Response of Differentiated Epithelial Cells to Commensal and Pathogenic Bacteria

The viability and barrier function of epithelial cells is important in maintaining homeostasis in the gingiva. Objective: To examine how multilayer well differentiated gingival epithelial cells respond to bacterial challenge. Methods: Human gingival epithelial cells were isolated from gingiva. After four passages, cells were seeded in collagen coated permeable membrane inserts and submerged in media until completely confluent after which the cells were incubated in an air-liquid-interface for 10-days. Epithelial cells were challenged with a pathogen, Porphyromonas gingivalis (Pg), a commensal, Streptococcus gordonii (Sg), or an opportunistic pathogen, Fusobacterium nucleatum (Fn) at 5x10^8/cm^2 for 24hrs. Apoptosis was examined by ELISA and barrier function was measured with FITC labelled dextran. Experiments were performed three times and significance was established by one way analysis of variance (ANOVA). Results: Histologic data showed that epithelial cells grown on permeable inserts formed stratified multilayers and had a 15-fold and 4-fold increase in mRNA levels of differentiation markers, keratin-1 and 10, respectively, compared to submerged cultures (P<0.05). When barrier function was tested, Pg caused a 5-fold increase in leakage, Fn a 2-fold increase and Sg no increase compared to cells alone (P<0.05). Pg interference with barrier function was primarily due to the production of proteases since the increased leakage was reversed with leupeptin (P<0.05). When apoptosis was measured, Pg and Fn stimulated slightly less than a 2-fold increase and Sg no increase compared to epithelial cells alone (P<0.05). In contrast Pg caused an 11-fold increase in apoptosis in submerged undifferentiated epithelial cultures (P<0.05). Conclusion: Pg and Fn caused barrier disruption and apoptosis in multilayer epithelial cultures while Sg had no effect. Thus, pathogenic bacteria may induce changes in the integrity of the epithelial barrier facilitating invasion into connective tissue. This work was supported by a grant from the NIDCR, R01DE018307.