IFN-Dependent Antiviral Immune Responses in

The Nucleotide-Binding Oligomerization Domain-Like Receptor NLRC5 Is Involved in IFN-Dependent Antiviral Immune Responses This information is current as of June 13, 2013.

Sven Kuenzel, Andreas Till, Michael Winkler, Robert Häsler, Simone Lipinski, Sascha Jung, Joachim Grötzinger, Helmut Fickenscher, Stefan Schreiber and Philip Rosenstiel J Immunol 2010; 184:1990-2000; Prepublished online 8 January 2010; doi: 10.4049/jimmunol.0900557 http://www.jimmunol.org/content/184/4/1990

References Subscriptions Permissions Email Alerts

http://www.jimmunol.org/content/suppl/2010/01/08/jimmunol.090055 7.DC1.html This article cites 43 articles, 10 of which you can access for free at: http://www.jimmunol.org/content/184/4/1990.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscriptions Submit copyright permission requests at: http://www.aai.org/ji/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/cgi/alerts/etoc

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

Downloaded from http://www.jimmunol.org/ by guest on June 13, 2013

Supplementary Material

The Journal of Immunology

The Nucleotide-Binding Oligomerization Domain-Like Receptor NLRC5 Is Involved in IFN-Dependent Antiviral Immune Responses Sven Kuenzel,*,1 Andreas Till,*,1 Michael Winkler,† Robert Ha¨sler,* Simone Lipinski,* Sascha Jung,‡ Joachim Gro¨tzinger,‡ Helmut Fickenscher,† Stefan Schreiber,*,x and Philip Rosenstiel*

T

he nucleotide-binding oligomerization (Nod)-like receptor (NLR) family represents a group of intracellular sensors of the innate immune system. At least 22 members have been identified within the human genome (1, 2). The family members are characterized by three distinct functional domains: an N-terminal effector-binding domain, a centrally located NAIP, CIITA, HET-E, and TP1 domain (NACHT) and a carboxy-terminal leucine-rich repeat (LRR) domain. The effector-binding domain is usually composed of caspase-recruitment domain(s) (CARD), a Pyrin domain, or a baculovirus inhibitor of apoptosis protein domain. Current knowledge suggests that pathogen-associated molecular

*Institute of Clinical Molecular Biology and ‡Biochemical Institute, ChristianAlbrechts University; and †Institute for Infection Medicine and xFirst Department for General Internal Medicine, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany

patterns (PAMPs) and other danger signals are directly or indirectly recognized by the LRRs. This recognition process induces NLR oligomerization and activation of NLR-dependent signal transduction pathways via induced-proximity signaling (3). In particular, activation of NF-kB, MAPKs, and proinflammatory caspases have been implicated as downstream events of NLR activation (4–6). Interestingly, several NLR family members (e.g., NOD1, NOD2, NLRP3/NALP3, NLRP1/NALP1, NLRA/MHC class II transcription activator) have been shown to contribute to susceptibility for chronic inflammatory diseases (7–15). Thus, it is assumed that NLR proteins represent pivotal components of the innate immune system and that impaired NLR function is closely linked to pathological conditions. In this report, we describe the genomic organization, regulation, and cellular function of NLRC5 (also referred to as NOD27).

1

Materials and Methods

Received for publication February 17, 2009. Accepted for publication December 5, 2009.

Cell lines and transfection

S.K. and A.T. contributed equally to this work.

This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 617-A20 (to P.R.) and Grant SFB 617-A24 (to H.F.) and the Clusters of Excellence “Inflammation at Interfaces” and “The Future Ocean” (to A.T., S.S., H.F., and P.R.). Work on CMV is supported by Deutsche Forschungsgemeinschaft Grant Wi1725 (to M.W.). Address correspondence and reprint requests to Dr. Philip Rosenstiel, Institute of Clinical Molecular Biology, Christian-Albrechts University, Schittenhelmstr. 12, D24105 Kiel, Germany. E-mail address: [email protected] The online version of this paper contains supplemental material. Abbreviations used in this paper: CARD, caspase-recruitment domain; FKBP, FK506 binding protein; GAS, IFN-g activation sequence; ISRE, IFN-specific response element; LRR, leucine-rich repeat; NACHT, NAIP, CIITA, HET-E, and TP1 domain; HFF, human foreskin fibroblast; NLR, nucleotide-binding oligomerization domainlike receptor; Nod, nucleotide-binding oligomerization; PAMP, pathogen-associated molecular pattern; poly I:C, polyinosinic-polycytidylic acid; siRNA, small interfering RNA. Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900557

Human cervical carcinoma HeLaS3 cells (ACC161), human acute monocytic cell line THP-1 (ACC16), human colonic carcinoma cell lines CaCo2 (ACC169), and HT-29 (ACC299) were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). Human foreskin fibroblasts (HFFs) were isolated from human foreskin and cultivated up to passage number 25 in DMEM. HeLaS3 and THP-1 were cultured in RPMI 1640 and CaCo2 and HT-29 were cultured in DMEM. All media were from PAA Laboratories (Paschberg, Austria). Media were supplemented with 10% FCS and penicillin/streptomycin (each at 50 mg/ml); all cell lines were grown in 5% CO2 at 37˚C. One day before transfection, cells were typically seeded at a density of 5 3 105 cells/2 ml on six-well plates or at 2 3 104/100 ml on a 96-well plate. Transfection of plasmids was performed using Fugene6 (Roche, Basel, Switzerland), small interfering RNA (siRNA) transfection was performed using Lipofectamine2000 (Invitrogen, Carlsbad, CA). For all procedures, standard protocols were used according to the manufacturers’ manuals.


Nucleotide-binding oligomerization domain-like receptors (NLRs) are a group of intracellular proteins that mediate recognition of pathogen-associated molecular patterns or other cytosolic danger signals. Mutations in NLR genes have been linked to a variety of inflammatory diseases, underscoring their pivotal role in host defense and immunity. This report describes the genomic organization and regulation of the human NLR family member NLRC5 and aspects of cellular function of the encoded protein. We have analyzed the tissue-specific expression of NLRC5 and have characterized regulatory elements in the NLRC5 promoter region that are responsive to IFN-g. We show that NLRC5 is upregulated in human fibroblasts postinfection with CMV and demonstrate the role of a JAK/STAT-mediated autocrine signaling loop involving IFN-g. We demonstrate that overexpression and enforced oligomerization of NLRC5 protein results in activation of the IFN-responsive regulatory promoter elements IFN-g activation sequence and IFN-specific response element and upregulation of antiviral target genes (e.g., IFN-a, OAS1, and PRKRIR). Finally, we demonstrate the effect of small interfering RNA-mediated knockdown of NLRC5 on a target gene level in the context of viral infection. We conclude that NLRC5 may represent a molecular switch of IFN-g activation sequence/IFN-specific response element signaling pathways contributing to antiviral defense mechanisms. The Journal of Immunology, 2010, 184: 1990–2000.


