Fig. 3. IFNβ production from epidermal keratinocyte in response to LL37 and dsRNA is mediated by MAVS.
(A–B). RTqPCR of mRNA expression of MAVS, various TLRs and IFNAR1 in KC (NHEK), pDC analyses or cDC (A) (n=3; ratio to GAPDH mRNA is shown), or separated epidermis and dermis from normal human skin (B) (fold changes are shown relative to TLR3) (n=3). (C). RTqPCR analysis of IFNB1 mRNA expression in NHEKs transfected with control siRNA, TLR3 siRNA or MAVS siRNA then stimulated with polyIC with or without LL37 pretreatment. (n=3). (D). RTqPCR analysis of Ifnb1 mRNA expression in LL37/polyIC stimulated mouse KCs isolated from Mavs−/+ HET or Mavs−/− KO littermate mice (n=3). (E). RTqPCR analysis of IFNB1 mRNA expression in NHEKs transfected with control siRNA, TLR3 siRNA or MAVS siRNA then stimulated with LL37 and U1-RNA as indicated (n=3). (F). NHEKs treated with various combination of polyIC and LL37 were co-immunostained with anti-MAVS antibody and mitochondrial tracker (MitoV) or phosphor-tyrosine (pTyr) as indicated. (G). Time course analyses of ISG15 mRNA expression in NHEKs treated with LL37 and polyIC as indicated (n=3). (H–I). ISG15 expression in siRNA mediated silencing of TLR3 or MAVS (H) or IFNB1 or IFNAR1. (I) NHEKs treated with LL37 and polyIC (n=3). (J) Proposed scheme for induction of proinflammatory cytokines and type-1 IFN responsive genes mediated by LL37 and dsRNA mediated through MAVS or TLR3. All error bars indicate mean ± s.e.m. * P<0.05, ** P<0.01, *** P<0.001 (one way Anova). L/P, LL37 and polyIC.