Upon inflammasome assembly, caspase 1 cleaves IL-1 family precursors, such as IL-1β, into an active form. Caspase 1 also cleaves a cytosolic protein, gasdermin D; upon cleavage, gasdermin D oligomerizes and forms pores in the plasma membrane, ultimately resulting in cell death by pyroptosis and IL-1 release. The authors treated bone marrow-derived macrophages with lipopolysaccharide and nigericin to induce pyroptotic NLRP3 activation and, as expected, observed the extracellular release of IL-1β and cytoplasmic lactate dehydrogenase (LDH). Interestingly, glycine buffering of the extracellular medium prevented cell lysis and LDH release but did not prevent IL-1β release; however, this phenotype was not seen in gasdermin D-deficient macrophages. These data suggest that the presence of gasdermin D on intact macrophages facilitates the release of IL-1β.
Several microbial stimuli (a strain of Staphylococcus aureus that lacks O-acetyltransferase, the N-acetyl glucosamine fragment of bacterial peptidoglycan) and self-derived damage-associated molecular patterns (oxidized lipids derived from dead mammalian cells) have been previously shown to hyperactivate phagocytes, resulting in IL-1 release from living cells. Bone marrow-derived macrophages that were treated with these hyperactivating stimuli did not release LDH but did show evidence of pore formation (measured using a membrane-impermeable dye) and the extracellular release of IL-1β. Again, the release of IL-1β was dependent on the presence of gasdermin D. Immunofluorescence staining showed that bone marrow-derived macrophages treated with hyperactivating stimuli remained adherent, had evidence of inflammasome assembly, could undergo phagocytosis and maintained their mitochondrial membrane integrity. Together, these data suggest that multiple stimuli can induce inflammasome assembly and a state of hyperactivation in viable macrophages, which then release IL-1β in a manner dependent on gasdermin D.
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