1991


FIGURE 1. Characterization of NLRC5 gene and the resulting protein NLRC5. A, The NLRC5 gene consists of 39 exons spanning a region of 96 kbp. The resulting protein is characterized by an N-terminal CARD, a central NACHT, and a region containing LRRs that are scattered along the C-terminal region. B, Amino acid sequence of NLRC5. The dashed line indicates the predicted CARD, the underlined region represents the NACHT domain, and the numbered boxes illustrate the position of LRR units. C, Tissue-specific mRNA expression profile of NLRC5. NLRC5 displays highest expression in brain, lung, prostate, and tonsil.

1992

CHARACTERIZATION OF NLRC5

FIGURE 2. A, Subcellular localization of NLRC5. GFP-NLRC5 was overexpressed in HeLaS3 cells. Nuclear DNA was counterstained using DAPI. Fluorescence microscopy reveals a spot-like cytosolic localization. B, Characterization of an Ab raised against NLRC5. Overexpression of GFP-NLRC5 fusion protein followed by immunoblotting revealed a specific signal of the expected size (230 kDa). The presence of two individual bands is estimated to be caused by posttranslational modification of unknown nature. As control, GFP alone and NLR family member NOD2 fused to GFP were overexpressed in parallel. No signals could be detected using antiNLRC5 Ab, whereas anti-GFP Ab exhibited strong signals as expected. C, Using the evaluated Ab, cytosolic localization of endogenous NLRC5 protein could be confirmed. Irrelevant primary Ab was used as negative control.

Infection of different epithelial cell lines with Listeria monocytogenes serotype 1/2a strain EGD was performed as described before (16). HFF cells were infected with human CMV AD169-BAC as described (17, 18). To inactivate and remove extracellular viral particles, HFF cells were additionally washed with citrate buffer (pH 3.0).

Plasmid construction Expression plasmid for NLRC5 was generated by amplifying full-length cDNA coding for NLRC5 from pancreas cDNA and inserting cDNA coding for NLRC5 into destination vectors by Gateway cloning (Invitrogen). The following primers were used for full-length amplification: forward 59ATGGACCCCGTTGGCCTCCA-39 and reverse 59-TCAAGTACCCCAAGGGGCCTGG-39. The N-terminal region of NLRC5 coding region containing the predicted CARD domain (bases 1–663) was cloned into pC4M-Fv2E vector (Ariad, Cambridge, MA) to obtain an FK506 binding protein (FKBP) fusion construct (CARD-FKBP) by using restriction sites EcoRI and XbaI. Primers were as follows: forward 59-ATGGACCCCGTTGGCCTCCAGCT-39 and reverse 59-CGGGCCCTTGTTAACCCTGGT-39. Expression constructs for NLRC5 are illustrated in the Supplemental Fig. 2. The control plasmids encoding GFP and GFP-NOD2 have been described before (16). The promoter region of NLRC5 spanning basepairs 21 to 21673 (relative to the first exon) was amplified from human genomic DNA and inserted into pGL3-Basic vector (Promega, Madison, WI) by using restriction sites XhoI and HindIII and the following primers: forward 59-GAGGAGGTCACTCTTCCAGAACC-39 and reverse 59-GCTCTGCACGAAACTGAAAGTAGAAG-39. For bioinformatic analysis of the NLRC5 promoter sequence, the MatInspector software (Genomatix, Mu¨nchen, Germany; www.genomatix.de/) was used. For specific mutagenesis of the constructs, the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used. For deletion of the combined kB/ STAT binding site, the forward primer 59-GGACTTCATGCACAGTGGGACTTCATGTA-39 and reverse primer 59-TACATGAAGTCCCACTGTGCATGAAGTCC-39 were used; for deletion of the single STAT binding site, forward primer 59-TCAGCCAGGGGTAGGAGCGAAC-39 and reverse primer 59-GTTCGCTCCTACCCCTGGCTGA-39 were used. The resulting deletion constructs were termed pGL3-DkB/STAT and pGL3-DSTAT, respectively. An additional construct harboring both deletions was termed pGL3-DSTAT/STAT.

could be detected using the preimmune serum (data not shown). The antagonistic IFN-g Ab IP-500 was purchased from Genzyme (Cambridge, MA). MitoTracker and LysoTracker staining solutions were purchased from Invitrogen. DAPI was from Sigma-Aldrich (St. Louis, MO). JAK inhibitor 1 was purchased from Calbiochem (San Diego, CA) and was compared with its diluent DMSO as negative control. The oligomerizer AP20187 was from Ariad. Polyinosinic-polycytidylic acid (poly I:C) that serves as surrogate stimulus for CMV infection was provided by Calbiochem.

Luciferase assay For quantification of promotor transcactivation and pathway-specific reportergen activity, transient transfection was used. HeLaS3 cells were seeded in a 96-well plate at a density of 1.5 3 104 per well. The next day, cells were transfected with 40 ng wild-type promoter construct (pGL3-NLRC5) or the deletion constructs (pGL3-DkB/STAT, pGL3-DSTAT) in combination with 10 ng reference plasmid pRL-TK (Promega). After 24 h, cells were stimulated with epidermal growth factor (100 ng/ml), TNF-a (10 ng/ml), LPS (10 mg/ml), flagellin (500 ng/ml), TGF-b (25 ng/ml), muramyl dipeptide (50 mg/ml), peptidoglycan of Staphylococcus aureus (10 mg/ml), IFN-g (1000 U/ml), IFN-a (subtype A/D hybrid; 1000 U/ml), or IFN-b (1000 U/ml) or infected with Listeria monocytogenes (multiplicity of infection = 100 bacteria/cell). For analysis of signaling events downstream of NLRC5, pathway-specific cis-reporter constructs pAP1-LUC, pNF-kBLUC, pISRE-LUC, and pGAS-LUC (Clontech, Palo Alto, CA) were used. The pathway-specific reporter gene constructs (22.5 ng) were cotransfected with either GFP-NLRC5 fusion construct, NLRC5-FKBP fusion construct, or the appropriate empty control vectors (22.5 ng) in combination with pRLTK (5 ng). After 24 h, the oligomerizer AP20187 was added at 0.1 mM. Transfection of siRNA was performed 24 h prior to transfection of luciferase vectors. Intracellular delivery of poly I:C was performed as previously described (19). Dual luciferase assay was performed using the Dual-Luciferase Reporter Assay System (Promega) and a MicroLumatPlus Luminometer (Berthold Technologies, Bad Wildbad, Germany).

