Innate immune recognition of bacterial ligands by NAIPs determines inflammasome specificity

Inflammasomes are a family of cytosolic multiprotein complexes that initiate innate immune responses to pathogenic microbes by activating the caspase 1 protease. Although genetic data support a critical role for inflammasomes in immune defence and inflammatory diseases, the molecular basis by which individual inflammasomes respond to specific stimuli remains poorly understood. The inflammasome that contains the NLRC4 (NLR family, CARD domain containing 4) protein was previously shown to be activated in response to two distinct bacterial proteins, flagellin and PrgJ, a conserved component of pathogen-associated type III secretion systems. However, direct binding between NLRC4 and flagellin or PrgJ has never been demonstrated. A homologue of NLRC4, NAIP5 (NLR family, apoptosis inhibitory protein 5), has been implicated in activation of NLRC4 (refs 7–11), but is widely assumed to have only an auxiliary role, as NAIP5 is often dispensable for NLRC4 activation. However, Naip5 is amember of a small multigene family, raising the possibility of redundancy and functional specialization among Naip genes. Here we show inmice that different NAIP paralogues determine the specificity of the NLRC4 inflammasome for distinct bacterial ligands. In particular, we found that activation of endogenous NLRC4 by bacterial PrgJ requires NAIP2, a previously uncharacterized member of the NAIP gene family, whereas NAIP5 and NAIP6 activate NLRC4 specifically in response to bacterial flagellin. We dissected the biochemical mechanism underlying the requirement for NAIP proteins by use of a reconstituted NLRC4 inflammasome system. We found that NAIP proteins control ligand-dependent oligomerization of NLRC4 and that the NAIP2–NLRC4 complex physically associates with PrgJ but not flagellin, whereas NAIP5–NLRC4 associates with flagellin but not PrgJ. Our results identify NAIPs as immune sensor proteins and provide biochemical evidence for a simple receptor–ligand model for activation of the NAIP–NLRC4 inflammasomes. A fundamental question in immunology is how host defence is initiated in response to specific microbial ligands. The inflammasome containing theNLRC4protein activates caspase 1 (CASP1) in response to the carboxy terminus of bacterial flagellin, as well as in response to the inner rod protein of the type III secretion systems of diverse bacterial species (for example, PrgJ of Salmonella enterica serovar Typhimurium). Activated CASP1 processes interleukin (IL)-1b and IL-18 inflammatory cytokines and induces a rapid and inflammatory host cell death called pyroptosis. In certain cases, NLRC4 activation requires NAIP5, asNaip5mice fail to activate NLRC4 or CASP1 in response to infectionwithLegionella pneumophilaor in response to the C terminus of flagellin. Interestingly, however,NAIP5 is not essential for NLRC4 activation in response to S. enterica Typhimurium or PrgJ. In addition to Naip5, C57BL/6 mice express three otherNaip genes (Naip1, Naip2 and Naip6), the functions of which remain unknown. We hypothesized that each NAIP paralogue may have evolved to be specific for a unique bacterial ligand. We first focused on NAIP2, as it appeared to be highly expressed in C57BL/6 mice. We used specific short hairpin RNAs (shRNAs) to knock down Naip2 expression in primary bone-marrow-derived macrophages. ShRNA1 and shRNA2 specifically reduced NAIP2 protein levels without targeting other NAIP paralogues, whereas empty vector, shRNA3 or a scrambled control shRNA had little effect on NAIP2 protein levels (Supplementary Fig. 1a, b). Macrophages expressing these shRNAs were then infected with flagellin-deficient Listeria strains that inducibly express PrgJ (Listeria-PrgJ) or flagellin (Listeria-FlaA).AListeria-based system was chosen because it is an efficientmeans for delivering PrgJ tomacrophages, and because it allows for controlled comparisons of PrgJ and FlaAwithin a single experimental system.Notably, knockdownofNaip2 prevented pyroptosis and CASP1 activation by Listeria-PrgJ (Fig. 1a–c). By contrast, Naip2 knockdown did not affect inflammasome activation by Listeria-FlaA (Fig. 1b, c) or L. pneumophila, which expresses flagellin but not PrgJ (Supplementary Fig. 1c). Instead, flagellin-dependent inflammasome activation depended on Naip5, as previously shown. Inflammasome activation by wild-type Salmonella, which encodes both flagellin and PrgJ, was not significantly affected by Naip2 knockdown (Fig. 1d, e). However, knockdown of Naip2 in Naip5 macrophages significantly reduced or abolished inflammasome activation by wildtype Salmonella (Fig. 1d, e), indicating that both NAIP2 and NAIP5 recognize Salmonella. Interestingly, inflammasome activation by flagellin-deficient (FliCFljB) Salmonella, which still express PrgJ, depended entirely on Naip2 (Fig. 1d, e). Taken together, these data indicate thatNaip2 is specifically required for activation of the NLRC4 inflammasome by PrgJ, in contrast to Naip5, which seems to be specifically required for NLRC4 activation by flagellin. Biochemical analysis of the inflammasome in macrophages is complicated by the expression of multiple NAIP proteins and by their low expression levels. We therefore decided to reconstitute the NLRC4 inflammasome in non-immune 293T cells, which do not express NLRC4 or NAIPs, so that the functions of individual NAIP proteins could be analysed. 293T cells transiently transfected with green fluorescent protein (GFP)-marked vectors encoding wild-type NLRC4, NAIP5 and CASP1 did not exhibit significant spontaneous inflammasome activation, and instead, most cells expressed GFP (Fig. 2a). However, when flagellin (FlaA) from L. pneumophilawas co-expressed with NLRC4, NAIP5 and CASP1, we observed a significant loss of GFP cells and an increase in the number of dead (7AAD) cells (Fig. 2a). This result was highly reminiscent of flagellin-dependent activationof the endogenousNAIP5–NLRC4 inflammasome inmacrophages, which also results in a rapid CASP1-dependent cell death, loss of membrane integrity, and release of cytosolic contents and GFP. Similar to the genetic requirement forNlrc4,Naip5 andCasp1 inmacrophages, we found that NAIP5, NLRC4, catalytically active CASP1, and FlaA are all required to trigger cell death and loss of membrane integrity/GFP in reconstituted 293T cells (Fig. 2b, c). The reconstituted NAIP5–NLRC4 inflammasome also recapitulated the ability of native inflammasomes to process CASP1 and IL-1b in response to cytosolic flagellin (Supplementary Fig. 2). Consistent with a lack of a role forNAIP5 in recognitionofPrgJbymacrophages, the reconstituted NAIP5–NLRC4 inflammasome did not respond to PrgJ (Fig. 2d, e). By