Control of intracellular Shigella in human macrophages. - Macrophages are one of the first immune responders to bacterial infections. Macrophages recruit neutrophils that phagocytose and clear bacteria and infected cells. In addition, macrophages internalize and kill bacteria intracellularly. Inflammasomes, cytosolic innate signaling complexes that induce inflammation and pyroptosis, mediate macrophage-specific restriction as shown for some intracellular bacteria. Although macrophage restriction of at least some bacteria depends on inflammasomes, the specific mechanisms of macrophage bacterial killing are incompletely understood. I found that the NLRP3 inflammasome restricts Shigella flexneri (Shigella spp. are the leading cause of diarrheal disease and dysentery in the world) in the cytosol of human macrophages and that macrophage restriction is enhanced, independently of NLRP3, by priming with IFNγ. I found that this restriction is independent of macrophage cell death and thus is executed by a cytosolic mechanism. I also found that CASP1 and GSDMD, and in IFNγ-primed macrophages also CASP4, play a role in controlling S. flexneri burden. I propose to define the mechanisms of macrophage-specific killing of the intracellular bacterial pathogen S. flexneri. Macrophage responses to S. flexneri are mediated, at least in part, by recognition of lipopolysaccharide (LPS) – the glycolipid present in the outer membrane of Shigella spp. and other gram-negative bacteria. Upon recognition in the cytosol of infected macrophages, LPS activates the caspase-4/5 (CASP4/5, human) or CASP11 (mouse) inflammasome. LPS contains lipid A, a disaccharide of glucosamine linked to six acyl chains. Bacteria adapt to the intracellular environment in part by hypo-acylating their lipid A. Recognition of hypo-acylated LPS differs between mice and humans: CASP4 is activated by hexa- and hypo-acylated LPS, whereas CASP11 is activated only by hexa-acylated LPS. Our lab identified NLRP11 as a primate-specific pattern recognition receptor of cytosolic LPS. I will test my hypothesis that NLRP11 contributes to the observed species differences in inflammasome pathway recognition of bacterial hypo- acylated LPS, further enhancing our understanding of host-pathogen interactions in control of intracellular S. flexneri. To achieve my goals, I propose the following approaches: 1. Define the mechanism of IFNγ-mediated control of intracellular S. flexneri in human macrophages; 2. Test whether S. flexneri hypo-acylation of LPS alters bacterial recognition and survival in human macrophages. These studies will provide valuable insights into human macrophage innate immunity mechanisms against S. flexneri and host-pathogen interactions more generally. These findings will have broad implications in gram- negative pathogenesis and thus be of great interest to innate immunologists and microbiologists. The proposed training will provide me with new expertise in bacterial pathogenesis and innate immunity to bacterial pathogens and place me in an optimal position to become an independent NIH-funded investigator.
