The Journal of Immunology NLRC5 Limits the Activation of Inflammatory Pathways Szilvia Benko,* Joao G. Magalhaes, † Dana J. Philpott, † and Stephen E. Girardin* Nod-like receptors (NLRs) are intracellular sentinel proteins that are implicated in the detection of microbes and danger signals, thereby controlling several key innate immune pathways. The human genome encodes 22 NLR proteins, the function of many of which remains unknown. In this study, we present the identification and characterization of NLRC5, a NLR protein whose expres- sion is found predominantly in cells of the myeloid and lymphoid lineages. NLRC5 expression was strongly induced by IFN-g and more modestly by LPS and polyinosinic:polycytidylic acid. Overexpression of NLRC5 in HEK293T cells resulted in a global dampening of NF-kB–, AP-1–, and type I IFN-dependent signaling, most likely through transcriptional repression. Accordingly, NLRC5 was found to shuttle between the cytosol and the nucleus in a CrmA-dependent manner. Knocking down NLRC5 expression in RAW264.7 murine macrophages resulted in a potent upregulation of the proinflammatory responses to IFN-g and LPS, including increased secretion of TNF, IL-6, and IL-1b, as well as cell surface expression of CD40. Strikingly, NLRC5 expression was also found to be critical for LPS-induced IL-10 production in RAW264.7 macrophages. Collectively, our results identify NLRC5 as a negative modulator of inflammatory pathways. The Journal of Immunology, 2010, 185: 1681–1691. I n vertebrates, the innate immune system relies on several families of pattern recognition molecules (PRMs) that mediate the activation of defense pathways following detection of microbial- and danger-associated molecular patterns (MAMPs and DAMPs, respectively). Whereas TLRs sense MAMPs and DAMPs exposed to the extracellular milieu, Nod-like receptors (NLRs) and Rig-I–like receptors have recently been identified as intracellular PRMs (1, 2). In humans, the NLR family is composed of 22 mem- bers having in common the juxtaposition of a NACHT (for domain present in NAIP, CIITA, HET-E, andTP1) domain flanked on the carboxyl-terminal side by a leucine-rich repeat (LRR) domain (3). Subfamilies of NLRs can be defined on the basis of the N-terminal region, which displays more variability. For instance, the well- studied NLR proteins Nod1 and Nod2 contain an N-terminal cas- pase activation and recruitment domain (CARD), which triggers the recruitment of the adaptor protein Rip2 and the activation of down- stream signaling including NF-kB and MAPKs pathways (4). More recently, Nod1 and Nod2 have also been shown to induce the re- cruitment of the autophagy machinery at the site of bacterial in- vasion through a Rip2-independent interaction with ATG16L1 (5). Another large and relatively homogeneous subgroup of the NLR family is represented by the pyrin domain-containing NLR proteins (NLRPs), which display an N-terminal PYRIN domain. Studies on NLRP3 and NLRP1 have demonstrated the key role for NLRPs in the activation of caspase-1 inflammasomes in response to numerous MAMPs and DAMPs, resulting in the maturation and secretion of IL-1b and IL-18 (6). Other NLR proteins displaying well-defined N- terminal domains include NAIP (baculoviral inhibitory repeat do- main) (7), NLRC4 (CARD) (8), and CIITA (CARD and acid- transactivation domain) (9). Finally, NLRC3 and NLRC5 have CARD-like N-terminal domains. In addition to the well-established role of NLRs in triggering in- flammatory pathways in response to microbes or danger signals, sev- eral lines of evidence have demonstrated that mutations and polymorphisms in genes encoding members of the NLR family are associated with susceptibility to inflammatory disorders. NLR genes associated with inflammatory disorders include Nod2 (Crohn’s dis- ease and Blau syndrome), Nod1 (asthma and atopic disorders, in- flammatory bowel disease), Nlrp3 (Muckle-Wells syndrome, fa- milial cold autoinflammatory syndrome, chronic infantile neurologic cutaneous and articular syndrome, inflammatory bowel disease), and Nlrp1 (vitiligo) (10). Finally, mutations in CIITA cause bare lympho- cyte syndrome, a rare recessive condition in which the MHC class II expression is defective, resulting in severe immunodeficiency (11). CIITAwas the first identified member of the NLR family, and this protein displays unique features as compared with other NLRs. Contrary to Nod1, Nod2, NLRPs, and NLRC4, CIITA does not seem to trigger proinflammatory signaling pathways (such as NF-kB, MAPK, or caspase-1 inflammasome), but rather it acts as a tran- scriptional coactivator implicated in the regulation of MHC class II expression (12). Accordingly, CIITA has been shown to shuttle between the cytosol and the nucleus via a CrmA-dependent mech- anism (13), and CIITA remains, up until now, the only known NLR protein that targets the nucleus. Note also that there are no known MAMPs or DAMPs that modulate the function of CIITA. This sug- gests that CIITA, unlike most other NLRs, likely would not act as a bona fide PRM, although this point remains difficult to establish with certainty. In this study, we report the first characterization and analysis of NLRC5. Our results demonstrate that NLRC5 is highly expressed in cells of the myeloid and lymphoid lineages, and we establish that *Department of Laboratory Medicine and Pathobiology and † Department of Immu- nology, University of Toronto, Toronto, Ontario, Canada Received for publication December 4, 2009. Accepted for publication May 31, 2010. This work was supported by Grant MOP81360 from the Canadian Institutes of Health Research (to S.E.G.) and a grant from the Howard Hughes Medical Institute (to D.J.P.). J.G.M. is supported by a fellowship from the Canadian Institutes of Health Research. D.J.P. is a Howard Hughes International Scholar. Address correspondence and reprint requests to Dr. Stephen E. Girardin, Department of Laboratory Medicine and Pathobiology, University of Toronto, Medical Sciences Building, Room 6336, Toronto, ON M5S 1A8, Canada. E-mail address: Stephen. girardin@utoronto.ca The online version of this article contains supplemental material. Abbreviations used in this paper: aMf, alveolar macrophages; Bl, blood; BMDC, bone marrow-derived dendritic cell; BMDM, bone marrow-derived macrophage; CARD, caspase activation and recruitment domain; Con A, concanavalin A; c.pl, cytoplasmic fraction; DAMP, danger-associated molecular pattern; DC, dendritic cell; IoM, ionomycin; IPS-1, IFN-b promoter stimulator-1; ISRE, IFN-stimulated regulatory element; LMB, leptomycin B; LRR, leucine-rich repeat; MAMP, micro- bial-associated molecular pattern; n, nuclear fraction; NLR, Nod-like receptor; NLRP, pyrin domain-containing NLR protein; poly(I:C), polyinosinic:polycytidylic acid; PRM, pattern recognition molecule; qPCR, quantitative PCR; RT, reverse tran- scriptase; shRNA, short hairpin RNA. Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903900