Native gel electrophoresis Native glycine polyacrylamide gels were used for analysis of NLRC5FKBP oligomerization upon addition of exogenous dimerizer AP20187. Native protein complexes were blotted onto polyvinylidene difluoride membranes (Millipore, Billerica, MA) as described below.

Abs and reagents

SDS-PAGE and immunoblotting

Antiserum against NLRC5 was generated by a commercial supplier (Eurogentec, Seraing, Belgium). In short, the NLRC5-specific peptide CGQIENLSFKSRK was used for immunization of rabbit host animals to generate an mAb directedagainst NLRC5 protein. Specificity of the antiserum was tested by immunoblotting of lysates containing overexpressed fusion proteins GFP-NLRC5, GFP-NOD2, or GFP alone. Only GFP-NLRC5– containing lysates revealed a specific band using anti-NLRC5 Ab. No signal

Cells (5 3 105 cells/well) were grown in a six-well plate overnight, followed by transfection and stimulation as indicated. After washing in ice-cold PBS, cells were pelleted and lysed in buffer containing 1% SDS, 10 mM Tris (pH 7.6), 1 mM Na3VO4, and mixtures for inhibition of phosphatases and proteases (Sigma-Aldrich) followed by boiling for 5 min and sonification. Lysed cells were cleared from debris by centrifugation, and supernatants were collected. A total of 15 mg protein extract was separated by denaturing SDS-PAGE and


Bacterial and viral infection


1993


FIGURE 3. Regulation of NLRC5. A and B, HeLaS3 were transfected with NLRC5 promoter construct and stimulated for 24 h as indicated. Luciferase assay revealed a specific increase in NLRC5 promoter transactivation after stimulation with IFN-g, whereas no other stimulus induced NLRC5 promoter activity. All measurements were performed in triplicate. ppp , 0.01. C, Using RT-PCR and quantitative real-time PCR, a significant upregulation of NLRC5 mRNA was shown within 8 h after stimulation with IFN-g. ppp , 0.01. Note that similar results were obtained using THP-1, CaCo2, and HT-29 cells (see Supplemental Fig. 4). D, Dose dependency of IFN-g–mediated upregulation of NLRC5 mRNA levels as demonstrated by RT-PCR and real-time PCR (measurements in duplicate). E, NLRC5 protein level is upregulated by stimulation with IFN-g within 6–12 h after stimulation. transferred onto polyvinylidene difluoride membranes (Millipore). After blocking, membranes were probed with specific primary Abs, washed, and incubated with HRP-conjugated IgG as secondary Ab. Proteins were visualized by chemiluminescence (Amersham Biosciences, Piscataway, NJ). To determine even transfer and equal loading, membranes were stripped and reprobed with Ab specific for b-actin (Sigma-Aldrich).

Fluorescence microscopy HeLa S3 cells were seeded on sterile coverslips at 2 3 105 cells/well in six-well plates and grown overnight. The next day, cells were transiently transfected with overexpression plasmid encoding GFP-NLRC5. After 24 h, the cells were washed twice in ice-cold PBS, fixed for 20 min at room temperature with 4% paraformaldehyde (Carl Roth, Karlsruhe, Germany), and stained with DAPI (Roche) for 15 min. Alternatively, untransfected cells were fixed and stained with anti-NRLC5 Ab (1:250 in PBS/BSA) (or irrelevant primary Ab as neg-

ative control) followed by staining with FITC-conjugated secondary antirabbit Ab. Slips were transferred onto glass slides and examined using an apotome-equipped Axio Imager Z1 microscope (Zeiss, Jena, Germany).

RNA isolation and RT-PCR Total RNA was isolated from human cell lines using the RNeasy kit (Qiagen, Valencia,CA),and 250 ng total RNAwasreverse-transcribedto cDNA using the Advantage RT-for-PCR kit (Clontech). Resulting cDNA was analyzed using standard semiquantitative RT-PCR procedures and the following primers: 59TGACTTCCTTCTGCGTCTGCACAG-39 (NLRC5-for), 59-CATTGTCGGCAAGCTCCTGGAG-39 (NLRC5-rev), 59-ATTCAGCTCTCTGGGCTGTGATC-39 (IFN-a-for), 59-CATCACACAGGACTCCAGGTCATT-39 (IFN-a-rev), 59-ATGCCAGAAGTGGGTGGAGAAC-39 (PKRIR-for), 59-AGCCTCATGTCCATCCAGAGGTAT-39 (PKRIR-rev), 59-TGGCTCCTCAGGCAAGGGC-39 (OAS1-for), 59-TGGTACCAGTGCTTGACTAGGCGG-39 (OAS1-

1994


rev), 59-ACCGGAAGGAACCATCTCACT-39 (IL-8-for), 59-AAACTTCTCCACAACCCTCTGC-39 (IL-8-rev), 59-CCCTGATAATCCTGACGAGG-39 (CMV-IE2-for), and 59-GGTTCTCGTTGCAATCCTCGG-39 (CMV-IE2rev). Housekeeping gene GAPDH was used as control and amplified using the following primers: 59-CCAGCCGAGCCACATCGC-39 (GAPDH-for) and 59-ATGAGCCCCAGCCTTCTCCAT-39 (GAPDH-rev). To analyze the tissue-specific expression pattern of NLRC5, we used commercially available human cDNA tissue panels (Clontech) and RT-PCR. Relative expression was determined by normalization to housekeeping gene GAPDH using densitometry (ImageJ software, http://rsb.info.nih.gov/ij/).

