Cytosolic RECONnaissance: Host Strategies for Sensing and Controlling Bacteria
McFarland, Adelle Pauline
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Cyclic dinucleotides (CDNs) of bacterial and host origin mediate cytosolic immune responses through the stimulator of interferon genes (STING) signaling pathway, though evidence suggests alternative receptors exist. We identified the oxidoreductase RECON as a high-affinity host receptor specific for bacterial CDNs. RECON’s enzyme activity was inhibited by bacterial CDNs, which bound in the substrate and cosubstrate sites. During bacterial infection of macrophages, RECON antagonized STING activation by acting as a molecular sink for CDNs. In hepatocytes, RECON negatively regulated NF-kappaB activation via its enzymatic activity. Loss of RECON resulted in increased NF-kappaB activation and reduced bacterial survival. Therefore, CDN inhibition of RECON promotes a proinflammatory, antibacterial state. We investigated the molecular pathways by which RECON controls inflammation and found that several branches of the arachidonic acid inflammatory cascade were involved. At the top of the cascade, free arachidonic acid levels were significantly elevated in the absence of RECON, which correlated with the formation of large lipid droplets. Genetic deletion of the cytosolic phospholipase A2, which liberates arachidonic acid from phospholipids, in RECON-deficient cells restored the inflammatory response to WT levels. The reactive lipid aldehyde 4-hydroxynonenal (4-HNE), which is a byproduct of arachidonic acid peroxidation, was elevated in RECON-deficient cells following TLR stimulation. 4-HNE was determined to be a bonafide RECON substrate that specifically enhanced Nos2 expression. Interestingly, a separate branch of the arachidonic acid cascade was found to govern Il6 expression distinct from the pathway described for Nos2. Dysregulated COX-mediated prostaglandin synthesis was discovered in RECON-deficient cells by comprehensive eicosanoid profiling. Accordingly, inhibition of COX-2 activity reduced Il6 but not Nos2 expression. Finally, although RECON controlled Nos2 via augmented NF-kappaB activation, we found that the MEK-ERK pathway was critical for Il6 expression. Overall, RECON’s targeting of inflammatory arachidonic acid oxidation metabolites provides a molecular explanation for how this enzyme operates as a regulator of multiple gene induction programs. In addition to its role as a regulator of innate immune gene activation, we also characterized the effects of RECON on the infection cycle of the intracellular bacterium Listeria monocytogenes, which secretes cyclic diadenosine monophosphate (c-di-AMP) into the cytosol of infected host cells. Remarkably, L. monocytogenes exhibited significantly enhanced cell-to-cell spread in RECON-deficient hepatocytes. L. monocytogenes actin tail lengths were significantly longer and there was a larger number of faster moving bacteria in the absence of RECON. Complementation experiments demonstrated that the effect of RECON on L. monocytogenes spread and actin tail lengths was linked to its enzymatic activity. Augmented NF-kappaB activation in the absence of RECON was responsible for the enhanced L. monocytogenes cell-to-cell spread. Finally, we found that increased NF-kappaB-dependent iNOS expression and nitric oxide production was sufficient to drive L. monocytogenes spread. This work revealed a novel host-pathogen interaction, whereby L. monocytogenes secretion of c-di-AMP inhibits RECON, drives augmented NF-kappaB activation and nitric oxide production, and enhances intercellular spread. To gain insights into how RECON functions in vivo, we generated mice deficient in RECON. Following systemic challenge L. monocytogenes, RECON-deficient mice exhibited increased survival and reduced bacterial loads. Apart from its role in infection, we found that RECON deficiency promoted aberrant low-level inflammation in the small intestines. The absence of RECON was associated with alterations in the microbiota, with aberrant expansion of segmented filamentous bacteria. These findings demonstrate that RECON functions in intestinal homeostasis and that the inflammatory programs under its control are protective during systemic bacterial infection. Collectively, the work presented in this dissertation establishes RECON as an innate immune sensor and modulator of cell-intrinsic metabolites that promote antibacterial immunity.