Cutting Edge: NLRC5-Dependent Activation of the Inflammasome Beckley K. Davis,* Reid A. Roberts,† Max T. Huang,‡ Stephen B. Willingham,*,1 ,2 † x Brian J. Conti,* W. June Brickey, Brianne R.{ Barker,* Mildred Kwan,‖ Debra J. Taxman,† Mary-Ann Accavitti-Loper, Joseph A. Duncan,*, ,# and Jenny P.-Y. Ting*,† The nucleotide-binding domain leucine-rich repeat- immunity (1–5). One view suggests that NLRC5 is a positive containing proteins, NLRs, are intracellular sensors of regulator of the IFN pathway in HeLa and THP-1 cells and is pathogen-associated molecular patterns and damage- required for robust levels of IFN secretion (3, 5). However, associated molecular patterns. A subgroup of NLRs Benko et al. (1) demonstrated that NLRC5 is a negative regu- can form inflammasome complexes, which facilitate lator of the IFN, NF-kB, and AP-1 pathways in 293 cells. the maturation of procaspase 1 to caspase 1, leading to Furthermore, in the mouse monocytic cell line RAW 264.7, IL-1b and IL-18 cleavage and secretion. NLRC5 is pre- Nlrc5 functioned in an inhibitory manner. Cui et al. (2) pro- dominantly expressed in hematopoietic cells and has vided mechanistic detail by demonstrating that Nlrc5 interacts not been studied for inflammasome function. RNA with inhibitor of kBkinasea and inhibits its catalytic activity. interference-mediated knockdown of NLRC5 nearly Therefore, in its absence, there is a more robust proinflamma- eliminated caspase 1, IL-1b, and IL-18 processing tory response characterized by increased levels of TNF-a,IL-6, in response to bacterial infection, pathogen-associated and IL-1b. NLRC5 has also been shown to positively regulate molecular patterns, and damage-associated molecular MHC class I gene expression by directly binding to the pro- patterns. This was confirmed in primary human mono- moter region of MHC class I and associated genes in 293T and cytic cells. NLRC5, together with procaspase 1, pro–IL- Jurkat T cells (4). However, an opposite effect has been described 1b, and the inflammasome adaptor ASC, reconstituted in RAW 264.7 cells, as RNA interference-mediated knockdown inflammasome activity that showed cooperativity with on Nlrc5 induced MHC class I expression (1). Therefore, a com- NLRP3. The range of pathogens that activate NLRC5 in- plex and either cell type- or species-specific role for NLRC5 is flammasome overlaps with those that activate NLRP3. emerging. However, none of the published studies investigated Furthermore, NLRC5 biochemically associates with a role of NLRC5 in inflammasome function or formation. NLRP3 in a nucleotide-binding domain-dependent In this study, we delineated a new function for NLRC5 in but leucine-rich repeat-inhibitory fashion. These results inflammasome formation in response to pathogens, pathogen- invoke a model in which NLRC5 interacts with NLRP3 associated molecular patterns (PAMPs), or damage-associated to cooperatively activate the inflammasome. The molecular patterns (DAMPs). In the absence of NLRC5, Journal of Immunology, 2011, 186: 1333–1337. monocyte cell types process pro–IL-1b and pro–IL-18 in- effectively, and activation of caspase 1 is nearly eliminated in he role of NLRC5 is controversial and unresolved. response to NLRP3-specific agonists. Finally, we demonstrate Five recent publications have offered conflicting and that NLRC5 associates with itself and NLRP3. These data sug- T alternating roles of NLRC5 in innate and adaptive gest NLRC5 is a novel nucleotide-binding domain leucine-

*Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel National Cancer Institute Grant R21CA131645 (to B.K.D.), by the University of North Hill, Chapel Hill, NC 27599; †Department of Microbiology and Immunology, Uni- Carolina Clinical Translation Science Award/K12 Scholars Program, National Institutes versity of North Carolina at Chapel Hill, Chapel Hill, NC 27599; ‡Curriculum in Oral of Health Grants KL2RR025746 and R21CA131645, the Burroughs Wellcome Fund x Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; Divi- Career Award for Medical Scientists (to J.A.D.), and by National Institute of Allergy and sion of Rheumatology, Allergy and Immunology, Department of Medicine, University Infectious Diseases Grant R21-AI061059 (to W.J.B.). { of North Carolina at Chapel Hill, Chapel Hill, NC 27599; University of Alabama at ‖ Address correspondence and reprint requests to Dr. Jenny P.-Y. Ting, CB#7295, Line- Birmingham, Birmingham, AL 35233; Division of Infectious Disease, Department of berger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; and 27599. E-mail address: [email protected] #Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 The online version of this article contains supplemental material. 1Current address: Institute of Stem Cell Biology and Regenerative Medicine, Stanford Abbreviations used in this article: DAMP, damage-associated molecular pattern; LRR, University, Palo Alto, CA. leucine-rich repeat; MOI, multiplicity of infection; MSU, monosodium urate; NBD, nucleotide-binding domain; NLR, nucleotide-binding domain leucine-rich repeat- 2Current address: Department of Biochemistry and Molecular Biology Oregon Health containing protein; Nod, nucleotide-binding oligomerization domain; PAMP, and Science University, Portland, OR. pathogen-associated molecular pattern; shNLRC5, short hairpin RNA target sequences Received for publication September 17, 2010. Accepted for publication November 29, to NLRC5; shRNA, short hairpin RNA; siControl, control short interfering RNA; 2010. siRNA, short interfering RNA.

This work was supported by National Institutes of Health Grants AI63031, AI67798, Ó and AI077437 (to J.P.-Y.T.), by the Crohn’s and Colitis Foundation of America and Copyright 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003111 1334 CUTTING EDGE: INFLAMMASOME ACTIVATION BY NLRC5 rich repeat (LRR)-containing protein (NLR) that cooperates it the largest member of the NLRs (Supplemental Fig. 2A) with NLRP3 to induce inflammasome formation. with an approximate molecular mass of 200 kDa compared with the molecular masses of other NLRs ∼100 kDa each. Materials and Methods NLRC5 is well conserved in vertebrate evolution; orthologs are Quantitative PCR analysis of NLRC5 expression found in human, chimpanzee, cow, rat, and mouse (Supple- mental Fig. 2B) (8). Thus, NLRC5 is a conserved NLR mem- Human total RNA Master Panel II (Clontech) and mouse tissue RNA was used for cDNA synthesis using standard procedures. Quantitative PCR was per- ber with preferential expression in hematopoietic cells. formed with NLRC5 primers (Applied Biosystems). Transcripts were calcu- DD lated by Ct method or relative expression by Gapdh, Actin,or18s rRNA. NLRC5-dependent inflammasome activation Microarray data were mined from the Genomics Institute of the Novartis We explored the role of NLRC5 in monocytic cells due to their Research Foundation (http://biogps.gnf.org/). Inflammasome-related genes were cloned from THP-1 mRNA with primers designed to amplify the open prominent role in innate immunity and inflammation. Its reading frames. role in inflammasome activation was studied using sh- and siRNA to reduce its expression in the THP-1 cell line. shRNA Cell culture for NLRP3 and ASC served as positive controls. Due to the The human monocytic cell line THP-1 was transduced with short hairpin potential off-target effects of shRNA, a panel of shRNA target RNA (shRNA) (Supplemental Table I) containing lentivirus. Knockdown efficacies were determined by immunoblot analysis. A total of 2 3 106 cells was sequences to NLRC5 (shNLRC5) and shRNA controls in transfected by Amaxa with short interfering RNA (siRNA) oligonucleotides lentiviral constructs was tested. shNLRC5 effectively reduced (at 2–200 pM) using protocols T008 (THP-1) or V001 (monocytes). endogenous levels of NLRC5 transcript (Supplemental Fig. Bacteria 3A) and protein (Supplemental Fig. 3B). The latter was de- termined by immunoblot