Oral Epithelial Cell Cytokine Response to b(1,6)-N-acetyl-D-glucosamine Homopolymer (PGA)

Production of a polysaccharide matrix is a hallmark of bacterial biofilms, which is used by bacteria for attachment to surfaces. Aggregatibacter actinomycetemcomitans (Aa) uses pga gene locus for the production of a biofilm matrix exopolysaccharide. Phylogenetically diverse bacterial species including S. epidermidis and E. coli make this structurally similar exopolysaccharide consisting of a homopolymer of GlcNAc residues in b(1,6)-linkage (PGA). The role of PGA in the pathogenesis of Aa and its effect on human gingival epithelial cells (GEC) is unknown. Objective: The Gram negative periodontal pathogen, Aa, has been strongly associated with Localized Aggressive Periodontitis. Our goal was to determine the nature of the inflammatory responses to PGA in order to better understand the host-oral pathogen interaction. Hypothesis: PGA will elicit cytokines as part of immune defense by GEC. Methods: PGA (100 µg/mL as suspension), LPS (1 µg/mL) and synthetic PGA tetrasaccharide (10 µg/mL) were used to challenge GEC OKF6/TERT-2 cells for 6, 12 and 24h. IL-1a and the untreated cells were used as controls. The levels of cytokines in the supernatant were quantitated using a 22-plex multicytokine detection system (Upstate). mRNA isolated from the cells were quantitated using RT-QPCR for the relative expression of cytokines using b2 microglobulin as a housekeeping gene. Results: The GEC cells expressed IL-8 and IP-10 in response to the challenge to PGA and the tetrasaccharide at 6h and maximally at 24h. LPS did not have any effect. QPCR revealed that the relative RNA expression was 80-fold (±27) for IL-8 and 15-fold (±3) for IP-10 compared to b2 microglobulin. Conclusion: These results support the hypothesis that PGA is partly responsible for triggering the local inflammatory response via the production of IL-8 and IP-10. This work was supported by USPHS Grant DE16291.