Project Summary Immune defense against bacterial infection requires signaling pathways that activate expression of inflammatory mediators to control and clear infection. Many pathogens inhibit these signaling pathways in order to evade host immune defenses. In particular, Yersinia injects a virulence factor, YopJ, which potently blocks key aspects of NF-ΚB and MAPK signaling pathways. How immune defense is mediated against pathogens that block immune signaling pathways remains poorly understood. NF-κB blockade in macrophages exposed to bacterial PAMPs leads to cell death. Importantly, our recently published data demonstrate that RIPK1 kinase activity is required for Yersinia-induced cell death, and that RIPK1 kinase activity is critical for resistance 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 IKK phosphorylates RIPK1 to limit induction of cell death by inflammatory stimuli, suggesting that Yersinia blockade of IKK releases RIPK1 from this inhibitory effect. How RIPK1 kinase activity and cell death promote host defense against bacterial infection is not known. Our central hypothesis is that RIPK1-mediated cell death triggered in response to pathogen-mediated NF-κB and MAPK blockade releases pro-inflammatory signals, such as IL-1 cytokines and intracellular alarmins that enable uninfected bystander cells to produce key inflammatory 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 innate immune 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 the molecular basis for how RIPK1 kinase activation occurs in response to YopJ blockade of IKK signaling, and test the role of this pathway in anti-bacterial immune defense. Second, we will define immune responses of infected 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 to antibacterial immune defense and will define the cell-type specific requirement for RIPK1- and TNFR- dependent cell death in host immune defense against Yersinia.