using an mAb specific for the N Shigella flexneri (strain 12022), Klebsiella pneumoniae (strain 43816 serotype 2), Porphyromonas gingivalis (strain A7436), and Listeria monocytogenes (strain terminus of NLRC5 (Supplemental Fig. 3C,3D). E. coli- 43251) were obtained from American Type Culture Collection. Escherichia infected cells with shNLRC5 produced significantly reduced coli (strain LF82) was provided by Dr. R. Sartor (University of North Car- levels of secreted IL-1b compared with cells with control olina at Chapel Hill) and Staphylococcus aureus (strain RN6390) from J.A. shRNAs in two different lentivirus vectors (Fig. 1A,1B, Duncan. Supplemental Fig. 3E). To avoid potential effects of lentiviral Infections and stimulations transduction, THP-1 cells were also transiently transfected THP-1 cells were grown to log phase growth. Bacteria were grown to stationary with siRNA oligonucleotides (siNLRC5 and siNLRP3). Cells phase. Cultures were diluted 1:250 and grown for an additional 2 h then with control siRNA (siControl) secreted significantly more quantitated by OD. Cocultures were pulsed with gentamicin at 50 mg/ml (Life IL-1b than cells with either siNLRC5 or siNLRP3 upon E. Technologies) after 1 h. Infections were harvested after an additional 2 h. Student t tests were performed to determine significance. For PAMP stimu- lation, cells were plated and stimulated with indicated PAMPs (Invivogen) overnight. For monosodium urate (MSU) (Invivogen) and Alum (Sigma- Aldrich) stimulations, cells were primed with 5 ng/ml ultrapure LPS from E. coli for 2 h then pulsed with agonist for an additional 4–6 h. For nigericin (Invivogen), a-hemolysin (from J.A. Duncan) stimulation cells were primed as before but harvested after 2 h. Cytokine, immunoblot, and cell death analyses IL-1b, IL-18, and TNF-a were measured by ELISA (BD Biosciences and Binder Life Sciences). Immunoblot was performed as described and probed with anti–IL-1b Abs from Cell Signaling Technology (2022 and 2021) and Santa Cruz Biotechnology (sc-52865) (6). Cell-free supernatants were used to quantitate cell death by ToxiLight bioassay (Lonza) (7). Immunoprecipitations HEK293T cells were transfected with Lipofectamine 2000 (Invitrogen) or Fugene6 (Roche). Cells were lysed in RIPA buffer in the presence of protease inhibitors and Benzoase (Novagen). Immunoprecipitations were washed with RIPA buffer then resolved on 4–12% Bis-Tris gels. Immunoblots were pro- bed with HRP-conjugated M2 (Sigma-Aldrich), anti-hemagglutinin (Roche), and anti-V5 (Invitrogen) mAbs. FIGURE 1. NLRC5 is necessary for inflammasome activation in response to E. coli. A, Lentivirus transduced THP-1 cells were stimulated with E. coli. Results Supernatants were assayed for IL-1b by ELISA. Two shNLRC5 and their Expression, cloning, and characterization of NLRC5 control shRNA with sequence specific mutations (shControl#1 and shCon- trol#2) were analyzed. B, shRNA containing control cell lines were assayed for NLRC5 is expressed predominantly in the lymphocytic and IL-1b secretion in response to E. coli. C, THP-1 cells were nucleofected with macrophage/monocytic cell lineages with low expression in siRNAs specific for NLRP3 and NLRC5 and with a nonsilencing (siControl) nonhematopoetic cells based on public gene profile database control siRNA; 48 h cells were stimulated as in A. D, Human primary (Supplemental Fig. 1A,1C). By real-time PCR analysis of monocytes were transfected with siRNA to NLRC5 and a nonsilencing human and mouse tissues (Supplemental Fig. 1B,1D), NLRC5 siRNA (siControl) and stimulated as in C.InD, values were normalized to is expressed preferentially in immune tissues relative to non- nonsilencing control. In A–C, experiments were repeated at least three times. Untreated samples were below the level of detection. THP-1 lysates were immune tissues. The gene is encoded by 49 exons, and its probed for active IL-1b in response to E. coli infection (E) and active caspase open reading