Cigarette smoke activates NFκB-mediated Tnf-α release from mouse middle ear cells

: Cigarette smoke exposure is a significant risk factor in the development of otitis media. NF-κB is a transcription factor known to mediate cigarette smoke effects in multiple cell types. We hypothesized that stimulation of murine middle ear epithelial cells (MEEC) with cigarette smoke condensate (CSC) activates NF-κB resulting in upregulation of proinflammatory cytokines.

: In vitro model of cultured murine middle ear epithelial cells.

: Time course CSC stimulation of MEEC was performed. Antibody microarrays were then utilized to simultaneously measure 40 inflammatory cytokines. Enzyme-linked immunosorbent assay (ELISA) and quantitative reverse transcriptase-polymerase chain reaction were performed to validate and further evaluate array results. Luciferase reporter assays were performed to evaluate NF-κB activation with CSC in MEEC. Chromatin immunoprecipitation (ChIP) assays were performed to determine whether CSC induces NF-κB interaction with the Tnf-α promoter.

: Multiple cytokines showed significant increases with CSC exposure. ELISA studies demonstrated that Tnf-α secretion increased the most. CSC stimulation likewise increased Tnf-α mRNA abundance and induced promoter activity 4.8-fold in a Tnf-α reporter plasmid. Reporter assays demonstrated 4.84-fold activation of NF-κB with CSC. ChIP assays demonstrated NF-κB binding to canonical κB sites in the Tnf-α promoter with CSC stimulation.

: CSC activates NF-κB in MEEC. Furthermore, this activation results in CSC induced Tnf-α promoter activation, gene expression, and levels in cell secretions. Laryngoscope, 120:2508-2515, 2010.