Real-time PCR cDNA derived from stimulation and infection experiments was analyzed for NLRC5 and IL-8 mRNA levels using TaqMan Gene Expression Assays (NLRC5; Hs00260008_m1, IL-8; Hs00174103_m1; housekeeping gene ACTB; Hs99999903_m1; Applied Biosystems, Foster City, CA) on the ABI Prism 7900HT Fast Sequence Detection System (Applied Biosystems). All other target genes were quantified using the primer sets given above and the SYBR Green PCR Master Mix System (Applied Biosystems). Transcript amounts were normalized to those of the housekeeping gene b-actin using the following primer pair: ACTB-F 59-GATGGTGGGCATGGGTCAG-39 and ACTB-R 59-CTTAATGTCACGCACGATTTCC-39. Acquired real-time PCR data were analyzed using the 22DDCt method (20). Fold changes were calculated based on the ratio between treated sample and untreated control; p values were generated using the Mann-Whitney U test. Transcripts were considered differentially expressed when the p value was #0.05.

siRNA-mediated knockdown of NLRC5 Synthetic siRNAs targeting NLRC5 were purchased from Applied Biosystems/Ambion. Target sequences were as follows: siRNA1 (ID 141058), F: 59-CGGGUGGAAGAAUAUGUGAtt-39, R: 59-UCACAUAUUCUUCCACCCGtg-39; siRNA2 (ID 141060), F: 59-CCAACAUCCUAGAGCACAGtt-39, R: 59-CUGUGCUCUAGGAUGUUGGtc-39; siRNA3 (ID 141059), F: 59-GCAGACAGGCUAUGCUUUCtt-39, R: 59-GAAAGCAUAGCCUGUCUGCtg-39. As control, unspecific siRNA Negative Control #2 (Applied Biosystems/Ambion) was used.

Results

a single gene duplication event early in mammalian evolution (Supplemental Fig. 1). The predicted protein consists of 1866 aas and shows the typical tripartite domain structure characterizing NLR family members (21). The N terminus comprises a predicted CARD domain spanning amino acids 1–91. The predicted NACHT domain is located between position 220 and 383. The predicted Cterminal LRR region consists of 713 aa residues and shows an unusual architecture compared with other NLRs, because the 20 LRR units are scattered irregularly along the domain (Fig. 1A, 1B). To describe the tissue-specific expression pattern of NLRC5, we designed exon-spanning primer pairs and used human cDNA tissue panels for RT-PCR analysis. NLRC5 mRNA is expressed ubiquitously, with highest expression levels in the brain, lung, and prostate (Fig. 1C) and medium to high expression in a variety of tissues including the heart, digestive tract, and thymus. The lowest expression was detected in spleen, lymph nodes, and leukocytes. To assess the subcellular localization of the NLRC5 protein, we generated a GFPNLRC5 fusion construct for transient transfection of HeLaS3 cells. Fluorescence microscopy revealed a spot-like expression of the GFP-NLRC5 fusion protein in the cytosol (Fig. 2A). Using LysoTracker and MitoTracker staining (Invitrogen), we found no evidence for lysosomal or mitochondrial localization of NLRC5 (Supplemental Fig. 3 and data not shown). Next, we used peptide immunization to raise an Ab against the NLRC5 protein. Validation of the Ab using control plasmids (Fig. 2B) showed that neither GFP nor GFP-NOD2 were recognized by the NLRC5 Ab, but a specific signal was detectable that corresponds to the predicted 230-kDa size of GFP-NRLC5. Interestingly, all experiments revealed the existence of a double-band pattern that might be caused by translational modifications of yet unknown nature. Using this validated Ab, we could demonstrate that NLRC5 protein expression is restricted to the cytosolic compartment (Fig. 2C).

Genomic organization and expression of NLRC5 In the human genome, NLRC5 is located at the locus 16q13, spanning a region of ∼96 kbp. The gene contains 39 exons, and the translation start site is located in the third exon. The gene structure is conserved among many vertebrates and seems to have occurred by

NLRC5 expression is specifically regulated by IFN-g For characterization of the transcriptional regulation of NLRC5, the promoter region was cloned into the pGL3B luciferase reporter gene vector. The resulting reporter gene construct (pGL3-NLRC5)


FIGURE 4. Regulatory elements controlling NLRC5 promoter activity. A, The NLRC5 promoter region contains two predicted STAT consensus binding sites (one of which is overlapping with a predicted NF-kB consensus site). Individual and combined deletion of these two regulatory elements significantly impairs the IFN-g response of the promoter construct. pp , 0.05. Note that the empty vector backbone activity (vector control) accounts for ,10% of the NLRC5 promoter construct. B, Preincubation with pharmacological JAK inhibitor reduces the regulatory influence of IFN-g on NLRC5 promoter activity. pp , 0.05. All experiments were performed in triplicate.


1995


FIGURE 5. Role of viral infection for regulation of NLRC5. A, Infection of HFFs with human CMV, but not with heat-inactivated virus particles, upregulates NLRC5 mRNA levels within 24 h postinfection. This effect is abolished if cells are preincubated with JAK inhibitor (30 nM). B, Indicates a regulatory role of the JAK/STAT pathway. C, Supernatant (SN) of CMV-infected HFF is capable of inducing upregulation of NLRC5. ppp , 0.01 (A–C). D, Preincubation of supernatant derived from CMV-infected HFF with antagonizing IFN-g Ab impairs the upregulation of NLRC5. NLRC5 mRNA levels were quantified by TaqMan real-time PCR (Applied Biosystems; measurements in duplicate). pp , 0.05; ppp , 0.01.

