Anthrax lethal factor (LF) is the protease component of anthrax lethal toxin (LT). LT induces pyroptosis in macrophages of certain inbred mouse and rat strains, while macrophages from other inbred strains are resistant to the toxin. In rats, the sensitivity of macrophages to toxin-induced cell death is determined by the presence of an LF cleavage sequence in the inflammasome sensor Nlrp1. LF cleaves rat Nlrp1 of toxin-sensitive macrophages, activating caspase-1 and inducing cell death. Toxin-resistant macrophages, however, express Nlrp1 proteins which do not harbor the LF cleavage site. We report here that mouse Nlrp1b proteins are also cleaved by LF. In contrast to the situation in rats, sensitivity and resistance of Balb/cJ and NOD/LtJ macrophages does not correlate to the susceptibility of their Nlrp1b proteins to cleavage by LF, as both proteins are cleaved. Two LF cleavage sites, at residues 38 and 44, were identified in mouse Nlrp1b. Our results suggest that the resistance of NOD/LtJ macrophages to LT, and the inability of the Nlrp1b protein expressed in these cells to be activated by the toxin are likely due to polymorphisms other than those at the LF cleavage sites.

The inflammasomes are intracellular complexes that have an important role in cytosolic innate immune sensing and pathogen defense. Inflammasome sensors detect a diversity of intracellular microbial ligands and endogenous danger signals and activate caspase-1, thus initiating maturation and release of the proinflammatory cytokines interleukin-1β and interleukin-18. These events, although crucial to the innate immune response, have also been linked to the pathology of several inflammatory and autoimmune disorders. The natural isothiocyanate sulforaphane, present in broccoli sprouts and available as a dietary supplement, has gained attention for its antioxidant, anti-inflammatory, and chemopreventive properties. We discovered that sulforaphane inhibits caspase-1 autoproteolytic activation and interleukin-1β maturation and secretion downstream of the nucleotide-binding oligomerization domain-like receptor leucine-rich repeat proteins NLRP1 and NLRP3, NLR family apoptosis inhibitory protein 5/NLR family caspase-1 recruitment domain-containing protein 4 (NAIP5/NLRC4), and absent in melanoma 2 (AIM2) inflammasome receptors. Sulforaphane does not inhibit the inflammasome by direct modification of active caspase-1 and its mechanism is not dependent on protein degradation by the proteasome or de novo protein synthesis. Furthermore, sulforaphane-mediated inhibition of the inflammasomes is independent of the transcription factor nuclear factor erythroid-derived 2-like factor 2 (Nrf2) and the antioxidant response-element pathway, to which many of the antioxidant and anti-inflammatory effects of sulforaphane have been attributed. Sulforaphane was also found to inhibit cell recruitment to the peritoneum and interleukin-1β secretion in an in vivo peritonitis model of acute gout and to reverse NLRP1-mediated murine resistance to Bacillus anthracis spore infection. These findings demonstrate that sulforaphane inhibits the inflammasomes through a novel mechanism and contributes to our understanding of the beneficial effects of sulforaphane.

Lysosomal cathepsin B (CTSB) has been proposed to play a role in the induction of acute inflammation. We hypothesised that the presence of active CTSB in the cytosol is crucial for NLRP3-inflammasome assembly and, consequently, for mature IL-1β generation after mycobacterial infection in vitro. Elevated levels of CTSB was observed in the lungs of mice and rabbits following infection with Mycobacterium tuberculosis (Mtb) H37Rv as well as in plasma from acute tuberculosis patients. H37Rv-infected murine bone marrow-derived macrophages (BMDMs) displayed both lysosomal leakage, with release of CTSB into the cytosol, as well as increased levels of mature IL-1β. These responses were diminished in BMDM infected with a mutant H37Rv deficient in ESAT-6 expression. Pharmacological inhibition of cathepsin activity with CA074-Me resulted in a substantial reduction of both mature IL-1β production and caspase-1 activation in infected macrophages. Moreover, cathepsin inhibition abolished the interaction between NLRP3 and ASC, measured by immunofluorescence imaging in H37Rv-infected macrophages, demonstrating a critical role of the enzyme in NLRP3-inflammasome activation. These observations suggest that during Mtb infection, lysosomal release of activated CTSB and possibly other cathepsins inhibitable by CA07-Me is critical for the induction of inflammasome-mediated IL-1β processing by regulating NLRP3-inflammasome assembly in the cytosol.