frame is 5601 nt (46 exons). NLRC5 has an 1(F). *p , 0.05. mshASC, mutant ASC; mshNLRC5, mutant shNLRC5#1; N-terminal caspase recruitment domain-like domain and an shASC, shNLRP3, shNLRC5, sequence-specific shRNA; shEV, empty vector; extended C terminus consisting of an expanded LRR, making shLuc, shRNA to luciferase. The Journal of Immunology 1335 coli infection (Fig. 1C). Importantly, human primary mono- toxins, all of which can activate the NLRP3 inflammasome, cytes from two anonymous donors transfected with siRNA possibly via the release of endogenous secondary signals such oligonucleotides specific for NLRC5 also failed to mount as ATP (9) or reactive oxygen species (10). The PAMPs in- robust IL-1b in response to E. coli (Fig. 1D). IL-1b pro- duce significantly less IL-1b in the absence of NLRC5 (Fig. cessing (Fig. 1E) and caspase 1 (Fig. 1F) maturation are also 2D). Treatment with MSU or Alum induces IL-1b secretion greatly reduced in the absence of NLRC5. Real-time PCR in an NLRC5-dependent fashion (Fig. 2D). Nigericin from analysis demonstrates that in the absence of NLRC5, tran- Streptomyces hygroscopicus and a-hemolysin from S. aureus scription of NLRP3 and ASC are unaffected (Supplemental activate the NLRP3 inflammasome by pore formation (6, 11, Fig. 4A–C). Additionally pro–IL-1b levels are comparable 12). Interestingly, IL-1b secretion in response to these toxins (Supplemental Fig. 4D). These data suggest that NLRC5 has is independent of NLRC5 (Fig. 2E). These data revealed that a role in IL-1b inflammasome formation in transformed and NLRC5 is not required for inflammasome activation by the nontransformed human monocytic cells. pore-forming toxins tested. The fact that purified S. aureus a-hemolysin induces NLRC5-independent IL-1b production NLRC5-dependent response to pathogens is compatible with earlier results showing that high multi- To determine the specificity of signals that activate the plicities of infection (MOIs) of S. aureus causes NLRC5- NLRC5-dependent inflammasome, THP-1 cell lines stably independent inflammasome activation and is consistent with a expressing shNLRC5 or control shRNA were infected with -hemolysin being a synergistic but not sole mechanism for b a panel of bacteria. Candidate pathogens E. coli, S. flexneri, robust IL-1 secretion (6, 12). These findings suggest that and S. aureus require NLRP3 and ASC to activate the inflammasome activation by bacterial PAMPs and crystals, inflammasome (Fig. 2A–C), and they induced significantly but not pore-forming toxins, requires NLRC5. less IL-1b secretion in cells expressing shNLRC5 relative to cells expressing shControl. The decrease in IL-1b and IL-18 NLRC5-independent regulation of TNF-a and cell death with different bacteria was observed over various concen- To assess the specificity of NLRC5 function, we assayed trations (Supplemental Fig. 5A–D) except at high concen- supernatants from infected cells for TNF-a. TNF-a levels b trations of S. aureus, IL-1 /IL-18 secretion is independent of from shNLRC5-containing cells are not significantly different NLRC5 (Supplemental Fig. 5B,5D). These data suggest that from control cell lines postinfection with bacteria (Fig. 2F). NLRC5 modulates inflammasome activation to a wide array Pathogen-mediated activation of caspase 1 and/or NLRs can of bacterial pathogens. induce several cell death pathways that are broadly charac- terized as proinflammatory because they result in the further NLRC5 is required for inflammasome activation by PAMPs, DAMPs, release of inflammatory molecules (13, 14). Interestingly, and toxins pathogen-induced cell death that is documented to require To further dissect possible agonists that activate the NLRC5 NLRP3 in THP-1 cells is not affected by the absence of inflammasome, THP-1 cells with shNLRC5 or control shRNA NLRC5 (Fig. 2G), suggesting that the function of NLRC5 is were stimulated with PAMPs, DAMPs, and pore-forming more restricted to inflammasome activation.

