Interferon-γ stimulates CD14, TLR2 and TLR4 mRNA expression in gingival fibroblasts increasing responsiveness to bacterial challenge

• IFN-γ induces mRNA expression of CD14, TLR2 and TLR4 in human gingival fibroblasts.
• IFN-γ induces expression of membrane-bound CD14 in human gingival fibroblasts.
• Pre-incubation with IFN-γ followed by LPS stimulation increases cytokine production.
• LPS-induced cytokine production is abrogated by a blocking antibody to CD14.
• IFN-γ primes human gingival fibroblasts, through up-regulated CD14 expression.

ObjectiveTo investigate the potential effects of IFN-03A5 on the responsiveness of human gingival fibroblasts to bacterial challenge.DesignmRNA and protein expression of CD14, TLR2 and TLR4 in human gingival fibroblasts was detected by quantitative polymerase chain reaction (Q-PCR) and flow cytometry. The effect of preincubation with IFN-03A5 on subsequent bacterial LPS-induced expression of IL-6 and IL-8 by gingival fibroblasts was determined by ELISA. Bacterial LPS-induced IκBα degradation in human gingival fibroblasts was investigated by western blot.ResultsHuman gingival fibroblasts express CD14, TLR2 and TLR4 mRNAs. IFN-03A5, but not IL-103B2, induced mRNA expression of all three receptors and the expression of membrane bound CD14 protein. Pre-incubation of fibroblasts with IFN-03A5 and subsequent stimulation with Escherichia coli LPS or Porphyromonas gingivalis LPS led to increased production of IL-6 and IL-8. LPS-induced pro-inflammatory cytokine production was abrogated by a blocking antibody to CD14. Both E. coli LPS and P. gingivalis LPS induced IκBα degradation in human gingival fibroblasts.ConclusionOur data indicate that IFN-03A5 primes human gingival fibroblasts, through the upregulation of CD14 expression, which results in increased responsiveness to bacterial LPS challenge, as determined by pro-inflammatory cytokine production.