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.
Author summary Periodontitis, or gum disease, is characterized by chronic inflammation and erosion of the tooth-supporting tissues. The condition is fuelled by bacterial accumulation on the tooth surface below the gum line that resists the host innate immune response, including massive accumulation of neutrophils. Despite possessing a formidable array of bactericidal machineries, including neutrophil extracellular traps (NETs) formation whereby neutrophils release DNA-composed fibers decorated with bactericidal proteins and peptides to efficiently trap and kill bacteria. Nevertheless, neutrophils in periodontitis are unable to clear the infection due to the presence of key periodontal pathogens, including Porphyromonas gingivalis. This bacterium secretes a variety of virulence factors, including proteases (gingipains) that allow the organism to manipulate the host immune response to benefit the entire dysbiotic microbial community. Here, we describe a unique strategy whereby P. gingivalis trigger NET formation through gingipain-dependent cleavage of Protease Activated Receptor (PAR)-2 on the neutrophil surface. Importantly, NETs formed in this way are deficient in antibacterial activity but instead, supports bacterial growth due to degradation of bactericidal components by gingipains. This finding may explain a paradox that dysbiotic bacteria flourished in periodontal pockets in spite of massive accumulation of neutrophils and abundant NETs formation.