FIGURE 2. NLRC5 is necessary for IL-1b production in response to bacterial pathogens and DAMPs. E. coli (MOI of 5) (A), S. aureus (MOI 5) (B), and S. flexneri (MOI 5) (C) were used to infect THP-1 cells. Supernatants were analyzed for IL-1b. D and E, THP-1 cells were stimulated with PAMPs, DAMPs, and toxins. MSU (100 mg/ml), Alum (100 mg/ml), nigericin (40 mM), and a-hemolysin 1 mg/ml (a-HL). Supernatants were harvested and assayed for IL-1b (D, E). F, TNF-a secretion in response to pathogenic bacteria infection. G, Cell death measured by release of ATP by Toxilight assay during infection with high (50) and low (5) MOIs. D–G, Values represent mean 6 SD of duplicate samples of shNLRC5 (open bars) or shControl (closed bars). All experiments were done at least three times. *p , 0.05. FSL, FSL-1; Lman, lipomannan; LTA, lipoteichoic acid; P3Cys, Pam3Cys; UnTx, untreated. 1336 CUTTING EDGE: INFLAMMASOME ACTIVATION BY NLRC5

Interactions of NLRC5 with NLRP3 and ASC but not other concept that the LRR is inhibitory in function. To note, we inflammasome components have consistently observed decreased levels of NLRP3 in the The overlapping biologic functions and pathogen specificity presence of the LRR of NLRC5 and are currently investi- of NLRC5 with NLRP3 suggests that these proteins might gating this phenomenon. act in a cooperative manner during the inflammasome as- Others have demonstrated that reconstitution of inflam- sembly of ASC, NLRP3, and caspase 1. To test if NLRC5 in- masome components in epithelial cells can lead to spontane- b teracts with inflammasome components, we performed co- ous or PAMP-induced IL-1 processing (15). To test whether immunoprecipitation experiments with NLRC5 and itself, NLRC5 can facilitate inflammasome function, we transfec- NLRP3, AIM2, ASC, procaspase 1, and pro–IL-1b. Epitope- ted these inflammasome components into HEK293T cells b tagged NLRC5 interacts with itself, NLRP3, and ASC (Fig. and measured IL-1 processing. Transient transfection with b 3A, Supplemental Fig. 6A). In contrast, it did not interact NLRC5, procaspase 1, pro–IL-1 ,andASCresultsinIL- b with the other inflammasome components AIM2, procaspase 1 processing that was dependent on the inflammasome b 1, pro–IL-1b, or additional cytosolic proteins such as TNFR- component expression as measured by secreted IL-1 (Fig. b associated factor 2 and JNK (Supplemental Fig. 6B,6C). To 3C). The level of IL-1 induced by nucleotide-binding olig- determine if the interactions of NLRC5 are specific to inflam- omerization domain 1 (NOD1) is significantly less than the masome-forming NLRs, we show that NLRC5 does not in- amount seen with NLRC5. When NLRC5 was cotransfected teract with another NLR, CIITA (Supplemental Fig. 6D). with increasing amounts of NLRP3 or vice versa, a synergis- b To determine the domain of NLRC5 that is necessary and tic increase in IL-1 secretion is observed (Fig. 3D). These sufficient for NLRP3 binding, truncation mutants were data suggest that NLRC5 is a component of the inflamma- used in coimmunoprecipitation experiments (Fig. 3B). The some in a reconstituted and ectopic system either by itself or nucleotide-binding domain (NBD) appears to be necessary in conjunction with NLRP3. and sufficient to bind NLRP3, as it coimmunoprecipitates with NLRP3. The