Anthrax Lethal Toxin Triggers the Formation of a Membrane-Associated Inflammasome Complex in Murine Macrophages

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Abstract
Multiple microbial components trigger the formation of an inflammasome complex that contains pathogen-specific nucleotide oligomerization and binding domain (NOD)-like receptors (NLRs), caspase-1, and in some cases the scaffolding protein ASC. The NLR protein Nalp1b has been linked to anthrax lethal toxin (LT)-mediated cytolysis of murine macrophages. Here we demonstrate that in unstimulated J774A.1 macrophages, caspase-1 and Nalp1b are membrane associated and part of approximately 200- and approximately 800-kDa complexes, respectively. LT treatment of these cells resulted in caspase-1 recruitment to the Nalp1b-containing complex, concurrent with processing of cytosolic caspase-1 substrates. We further demonstrated that Nalp1b and caspase-1 are able to interact with each other. Intriguingly, both caspase-1 and Nalp1b were membrane associated, while the caspase-1 substrate interleukin-18 was cytosolic. Caspase-1-associated inflammasome components included, besides Nalp1b, proinflammatory caspase-11 and the caspase-1 substrate alpha-enolase. Asc was not part of the Nalp1b inflammasome in LT-treated macrophages. Taken together, our findings suggest that LT triggers the formation of a membrane-associated inflammasome complex in murine macrophages, resulting in cleavage of cytosolic caspase-1 substrates and cell death.