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IFN-Dependent Antiviral Immune ResponsesinDomain-Like Receptor NLRC5 Is Involved

The Nucleotide-Binding Oligomerization

Helmut Fickenscher, Stefan Schreiber and Philip RosenstielHäsler, Simone Lipinski, Sascha Jung, Joachim Grötzinger, Sven Kuenzel, Andreas Till, Michael Winkler, Robert

http://www.jimmunol.org/content/184/4/1990doi: 10.4049/jimmunol.0900557January 2010;

2010; 184:1990-2000; Prepublished online 8J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2010 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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The Journal of Immunology

The Nucleotide-Binding Oligomerization Domain-LikeReceptor NLRC5 Is Involved in IFN-Dependent AntiviralImmune Responses

Sven Kuenzel,*,1 Andreas Till,*,1 Michael Winkler,† Robert Hasler,* Simone Lipinski,*

Sascha Jung,‡ Joachim Grotzinger,‡ Helmut Fickenscher,† Stefan Schreiber,*,x and

Philip Rosenstiel*

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.

The nucleotide-binding oligomerization (Nod)-like receptor(NLR) family represents a group of intracellular sensors ofthe innate immune system. At least 22 members have been

identified within the human genome (1, 2). The family members arecharacterized by three distinct functional domains: an N-terminaleffector-binding domain, a centrally located NAIP, CIITA, HET-E,and TP1 domain (NACHT) and a carboxy-terminal leucine-richrepeat (LRR) domain. The effector-binding domain is usuallycomposed of caspase-recruitment domain(s) (CARD), a Pyrin do-main, or a baculovirus inhibitor of apoptosis protein domain. Cur-rent knowledge suggests that pathogen-associated molecular

patterns (PAMPs) and other danger signals are directly or indirectlyrecognized by the LRRs. This recognition process induces NLRoligomerization and activation of NLR-dependent signal trans-duction pathways via induced-proximity signaling (3). In particular,activation of NF-kB, MAPKs, and proinflammatory caspases havebeen 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 transcrip-tion activator) have been shown to contribute to susceptibility forchronic inflammatory diseases (7–15). Thus, it is assumed that NLRproteins represent pivotal components of the innate immune systemand that impaired NLR function is closely linked to pathologicalconditions.In this report, we describe the genomic organization, regulation,

and cellular function of NLRC5 (also referred to as NOD27).

Materials and MethodsCell lines and transfection

Human cervical carcinoma HeLaS3 cells (ACC161), human acutemonocytic cell line THP-1 (ACC16), human colonic carcinoma cell linesCaCo2 (ACC169), and HT-29 (ACC299) were purchased from the Ger-man Collection of Microorganisms and Cell Cultures (DSMZ,Braunschweig, Germany). Human foreskin fibroblasts (HFFs) were iso-lated from human foreskin and cultivated up to passage number 25 inDMEM. HeLaS3 and THP-1 were cultured in RPMI 1640 and CaCo2 andHT-29 were cultured in DMEM. All media were from PAA Laboratories(Paschberg, Austria). Media were supplemented with 10% FCS andpenicillin/streptomycin (each at 50 mg/ml); all cell lines were grown in5% CO2 at 37˚C. One day before transfection, cells were typically seededat a density of 5 3 105 cells/2 ml on six-well plates or at 2 3 104/100 mlon a 96-well plate. Transfection of plasmids was performed using Fu-gene6 (Roche, Basel, Switzerland), small interfering RNA (siRNA)transfection was performed using Lipofectamine2000 (Invitrogen,Carlsbad, CA). For all procedures, standard protocols were used ac-cording to the manufacturers’ manuals.

*Institute of Clinical Molecular Biology and ‡Biochemical Institute, Christian-Albrechts University; and †Institute for Infection Medicine and xFirst Departmentfor General Internal Medicine, University Hospital Schleswig-Holstein, CampusKiel, Kiel, Germany

1S.K. and A.T. contributed equally to this work.

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

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 “Inflam-mation at Interfaces” and “The Future Ocean” (to A.T., S.S., H.F., and P.R.). Work onCMV is supported by Deutsche Forschungsgemeinschaft Grant Wi1725 (to M.W.).

Address correspondence and reprint requests to Dr. Philip Rosenstiel, Institute ofClinical Molecular Biology, Christian-Albrechts University, Schittenhelmstr. 12, D-24105 Kiel, Germany. E-mail address: p.rosenstiel@mucosa.de

The online version of this paper contains supplemental material.

Abbreviations used in this paper: CARD, caspase-recruitment domain; FKBP, FK506binding protein; GAS, IFN-g activation sequence; ISRE, IFN-specific response ele-ment; LRR, leucine-rich repeat; NACHT, NAIP, CIITA, HET-E, and TP1 domain;HFF, human foreskin fibroblast; NLR, nucleotide-binding oligomerization domain-like receptor; Nod, nucleotide-binding oligomerization; PAMP, pathogen-associatedmolecular pattern; poly I:C, polyinosinic-polycytidylic acid; siRNA, small interferingRNA.

Copyright� 2010 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/10/$16.00

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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.