N-terminal caspase recruitment domain Discussion (N-term) or C-terminal LRR of NLRC5 failed to coimmu- Inflammasome activation represents a major function of noprecipitate with NLRP3. The presence of the N terminus some NLR proteins (10), although the function of a majority to the NBD domain in the N–NBD construct did not in- of NLR proteins remains unclear. NLRC5 is primarily ex- fluence the binding capacity of the NBD. However, the pressed in adaptive and innate immune systems. The con- presence of LRR in the NBD–LRR construct reduced in- served expression pattern of NLRC5 orthologs suggests a criti- teraction with NLRP3. This result agrees with the general cal biological role in immunity. As its expression in monocytic cells might be indicative of a role in innate immunity to pathogens, we examined the role of NLRC5 in inflammasome function. We now describe NLRC5 as a critical component of inflammasome-dependent IL-1b secretion in response to a repertoire of stimuli that also activate NLRP3. This func- tion is supported by the interactions of NLRC5 with NLRP3 and ASC as well as its ability to form inflammasome com- plexes in an ectopic system. These interactions might greatly increase the repertoire of biological moieties that can be sensed by NLR inflammasomes as has been previously pre- dicted (8). In THP-1 cells infected with higher concentrations of S. aureus or stimulated with purified a-hemolysin, secretion of IL-1b is independent of NLRC5. S. aureus is known to elaborate many pore-forming and immune-modulating toxins. Thus, at high concentrations, it is possible that these toxins activate an NLRC5-independent but NLRP3-depen- dent inflammasome. Interestingly, many of these toxins dis- rupt the cell membrane and ultimately induce cell death. In our studies, NLRC5 appears to be dispensable in pathogen- induced cell death, whereas NLRP3 (7) is essential. This di- vergence of function may signify segregation between multiple FIGURE 3. NLRC5 interacts with inflammasome components. A, FLAG- pathways. tagged NLRP3 and ASC were cotransfected with NLRC5-V5. Lysates were The mechanisms that govern pathogen-induced inflam- immunoprecipitated with anti-V5 and probed with anti-FLAG mAbs. B, masome activation remain poorly characterized, and the con- Truncation mutants of NLRC5 were cotransfected with NLRP3-FLAG. tribution of individual NLRs is controversial. For example, Lysates were immunoprecipitated with anti-V5 mAb and probed with anti- early studies demonstrated an absolute role for Nlrc4 in Sal- b FLAG. C, HEK293T cells were transfected with caspase 1 and IL-1 in the monella-dependent IL-1b secretion (11); however, recent presence of increasing amounts of ASC in the presence or absence of NLRs. b Supernatants were analyzed for IL-1b by ELISA. D, HEK293T cells were studies have challenged the notion that IL-1 response to transfected with increasing concentrations of NLRP3 or NLRC5 (ng) in the Salmonella relies exclusively upon Nlrc4 (16). Similarly, Miao presence of ASC, procaspase 1, and pro–IL-1b. Lysates were probed for et al. (17) has recently demonstrated Nlrc4-dependent inflam- cleaved IL-1b. masome activation in response components of the type 3 se- The Journal of Immunology 1337 cretion system in a broad panel of Gram-negative bacteria. human NLR family member NLRC5 in antiviral responses. J. Biol. Chem. 285: 26223–26232. Likewise, both Nlrp3- and Nlrc4-dependent inflammasome 6. Craven, R. R., X. Gao, I. C. Allen, D. Gris, J. Bubeck Wardenburg, E. McElvania- activation in response to S. flexneri infection are reported (7, Tekippe, J. P. Ting, and J. A. Duncan. 