs and galectin-10 are similar, inhibitors of citrullination block EETosis in a dose dependent manner but fail to inhibit galectin-10 release. The importance of citrullination is further exemplified with CIT-013, a monoclonal antibody specific for citrullinated histones H2A and H4. CIT-013 potently inhibits release of EETs (half-maximal inhibitory concentration of 2.5 nM) without inhibiting other eosinophil functions such as degranulation, adhesion, superoxide production and induction of chemokine expression. Together, this study provides new insights into the requirement of protein arginine deiminase 4 (PAD4) for EETosis, differentiates requirements of EETosis from galectin-10 release, and identifies a novel therapeutic approach for EETosis inhibition by targeting citrullinated histones in eosinophil-driven diseases such as ECRS.

嗜酸性粒细胞（Eosinophils）是粒细胞的一个亚群，可通过释放其颗粒内容物帮助宿主抵御真菌与寄生虫感染。在特定刺激下，嗜酸性粒细胞还会发生一类溶解性细胞死亡，即嗜酸性粒细胞胞外陷阱（eosinophil extracellular trap, EET）相关细胞死亡（EETosis）：此过程中，组蛋白瓜氨酸化可促进染色质解聚、细胞破裂，并将促炎的解聚染色质以EET的形式释放至细胞外环境。本研究证实，在嗜酸性粒细胞性慢性鼻窦炎（eosinophilic chronic rhinosinusitis, ECRS）患者的鼻息肉组织中，嗜酸性粒细胞与瓜氨酸化组蛋白大量存在。研究人员对纯化的人嗜酸性粒细胞开展活细胞成像显微镜观察，发现生理相关刺激可诱导瓜氨酸化EET的释放，以及嗜酸性粒细胞活化标志物半乳糖凝集素-10（galectin-10）的释放。尽管EET与半乳糖凝集素-10的释放动力学相似，但瓜氨酸化抑制剂可通过剂量依赖性方式阻断EETosis，却无法抑制半乳糖凝集素-10的释放。靶向瓜氨酸化组蛋白H2A与H4的单克隆抗体CIT-013进一步验证了瓜氨酸化的重要性。CIT-013可强效抑制EET的释放（半最大抑制浓度为2.5 nM），却不会影响嗜酸性粒细胞的其他功能，如脱颗粒、黏附、超氧化物产生以及趋化因子表达的诱导。综上，本研究揭示了肽酰精氨酸脱亚胺酶4（protein arginine deiminase 4, PAD4）在EETosis中的必要性，区分了EETosis与半乳糖凝集素-10释放的不同调控需求，并为嗜酸性粒细胞介导的疾病（如ECRS）中通过靶向瓜氨酸化组蛋白抑制EETosis提供了全新的治疗策略。