Defining the mechanism and functions of RIPK1-induced cell death in anti-bacterial immune defense

Project SummaryImmune defense against bacterial infection requires signaling pathways that activate expression ofinflammatory mediators to control and clear infection. Many pathogens inhibit these signaling pathways inorder to evade host immune defenses. In particular, Yersinia injects a virulence factor, YopJ, which potentlyblocks key aspects of NF-?B and MAPK signaling pathways. How immune defense is mediated againstpathogens that block immune signaling pathways remains poorly understood. NF-?B blockade in macrophagesexposed to bacterial PAMPs leads to cell death. Importantly, our recently published data demonstrate thatRIPK1 kinase activity is required for Yersinia-induced cell death, and that RIPK1 kinase activity is critical forresistance to Yersinia infection and innate inflammatory cytokine production in vivo. This suggests that RIPK1-induced death triggered by Yersinia is a key immune protective mechanism. Our new studies indicate that IKKphosphorylates RIPK1 to limit induction of cell death by inflammatory stimuli, suggesting that Yersinia blockadeof IKK releases RIPK1 from this inhibitory effect. How RIPK1 kinase activity and cell death promote hostdefense against bacterial infection is not known. Our central hypothesis is that RIPK1-mediated cell deathtriggered in response to pathogen-mediated NF-?B and MAPK blockade releases pro-inflammatory signals,such as IL-1 cytokines and intracellular alarmins that enable uninfected bystander cells to produce keyinflammatory mediators such as TNF, which control infection by pathogens that interfere with innate signaling.This is an important problem as this pathway likely responds to many pathogens that block critical innateimmune signaling pathways and in the context of pathological stimuli that lead to RIPK1-induced cell death.We propose three Specific Aims to address this important gap in our knowledge. First we will define themolecular basis for how RIPK1 kinase activation occurs in response to YopJ blockade of IKK signaling, andtest the role of this pathway in anti-bacterial immune defense. Second, we will define immune responses ofinfected and bystander cells, and will define the contribution of RIPK1-dependent cell death pathways to anti-bacterial host defense in vivo. Finally, we will determine the contribution of TNF signaling by bystander cells toantibacterial immune defense and will define the cell-type specific requirement for RIPK1- and TNFR-dependent cell death in host immune defense against Yersinia.