Expression of Nlrp1b Inflammasome Components in Human Fibroblasts Confers Susceptibility to Anthrax Lethal Toxin

Abstract

Anthrax lethal toxin causes macrophages and dendritic cells from some mouse strains to undergo caspase-1-dependent cell death. Central to this process is the NOD-like receptor Nlrp1b (Nalp1b), which detects intoxication and then self-associates to form a complex, termed an inflammasome, that is capable of activating the procaspase-1 zymogen. The nature of the signal detected directly by Nlrp1b is not known, and the mechanisms of inflammasome assembly are poorly understood. Here, we demonstrate that transfection of human fibroblasts with plasmids encoding murine Nlrp1b and procaspase-1 was sufficient to confer susceptibility to lethal toxin-mediated death on the cells. As has been observed in murine macrophages, the enzymatic activities of lethal toxin and the proteasome were both required for activation of the Nlrp1b inflammasome and this activation led to prointerleukin-1␤ processing. Release of interleukin-1␤ from cells was not dependent on cell lysis, as its secretion was not affected by an osmoprotectant that prevented the appearance of lactate dehydrogenase in the culture medium. We generated constitutively active mutants of Nlrp1b by making amino-terminal deletions to the protein and observed that the ability to activate procaspase-1 was dependent on the CARD domain, which bound procaspase-1, and a region adjacent to the CARD domain that promoted self-association. Our results demonstrate that lethal toxin can activate Nlrp1b in a nonmyeloid cell line and are consistent with work that suggests that activation induces proximity of procaspase-1.

Identification of the Inflammasome Nlrp1b as the Candidate Gene Conferring Diabetes Risk at the Idd4.1 Locus in the Nonobese Diabetic Mouse

“…cDNA synthesis with 1 mg RNA was performed with a SuperScript VILO cDNA synthesis kit (Invitrogen). Nlrp1b was amplified with previously published forward primer 59-CGC GGA TCC TAT GGA AGA ATC CCC ACC CAA G-39 and the reverse primer 59-CGC CTC GAG TCA TGA TCC CAA AGA GAC CCC ACC TG-39 (19). A shorter fragment of the Nlrp1b cDNA fragment flanking exon 9 was amplified using the forward primer 59-GAC CTG TCC TCT CTC AGT GC-39 and the same reverse primer to better visualize the polymorphism between NOD and NOR alleles.…”

“…The procaspase-1 cDNA was amplified with forward primer 59-CGC GGA TTC TAT GGC TGA CAA GAT CCT GAG G-39 and reverse primer 59-CGC CTC GAG ATG TCC CGG GAA GAG GTA G-39 and the PCR product was digested with BamHI and XhoI and then ligated into pcDNA3-T7 (19).…”

“…Primer sequences previously described (19) were used to amplify the coding sequence region of the gene: forward primer, 59-CGC GGA TCC TAT GGA AGA ATC CCC ACC CAA G-39, reverse primer, 59-CGC CTC GAG TCA TGA TCC CAA AGA GAC CCC ACC TG-39.…”

“…PCR products were digested with BamHI and XhoI and ligated into pNTAP-A (Stratagene). Nlrp1b allele 3 (20) was cloned in two steps, as reported (19), using cDNA from TIB-47 cells (from AKR/J mice). The 59 fragment was amplified with forward primer 59-CGC GGA TCC TAT GGA AGA ATC CCC ACC CAA G-39 and reverse primer 59-CGC CTC GAG TCA TGA TCC CAA AGA GAC CCC ACC TG-39 and the product was digested with BamHI and XhoI and ligated into pNTAP-A.…”

“…The Nlrp1b protein contains several domains crucial to its activity (18,19). The NACHT and FIIND domains mediate oligomerization, resulting in proteolysis of procaspase-1 that is bound to the CARD domain (20,21).…”