2009. Staphylococcus aureus a-hemolysin activates the NLRP3-inflammasome in human and mouse monocytic cells. PLoS 18). These differences might reflect differences in bacterial ONE 4: e7446. strains, host species, infected cell types, or duration and multi- 7. Willingham, S. B., D. T. Bergstralh, W. O’Connor, A. C. Morrison, D. J. Taxman, plicities of infection. Thus, the emerging evidence suggests J. A. Duncan, S. Barnoy, M. M. Venkatesan, R. A. Flavell, M. Deshmukh, et al. 2007. Microbial pathogen-induced necrotic cell death mediated by the inflammasome that during bacterial infections, multiple NLR inflamma- components CIAS1/cryopyrin/NLRP3 and ASC. Cell Host Microbe 2: 147–159. somes have the potential to become activated. 8. Ting, J. P., and B. K. Davis. 2005. CATERPILLER: a novel gene family important in immunity, cell death, and diseases. Annu. Rev. Immunol. 23: 387–414. NLRs have been shown to have different roles in separate cell 9. Netea, M. G., C. A. Nold-Petry, M. F. Nold, L. A. Joosten, B. Opitz, J. H. van der types (19). A prime example is NOD2, which was initially Meer, F. L. van de Veerdonk, G. Ferwerda, B. Heinhuis, I. Devesa, et al. 2009. described as an epithelial cell and/or macrophage sensor of Differential requirement for the activation of the inflammasome for processing and release of IL-1b in monocytes and macrophages. Blood 113: 2324–2335. muramyl dipeptide that induces NF-kB activation (20, 21). 10. Martinon, F., A. Mayor, and J. Tschopp. 2009. The inflammasomes: guardians of Others have shown that NOD2 functions as an inflammasome the body. Annu. Rev. Immunol. 27: 229–265. 11. Mariathasan, S., D. S. Weiss, K. Newton, J. McBride, K. O’Rourke, M. Roose- component that facilitates NLRP1-dependent activation in Girma, W. P. Lee, Y. Weinrauch, D. M. Monack, and V. M. Dixit. 2006. Cry- monocytic cells (22). The multiple functions of NLRs are also opyrin activates the inflammasome in response to toxins and ATP. Nature 440: 228–232. illustrated in the analysis of NLRC5. NLRC5 is reported to 12. Mun˜oz-Planillo, R., L. Franchi, L. S. Miller, and G. Nu´n˜ez. 2009. A critical role for have a positive role in antiviral immunity in human epithelial hemolysins and bacterial lipoproteins in Staphylococcus aureus-induced activation of cell lines and primary fibroblasts (3, 5), as a negative regulator the Nlrp3 inflammasome. J. Immunol. 183: 3942–3948. 13.Fernandes-Alnemri,T.,J.Wu,J.W.Yu,P.Datta,B.Miller,W.Jankowski, of both type I IFN and NF-kB–dependent cytokines via in- S. Rosenberg, J. Zhang, and E. S. Alnemri. 2007. The pyroptosome: a su- teractions in epithelial and monocytic cells (1, 2), and as a pramolecular assembly of ASC dimers mediating inflammatory cell death via caspase-1 activation. Cell Death Differ. 14: 1590–1604. positive regulator of MHC class I gene transcription in human 14. Ting, J. P., S. B. Willingham, and D. T. Bergstralh. 2008. NLRs at the intersection cells (4) but not mouse cell lines (1). Our data demonstrate of cell death and immunity. Nat. Rev. Immunol. 