Differential utilization of nuclear factor-κB signaling pathways for gingival epithelial cell responses to oral commensal and pathogenic bacteria

Introduction: Human beta-defensin-2 (hBD-2) is an antimicrobial peptide, induced by bacterial stimuli and inflammation, that plays a role in mucosal and skin innate immune defense. The nuclear factor-kappa B (NF-kappa B) transcription factor family is important in innate and adaptive immune responses to bacteria and proinflammatory cytokines. NF-kappa B operates via the traditional IKK beta signaling, as well as an alternative pathway utilizing IKK alpha signaling, which is important in keratinocyte differentiation. Our previous studies showed that pathogenic, but not commensal, bacteria used NF-kappa B signaling in hBD-2 induction. The objective of this study was to understand which arm of the NF-kappa B pathway is involved in gingival epithelial cell responses to pathogenic bacteria, including hBD-2 induction. Methods: Cultured oral epithelial cells were transfected with synthetic small interfering RNAs (siRNAs) specific for various steps in each pathway, namely IKK beta, TRAF6 and MyD88 in the canonical, and IKK alpha and TRAF3 in the alternative pathway, and subsequently stimulated with various oral bacteria. Results: The hBD-2 induction level was reduced to 21-61% in cells in which the alternative NF-kappa B pathway was blocked and subsequently stimulated with pathogenic bacteria, while cells in which the canonical pathway was blocked showed reduction to 78-99%. Cells stimulated with commensals showed little change in hBD-2 induction level regardless of the siRNA used. Microarray analysis showed that oral epithelia differentially regulated numerous innate immune markers in response to pathogens and commensals. Conclusion: Our data suggest a role for the IKK alpha/TRAF3 pathway in NF-kappa B activation by pathogenic bacteria, while commensal bacteria do not utilize either NF-kappa B pathway, for hBD-2 induction.