Macpherson_2014_Inflamm.Res_63_557

OBJECTIVE: The alpha 7 nicotinic receptor (alpha7nAChR) is expressed by oral keratinocytes. alpha7nAChR activation mediates anti-inflammatory responses. The objective of this study was to determine if alpha7nAChR activation inhibited pathogen-induced interleukin-8 (IL-8) expression by oral keratinocytes. MATERIALS AND
METHODS: Periodontal tissue expression of alpha7nAChR was determined by real-time PCR. OKF6/TERT-2 oral keratinocytes were exposed to Porphyromonas gingivalis in the presence and absence of a alpha7nAChR agonist (PHA-543613 hydrochloride) alone or after pre-exposure to a specific alpha7nAChR antagonist (alpha-bungarotoxin). Interleukin-8 (IL-8) expression was measured by ELISA and real-time PCR. Phosphorylation of the NF-kappaB p65 subunit was determined using an NF-kappaB p65 profiler assay and STAT-3 activation by STAT-3 in-cell ELISA. The release of ACh from oral keratinocytes in response to P. gingivalis lipopolysaccharide was determined using a GeneBLAzer M3 CHO-K1-bla cell reporter assay.
RESULTS: Expression of alpha7nAChR mRNA was elevated in diseased periodontal tissue. PHA-543613 hydrochloride inhibited P. gingivalis-induced expression of IL-8 at the transcriptional level. This effect was abolished when cells were pre-exposed to a specific alpha7nAChR antagonist, alpha-bungarotoxin. PHA-543613 hydrochloride downregulated NF-kappaB signalling through reduced phosphorylation of the NF-kappaB p65-subunit. In addition, PHA-543613 hydrochloride promoted STAT-3 signalling by maintenance of phosphorylation. Furthermore, oral keratinocytes upregulated ACh release in response to P. gingivalis lipopolysaccharide. CONCLUSION: These data suggest that alpha7nAChR plays a role in regulating the innate immune responses of oral keratinocytes.