Role of IRF6 in epithelial cell-mediated host defence and inflammation

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Members of the Interferon Regulatory Factor (IRF) family of transcription factors have essential roles in developmental pathways, as well as immune function and host defence. IRF6 has a well-characterized role in epithelial cell differentiation, but has not been studied in the context of immune function. In this thesis, I investigated IRF6 involvement in Toll-like Receptor (TLR) responses in epithelial cells. In contrast to the ubiquitous and myeloid-restricted expression patterns of IRF3 and IRF8, respectively, IRF6 was selectively expressed in a range of human epithelial cell types, particularly keratinocytes. Keratinocyte differentiation also increased IRF6 mRNA and protein expression. Primary human keratinocytes, as well as the keratinocyte cell line HaCaT, responded weakly to most TLR ligands, with the exception of the TLR3 agonist poly(IC). Poly(IC) robustly upregulated mRNA expression of IFN-β, as well as the chemokines IL-8, CCL5 and CCL20 in both of these cell types. Surprisingly, poly(IC) also selectively upregulated mRNAs encoding IL-23p19 and EBI3, two members of the IL-12 family that have not been shown to heterodimerize. Although IRF6 enhanced poly(IC)-inducible IFN-β promoter activity in HEK-TLR3 cells, silencing IRF6 expression did not decrease poly(IC)-inducible IFN-β mRNA expression in primary keratinocytes. Several other inducible genes were also unaffected, however knock-down of IRF6 did impair poly(IC)-inducible IL-23p19 mRNA expression in primary keratinocytes. Conversely, ectopic expression of IRF6 in the bladder epithelial cell line T24 conferred poly(IC)-inducible IL-23p19 mRNA expression. To investigate the mechanisms responsible, the IL-23p19 promoter was cloned and transient transfection analyses were performed. These experiments indicated that IRF6 enhanced IL-23p19 promoter activity upon poly(IC) stimulation. Poly(IC) also trigged nuclear translocation of IRF6 in keratinocytes. However, IL-23p19 protein was not detected in supernatants or cell lysates from poly(IC)-activated keratinocytes. Therefore, the effect of inflammatory stimuli, like IFN-γ, on keratinocyte responses to poly(IC) was examined. IFN-γ priming enhanced poly(IC)-inducible gene expression but still did not license IL-23p19 secretion. Interestingly, keratinocyte differentiation increased poly(IC)-inducible IL-23p19 mRNA expression, but decreased poly(IC)-inducible IFN-β mRNA expression upon IFN-γ priming. Collectively, these data suggest that IRF6 promotes a sub-set of TLR3 responses in human keratinocytes. Studies were also undertaken to determine whether IRF6 is involved in MyD88-dependent signalling responses in keratinocytes. Most MyD88-dependent TLR agonists were generally ineffective at triggering inflammatory responses in these cells. Investigation of other MyD88-dependent ligands revealed that the TLR7 agonist imiquimod and the TLR2/6 agonist FSL-1 did upregulate inflammatory chemokine expression in keratinocytes. Knock-down studies suggested that IRF6 contributed to TLR7-inducible CCL5 and IFN-β mRNA expression in these cells. Given the modest effects of most MyD88-dependent TLR agonists on inflammatory responses in keratinocytes, factors that may enhance these responses were also investigated. Keratinocyte differentiation increased imiquimod-inducible CCL5 mRNA expression in keratinocytes, whereas there was no clear effect of IFN-γ priming. The role of IRF6 in promoting TLR2/6 responses in keratinocytes remains unresolved. In total, my findings posit IRF6 as a transducer of a sub-set of MyD88-dependent and -independent TLR responses in keratinocytes, implicating it as a sentinel transcription factor in pathogen sensing by the skin.