5A–D); this effect was significantly enhanced by TRIF (Fig. 5A and C). Also, suppression of IRF7 expression impaired poly(I:C)-mediated IFN-β gene induction, confirming that IRF7 is involved in poly(I:C)-mediated induction of IFN-β (data not shown). Interestingly, we demonstrate that although ectopic expression of Mal or the TIR domain of Mal dose-dependently inhibited IRF7:TRIF-induced activation of the IFN-β and PRDI-III reporter genes, the N-terminal region of Mal did not (Fig. 5A and C). Additionally, Mal did not affect TBK1/IKKε-induced activation of the IFN-β and PRDI-III reporter genes nor the IRF3/IRF7 transactivation reporter gene induction (Supporting Information Fig. 3).
We also show that Mal and its variants did not significantly affect IRF3:TRIF-induced activation Natural Product Library clinical trial of the IFN-β and PRDI-III reporters (Fig. 5B and D). Given that our data suggest that R428 nmr the TIR domain of Mal negatively regulates TLR3:TRIF:IRF7-induced IFN-β gene induction, we sought to further explore the mechanism involved. Thus, we examined the ability of Mal to modulate poly(I:C)-mediated IRF7 phosphorylation and nuclear translocation 28. We clearly demonstrate that IRF7 undergoes poly(I:C)-induced phosphorylation
and this effect is blocked by Mal (Fig. 6A). Moreover, poly(I:C) induced the phosphorylation of endogenous IRF7 to a greater extent in BMDM lacking Mal (Fig. 6B) and densitometric analysis revealed that ∼50% greater phosphorylation of IRF7 was evident in Mal-deficient cells when compared with WT cells following poly(I:C) stimulation. On the contrary, equivalent IRF3 phosphorylation is evident in WT and Mal-deficient
BMDM following poly(I:C) stimulation (Fig. 6B, lower). As a further test of the negative role of Mal on IRF7 activation, we examined the effect of Mal www.selleck.co.jp/products/hydroxychloroquine-sulfate.html on the nuclear translocation of IRF7. We demonstrate that over-expression of Mal blocked poly(I:C)-induced nuclear translocation of IRF7 (Fig. 6E). As expected, Mal did not affect the nuclear translocation of IRF3 following ligand stimulation (Fig. 6E). We also show that Mal colocalises with IRF7, not IRF3 within the cytosol of HEK293:TLR3 cells (Supporting Information Fig. 4). Together, these data show that Mal inhibits IRF7, but not IRF3, functionality and concomitant IFN-β gene induction. Given that previous studies show an interaction between IRF and Mal 27, we hypothesised that Mal may be directly binding to IRF7 and thus prevent its phosphorylation and translocation. We found that full-length Mal co-immunoprecipitates with IRF7, but not IRF3 (Fig. 6C and D). Further, co-immunoprecipitation experiments show that the TIR-domain of Mal, but not the N-terminal domain of Mal, co-immunoprecipitates with IRF7, but not with IRF3 (Supporting Information Fig. 5) and supports the hypothesis that Mal impacts on TLR3:IRF7, not TLR3:IRF3-mediated IFN-β induction.