Regulation of Caspase-1-associated Activities in Macrophages During Legionella pneumophila Infection

Pathogens trigger a large repertoire of innate immune signaling pathways upon detection of their products or virulence-associated activities in host cell cytosols. One major arm of the innate immune response involves activation of the cysteine protease caspase-1. Caspase-1 is a critical factor for cleavage and secretion of the proinflammatory cytokines IL-1beta and IL-18, as well as induction of a specialized form of cell death termed pyroptosis. Regulation of caspase-1 activation occurs through upstream sensor proteins and adaptors, including the nucleotide-binding domain, leucine-rich repeat containing proteins (NLRs), absent in melanoma 2 (AIM2) and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). These proteins regulate formation of specialized complexes called inflammasomes which promote caspase-1 activation. The facultative intracellular bacterial pathogen Legionella pneumophila activates caspase-1 in mouse macrophages through mechanisms requiring the host NLRs NLRC4 and NAIP5, and the adaptor protein ASC. NLRC4 and NAIP5 function epistatically in the caspase-1 activation pathway involved in detection of bacterial flagellin during L. pneumophila infection. Defects in this pathway result in impaired caspase-1 activation and deficiencies in IL-1beta/IL-18 production and cell death. Furthermore, absence of this pathway enables intracellular growth of L. pneumophila, which is normally restricted in wild-type macrophages. Interestingly, ASC is required for efficient caspase-1 activation and IL-1beta/IL-18 secretion, but is dispensable for restriction of L. pneumophila growth. These data suggest that ASC may direct a NAIP5/NLRC4/flagellin-independent pathway leading to caspase-1 activation, and that caspase-1-associated activities may be regulated by upstream components in order to promote distinct downstream consequences. To address these possibilities, I have examined the role of ASC more closely during caspase-1 activation in response to L. pneumophila infection in macrophages in order to identify whether alternate pathways exist. In addition, I have investigated the spatial organization of components involved in caspase-1 activation, as well as the temporal characteristics of activation in order to better understand the differential regulation of caspase-1-associated activities. I have determined that L. pneumophila induces caspase-1 activation through multiple pathways, each resulting in formation of caspase-1 activation complexes requiring the adaptor protein ASC. These complexes are required for efficient processing of caspase-1, and are formed through NLRC4-dependent and -independent mechanisms. In addition, I have shown that NLRC4/flagellin-mediated signaling results in activation of caspase-1 in the absence of ASC. ASC-independent activation of caspase-1 occurs without efficient cleavage of caspase-1, but is sufficient for pore formation and induction of cell death in infected cells. Finally, I showed that NLRC4/flagellin-independent activation of caspase-1 occurs through a caspase-11 dependent mechanism. I also demonstrated that L. pneumophila induces a caspase-11-dependent cell death pathway, independent of NLRC4, flagellin and caspase-1. Together, these studies have revealed multiple mechanisms involved in the innate immune response to L. pneumophila which are important for proinflammatory cytokine production and cell death in macrophages. Furthermore, this work has contributed to the overall understanding of caspase-1-activation and the regulation of caspase-1-associated activities.