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Inflammasomes are a family of cytosolic multiprotein complexes that initiate innate immune responses to pathogenic microbes by activating the CASPASE1 (CASP1) protease1,2. Although genetic data support a critical role for inflammasomes in immune defense and inflammatory diseases3, 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 C4) protein was previously shown to be activated in response to two distinct bacterial proteins, flagellin4,5 and PrgJ6, a conserved component of pathogen-associated type III secretion systems. However, direct binding between NLRC4 and flagellin or PrgJ has never been demonstrated. A homolog of NLRC4, NAIP5 (NLR family, Apoptosis Inhibitory Protein 5), has been implicated in activation of NLRC47–11, but is widely assumed to play only an auxiliary role1,2, since NAIP5 is often dispensable for NLRC4 activation7,8. However, Naip5 is a member of a small multigene family12, raising the possibility of redundancy and functional specialization among Naip genes. Indeed, we show here that different NAIP paralogs dictate 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 NAIP2/NLRC4 physically associates with PrgJ but not flagellin, whereas NAIP5/NLRC4 associates with flagellin but not PrgJ. Taken together, 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 defense is initiated in response to specific microbial ligands. The inflammasome containing the NLRC4 protein activates CASP1 in response to the C-terminus of bacterial flagellin6,7, as well as in response to the inner rod protein of the type III secretion systems of diverse bacterial species (e.g., PrgJ of Salmonella Typhimurium)6. Activated CASP1 processes interleukin-1β and −18 inflammatory cytokines and induces a rapid and inflammatory host cell death called pyroptosis13. In certain cases, NLRC4 activation requires NAIP5, as Naip5−/− mice fail to activate NLRC4 or CASP1 in response to infection with Legionella pneumophila or in response to the C-terminus of flagellin7,8. Interestingly, however, NAIP5 is not essential for NLRC4 activation in response to Salmonella Typhimurium or PrgJ7,8.
In addition to Naip5, C57BL/6 mice express three additional Naip genes (Naip1, Naip2, and Naip6), the functions of which remain unknown12. We hypothesized that each NAIP paralog 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 mice14. We used specific shRNAs to knock down Naip2 expression in primary bone marrow-derived macrophages. ShRNA#1 and #2 specifically reduced NAIP2 protein levels without targeting other NAIP paralogs, whereas empty vector, shRNA#3 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)8. A Listeria-based system was chosen because it is an efficient means for delivering PrgJ to macrophages8, and because it allows for controlled comparisons of PrgJ and FlaA within a single experimental system. Remarkably, knockdown of Naip2 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 shown7–11. 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 wild-type Salmonella (Fig. 1d, e), indicating that both NAIP2 and NAIP5 recognize Salmonella. Interestingly, inflammasome activation by flagellin-deficient (FliC/FljB−) Salmonella, which still express PrgJ, depended entirely on Naip2 (Fig. 1d, e). Taken together, these data indicate that Naip2 is specifically required for activation of the NLRC4 inflammasome by PrgJ, in contrast to Naip5, which appears 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 analyzed. 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, a majority of cells expressed GFP (Fig. 2a). However, when flagellin (FlaA) from L. pneumophila was co-expressed with NLRC4/NAIP5/CASP1, we observed a significant loss of GFPhigh cells and an increase in the number of dead (7AAD+) cells (Fig. 2a). This result was highly reminiscent of flagellin-dependent activation of the endogenous NAIP5/NLRC4 inflammasome in macrophages, which also results in a rapid CASP1-dependent cell death, loss of membrane integrity, and release of cytosolic contents and GFP7. Similar to the genetic requirement for Nlrc4, Naip5 and Casp1 in macrophages4,5,7–11,15, 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-1β in response to cytosolic flagellin (Supplementary Fig. 2). Consistent with a lack of a role for NAIP5 in recognition of PrgJ by macrophages8, the reconstituted NAIP5/NLRC4 inflammasome did not respond to PrgJ (Fig. 2d, e). By contrast, a reconstituted NAIP2/NLRC4 inflammasome responded specifically to PrgJ but not flagellin (Fig. 2d, e). Taken together, we conclude that we have successfully reconstituted NAIP2/NLRC4 and NAIP5/NLRC4 inflammasomes that exhibit all the known requirements and specificities of the native inflammasomes.
