Investigating Oral Epithelial Damage by Transmigrating Neutrophils

Abstract

Neutrophils are integral to the innate immune system, serving as the primary defense against infections and inflammation. However, excessive neutrophil activity can result in tissue damage. Neutrophils play a significant role in amplifying inflammation through the release of granule proteases, which activate various interleukin-1(IL-1) family cytokines, including IL-36. Initially, IL-36 cytokines are produced in an inactive form and require proteolytic processing to become biologically active and exert their pro-inflammatory effects. Specifically, neutrophil-derived proteases such as neutrophil elastase (NE), Cathepsin G (Cat G), and proteinase 3 (PR3) have been demonstrated to activate IL-36γ. IL-36γ, part of the IL-1 superfamily, is pivotal in the oral immune response to pathogens such as Porphyromonas gingivalis. This study investigates the role of P.gingivalis lipopolysaccharide (LPS) and the viral mimic polyinosinic:polycytidylic acid (Poly (I:C)) in stimulating IL-36γ release from oral epithelial cells (OECs). Additionally, it examines the release of pro-inflammatory mediators IL-6 cytokine and IL-8 chemokine, involved in neutrophil recruitment and migration in OECs and oral fibroblasts following stimulation with IL-36γ. Normal human oral keratinocytes (OKF6) were cultured and treated with different concentrations of P. gingivalis LPS (1, 10, and 25 μg/ml) and 25 μg/ml Poly (I:C). qPCR analysis showed IL-36γ upregulation in OKF6 cells in response to LPS and Poly (I:C), with significant mRNA expression following 25 μg/ml Poly (I:C) treatment. Further analysis of OKF6 cell supernatants using ELISA revealed a significant increase in IL-36γ levels after 24 hours with 25 μg/ml Poly (I:C). These findings indicate that IL-36γ mRNA expression and cytokine levels are induced in OECs in response to P. gingivalis and Poly (I:C). OKF6 and normal human oral fibroblast (NHOF) cells were also stimulated with 1, 10, 100, and 200 ng/ml IL-36γ (aa18-169) and full-length IL-36γ (inactive) for 24 hours, we observed increased levels of IL-6 and IL-8 release in OKF6 and NHOF stimulated with higher IL-36γ concentrations (200 and 100 ng/ml) in comparison with inactive full-length IL-36γ and untreated samples. Our results suggest that IL-36γ may be a crucial inflammatory mediator in oral mucosal immunity, modulating neutrophil responses and potentially exacerbating oral epithelial damage by activating pro-inflammatory mediators such as IL-6 and IL-8.