1996


was transfected into HeLaS3. Baseline reporter gene expression by the empty vector backbone accounted for ,10% of the NLRC5 promoter construct (data not shown). Cells were stimulated with 10 different stimuli, including cytokines and a panel of PAMPs. Alternatively, cells were infected with cytoinvasive Listeria monocytogenes to check for direct response to bacterial infection. As shown in Fig. 3A and 3B, only stimulation with IFN-g resulted in a significant increase in luciferase activity, whereas none of the other stimuli, including IFN-a and IFN-b, affected NLRC5 promoter transactivation. These findings were supported by quantitative real-time PCR data showing that NLRC5 mRNA levels were significantly increased at 8 h after stimulation with IFN-g (Fig. 3C). Similar findings were obtained in a variety of other cell lines (THP-1, CaCo2, and HT-29; Supplemental Fig. 4). Moreover, this effect was dose-dependent, as shown by RT-PCR and quantitative real-time PCR (Fig. 3D). As control, quantification of IL-8 mRNA levels was used to demonstrate responsiveness of the cells to the various stimuli (Supplemental Fig. 5A–C). Using the validated anti-NLRC5 Ab, we could demonstrate that NLRC5 protein ex-


FIGURE 6. Signal transduction events downstream of NLRC5. Pathway-specific cis-reporter gene constructs (A–D) were cotransfected in combination with either GFP-tagged NLRC5, CARD-FKBP fusion construct, or the appropriate control vectors. In addition, FKBP-expressing cells were stimulated with exogenous oligomerizer AP20187 to enforce oligomerization of the (FKBP)2 domain. Although NLRC5 does not influence AP-1 or NF-kB activity (A and B), our data demonstrate a significant enhancement of ISRE- and GAS-dependent reporter gene activity by overexpression and by enforced oligomerization of the CARD domain of NLRC5 (C and D). Values represent mean of relative luciferase activity. pp , 0.05; ppp , 0.01. All experiments were performed in triplicate.

pression exhibits the same response to IFN-g stimulation because NLRC5 protein levels were remarkably increased 6 to 12 h after stimulation with IFN-g (Fig. 3E). Next, we aimed to understand the regulatory mechanisms underlying the IFN-g–dependent effect on NLRC5 expression. Using bioinformatical prediction tools, the NLRC5 promoter region was analyzed for the presence of potential cis-regulatory elements. The computational analysis revealed the presence of two STAT consensus binding sites. The STAT family of transcription factors represents a central component of multiple signal transduction cascades, including the IFN and IL-6 pathways (22, 23). The NLRC5 promoter region contains two individual potential STAT binding sites located at positions 21336 and 2452, respectively. Interestingly, the STAT site at 21336 partially overlaps with a predicted NF-kB consensus binding site. To further analyze a possible involvement of these binding sites in NLRC5 promoter activation, we created deletion constructs by site-directed mutagenesis. Individual or combined deletion of the predicted regulatory elements resulted in significant decrease of IFN-g–dependent promoter transactivation (Fig. 4A).


1997

Moreover, pretreatment with a specific inhibitor of JAK activity prior to IFN-g stimulation decreased promoter transactivation almost completely (Fig. 4B). Because JAK activity is essential for phosphorylation and nuclear translocation of STATs (24), these data further emphasize the role of IFN-g–dependent JAK/STAT signaling for NLRC5 transcriptional regulation . Viral infection contributes to transcriptional regulation of NLRC5 via autocrine IFN-g The secretion of the cytokine IFN-g is a pivotal cellular response to viral infection (25). In order to elucidate whether the expression of NLRC5 is directly linked to viral infection, we used a well-established viral infection model that is based on the infection of HFFs with human CMV (17, 18). To differentiate between effects that originate from direct viral infection and effects depending on sole presence of inactive virus particles, heat-inactivated virus particles were used in a parallel experimental setup. Six, 12, or 24 h postinfection with CMV, expression levels of NLRC5 mRNA were checked by quantitative real-time PCR. Efficiency of the viral infection was checked by

concomitant detection of CMV-IE2. As shown in Fig. 5A, a strong increase in NLRC5 expression was detectable that was peaking at 12 h postinfection. Remarkably, only intact virus particles were capable of inducing this effect, whereas heat-inactivated CMV did not alter NLRC5 mRNA levels within 24 h (Fig. 5A). Thus, the ability to efficiently infect human cells appears to be a prerequisite for regulation of NLRC5 expression. To investigate if autocrine JAK/STAT signaling in response to infection is involved in these regulatory events, we infected cells in presence of JAK inhibitor to block JAK/STAT signaling. Blockade of JAK-dependent pathways resulted in almost complete abrogation of NLRC5 upregulation (Fig. 5B). These results were confirmed by the fact that the supernatant of infected cells was able to upregulate NLRC5 mRNA in cells that were not infected (Fig. 5C). To analyze whether autocrine IFN-g secretion initiated by viral infection is responsible for regulation of NLRC5, we blocked endogenous IFN-g by addition of an inhibitory Ab. As shown in Fig. 5D, supernatant of infected cells preincubated with anti–IFN-g Ab resulted in impaired induction of NLRC5.


FIGURE 7. NLRC5-dependent regulation of antiviral target genes. A, Overexpression of GFP-NLRC5 in HeLaS3 cells results in increased expression of IFN-a, PRKRIR, and OAS1, whereas expression of chemokine IL-8 is unaffected. pp , 0.05. B, siRNA-mediated knockdown of NLRC5 results in reduction of endogenous mRNA levels of target genes, as demonstrated by RT-PCR and quantitative RT-PCR. pp , 0.05; ppp , 0.01.

1998 NLRC5 oligomerization activates IFN-specific response element- and IFN-g activation sequence-dependent signal transduction pathways

In order to analyze whether this activation profile was also detectable on the target gene level, we overexpressed GFP-NLRC5 in epithelial cells and used RT-PCR and real-time PCR to characterize potential NLRC5-dependent downstream target genes. As shown in Fig. 7A, the overexpression resulted in remarkable increase in mRNA levels of IFN-a, PRKRIR, and OAS1 24 h after transfection, whereas expression of IL-8 was not affected. Next, we checked the influence of siRNA-mediated knockdown of NLRC5 on the target gene level. As demonstrated in Fig. 7B, efficient knockdown of NLRC5 by three individual siRNAs results in reduction of endogenous mRNA levels of the investigated target genes as compared with control. To analyze whether this effect is also relevant in the context of viral infection, we next transfected HFF cells with siRNA and quantified expression of IFN-a in response to CMV. siRNA-mediated knockdown of NLRC5 significantly reduces induction of IFN-a after CMV infection (Fig. 8A), further emphasizing the role of NLRC5 as an essential component of antiviral signaling pathways. Supporting this view, NLRC5 knockdown also significantly reduces induction of GAS-driven luciferase activity in response to poly I:C, as shown in Fig. 8B. Taken together, our data argue for the NLR family member NLRC5 as being both target and activator of signaling pathways contributing to antiviral defense mechanisms.

