ABSTRACT Innate immunity provides the first line of host defense in response to invading pathogens. Pattern recognition receptors (PRRs) sense pathogen-associated molecular patterns (PAMPs) in viruses and other pathogens. RIG-I/MDA-5 are DExD/H box RNA helicases in the RIG-I-like receptor (RLR) pathway that detect double stranded RNA viral genomes and activate the mitochondrial adaptor protein MAVS. The DNA sensor cGAS detects double stranded DNA and activates STING, a transmembrane endoplasmic reticulum adaptor. Both MAVS and STING activate canonical (IKKa and IKKb), and noncanonical IKK kinases (TBK1 and IKKi) to activate transcription factors NF-kB and IRF3 respectively. Together, IRF3 and NF-kB regulate the expression of type I interferon (IFN) and inflammatory genes that coordinate the innate response and initiate the adaptive immune response against pathogens. The NLRP3 inflammasome also plays a critical role in inflammatory responses by triggering caspase-1-mediated pro-IL-1b cleavage to yield the biologically active form of IL- 1b that drives inflammation and adaptive immunity. NLRP3 also induces a highly lytic form of inflammatory cell death termed pyroptosis via cleavage of gasdermin-D to form plasma membrane pores. The RLR, cGAS- STING and NLRP3 inflammasome pathways are potent inducers of inflammation that must be tightly regulated to avert overexuberant inflammation and tissue damage. TAX1BP1 was first identified as an anti-apoptotic protein that interacts with the zinc finger deubiquitinase A20/TNFAIP3. Our previous work has established that TAX1BP1 restricts cytokine-induced NF-kB activation as well as RLR-induced type I IFN production and apoptosis. TAX1BP1 functions as a selective autophagy receptor by recruiting ubiquitinated cargo to developing autophagosomes via two LC3 interaction regions (LIRs). However, it remains unclear how TAX1BP1 autophagy function is regulated and if TAX1BP1 inhibits other innate immune signaling pathways. In preliminary studies we provide experimental evidence that TAX1BP1 is phosphorylated by both noncanonical and canonical IKK kinases which controls both basal and virus-induced TAX1BP1 autophagic degradation respectively. Using TAX1BP1-deficient macrophages we have demonstrated that TAX1BP1 is a novel inhibitor of both cGAS-STING and NLRP3 pathways. Furthermore, MAVS protein aggregates accumulate in TAX1BP1- deficient cells suggesting a potential aggrephagy function in the regulation of innate immune signaling. The central hypothesis driving the proposed investigations is that TAX1BP1 inhibits RLR, cGAS-STING and NLRP3 pathways by autophagy-mediated clearance of signaling protein aggregates. We will test this hypothesis experimentally with the following Specific Aims: (1) determine the role of TAX1BP1 phosphorylation in its autophagy function, (2) determine the mechanisms of TAX1BP1 inhibition of the cGAS-STING pathway, and (3) determine the mechanisms of TAX1BP1 inhibition of the NLRP3 pathway. Completion of the proposed studies will provide new insights into innate immune regulation and immune homeostasis.

NARRATIVE Innate immune signaling pathways including the RIG-I-like receptor (RLR), cGAS-STING and NLRP3 inflammasome pathways play crucial roles in the host response to pathogenic microbes. These pathways are potent inducers of inflammation when triggered and must be tightly regulated to avoid overexuberant inflammation and tissue damage. This proposal will investigate the mechanisms by which the selective autophagy receptor TAX1BP1 regulates innate immune signaling and immune homeostasis.