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

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. 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.Anthrax is a zoonotic disease caused by the Gram-positive bacterium Bacillus anthracis. B. anthracis provokes a shock-like syndrome that can prove fatal to the host (1) and has recently gained notoriety as a potential bioterrorism agent. Anthrax pathogenicity relies on its ability to secrete three virulence proteins, which combine with each other to form two toxins. The protective antigen (PA) combines with the edema factor (EF) to form the edema toxin (2, 3). EF is an adenylate cyclase that causes edema of the infected tissue. The binary combination of PA with lethal factor (LF) gives rise to the most virulent factor, called lethal toxin (LT), responsible for the systemic symptoms and death of the infected animal. To escape the host immune response, LT impairs the host innate immunity by killing macrophages (4–6). The PA protein interacts with LF and binds to cell surface receptors, enabling endocytosis of the LT complex. In the acidic compartment, PA forms pores allowing the delivery of LF to the cytosol. LF is a zinc metalloprotease that was shown to cleave the N-terminal region of many MAP kinase kinases and to induce apoptosis of macrophages. LT also triggers pyroptosis through the formation of a caspase-1–activating platform, named “inflammasome” (6–8).Inflammasomes are multiprotein complexes of the innate immune response that control caspase-1 activity and pro–IL-1β and pro–IL-18 maturation. Most inflammasomes are composed of specific cytosolic pathogen recognition receptors (PRRs), as well as the apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CARD) (ASC) adaptor protein that enables the recruitment and activation of the caspase-1 protease. Once caspase-1 is oligomerized within an inflammasome platform, the enzyme undergoes autoproteolysis to form heterodimers of active caspase-1 (9–12). In the mouse, at least five distinct inflammasomes have been described, distinguished by the PRR that induces the complex formation. The PRRs capable of participating in inflammasome platform formation are either members of the nod-like receptor (NLR) family (e.g., NLRP1, NLRP3, or NLRC4) or of the PYrin and HIN (PYHIN) family (e.g., AIM2) (13, 14). ASC is composed of a pyrin domain (PYD) and a caspase activation and recruitment domain (CARD). ASC interacts with a PYD-containing PRR via its PYD domain and recruits the CARD domain of caspase-1 via its CARD domain. Thus, ASC is essential to the formation of the inflammasome by receptors such as NLRP3 or AIM2 (15–18). However, its presence is dispensable for NLRC4, which contains a CARD in place of a PYD, allowing direct interaction with the CARD domain of caspase-1 (19, 20).Past studies have determined that certain mouse strains are more sensitive than others to LT cytotoxicity, and genetic studies identified NLRP1b as the factor conferring mouse strain susceptibility to anthrax LT (21). The mouse genome contains three different NLRP1 isoforms (a, b, and c) and a functional NLRP1b was found to be expressed by the mouse strains sensitive to LT (e.g., BALB/c or 129 background). Expression of NLRP1b was shown to mediate IL-1β release and caspase-1–mediated cell death in response to LT (7, 21, 22). Mouse NLRP1b differs structurally from human NLRP1 in that it lacks the N-terminal PYD (23). The absence of the PYD suggests that NLRP1b can directly engage caspase-1 without a requirement for ASC. However, studies dissecting the mechanism of NLRC4 inflammasome activation demonstrated that ASC is required for the amplification of caspase-1 autoprocessing and IL-1β secretion but not for pyroptosis (19, 20). Cell lysis mediated by LT was shown to be dependent on sodium and potassium fluxes (24), and high extracellular potassium inhibited IL-1β secretion upon LT treatment, suggesting a role for the NLRP3 inflammasome in LT sensing (22, 25). Therefore, we investigated whether NLRP3 and/or ASC were required for caspase-1 activation in response to LT. The NLRP3, ASC, and caspase-1 mouse knockout strains were backcrossed into the BALB/c background and the response of macrophages and dendritic cells (DCs) to LT intoxication was studied. Our data reveal that (i) in response to LT, ASC is dispensable for caspase-1 activation, but uncleavable caspase-1 is fully active; and (ii) upon activation of the NLRP3 inflammasome, uncleavable caspase-1 is inactive.