TLR3 drives IRF6‐dependent IL‐23p19 expression and p19/EBI3 heterodimer formation in keratinocytes

Abstract

Interferon regulatory factor (IRF) family members impart cell-type specificity to toll-like receptor (TLR) signalling, and we recently identified a role for IRF6 in TLR2 signalling in epithelial cells. TLR3 has a well-characterized role in wound healing in the skin, and here, we examined TLR3-dependent IRF6 functions in human keratinocytes. Primary keratinocytes responded robustly to the TLR3 agonist poly(IC) with upregulation of mRNAs for interferon-β (IFN-β), the interleukin-12 (IL-12) family member IL-23p19 and the chemokines IL-8 and chemokine (C-C motif) ligand 5 (CCL5). Silencing of IRF6 expression enhanced poly(IC)-inducible IFN-β mRNA levels and inhibited poly(IC)-inducible IL-23p19 mRNA expression in primary keratinocytes. Consistent with these data, co-transfection of IRF6 increased poly(IC)-inducible IL-23p19 promoter activity, but inhibited poly(IC)-inducible IFN-β promoter activity in reporter assays. Surprisingly, poly(IC) did not regulate IL-12p40 expression in keratinocytes, suggesting that TLR3-inducible IL-23p19 may have an IL-23-independent function in these cells. The only other IL-12 family member that was strongly poly(IC) inducible was EBI3, which has not been shown to heterodimerize with IL-23p19. Both co-immunoprecipitation and proximity ligation assays revealed that IL-23p19 and EBI3 interact in cells. Co-expression of IL-23p19 and EBI3, as compared with IL-23p19 alone, resulted in increased levels of secreted IL-23p19, implying a functional role for this heterodimer. In summary, we report that IRF6 regulates a subset of TLR3 responses in human keratinocytes, including the production of a novel IL-12 family heterodimer (p19/EBI3). We propose that the TLR3-IRF6-p19/ EBI3 axis may regulate keratinocyte and/or immune cell functions in the context of cell damage and wound healing in the skin.

The toll-like receptor 3 pathway in homeostasis, responses to injury and wound repair

“…In oral keratinocytes, IRF6 regulates Grainyhead-like 3 expression [57,58], which has been reported to directly regulate TGM1 transcription [59]. Thus, it is conceivable that TLR3-inducible TGM1 expression occurs via the IRF6-dependent pathway [18]. Finally, the importance of such responses in wound repair is highlighted by the observation that Tlr3 -/-mice exhibit chronic wounds and impaired re-epithelialization following acute UVB-irradiation [56].…”

“…Therefore, IRF6-mediated IFN- inhibition may enable a proliferative and reparative response in the skin. Intriguingly, we recently showed that poly(IC) selectively upregulates the expression of the IL-12 family genes IL-23p19 ( sub-unit) and Epstein-Barr virus induced 3 (EBI3) ( sub-unit) in keratinocytes, and that these two sub-units interact when co-expressed in cells [18]. This suggests that IL-23p19 and EBI3 may function as a novel IL-12 family cytokine.…”

“…Not surprisingly then, keratinocytes express TLRs, responding to PAMPs with inflammatory cytokine production. These cells are especially responsive to TLR3 agonists [18,[44][45][46][47], and an initial study on human keratinocytes revealed an unexpected role for this PRR in detecting RNA released from damaged cells [48]. More specifically, UVB-damaged keratinocytes release U1 spliceosomal non-coding RNA that activates TLR3 in non-irradiated cells, resulting in the release of inflammatory cytokines, such as TNF-α and IL-6 [49].…”

“…IRF6 suppressed poly(IC)-inducible IFN- mRNA, while promoting inducible IL-23p19 mRNA expression in human keratinocytes [18]. IFN- has been shown to inhibit proliferation and promote terminal differentiation of keratinocytes [62,63].…”

“…Specifically, it inhibits poly(IC)-inducible IFN- expression, while promoting poly(IC)-inducible IL-23p19 expression in primary human keratinocytes [18].…”