Discussion In this report, we describe the genomic organization, regulation, and aspects of cellular function of the NLR family member NLRC5. This gene family has attracted much attention because mutations in several NLR genes have been linked to a variety of inflammatory diseases (29). For example, different genetic variants

FIGURE 8. Role of NLRC5 knockdown in antiviral signaling. A, siRNA-mediated knockdown of NLRC5 impairs upregulation of IFN-a in HFF cells in response to CMV infection. Cells were transfected with specific siRNA or control siRNA for 24 h and followed by infection with CMV for 24 h. NLRC5 and IFN-a mRNA levels were quantified by TaqMan real-time PCR (Applied Biosystems; measurements in duplicate). pp , 0.05; ppp , 0.01. B, NLRC5 knockdown results in diminished activation of GAS reporter gene activity upon stimulation with poly I:C. HeLaS3 cells were transfected with siRNA (24 h) and reporter gene constructs (additional 24 h), followed by stimulation with poly I:C (8 h) that serves as surrogate stimulus for CMV. GAS reporter gene activity was quantified by dual luciferase assay. Experiments were performed in triplicate. pp , 0.05.


Next we asked which signaling pathways are activated downstream of NLRC5 activation. Because it is a common phenomenon of NLRs that ectopic overexpression leads to ligand-independent autoactivation of downstream signaling events (3), we used overexpression of GFP-NLRC5 to screen for potential signaling cascades initiated by NLRC5 activity. In addition, we overexpressed a fusion protein consisting of the N-terminal CARD of NLRC5 fused (FKBP)2, whose oligomerization could be exogenously triggered by addition of the synthetic oligomerizer AP20187 (for illustration, see Supplemental Fig. 6). As shown by native glycine gel analyses, addition of the exogenous oligomerizer resulted in enforced oligomerization of CARD-FKBP monomers (Supplemental Fig. 7). Next, both GFPNLRC5 and CARD-FKBP constructs were expressed in parallel with a number of pathway-specific reporter gene constructs that could monitor the activation of specific regulatory promoter elements (Fig. 6). No activation of AP-1 or NF-kB signaling pathways was detectable (Fig. 6A, 6B). This was surprising because several members of the NLR family have been shown to directly activate NF-kB and/or MAPK signaling cascades (26–28). In contrast, our data point to a specific role of NLRC5 for the activation of signaling pathways that uses the consensus binding sites IFN-specific response element (ISRE) and IFN-g activation sequence (GAS) (Fig. 6C, 6D). These regulatory elements represent the major binding sites for STAT family members. Supporting these data, we demonstrate increased and prolonged phosphorylation of STAT1 downstream of IFN-g in the presence of overexpressed NLRC5 (Supplemental Figs. 8, 9).



By now, only few members of the NLR family have been associated with antiviral immune responses, whereas the majority of NLRs have been shown to contribute to innate defense mechanisms against pathogenic bacteria (38–40). The recently characterized NLR family member NLRX1 interacts with mitochondrial antiviral signaling adaptor in the outer membrane of mitochondria and represents a checkpoint modulating mitochondrial antiviral responses (41). Furthermore, NLRX1 is capable of amplifying NFkB and JNK pathways activated by different proinflammatory stimuli via production of reactive oxygen species (42). A recent study reports on the specific detection of influenza virus by components of the ASC/NLRP3 inflammasome, but the direct requirement for NLRP3 in the sensing process is still under debate (43). Our data add NLRC5 to the list of NLR family members involved in mechanisms that respond to viral infection and emphasize the role of NLRs as general cytosolic surveillance proteins involved in both bacterial and viral infection and endogenous danger response. In summary, we have characterized the genomic organization of NLRC5 and describe complex regulatory mechanisms identifying NLRC5 as target gene of IFN-g and mediator of IFN-mediated antiviral signaling pathways. Further analyses on NLRC5 regulation and function will help us to fully understand the impact of NLR family members on molecular defense strategies against viral pathogens.

Acknowledgments We thank Tanja Kaacksteen, Melanie Schlapkohl, Dorina Oelsner, Yasmin Brodtmann, and Alina Gra¨ff for their technical assistance.

Disclosures The authors have no financial conflicts of interest.

References 1. Ting, J. P.-Y., and B. K. Davis. 2005. CATERPILLER: a novel gene family important in immunity, cell death, and diseases. Annu. Rev. Immunol. 23: 387– 414. 2. Inohara, N., and G. Nun˜ez. 2003. NODs: intracellular proteins involved in inflammation and apoptosis. Nat. Rev. Immunol. 3: 371–382. 3. Inohara, N., T. Koseki, J. Lin, L. del Peso, P. C. Lucas, F. F. Chen, Y. Ogura, and G. Nuń˜ez. 2000. An induced proximity model for NF-kappa B activation in the Nod1/RICK and RIP signaling pathways. J. Biol. Chem. 275: 27823–27831. 4. Franchi, L., N. Warner, K. Viani, and G. Nun˜ez. 2009. Function of Nod-like receptors in microbial recognition and host defense. Immunol. Rev. 227: 106– 128. 5. Ting, J. P., and K. L. Williams. 2005. The CATERPILLER family: an ancient family of immune/apoptotic proteins. Clin. Immunol. 115: 33–37. 6. Creagh, E. M., and L. A. J. O’Neill. 2006. TLRs, NLRs and RLRs: a trinity of pathogen sensors that co-operate in innate immunity. Trends Immunol. 27: 352– 357. 7. Hugot, J. P., M. Chamaillard, H. Zouali, S. Lesage, J. P. Ce´zard, J. Belaiche, S. Almer, C. Tysk, C. A. O’Morain, M. Gassull, et al. 2001. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease. Nature 411: 599–603. 8. Ogura, Y., D. K. Bonen, N. Inohara, D. L. Nicolae, F. F. Chen, R. Ramos, H. Britton, T. Moran, R. Karaliuskas, R. H. Duerr, et al. 2001. A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease. Nature 411: 603–606. 9. Hampe, J., A. Cuthbert, P. J. Croucher, M. M. Mirza, S. Mascheretti, S. Fisher, H. Frenzel, K. King, A. Hasselmeyer, A. J. MacPherson, et al. 2001. Association between insertion mutation in NOD2 gene and Crohn’s disease in German and British populations. Lancet 357: 1925–1928. 10. Miceli-Richard, C., S. Lesage, M. Rybojad, A. M. Prieur, S. Manouvrier-Hanu, R. Ha¨fner, M. Chamaillard, H. Zouali, G. Thomas, and J. P. Hugot. 2001. CARD15 mutations in Blau syndrome. Nat. Genet. 29: 19–20. 11. Kastner, D. L., and J. J. O’Shea. 2001. A fever gene comes in from the cold. Nat. Genet. 29: 241–242. 12. Hoffman, H. M., J. L. Mueller, D. H. Broide, A. A. Wanderer, and R. D. Kolodner. 2001. Mutation of a new gene encoding a putative pyrin-like protein causes familial cold autoinflammatory syndrome and Muckle-Wells syndrome. Nat. Genet. 29: 301–305. 13. Feldmann, J., A. M. Prieur, P. Quartier, P. Berquin, S. Certain, E. Cortis, D. Teillac-Hamel, A. Fischer, and G. de Saint Basile. 2002. Chronic infantile


