A20 Restricts Inflammatory Response and Desensitizes Gingival Keratinocytes to Apoptosis

Abstract

The pathophysiology of periodontal disease involves a perturbed immune system to a dysbiotic microflora leading to unrestrained inflammation, collateral tissue damage, and various systemic complications. Gingival epithelial cells function as an important part of immunity to restrict microbial invasion and orchestrate the subsequent innate responses. A20 (TNFAIP3), an ubiquitin-editing enzyme, is one of the key regulators of inflammation and cell death in numerous tissues including gastrointestinal tract, skin, and lungs. Emerging evidence indicates A20 as an essential molecule in the oral mucosa as well. In this study, we characterized the role of A20 in human telomerase immortalized gingival keratinocytes (TIGKs) through loss and gain of function assays in preclinical models of periodontitis. Depletion of A20 through gene editing in TIGKs significantly increased IL-6 and IL-8 secretion in response to Porphyromonas gingivalis infection while A20 over-expression dampened the cytokine production compared to A20 competent cells through modulating NF-κB signaling pathway. In the subsequent experiments which assessed apoptosis, A20 depleted TIGKs displayed increased levels of cleaved caspase 3 and DNA fragmentation following P. gingivalis infection and TNF/CHX challenge compared to A20 competent cells. Consistently, there was reduced apoptosis in the cells overexpressing A20 compared to the control cells expressing GFP further substantiating the role of A20 in regulating gingival epithelial cell fate in response to exogenous insult. Collectively, our findings reveal first systematic evidence and demonstrate that A20 acts as a regulator of inflammatory response in gingival keratinocytes through its effect on NF-κB signaling and desensitizes cells to bacteria and cytokine induced apoptosis in the oral mucosa. As altered A20 levels can have profound effect on different cellular responses, future studies will determine whether A20-targeted therapies can be exploited to restrain periodontal inflammation and maintain oral mucosa tissue homeostasis.

The Ubiquitin System and A20: Implications in Health and Disease

“…In the oral mucosa, the stringent regulation of immune cell life span and turnover is an important dimension of immune homeostasis, and a disruption of apoptosis can contribute to chronic inflammation seen in periodontitis (Meghil and Cutler 2020). In fact, insufficient A20 levels sensitize gingival keratinocytes to bacteria and TNF-induced apoptosis, supporting the notion that A20-targeted therapies can help maintain the integrity of the gingival epithelium and restore impaired periodontal tissue homeostasis (Li et al 2020).…”

“…In support of plausible biological functions of A20 in promoting oral cavity health, A20 has been specifically shown to regulate inflammation, autophagy, and cell death responses in the oral mucosa (Hong et al 2016; Crump et al 2017; Li et al 2019; 2020; Yan et al 2020). A20 insufficiency was associated with a more severe disease phenotype in a murine periodontitis model (Li et al 2019).…”

“…Particularly, abnormal expression and/or function of ubiquitin-editing protein A20 (tumor necrosis factor α–induced protein 3 or TNFAIP3) has been associated with chronic inflammation and tissue damage, contributing to the immunopathology of multiple human autoimmune and inflammatory diseases, including diabetes and cancer, as well as pulmonary, neurological, skin, and gastrointestinal disorders (Fukaya et al 2016; Zhou et al 2016; Aeschlimann et al 2018; Kadowaki et al 2018; Devos et al 2019; Momtazi et al 2019). Recent evidence also reveals the ubiquitin system and A20 as a critical regulator in the oral cavity and particularly in periodontal disease pathogenesis (Hong et al 2016; Zhou et al 2016; Crump et al 2017; Li et al 2019; 2020; Yan et al 2020).…”

The Ubiquitin Proteasome System in Periodontal Disease: A Comprehensive Review

“…Still, in mice infected with Porphyromonas gingivalis, bone marrow-derived macrophages from A20-deficient showed increased NF-kB activity and cytokine production compared with cells isolated from A20-competent mice (84). Recently, Li et al (85) in 2020 reported a specific role of an ubiquitin editing molecule in gingival keratinocyte inflammatory response, indicated that sufficient A20 levels is a major determinant to control apoptosis in response to microbial and inflammatory injury. Also, the A20 expression in response to oral bacterial depends among different cells and, therefore, it is essential studying the cell-type specific A20 responses and their effect on disease phenotype to develop targeted therapies (85).…”

“…Recently, Li et al (85) in 2020 reported a specific role of an ubiquitin editing molecule in gingival keratinocyte inflammatory response, indicated that sufficient A20 levels is a major determinant to control apoptosis in response to microbial and inflammatory injury. Also, the A20 expression in response to oral bacterial depends among different cells and, therefore, it is essential studying the cell-type specific A20 responses and their effect on disease phenotype to develop targeted therapies (85). Another mechanism that could explain the ability of transforming growth factor β (TGFβ) to inhibit Toll-like receptor (TLR)-NFκB signaling is protein arginine methyltransferase 1 (PRMT1)-induced Smad6 methylation.…”