Impacts Of FOXO1and Periopathogens On Oral Mucosal Wound Healing

Oral mucosal surface is covered with epithelium, which is an interface between the external environment and underlying connective tissue and serves as an important barrier against pathogenic microbes. Re-epithelialization by mucosal epithelial cells is important for normal mucosal wound healing and is driven by migration and proliferation of keratinocytes. Previous reports have shown that diabetes-induced high glucose environement negatively impacts cutaneous healing primarily by decreasing cutaneous epithelial cell migration and proliferation. FOXO1, which belongs to a large family of forkhead transcription factors, participates in a wide range of cellular processes including apoptosis, oxidative stress resistance and glucose metabolism. Compared with normoglycemic mice skin wounds, the level of activated FOXO1is increased in diabetic skin wounds. However, few studies have directly examined the mechanism of impaired oral mucosal wound healing in diabetes. To study mucosal wound healing, we compared normoglycemic and diabetic wound healing process and determined whether FOXO1deletion played an important role in the healing process. Furthermore, as a local influencing factor, the impact of periopathogens on re-epithelialization of oral mucosal epithelial cells was investigated in this study.(1) Cellular mechanisms of impacts of FOXO1on oral mucosal wound healing:To determine the role of FOXO1in wound healing, we generated mice with deletion of FOXO1in epithelial cells by breeding floxed FOXO1mice with keratin14Cre-expressing mice. Type1diabetes was induced by multiple intraperitoneal injection of streptozotocin, and small excisional wounds of1.5mm were made with punch on tongue dorsum of mice. The oral mucosal wound healing of different types of mice was analyzed in the present study. The results indicated that diabetic mice exhibited delayed oral mucosal wound healing with an83%reduction in migration and a54%reduction in proliferation of mucosal epithelial cells. The in vivo results were supported by in vitro experiments. To identify the mechanism for altered mucosal epithelial cell migration and proliferation, we examined mice with keratinocyte-specific deletion of the transcription factor FOXO1. Activated FOXO1was found to increase2.6fold in mucosal epithelium of diabetic compared to matched normoglycemic mice, and the deletion of FOXO1in mucosal epithelial cells in diabetic mice largely reversed the negative effect of hyperglycemia of the number of migrating and proliferating mucosal epithelial cells in vivo. Interestingly, deletion of FOXO1had the opposite effect in normoglycemic animals, impairing the healing process and reducing mucosal epithelial migration.(2) Screening and identification of down-stream target genes of FOXO1:To understand the molecular mechanism of effects of FOXO1on oral mucosal wound healing, microarray and real-time PCR studies were carried out and CCL20and IL1F9were identified as potential genes that were induced by high glucose in a FOXO1dependent manner. The results revealed that high glucose significantly increased the mRNA levels of CCL20by64%and IL1F9by33%, and FOXO1knockdown with siRNA significantly decreased levels of CCL20and IL1F9in primary human oral epithelial cells in vitro. Our in vivo studies confirmed that CCL20and IL1F9expression was elevated in mucosal epithelium of diabetic mice compared to normoglycemic. Moreover, FOXO1deletion in vivo reduced both CCL20and IL1F9in diabetic animals suggesting that their expression is driven by FOXO1during healing.(3) Impacts of CCL20and IL1F9on migration of oral mucosal epithelial cells:The possiblitiy that CCL20and IL1F9are regulators of oral mucosal epithelial cell migration released by these cells under high glucose stimulation was determined by using neutralizing antibodies. When CCL20or IL1F9was blocked with neutralizing antibody, the migration of oral mucosal epithelial cells was increased by60%and110%respectively. Moreover, CCL20and IL1F9had an additive effect on inhibiting mucosal epithelial cell migration since there was significantly greater improvement when both were blocked compared to each singly. The negative effect of high levels of CCL20and IL1F9on mucosal epithelial cell migration was also shown by adding CCL20or IL1F9to factors released by mucosal epithelial cells incubated in standard low glucose media. These results revealed the molecular mechanism of the effects of high glucose conditions on impaired oral mucosal wound healing through a FOXO1mediated pathway.(4) Impacts of periopathogens on re-epithelialization of mucosal epithelial cells and effectiveness of IgY against periopathogens:With evaluation of the impacts of periopathogens, F. nucleatum and P. gingivalis, on re-epithelialization of mucosal epithelial cells, both bacteria were found to reduce wound closure by inducing cell apoptosis. To inhibit the bacterial infection, specific egg yolk immunoglobulin (IgY) was produced from egg yolks obtained from hens immunized with F. nucleatum or P. gingivalis respectively. Growth of both bacteria were inhibited by specific IgYs in vitro, the immunofluorescence and immunoelectron microscope assays revealed a high binding ability of IgYs, which preliminarily explored the mechanisms of the effectiveness of IgYs against periopathogens. Furthermore, in an in vivo study, IgY treatment resulted in a marked decrease in alveolar bone loss after F.nucleatum infection in a mouse model confirming the effectiveness of IgY against periodontal disease-causing F. nucleatum. With the inhibitory effects against periopathogens, IgY may facilitate the improvement of oral mucosal wound healing.In summary, diabetic mice showed delayed oral mucosal wound healing compared with normoglycemic mice. Oral mucosal epithelial cells under high glucose conditions exhibited increased expression of CCL20and IL1F9that was due to FOXO1activation. Moreover, rescue experiments with function-blocking antibodies demonstrated that CCL20and IL1F9were largely responsible for the negative effect of FOXO1on mucosal epithelial cell migration. In contrast, FOXO1deletion in normoglycemic mice delayed oral mucosal wound healing, which was caused by impaired oral mucosal epithelial cells migration with FOXO1knockdown. Moreover, impacts of periopathogens on oral mucosal wound healing were investigated. F. nucleatum and P. gingivalis were found to reduce re-epithelialization by inducing cell apoptosis, while the growth and clononization of bacteria were inhibited by specific IgYs produced from egg yolks obtained from hens immunized with F. nucleatum or P. gingivalis, which offers an opportunity for IgY to accelerate oral mucosal wound healing impaired by periopathogens.