8: 372–379. 15.Yu,J.W.,J.Wu,Z.Zhang,P.Datta,I.Ibrahimi,S.Taniguchi,J.Sagara,T. a separate function of NLRC5 in monocytic cells. Whether Fernandes-Alnemri, and E. S. Alnemri. 2006. Cryopyrin and pyrin activate these diverse functions relate to the unique structure of NLRC5 caspase-1, but not NF-kB, via ASC oligomerization. Cell Death Differ. 13: or the biological systems used remains to be seen. The genera- 236–249. 16. Broz, P., K. Newton, M. Lamkanfi, S. Mariathasan, V. M. Dixit, and D. M. tion of mice deficient in Nlrc5 might shed light on the in vivo Monack. 2010. Redundant roles for inflammasome receptors NLRP3 and NLRC4 role of this gene. in host defense against Salmonella. J. Exp. Med. 207: 1745–1755. 17. Miao, E. A., D. P. Mao, N. Yudkovsky, R. Bonneau, C. G. Lorang, S. E. Warren, I. A. Leaf, and A. Aderem. 2010. Innate immune detection of the type III secretion Disclosures apparatus through the NLRC4 inflammasome. Proc. Natl. Acad. Sci. USA 107: The authors have no financial conflicts of interest. 3076–3080. 18. Suzuki, T., L. Franchi, C. Toma, H. Ashida, M. Ogawa, Y. Yoshikawa, H. Mimuro, N. Inohara, C. Sasakawa, and G. Nun˜ez. 2007. Differential regulation of caspase-1 References activation, pyroptosis, and autophagy via Ipaf and ASC in Shigella-infected mac- 1. Benko, S., J. G. Magalhaes, D. J. Philpott, and S. E. Girardin. 2010. NLRC5 limits rophages. PLoS Pathog. 3: e111. the activation of inflammatory pathways. J. Immunol. 185: 1681–1691. 19. Ting, J. P., J. A. Duncan, and Y. Lei. 2010. How the noninflammasome NLRs 2. Cui, J., L. Zhu, X. Xia, H. Y. Wang, X. Legras, J. Hong, J. Ji, P. Shen, S. Zheng, function in the innate immune system. Science 327: 286–290. Z. J. Chen, and R. F. Wang. 2010. NLRC5 negatively regulates the NF-kappaB and 20.Ogura,Y.,D.K.Bonen,N.Inohara,D.L.Nicolae,F.F.Chen,R.Ramos, type I interferon signaling pathways. Cell 141: 483–496. H. Britton, T. Moran, R. Karaliuskas, R. H. Duerr, et al. 2001. A frameshift 3.Kuenzel,S.,A.Till,M.Winkler,R.Ha¨sler,S.Lipinski,S.Jung,J.Gro¨tzinger, mutation in NOD2 associated with susceptibility to Crohn’s disease. Nature H. Fickenscher, S. Schreiber, and P. Rosenstiel. 2010. The nucleotide-binding 411: 603–606. oligomerization domain-like receptor NLRC5 is involved in IFN-dependent 21. Girardin, S. E., I. G. Boneca, J. Viala, M. Chamaillard, A. Labigne, G. Thomas, D. antiviral immune responses. J. Immunol. 184: 1990–2000. J. Philpott, and P. J. Sansonetti. 2003. Nod2 is a general sensor of peptidoglycan 4.Meissner,T.B.,A.Li,A.Biswas,K.H.Lee,Y.J.Liu,E.Bayir,D.Iliopoulos, through muramyl dipeptide (MDP) detection. J. Biol. Chem. 278: 8869–8872. P.J.vandenElsen,andK.S.Kobayashi.2010.NLRfamilymemberNLRC5is 22. Hsu, L. C., S. R. Ali, S. McGillivray, P. H. Tseng, S. Mariathasan, E. W. Humke, a transcriptional regulator of MHC class I genes. Proc. Natl. Acad. Sci. USA L. Eckmann, J. J. Powell, V. Nizet, V. M. Dixit, and M. Karin. 2008. A NOD2- 107: 13794–13799. NALP1 complex mediates caspase-1-dependent IL-1b secretion in response to 5. Neerincx, A., K. Lautz, M. Menning, E. Kremmer, P. Zigrino, M. Hosel, Bacillus anthracis infection and muramyl dipeptide. Proc. Natl. Acad. Sci. USA 105: H. Buning, R. Schwarzenbacher, and T. A. Kufer. 2010. A role for the 7803–7808.