of NOD2 are associated with susceptibility for Crohn’s disease or Blau syndrome (7–10, 30), whereas NLRP3/NALP3 has been linked to rare hereditary fever syndromes and Crohn’s disease (11–14, 31, 32). These genetic findings underscore the importance of NLRs as surveillance proteins for host defense and immunity and point to their potential as novel therapeutic targets (33). Among the NLR family members identified and characterized so far, NLRC5 displays an unusual architecture of its carboxyterminal LRR because its repeat units are irregularly scattered along the domain. The LRR domain of NLR is generally considered to represent the sensor region of the proteins. The molecular structures that are recognized by NLR proteins represent either conserved molecular signatures of bacteria (PAMPs) or endogenous molecules that arise from conditions associated with cellular danger (danger-associated molecular patterns) (34). Interestingly, some NLRs are described to constitute highly selective sensor platforms that only respond to a specific molecular elicitor, whereas others exhibit a promiscuous selectivity by responding to a variety of stimuli. For example, NLRC4/IPAF responds to cytosolic presence of the bacterial protein flagellin, whereas NLRP3/ NALP3 is activated by various endogenous danger-associated molecular patterns like monosodium urate crystals or low potassium ion concentration (35). Nevertheless, it is still unclear whether this response is caused by direct physical interactions of the respective NLR with its elicitor or indirectly by mediators of unknown nature. The identification of physiological elicitors of NLR signaling and the dissection of the exact interaction mode of putative ligand and cognate receptor still remain important tasks for future studies. Because both the identity of a potential physiological elicitor of NLRC5 and the downstream signaling events were unknown, we used enforced oligomerization of an FKBP fusion protein and overexpression to simulate cellular events that trigger oligomerization of NLRC5. Our data demonstrate that NLRC5 oligomerization can efficiently trigger GAS- and ISREdependent promoter activity. In subsequent experiments, a panel of known PAMPs was used to test stimulus specificity of NLRC5dependent GAS/ISRE activation. However, from the tested compounds (e.g., LPS, flagellin, single-stranded RNA, muramyl dipeptide), the biochemical identity of a potential endogenous NLRC5 elicitor could not be elucidated (data not shown). Our data demonstrate a direct link between signaling events triggered by viral infection and transcriptional regulation of NLRC5 and argue for autocrine IFN-g activating JAK/STAT signaling as a key player of this autoregulatory loop. Moreover, we show that activation of NLRC5 implies specific activation of ISRE and GAS elements and upregulation of IFN-a, PRKRIR, and OAS1 without affecting transcription of chemokine IL-8. These findings are underscored by our data demonstrating that knockdown of NLRC5 significantly impairs induction of these target genes in the context of CMV infection. IFN-a transcriptional regulation has been shown to be dependent on JAK/STAT signaling via the IFN-stimulated gene factor 3 transcription factor complex (36). PRKRIR encodes for a protein that was first identified as a regulator of P58(IPK), a cellular inhibitor of the RNAdependent protein kinase (37). In addition, OAS proteins represent a group of IFN-inducible enzymes contributing to activation of RNAseL that degrade viral RNAs and thus inhibit viral replication. Conversely, IL-8 transcriptional regulation, which was used as a control, is mainly dependent on NF-kB and AP-1 transactivation activity, thus supporting our data on a specific induction of GAS/ISRE by NLRC5 activation. Given the regulatory role of IFN-g on NLRC5 gene regulation, these data argue for a specific role of NLRC5 as an endogenous amplifier of antiviral signaling pathways.

1999

2000

14.

15.

16.

17.

18.

19.

20.

22. 23. 24. 25. 26.

27.

28.

29. 30.

31.

32.

33. 34. 35.

36.

37.

38. 39.

40.

41.

42.

43.

