ABSTRACT Hypomorphic variants of the Immunity Related GTPase (IRG) M gene are associated with poor outcomes for sepsis patients; however, the underlying mechanism is not known. IRGM and its mouse orthologues are produced at high levels in response to LPS and/or interferons, particularly in macrophages. Although prior work has shown that IRGM proteins bind intracellular membranes and modulate immune responses that restrict survival of intracellular pathogens, their primary roles in inflammation - independent of their roles in cell- autonomous host defense - are mostly unexplored. As instructed by extensive preliminary data, this proposal will address the novel hypothesis that IRGM proteins directly downmodulate the production of inflammatory cytokines in response to LPS, and that they do so through at least two distinct mechanisms: (1) by shaping metabolic pathways that regulate activation of the extracellular LPS sensor TLR4, and (2) by modulating intracellular processing of ingested LPS to limit activation of the cytosolic LPS sensor Casp11 (CASP4). We will test these two non-exclusive mechanisms by which IRGM proteins dampen pro-inflammatory signaling in mouse and human macrophages in vitro, as well as define the roles of IRGM proteins in sepsis and antimicrobial inflammation in vivo. Aim1 will determine the impact of IRGM proteins on mitochondrial function, metabolite levels, glucose metabolism, and cytokine production in response to TLR4 activation. Causation between altered metabolism and/or mitochondrial function and altered cytokine expression will be established through pharmacological and genetic interventions. Aim2 will explore the role of IRGM proteins in the processing and sensing of cytoplasmic LPS and define the mechanism(s) by which IRGM deficiencies lead to LPS-triggered Casp11 hyperactivation. Aim3 will determine how changes in IRGM protein function impact TLR4- and /or Casp11-dependent sepsis in vivo. Instructed by our finding that IRGM insufficiency leads to excessive inflammatory cytokine production to LPS in vivo, we will define the consequences of IRGM protein insufficiencies on morbidity and survival in clinically relevant sepsis mouse models. Newly generated conditional Irgm knockout models and a pan-Irgm knockout mouse will be used to determine cell-specific roles for Irgm proteins and the interdependent functions of Irgm isoforms. Collectively, our studies will not only provide a mechanistic understanding of IRGM proteins as negative regulators of LPS-activated proinflammatory signaling pathways but also define the relevance of these regulatory processes in determining the outcome of different sepsis subtypes.