RIPK3 Activates Parallel Pathways of MLKL-Driven Necroptosis and FADD-Mediated Apoptosis to Protect against Influenza A Virus

Abstract

Summary Influenza A virus (IAV) is a lytic virus in primary cultures of many cell types and in vivo. We report that the kinase RIPK3 is essential for IAV-induced lysis of mammalian fibroblasts and lung epithelial cells. Replicating IAV drives assembly of a RIPK3-containing complex that includes the kinase RIPK1, the pseudokinase MLKL, and the adaptor protein FADD, and forms independently of signaling by RNA-sensing innate immune receptors (RLRs, TLRs, PKR), or the cytokines type I interferons and TNF-α. Downstream of RIPK3, IAV activates parallel pathways of MLKL-driven necroptosis and FADD-mediated apoptosis, with the former reliant on RIPK3 kinase activity and neither on RIPK1 activity. Mice deficient in RIPK3 or doubly-deficient in MLKL and FADD, but not MLKL alone, are more susceptible to IAV than their wild-type counterparts, revealing an important role for RIPK3-mediated apoptosis in antiviral immunity. Collectively, these results outline RIPK3-activated cytolytic mechanisms essential for controlling respiratory IAV infection.

ZBP1/DAI is an innate sensor of influenza virus triggering the NLRP3 inflammasome and programmed cell death pathways

“…Moreover, ZBP1 regulation of cell death in IAV-infected cells is mediated via RIPK3. Of note, while our manuscript was in revision Nogusa et al reported RIPK3-dependent activation of parallel necroptotic and apoptotic pathways in IAV-infected cells (33). Although this study demonstrate the importance of RIPK3 in driving cell death during IAV-infection, the upstream receptors and signaling pathways regulating RIPK3 were not identified.…”

Programmed cell death as a defence against infection

“…The RIPK3 pathway also defends against a mouse adapted influenza infection. Zbp1 −/− and Ripk3 −/− mice have increased mortality during infection influenza A PR8 54,64 . However, the susceptibility of Ripk3 −/− mice to influenza virus was not replicated in another study 70 and a third study shows the reverse, where Zbp1 −/− mice have higher viral titers but reduced tissue damage and mortality 53 , suggesting a simultaneously beneficial role in defense paired with a detrimental role in immunopathology.…”

“…The morphologies in comparison deserve further study. Necroptosis can be investigated in vivo by using Ripk3 −/− or Mlkl −/− mice; however, RIPK3 has been suggested to have non-necroptotic functions during influenza infection 64 and during inflammatory disease models 65 . Therefore, before attributing a phenotype to necroptosis, it is important to verify that phenotypes observed in Ripk3 −/− mice are also seen in Mlkl −/− mice.…”

“…Defective TLR4 signalling would also explain the resistance of Casp8 −/− background mice to endotoxic shock 88 . Fadd −/− Mlkl −/− mice (which are similar to Casp8 −/− Ripk3 −/− mice, see Box 2) have increased mortality to influenza A virus infection 64 , perhaps due to defects in TLR3 signalling.…”

Complex Pathologic Roles of RIPK1 and RIPK3: Moving Beyond Necroptosis

“…Nogusa et al examined pathways of Influenza A virus induced death and provided a striking example of RIPK3 acting in a kinase-independent manner to promote clearance of virally infected cells through activation of caspase-8-mediated apoptosis [84]. It has also been shown that RIPK3 can induce inflammatory signaling in a kinase-independent manner through the activation of a non-canonical caspase-8-dependent inflammasome [85].…”