granulomatosis syndromes: a study of the original Blau syndrome kindred and other families with large-vessel arteritis and cranial neuropathy. Arthritis Rheum. 46: 3041–3045. Aganna, E., F. Martinon, P. N. Hawkins, J. B. Ross, D. C. Swan, D. R. Booth, H. J. Lachmann, A. Bybee, R. Gaudet, P. Woo, et al. 2002. Association of mutations in the NALP3/CIAS1/PYPAF1 gene with a broad phenotype including recurrent fever, cold sensitivity, sensorineural deafness, and AA amyloidosis. Arthritis Rheum. 46: 2445–2452. Aksentijevich, I., M. Nowak, M. Mallah, J. J. Chae, W. T. Watford, S. R. Hofmann, L. Stein, R. Russo, D. Goldsmith, P. Dent, et al. 2002. De novo CIAS1 mutations, cytokine activation, and evidence for genetic heterogeneity in patients with neonatal-onset multisystem inflammatory disease (NOMID): a new member of the expanding family of pyrin-associated autoinflammatory diseases. Arthritis Rheum. 46: 3340–3348. Mathews, R. J., M. B. Sprakes, and M. F. McDermott. 2008. NOD-like receptors and inflammation. Arthritis Res. Ther. 10: 228. Benko, S., D. J. Philpott, and S. E. Girardin. 2008. The microbial and danger signals that activate Nod-like receptors. Cytokine 43: 368–373. Martinon, F., V. Pe´trilli, A. Mayor, A. Tardivel, and J. Tschopp. 2006. Goutassociated uric acid crystals activate the NALP3 inflammasome. Nature 440: 237–241. Harada, H., M. Matsumoto, M. Sato, Y. Kashiwazaki, T. Kimura, M. Kitagawa, T. Yokochi, R. S. Tan, T. Takasugi, Y. Kadokawa, et al. 1996. Regulation of IFNalpha/beta genes: evidence for a dual function of the transcription factor complex ISGF3 in the production and action of IFN-alpha/beta. Genes Cells 1: 995–1005. Gale, M., Jr., C. M. Blakely, D. A. Hopkins, M. W. Melville, M. Wambach, P. R. Romano, and M. G. Katze. 1998. Regulation of interferon-induced protein kinase PKR: modulation of P58IPK inhibitory function by a novel protein, P52rIPK. Mol. Cell. Biol. 18: 859–871. Inohara, N., and G. Nun˜ez. 2001. The NOD: a signaling module that regulates apoptosis and host defense against pathogens. Oncogene 20: 6473–6481. Chamaillard, M., M. Hashimoto, Y. Horie, J. Masumoto, S. Qiu, L. Saab, Y. Ogura, A. Kawasaki, K. Fukase, S. Kusumoto, et al. 2003. An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid. Nat. Immunol. 4: 702–707. Bonen, D. K., Y. Ogura, D. L. Nicolae, N. Inohara, L. Saab, T. Tanabe, F. F. Chen, S. J. Foster, R. H. Duerr, S. R. Brant, et al. 2003. Crohn’s diseaseassociated NOD2 variants share a signaling defect in response to lipopolysaccharide and peptidoglycan. Gastroenterology 124: 140–146. Moore, C. B., D. T. Bergstralh, J. A. Duncan, Y. Lei, T. E. Morrison, A. G. Zimmermann, M. A. Accavitti-Loper, V. J. Madden, L. Sun, Z. Ye, et al. 2008. NLRX1 is a regulator of mitochondrial antiviral immunity. Nature 451: 573–577. Tattoli, I., L. A. Carneiro, M. Jehanno, J. G. Magalhaes, Y. Shu, D. J. Philpott, D. Arnoult, and S. E. Girardin. 2008. NLRX1 is a mitochondrial NOD-like receptor that amplifies NF-kappaB and JNK pathways by inducing reactive oxygen species production. EMBO Rep. 9: 293–300. Ichinohe, T., H. K. Lee, Y. Ogura, R. Flavell, and A. Iwasaki. 2009. Inflammasome recognition of influenza virus is essential for adaptive immune responses. J. Exp. Med. 206: 79–87.


21.

neurological cutaneous and articular syndrome is caused by mutations in CIAS1, a gene highly expressed in polymorphonuclear cells and chondrocytes. Am. J. Hum. Genet. 71: 198–203. Villani, A. C., M. Lemire, G. Fortin, E. Louis, M. S. Silverberg, C. Collette, N. Baba, C. Libioulle, J. Belaiche, A. Bitton, et al. 2009. Common variants in the NLRP3 region contribute to Crohn’s disease susceptibility. Nat. Genet. 41: 71–76. Jin, Y., C. M. Mailloux, K. Gowan, S. L. Riccardi, G. LaBerge, D. C. Bennett, P. R. Fain, and R. A. Spritz. 2007. NALP1 in vitiligo-associated multiple autoimmune disease. N. Engl. J. Med. 356: 1216–1225. Till, A., P. Rosenstiel, K. Braütigam, C. Sina, G. Jacobs, H. H. Oberg, D. Seegert, T. Chakraborty, and S. Schreiber. 2008. A role for membrane-bound CD147 in NOD2-mediated recognition of bacterial cytoinvasion. J. Cell Sci. 121: 487–495. Hobom, U., W. Brune, M. Messerle, G. Hahn, and U. H. Koszinowski. 2000. Fast screening procedures for random transposon libraries of cloned herpesvirus genomes: mutational analysis of human cytomegalovirus envelope glycoprotein genes. J. Virol. 74: 7720–7729. Schierling, K., C. Buser, T. Mertens, and M. Winkler. 2005. Human cytomegalovirus tegument protein ppUL35 is important for viral replication and particle formation. J. Virol. 79: 3084–3096. Hirata, Y., A. H. Broquet, L. Mencheń, and M. F. Kagnoff. 2007. Activation of innate immune defense mechanisms by signaling through RIG-I/IPS-1 in intestinal epithelial cells. J. Immunol. 179: 5425–5432. Livak, K. J., and T. D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402–408. Albrecht, M., and F. L. W. Takken. 2006. Update on the domain architectures of NLRs and R proteins. Biochem. Biophys. Res. Commun. 339: 459–462. Schindler, C., D. E. Levy, and T. Decker. 2007. JAK-STAT signaling: from interferons to cytokines. J. Biol. Chem. 282: 20059–20063. Shuai, K., and B. Liu. 2003. Regulation of JAK-STAT signalling in the immune system. Nat. Rev. Immunol. 3: 900–911. Platanias, L. C. 2005. Mechanisms of type-I- and type-II-interferon-mediated signalling. Nat. Rev. Immunol. 5: 375–386. Katze, M. G., Y. He, and M. Gale. 2002. Viruses and interferon: a fight for supremacy. Nat. Rev. Immunol. 2: 675–687. Inohara, N., T. Koseki, L. del Peso, Y. Hu, C. Yee, S. Chen, R. Carrio, J. Merino, D. Liu, J. Ni, and G. Nuń˜ez. 1999. Nod1, an Apaf-1-like activator of caspase-9 and nuclear factor-kappaB. J. Biol. Chem. 274: 14560–14567. Ogura, Y., N. Inohara, A. Benito, F. F. Chen, S. Yamaoka, and G. Nunez. 2001. Nod2, a Nod1/Apaf-1 family member that is restricted to monocytes and activates NF-kappaB. J. Biol. Chem. 276: 4812–4818. Girardin, S. E., R. Tournebize, M. Mavris, A. L. Page, X. Li, G. R. Stark, J. Bertin, P. S. DiStefano, M. Yaniv, P. J. Sansonetti, and D. J. Philpott. 2001. CARD4/Nod1 mediates NF-kappaB and JNK activation by invasive Shigella flexneri. EMBO Rep. 2: 736–742. Chen, G., M. H. Shaw, Y. G. Kim, and G. Nun˜ez. 2009. NOD-like receptors: role in innate immunity and inflammatory disease. Annu. Rev. Pathol. 4: 365–398. Wang, X., H. Kuivaniemi, G. Bonavita, L. Mutkus, U. Mau, E. Blau, N. Inohara, G. Nunez, G. Tromp, and C. J. Williams. 2002. CARD15 mutations in familial