It is believed that activated inflammasomes assemble into high molecular-weight multiprotein complexes16, but this has not been demonstrated for the NLRC4 inflammasome. To visualize inflammasome assembly, 293T cells were transfected with NAIP5, NLRC4, and FlaA in various combinations, but CASP1 was omitted so that cell death and loss of cellular contents (and assembled inflammasomes) would not occur. Digitonin-solubilized cell lysates were resolved on Blue Native (BN) PAGE gels17. A dramatic shift of NLRC4 from a monomer (~120kDa) to an oligomeric complex (~1000kDa) was seen in the presence of NAIP5 and FlaA. NAIP5 was also contained within the high molecular weight oligomeric complex (Fig. 3a). The association of NAIP5 and NLRC4 in the same complex was validated by co-immunoprecipitation (Supplementary Fig. 3)11,18. NLRC4 oligomerization was induced by either untagged FlaA or a GFP-FlaA fusion protein (Fig. 3a), both of which activate NLRC4/CASP1. Importantly, assembly of the NLRC4 inflammasome required FlaA (Supplementary Fig. 4a) and was not observed in the absence of NAIP5 (Fig. 3a), indicating that a biochemical function of NAIP5 is to promote NLRC4 oligomerization.
Despite strong genetic evidence that NLR proteins, such as NAIP5 and NLRC4, function as microbial ‘sensors’, there is no biochemical evidence that NLRs interact directly with microbial ligands. In fact, some studies of the NLRP3 inflammasome19–21, as well as analyses of analogous proteins from plants22, suggest that at least some NLRs recognize pathogens indirectly. In order to determine if the oligomerized NAIP5/NLRC4 complex also contains flagellin, we subjected samples separated in the first dimension by native PAGE to a second dimension of SDS-PAGE. To facilitate detection of flagellin, we used a 6x-Myc-tagged flagellin, which activates the inflammasome identically to native flagellin (data not shown). This approach revealed that FlaA was indeed present in a high-molecular weight complex, along with NAIP5 and NLRC4 (Fig. 3b). NAIP5 exhibited a weak flagellin-dependent mobility shift in the absence of NLRC4 (Supplementary Fig. 4b) suggesting that NLRC4 is not essential for flagellin recognition, though formation/stabilization of the oligomerized complex appears to be significantly enhanced by NLRC4. FlaA expressed alone was present in cell extracts only as a monomer (Supplementary Fig. 4c). Taken together, these observations provide evidence for a simple receptor-ligand model of NAIP5/NLRC4 activation by flagellin.
Consistent with the autoinhibitory function of the leucine rich repeats (LRRs) in other NLRs, we found that NAIP5ΔLRR and NLRC4ΔLRR constitutively activated CASP1-dependent cell death, independent of the presence of flagellin (Supplementary Fig. 5). Interestingly, NLRC4ΔLRR was able to activate CASP1 in the absence of NAIP5, whereas constitutively active NAIP5ΔLRR required wild-type NLRC4 in order to activate CASP1. This result suggests that NAIP5 functions upstream of NLRC4. Indeed, NAIP5ΔLRR was able to induce the oligomerization of wild-type NLRC4 (Fig. 3c), whereas the spontaneous oligomerization of NLRC4ΔLRR did not require NAIP5 (Fig. 3d). Spontaneous oligomerization of NLRC4ΔLRR did require the nucleotide binding domain (NBD) of NLRC4, as a K175R mutation previously shown to disrupt NBD function23 abolished NLRC4ΔLRR auto-oligomerization (Fig. 3d). The ability of NAIP5 to induce oligomerization of NLRC4 in response to flagellin required both the NBD and N-terminal BIRs of NAIP5, but did not require the N-terminal CARD of NLRC4 (Supplementary Fig. 4d, e), whereas functional CASP1 activation required all these domains (Supplementary Fig. 5, 6). Taken together, these data suggest a working model (Supplementary Fig. 7) in which NAIP5 is activated by flagellin and induces downstream NLRC4 oligomerization and CASP1 activation.
