The Roles Of Toll-like Recepror 2 And 4 In Endotoxin Tolerance In Human Gingival Epithelial Cells:A Preliminary Study

Human gingival epithelial cells(HGECs)are exposed to numerous bacteria persistently and are the first barriers of periodontal tissues.Periodontitis is a chronic inflammatory disease.The repeated bacterial lipopolysaccharides(LPS)stimuli might contribute to the reduced responses to the subsequent stimuli,namely endotoxin tolerance.The mechanisms of endotoxin tolerance are still not clear.In addition,it is also not fully understood the influences of endotoxin tolerance on the development of periodontitis.OBJECTIVE:The aim of this study is to develop an endotoxin tolerance model in HGECs and to explore the roles and mechanisms of endotoxin tolerance in periodontal inflammation.METHODS:1.HGECs were prepared from gingival samples collected in crown lengthening procedures and third molar extraction.Expressions of keratin and vimentin in HGECs were confirmed by immunohistochemical staining,and growth curve was made according to the results of MTT assay.2.Endotoxin tolerance in HGECs was developed by repeated 1μg/ml Porphyromonas gingivalis(P.gingivalis)LPS and(or)1μg/ml Escherichia coli(E.coli)LPS stimulation.And the changes of the levels of IL-1β、IL-6 and IL-8 secreted by HGECs were detected by ELISA.3.Reverse transcription PCR was used to confirm the mRNA expressions of TLR2,4 in HGECs.The change of gene and protein expressions of TLR2,4 were detected in HGECs stimulated with repeated P.gingivalis LPS and(or)E.coli LPS by real-time PCR and flow cytometry(FCM)respectively.RESULTS:1.HGECs obtained from the gingival samples constitutively expressed keratin and could be subcultured well.2.The amounts of IL-1β、IL-6 and IL-8 secreted by HGECs stimulated with 1μg/ml P.gingivalis LPS or 1μg/ml E.coli LPS were increased significantly after 24h(p<0.05).After the second stimulations for another 24h(homotolerance and heterotolerance),IL-6 and IL-8 productions were decreased significantly compared with those secreted by HGECs stimulated with 1μg/ml P.gingivalis LPS or 1 μg/ml E.coli LPS only once(p<0.05).In addition,after repeated 1μpg/ml E.coli LPS stimulations,the secretion levels of IL-1β were also reduced significantly(p<0.05)compared with those secreted by the cells stimulated only once(p>0.05).3.HGECs from different individuals constitutively expressed TLR2,4 mRNA.The expression levels of TLR2 mRNA and protein were increased significantly in HGECs stimulated with 1μg/ml P.gingivalis LPS(p<0.05),and the expression levels of TLR4 mRNA and protein were also increased obviously in HGECs stimulated with 1μg/ml E.coli LPS(p<0.05).Moreover,the expression levels of TLR2 mRNA and protein were reduced significantly in HGECs stimulated with repeated 1μg/ml P.gingivalis LPS compared with those in HGECs stimulated only once(p<0.05),and the expression levels of TLR4 mRNA and protein were also reduced obviously in HGECs stimulated with repeated 1μg/ml E.coli LPS compared with those in HGECs stimulated only once(p<0.05).CONCLUSIONS:This is the first report on the development of endotoxin tolerance in HGECs and the roles and mechanisms of endotoxin tolerance in periodontal inflammation were explored at the same time.This study indicated that repeated P.gingivalis LPS and(or)E.coli LPS stimulations could induce endotoxin tolerance in HGECs,and contributed to the decreased secretions of IL-1β,IL-6 and IL-8.In addition,TLR 2,4 might play a role in triggering endotoxin tolerance.