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

Abstract

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.

Neutrophil Extracellular Traps in Periodontitis

“…As P. gingivalis is one of the most important periodontal pathogens, the DNase expression of six different strains was analyzed, showing all of them had different degrees of DNase activity [54]. P. gingivalis is a potent inducer of NET formation that is mediated by gingipains, but its proteolytical activity has shown to inactivate the bactericidal components of NETs through the activation of protease-activated receptor-2 [55]. Several mutant and wild-type strains of P. gingivalis have been analyzed and their results showed that mutant strains induced a characteristic NET formation [56].…”

“…P. gingivalis is found in the oral cavity, where it is implicated in periodontal disease. Furthermore, a novel role has also been demonstrated for proteases as bacterial virulence factors antagonizing the antibacterial activity of NETs [55]. Additionally, the suggested generation of NETs in the periodontium leads to increased inflammation and can be considered another virulence strategy used by P. gingivalis.…”

“…P. gingivalis is the most important pathogen responsible for periodontitis. Further, it was shown that P. gingivalis could induce NET generation [55]. Interestingly, a study has demonstrated that patients with periodontitis and RA showed significantly higher serum levels of NETs than the control group.…”

Application of the In Vitro HoxB8 Model System to Characterize the Contributions of Neutrophil–LPS Interaction to Periodontal Disease

“…Alternatively, neutrophils ( Figure 3 ) were isolated as described before from granulocyte-enriched fractions, applied to centrifugation over a density gradient using a lymphocyte separation medium [ 30 ]. Neutrophils and erythrocytes were harvested as the high-density fraction and separated after 30 min of incubation with 1% polyvinyl alcohol (POCH, Warsaw, Poland).…”

“…In relation to Figure 3 , Human blood for PMN isolation was purchased from Red Cross, Krakow, Poland as described before [ 30 ]. The Red Cross de-identified blood materials as appropriate for the confidentiality assurance of human subjects.…”