Smoking Differentially Modulates Epithelial Responses To Commensal And Pathogenic Biofilms

Objectives: We have previously demonstrated that smoking alters host-bacterial interactions in health and disease. However, it is not known whether smoking alters this interaction by preferentially acting on the host cells, the bacterial biofilm, or both. The purpose of the present study was to examine the relative contributions of smoking to the inflammatory potential of the bacterial biofilm and to the immune response of host epithelial cells. 

Methods: Human oral keratinocytes (TERT-OKF6 cell lines) were grown to near-confluence in cigarette smoke-conditioned or normal media and challenged with commensal or pathogenic biofilms grown under normal conditions or in media conditioned with 1% smoke in a 1:100 multiplicity of infection. Supernatant was collected at 2,4,6 and 8 hours and the levels of 27 immune mediators were analyzed by multiplexed bead-based flow cytometry. Cytokines levels were compared between groups using parametric tests.

Results: Pathogenic and commensal biofilms elicited a time-dependent immune response from the epithelial cells (p<0.05, repeated measures ANOVA). Smoke-conditioned commensal biofilms elicited similar levels of IL1-RA, TNF-α, IL-6, IL-8, IL-9, IL-12, G-CSF, and VEGF responses when compared to non-smoked controls.  Smoke-conditioned pathogenic biofilms elicited significantly greater concentrations of IL-6, IL-8, IL-12, IL-13, TNF-α, G-CSF and VEGF and lower levels of PDGF and IL-9 after 4 hours when compared to controls (p<0.05). Smoke-conditioned host cells responded with lower levels of IL-6, IL-8, IL-9, IL-12 and VEGF to the commensal biofilm at all time points when compared to the controls (p<0.05, 2-sample t-test), however, there was no difference in the immune response to a pathogenic biofilm between smoke-conditioned host cells and controls.

Conclusions: Smoking increases the pro-inflammatory potential of pathogenic biofilms while promoting a suppression of the immune response to a commensal biofilm. These findings suggest a mechanism by which smoking affects subgingival host-bacterial interactions and increases susceptibility to disease.