ABSTRACT In periodontal disease (PD), bacterial biofilms cause gingival inflammation, leading to bone loss. In healthy individuals, αvβ6 integrin in junctional epithelium maintains anti-inflammatory transforming growth factor-β1 (TGF-β1) signaling, whereas its expression is lost in individuals with PD. Bacterial biofilms suppress β6 integrin expression in cultured gingival epithelial cells (GECs) by attenuating TGF-β1 signaling, leading to an enhanced pro-inflammatory response. In the present study, we show that GEC exposure to biofilms induced activation of mitogen-activated protein kinases and epidermal growth factor receptor (EGFR). Inhibition of EGFR and ERK stunted both the biofilm-induced ITGB6 suppression and IL1B stimulation. Furthermore, biofilm induced the expression of endogenous EGFR ligands that suppressed ITGB6 and stimulated IL1B expression, indicating that the effects of the biofilm were mediated by autocrine EGFR signaling. Biofilm and EGFR ligands induced inhibitory phosphorylation of the TGF-β1 signaling mediator Smad3 at S208. Overexpression of a phosphorylation-defective mutant of Smad3 (S208A) reduced the β6 integrin suppression. Furthermore, inhibition of EGFR signaling significantly reduced bone loss and inflammation in an experimental PD model. Thus, EGFR inhibition may provide a target for clinical therapies to prevent inflammation and bone loss in PD. This article has an associated First Person interview with the first author of the paper.

Epithelial alpha v beta 6 integrin participates in immune surveillance in many organs, including the gastrointestinal track. Expression of alpha v beta 6 integrin is reduced in the junctional epithelium of the gingiva in periodontal diseases, and mutations in the ITGB6 gene are associated with these diseases in humans and mice. The aim of this study was to unravel potential differences in the inflammatory responses in the periodontal tissues of FVB wild-type (WT) and beta 6 integrin-null (Itgb6(-/-)) mice, using a ligature-induced periodontitis model and assessing inflammation, bone loss and expression profiles of 34 genes associated with periodontal disease. Using micro-CT and histology, we demonstrated more advanced inflammation and bone loss in the control and ligatured Itgb6(-/-) mice compared to the WT animals. Neutrophil and macrophage marker genes were significantly upregulated by ligation in both WT and Itgb6(-/-) mice while the expression of T-cell and B-cell markers was downregulated, suggesting acute-type of inflammation. Expression of inflammasome NLRP3-related genes Nlpr3 and Illb was also significantly increased in both groups. However, the expression of 1118 was significantly lower in non ligatured Itgb6(-/-) mice than in the WT mice and was further downregulated in both groups by the ligatures. IL 18 mediates many effects of the AIM2 inflammasome, including regulation of the microbiome. Interestingly, expression of Aim2 was significantly lower in both control and ligatured Itgb6(-/-) mice than in WT animals. Overall, ligature-induced periodontitis was associated with increased expression of pro-inflammatory cytokines, chemokines and osteoclastogenic regulatory molecules. Another significant difference between the Itgb6(-/-) and WT mice was that mRNA expression of the anti-inflammatory cytokine IL-10 was increased in ligatured WT mice but reduced in the Itgb6(-/-) mice. In conclusion, alpha v beta 6 integrin in junctional epithelium of the gingiva appears to positively regulate the expression of the AIM2 inflammasome and anti-inflammatory IL-10, thus providing protection against periodontal inflammation.