Objectives: To investigate cytokine responses to P. gingivalis invasion of an organotypic oral mucosal model (OMM) and monolayer culture of oral epithelial cells.
Methods: Normal oral keratinocytes (NOK) or the H357 oral keratinocyte cell line were seeded onto fibroblast-populated type I collagen and cultured at an air-liquid interface for 7-10days. OMMs and normal oral biopsy were stained immunohistochemically for cytokeratin 13, laminin V and pan-cytokeratin. H357 and NOK cells were also grown as monolayers. NOK-monolayers or OMM were pre-stimulated with 25ng/ml TNFα for 4 hours then exposed to live P. gingivalis NCTC11834 (MOI 100) for 1.5 hours. Extracellular P. gingivalis were killed with 200µg/ml metronidazole for 4 hours and conditioned media were analysed using RayBio® Human Inflammation Antibody Arrays. Conditioned media and cell lysates following overnight infection of H357-monolayers with P. gingivalis W50, DRgpA:RgpB and DKgp gingipain mutants were assessed for interleukin 8 (CXCL8) protein and mRNA expression.
Results: NOK-OMM resembled normal oral mucosa in all immunohistochemical staining, H357-OMM did not stain for cytokeratin 13. There was a significant reduction in levels of cytokines detected at the protein level for both monolayers and OMMs following P.gingivalis invasion compared to uninfected controls. However, the levels of CXCL8, IL-6, CCL2 and CCL5 were higher in OMM compared to monolayer cultures (4.99, 688.0, 60.73 and 29.97 fold respectively), suggesting a multilayered epithelium behaves differently from cells cultured as monolayers. Use of DRgpA:RgpB and DKgp P.gingivalis mutants confirmed a role for intracellular gingipains in reducing CXCL8 in the extracellular medium. However, real-time PCR showed a 50-fold increase in CXCL8 mRNA expression on P. gingivalis invasion.
Conclusion: The cytokine responses following invasion of P.gingivalis were reduced, which may occur post-transcriptionally or post-translationally, and cytokine responses in OMM differ from monolayers, which may be due to their multilayered epithelium.
Funded by GlaxoSmithKline