Date Published: January 3, 2013
Publisher: Public Library of Science
Author(s): Helen M. Lazear, Alissa Lancaster, Courtney Wilkins, Mehul S. Suthar, Albert Huang, Sarah C. Vick, Lisa Clepper, Larissa Thackray, Margaret M. Brassil, Herbert W. Virgin, Janko Nikolich-Zugich, Ashlee V. Moses, Michael Gale, Klaus Früh, Michael S. Diamond, Christopher F. Basler.
Although the transcription factors IRF-3 and IRF-7 are considered master regulators of type I interferon (IFN) induction and IFN stimulated gene (ISG) expression, Irf3−/−×Irf7−/− double knockout (DKO) myeloid dendritic cells (mDC) produce relatively normal levels of IFN-β after viral infection. We generated Irf3−/−×Irf5−/−×Irf7−/− triple knockout (TKO) mice to test whether IRF-5 was the source of the residual induction of IFN-β and ISGs in mDCs. In pathogenesis studies with two unrelated positive-sense RNA viruses (West Nile virus (WNV) and murine norovirus), TKO mice succumbed at rates greater than DKO mice and equal to or approaching those of mice lacking the type I IFN receptor (Ifnar−/−). In ex vivo studies, after WNV infection or exposure to Toll-like receptor agonists, TKO mDCs failed to produce IFN-β or express ISGs. In contrast, this response was sustained in TKO macrophages following WNV infection. To define IRF-regulated gene signatures, we performed microarray analysis on WNV-infected mDC from wild type (WT), DKO, TKO, or Ifnar−/− mice, as well as from mice lacking the RIG-I like receptor adaptor protein MAVS. Whereas the gene induction pattern in DKO mDC was similar to WT cells, remarkably, almost no ISG induction was detected in TKO or Mavs−/− mDC. The relative equivalence of TKO and Mavs−/− responses suggested that MAVS dominantly regulates ISG induction in mDC. Moreover, we showed that MAVS-dependent induction of ISGs can occur through an IRF-5-dependent yet IRF-3 and IRF-7-independent pathway. Our results establish IRF-3, -5, and -7 as the key transcription factors responsible for mediating the type I IFN and ISG response in mDC during WNV infection and suggest a novel signaling link between MAVS and IRF-5.
The type I interferon (IFN) signaling network is an essential component of the innate immune response against viral infections, and also functions to shape adaptive immunity –. Infected cells initiate an antiviral response upon recognition of non-self pathogen-associated molecular patterns (PAMPs), which are detected by host pattern recognition receptors (PRRs) , . PRRs that recognize RNA viruses include members of the Toll-like receptor (TLR3 and TLR7) and the RIG-I-like receptor (RLR; RIG-I and MDA5) families. TLRs and RLRs recognize distinct PAMPs in different locations (extracellular/endosomes and cytoplasm, respectively) and activate signaling cascades to initiate antiviral and inflammatory responses. TLR3 binds to double-stranded RNA and recruits the adaptor molecule TRIF to activate the kinases TRAF and IKK-ε, which in turn activates the latent transcription factors IRF-3, IRF-7, and NF-κB. Single-stranded RNA is recognized by TLR7, which uses the adaptor molecule MyD88 to activate TRAF and IKK-ε, and subsequently NF-κB- and IRF-7-dependent transcription. RLRs interact with the mitochondria-associated adapter molecule MAVS (also called IPS-1, VISA, or CARDIF), which signals through the kinases TBK1 and IKK-ε to activate IRF-3, IRF-7, and NF-κB and initiate type I IFN production.
In the present study, we generated Irf3−/−×Irf5−/−×Irf7−/− TKO mice to establish that these three IRF family transcription factors coordinately regulate IFN-β production and ISG expression in mDC. We found that antiviral gene induction was ablated almost entirely in mDC from TKO or Mavs−/− mice, suggesting a dominant role for MAVS in initiating the antiviral response and pointing to a novel signaling interaction between IRF-5 and the RLR signaling pathway.