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.

Chlorin e6-mediated photodynamic effect diminishes therapeutic potential of 5-aza-2'-deoxycytidine-based whole-tumour-cell vaccine in mice bearing squamous cell carcinoma SCCVII.

After years of setbacks, therapeutic cancer vaccines have become an alternative treatment option. Among the diversity of targeted tumour associated antigens (TAA), cancer-testis antigens (CTAs) are promising targets for cancer immunotherapy because they are highly immunogenic; meanwhile, they are expressed in human tumours of different histological origin but not in adult somatic tissues. Epigenetic modifications, such as DNA methylation, regulate CTAs expression both in normal and cancer cells. 5-Aza-2'-deoxycytidine (5-AZA-CdR), a DNA hypomethylating drug, induces the expression of CTAs in neoplastic cells. In these studies, we used 5-AZA-CdR-mediated up-regulation of CTAs and chlorin e6-mediated photodynamic effect in the production of a whole-tumour-cell vaccine against murine squamous cell carcinoma SCCVII in C3H/HeN mice. The results show that 5-AZA-CdR can be used to elevate levels of diverse CTAs in SCCVII cells. The 5-AZA-CdR-based vaccine, combined with the systemic administration of 5-AZA-CdR, delayed tumour growth. However, the treatment had no effect on survival in mice, most likely because of the toxicity of systemic treatment with 5-AZA-CdR. The photodynamic effect diminished therapeutic potential of 5-AZA-CdR-based vaccine. Chemo-immunotherapy with 5-AZA-CdR and therapeutic cancer vaccines may be an alternative approach to cancer therapy. However, further studies are needed to optimize treatment and vaccine preparation protocols.