Consistent with a specific role for NAIP2 in recognition of PrgJ, we found that PrgJ did not induce the oligomerization of NAIP5/NLRC4 (Fig. 4a), but did induce oligomerization of NAIP2/NLRC4. Oligomerization of NLRC4 did not occur when co-expressed with NAIP2 alone or with NAIP2/FlaA (Fig. 4b). Interestingly, NAIP6 resembled NAIP5 and supported NLRC4 oligomerization in response to FlaA but not PrgJ (Fig. 4c), perhaps providing an explanation for the previously puzzling observation that Naip5−/− cells can respond to high levels of flagellin7. In contrast, NAIP1 is an ‘orphan’ NAIP since it responded neither to PrgJ or flagellin (Fig. 2d, e; Supplementary Fig. 8).
Our results demonstrate that the ability of the NLRC4 inflammasome to assemble and functionally activate CASP1 in response to specific bacterial ligands is dictated by NAIP family members. The most parsimonious model to account for our results is that NAIP proteins function as direct receptors for bacterial ligands (Supplementary Fig. 7). Although NLRC4 was previously suspected to be the cytosolic flagellin sensor1,2, we hypothesize that a main function of NLRC4 may instead be to serve as an adaptor, downstream of NAIP proteins, to recruit CASP1 via a CARD-CARD interaction. NLRC4 may also play an important role in ligand binding or in stabilizing NAIP/NLRC4/ligand complexes, but the specificity of the complexes for particular ligands appears to be controlled by NAIP proteins.
The number and sequence of Naip paralogs varies significantly among inbred mouse strains, and has been suggested to be evolving rapidly24. Indeed, the murine Naip locus was originally identified by a forward genetic approach which took advantage of the widely varying susceptibility of inbred mouse strains to L. pneumophila infection14,25. The single known human NAIP ortholog may also exist within a rapidly evolving locus24; our results suggest that it will be of great interest to establish the specificity of the human NAIP protein. We propose that Naip gene evolution represents a fascinating example of the molecular arms race between bacteria and their hosts.
Primary C57BL/6 bone marrow cells were transduced with pLKO.1-based lentivirus encoding shRNAs that specifically target Naip2 or controls. Bone marrow cells were differentiated into macrophages by culture in media containing MCSF. On day 4 of culture, transduced macrophages were selected by addition of puromycin (5μg/ml). On day 8 of culture, macrophages were replated and infected the next day with Listeria monocytogenes or Salmonella Typhimurium expressing flagellin or PrgJ8 and inflammasome activation was measured by assaying release of the cytosolic enzyme lactate dehydrogenase (LDH)7 or by western blotting for processed (p10) CASP1.
The inflammasome was reconstituted by transfection of 293T cells with MSCV2.2-IRES-GFP-based expression vectors encoding various mouse (C57BL/6-derived) Naip genes, Nlrc4 and Caspase-1. Inflammasome oligomerization was assessed in digitonin (1%) lysates using a Bis-Tris NativePAGE system (Invitrogen) followed by western blotting.
Statistical differences were calculated with an unpaired two-tailed Student’s t-test using GraphPad Prism 5.0b.
Work in R.E.V.’s laboratory is supported by Investigator Awards from the Burroughs Wellcome Fund and the Cancer Research Institute and by NIH grants AI075039, AI080749, and AI063302. We thank Jakob von Moltke, Alex Kintzer and Bryan Krantz for provision of LFn-FlaA and PA, S. Mariathasan and V. Dixit for the gift of anti-NLRC4 antibodies and Nlrc4−/− mice, J.D. Sauer and D. Portnoy for development of Listeria strains to deliver PrgJ and FlaA, and E. Michelle Long and W. Dietrich for pCDNA3-NAIP constructs and anti-NAIP antibodies. We thank A. Roberts for her initial efforts to knock down NAIP2, M. Fontana for validating NAIP knockdowns, and Jakob von Moltke and members of the Barton and Vance Labs for helpful discussions.
Author Contributions: E.M.K. and R.E.V. conceived experiments and wrote the paper. E.M.K. performed the experiments.
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The authors declare no competing financial interests.