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. 2014 Dec 2;111(48):17254-9.
doi: 10.1073/pnas.1415756111. Epub 2014 Nov 17.

Caspase-1 autoproteolysis is differentially required for NLRP1b and NLRP3 inflammasome function

Baptiste Guey  1 , Mélanie Bodnar  1 , Serge N Manié  1 , Aubry Tardivel  2 , Virginie Petrilli  3
Affiliations

Caspase-1 autoproteolysis is differentially required for NLRP1b and NLRP3 inflammasome function

Baptiste Guey et al. Proc Natl Acad Sci U S A. .

Abstract

Inflammasomes are caspase-1-activating multiprotein complexes. The mouse nucleotide-binding domain and leucine rich repeat pyrin containing 1b (NLRP1b) inflammasome was identified as the sensor of Bacillus anthracis lethal toxin (LT) in mouse macrophages from sensitive strains such as BALB/c. Upon exposure to LT, the NLRP1b inflammasome activates caspase-1 to produce mature IL-1β and induce pyroptosis. Both processes are believed to depend on autoproteolysed caspase-1. In contrast to human NLRP1, mouse NLRP1b lacks an N-terminal pyrin domain (PYD), indicating that the assembly of the NLRP1b inflammasome does not require the adaptor apoptosis-associated speck-like protein containing a CARD (ASC). LT-induced NLRP1b inflammasome activation was shown to be impaired upon inhibition of potassium efflux, which is known to play a major role in NLRP3 inflammasome formation and ASC dimerization. We investigated whether NLRP3 and/or ASC were required for caspase-1 activation upon LT stimulation in the BALB/c background. The NLRP1b inflammasome activation was assessed in both macrophages and dendritic cells lacking either ASC or NLRP3. Upon LT treatment, the absence of NLRP3 did not alter the NLRP1b inflammasome activity VSports手机版. Surprisingly, the absence of ASC resulted in IL-1β cleavage and pyroptosis, despite the absence of caspase-1 autoprocessing activity. By reconstituting caspase-1/caspase-11(-/-) cells with a noncleavable or catalytically inactive mutant version of caspase-1, we directly demonstrated that noncleavable caspase-1 is fully active in response to the NLRP1b activator LT, whereas it is nonfunctional in response to the NLRP3 activator nigericin. Taken together, these results establish variable requirements for caspase-1 cleavage depending on the pathogen and the responding NLR. .

Keywords: dendritic cell; interleukin-1beta; lethal toxin; macrophage; pyroptosis V体育安卓版. .

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Conflict of interest statement

The authors declare no conflict of interest.

V体育ios版 - Figures

Fig. 1.
Fig. 1.
LT induces IL-1β secretion but not caspase-1 autoprocessing in ASC-deficient macrophages and BMDCs. Nonprimed or LPS-primed mouse peritoneal macrophages (A) or BMDMs (B) or BMDCs (C) of indicated genotypes were treated with B. anthracis lethal toxin (LT, 0.5 μg/mL, 6 h) or nigericin (Nig, 10 μM, 2 h) or left untreated (−); caspase-1 and IL-1β cleavage, and ASC and NLRP3 protein levels were assessed by Western blot. Actin was used as a loading control. These results were replicated three times independently.
Fig. 2.
Fig. 2.
ASC and NLRP3 are dispensable for LT-induced cell death. LPS-primed mouse peritoneal macrophages (A) or BMDMs (B) or BMDCs (C) of indicated genotypes were treated with LT (0.5 μg/mL) for 6 h. Cell death was measured by cytotoxic assay (lactate dehydrogenase, LDH release) upon LT treatment over a time course of 6 h. Quantification by flow cytometry of peritoneal macrophages and neutrophils in the peritoneum of WT and ASC−/− mice (Left) or WT and caspase-1/caspase-11−/− mice (Right) injected with PBS or LT for 3 h is shown (D). Data represent mean ± SEM *P < 0.01 (Mann–Whitney test) (N.S., nonsignificant).
Fig. 3.
Fig. 3.
LT-induced cell death and IL-1β secretion rely on caspase-1 catalytic activity in ASC-deficient cells. LPS-primed mouse peritoneal macrophages were treated or not (−, NT) with LT (0.5 μg/mL) in the presence or absence of the pan-caspase inhibitor z-VAD-fmk (50 μM) or caspase-1 inhibitor z-YVAD-fmk (50 μM) for 6 h. Protein expression was assessed by Western blot (A) and cell death was determined by measuring the LDH released in cell supernatants (B). Peritoneal macrophages were isolated from WT or ASC−/− mice.
Fig. 4.
Fig. 4.
Caspase-1 autoproteolysis is not required to induce cell death nor to induce IL-1β cleavage and secretion upon LT treatment. LPS-primed BMDMs (A and B) or BMDCs (C and D) from caspase-1/caspase-11−/− mice, transduced or not (−) with the indicated caspase-1 constructs were treated with LT (0.5 μg/mL) for 6 h or with nigericin (Nig, 10 μM) for 2 h or left untreated (−). Protein expression was assessed by Western blot (A and C), LDH release was measured in cell supernatants of LT-treated cells (B and D).
Fig. 5.
Fig. 5.
LT induces ASC speck formation in a caspase-1–independent manner. LPS-primed peritoneal macrophages were treated with LT (0.5 μg/mL) or not (−) for 2 h. Cells were then fixed in 2% (wt/vol) paraformaldehyde and immunostained for ASC (green, A) or caspase-1 (red, B). Confocal microscopy sequential imaging of ASC immunostaining (green) and caspase-1 immunostaining (red) was performed on LPS-primed macrophages treated or not (−) with LT (0.5 μg/mL) (C). Hoechst (blue) was used to stain nuclei.
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