Triggering NETosis via protease-activated receptor (PAR)-2 signaling as a mechanism of hijacking neutrophils function for pathogen benefits

Neutrophil-derived networks of DNA-composed extracellular fibers covered with antimicrobial molecules, referred to as neutrophil extracellular traps (NETs), are recognized as a physiological microbicidal mechanism of innate immunity. The formation of NETs is also classified as a model of a cell death called NETosis. Despite intensive research on the NETs formation in response to pathogens, the role of specific bacteria-derived virulence factors in this process, although postulated, is still poorly understood. The aim of our study was to determine the role of gingipains, cysteine proteases responsible for the virulence of P. gingivalis, on the NETosis process induced by this major periodontopathogen. We showed that NETosis triggered by P. gingivalis is gingipain dependent since in the stark contrast to the wild-type strain (W83) the gingipain-null mutant strain only slightly induced the NETs formation. Furthermore, the direct effect of proteases on NETosis was documented using purified gingipains. Notably, the induction of NETosis was dependent on the catalytic activity of gingipains, since proteolytically inactive forms of enzymes showed reduced ability to trigger the NETs formation. Mechanistically, gingipain-induced NETosis was dependent on proteolytic activation of protease-activated receptor-2 (PAR-2). Intriguingly, both P. gingivalis and purified Arg-specific gingipains (Rgp) induced NETs that not only lacked bactericidal activity but instead stimulated the growth of bacteria species otherwise susceptible to killing in NETs. This protection was executed by proteolysis of bactericidal components of NETs. Taken together, gingipains play a dual role in NETosis: they are the potent direct inducers of NETs formation but in the same time, their activity prevents P. gingivalis entrapment and subsequent killing. This may explain a paradox that despite the massive accumulation of neutrophils and NETs formation in periodontal pockets periodontal pathogens and associated pathobionts thrive in this environment.

由被抗菌分子包被的DNA构成的细胞外纤维所组成的中性粒细胞衍生网状结构，被称为中性粒细胞胞外陷阱（neutrophil extracellular traps, NETs），现已被认定为先天免疫的生理性杀菌机制。NETs的形成过程同时也被归类为一种名为NETosis的细胞死亡模式。尽管学界针对病原体诱导的NETs形成已开展了大量研究，但特定细菌来源的毒力因子在此过程中的作用，尽管已有相关假说，目前仍未得到充分阐明。本研究旨在明确牙龈素——即介导牙龈卟啉单胞菌（Porphyromonas gingivalis, P. gingivalis）毒力的半胱氨酸蛋白酶——在该主要牙周致病菌诱导的NETosis过程中的作用。我们证实，牙龈卟啉单胞菌诱导的NETosis依赖于牙龈素：与野生型菌株（W83）形成鲜明对比的是，牙龈素敲除突变株仅能微弱诱导NETs的形成。此外，通过使用纯化的牙龈素，我们证实了该蛋白酶对NETosis的直接调控作用。值得注意的是，NETosis的诱导依赖于牙龈素的催化活性，丧失蛋白水解活性的酶变体仅能微弱触发NETs的形成。从机制层面来看，牙龈素诱导的NETosis依赖于蛋白酶激活受体-2（protease-activated receptor-2, PAR-2）的蛋白水解激活。有趣的是，牙龈卟啉单胞菌与纯化的精氨酸特异性牙龈素（Arg-specific gingipains, Rgp）所诱导的NETs不仅不具备杀菌活性，反而会促进原本可被NETs杀灭的细菌的生长。这种保护效应通过降解NETs的杀菌组分得以实现。综上，牙龈素在NETosis过程中发挥双重作用：它们既是强效的NETs形成直接诱导剂，同时其活性又可阻碍牙龈卟啉单胞菌被捕获并后续被杀灭。这或许可以解释一个悖论：尽管牙周袋内存在中性粒细胞的大量聚集与NETs的形成，牙周致病菌及相关致病共生菌仍可在此环境中旺盛增殖。