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Bacterial and viral infection

Infection of different epithelial cell lines with Listeria monocytogenesserotype 1/2a strain EGD was performed as described before (16). HFFcells were infected with human CMVAD169-BAC as described (17, 18).To inactivate and remove extracellular viral particles, HFF cells wereadditionally washed with citrate buffer (pH 3.0).

Plasmid construction

Expression plasmid for NLRC5 was generated by amplifying full-lengthcDNA coding for NLRC5 from pancreas cDNA and inserting cDNA codingfor NLRC5 into destination vectors by Gateway cloning (Invitrogen). Thefollowing primers were used for full-length amplification: forward 59-ATGGACCCCGTTGGCCTCCA-39 and reverse 59-TCAAGTACCC-CAAGGGGCCTGG-39. The N-terminal region of NLRC5 coding regioncontaining the predicted CARD domain (bases 1–663) was cloned intopC4M-Fv2E vector (Ariad, Cambridge, MA) to obtain an FK506 bindingprotein (FKBP) fusion construct (CARD-FKBP) by using restriction sitesEcoRI and XbaI. Primers were as follows: forward 59-ATGGACCCC-GTTGGCCTCCAGCT-39 and reverse 59-CGGGCCCTTGTTAACCC-TGGT-39. Expression constructs for NLRC5 are illustrated in theSupplemental Fig. 2. The control plasmids encoding GFP and GFP-NOD2have been described before (16). The promoter region of NLRC5 spanningbasepairs 21 to 21673 (relative to the first exon) was amplified fromhuman genomic DNA and inserted into pGL3-Basic vector (Promega,Madison, WI) by using restriction sites XhoI and HindIII and the followingprimers: forward 59-GAGGAGGTCACTCTTCCAGAACC-39 and reverse59-GCTCTGCACGAAACTGAAAGTAGAAG-39. For bioinformatic analysisof the NLRC5 promoter sequence, the MatInspector software (Genomatix,Munchen, Germany; www.genomatix.de/) was used. For specific muta-genesis 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-GGACTTCATGCACAGTG-GGACTTCATGTA-39 and reverse primer 59-TACATGAAGTCCCACT-GTGCATGAAGTCC-39 were used; for deletion of the single STAT bindingsite, forward primer 59-TCAGCCAGGGGTAGGAGCGAAC-39 and reverseprimer 59-GTTCGCTCCTACCCCTGGCTGA-39 were used. The resultingdeletion constructs were termed pGL3-DkB/STAT and pGL3-DSTAT, re-spectively. An additional construct harboring both deletions was termedpGL3-DSTAT/STAT.

Abs and reagents

Antiserum against NLRC5 was generated by a commercial supplier (Euro-gentec, Seraing, Belgium). In short, the NLRC5-specific peptideCGQIENLSFKSRK was used for immunization of rabbit host animals togenerateanmAbdirectedagainstNLRC5protein.Specificityof theantiserumwas tested by immunoblotting of lysates containing overexpressed fusionproteins GFP-NLRC5, GFP-NOD2, or GFP alone. Only GFP-NLRC5–containing lysates revealed a specific band using anti-NLRC5 Ab. No signal

could be detected using the preimmune serum (data not shown). The an-tagonistic IFN-g Ab IP-500 was purchased from Genzyme (Cambridge,MA). MitoTracker and LysoTracker staining solutions were purchased fromInvitrogen. DAPI was from Sigma-Aldrich (St. Louis, MO). JAK inhibitor 1was purchased fromCalbiochem (SanDiego, CA) andwas comparedwith itsdiluent DMSO as negative control. The oligomerizer AP20187 was fromAriad. Polyinosinic-polycytidylic acid (poly I:C) that serves as surrogatestimulus for CMV infection was provided by Calbiochem.

Luciferase assay

For quantification of promotor transcactivation and pathway-specificreportergen activity, transient transfection was used. HeLaS3 cells wereseeded in a 96-well plate at a density of 1.53 104 perwell. The next day, cellswere transfectedwith 40 ngwild-type promoter construct (pGL3-NLRC5) orthe deletion constructs (pGL3-DkB/STAT, pGL3-DSTAT) in combinationwith 10 ng reference plasmid pRL-TK (Promega). After 24 h, cells werestimulated 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 di-peptide (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 in-fection = 100 bacteria/cell). For analysis of signaling events downstream ofNLRC5, pathway-specific cis-reporter constructs pAP1-LUC, pNF-kB-LUC, pISRE-LUC, and pGAS-LUC (Clontech, Palo Alto, CA) were used.The pathway-specific reporter gene constructs (22.5 ng) were cotransfectedwith either GFP-NLRC5 fusion construct, NLRC5-FKBP fusion construct,or the appropriate empty control vectors (22.5 ng) in combination with pRL-TK (5 ng). After 24 h, the oligomerizer AP20187 was added at 0.1 mM.Transfection of siRNAwas performed 24 h prior to transfection of luciferasevectors. Intracellular delivery of poly I:C was performed as previously de-scribed (19). Dual luciferase assaywas performed using the Dual-LuciferaseReporter Assay System (Promega) and a MicroLumatPlus Luminometer(Berthold Technologies, Bad Wildbad, Germany).

Native gel electrophoresis

Native glycine polyacrylamide gels were used for analysis of NLRC5-FKBP oligomerization upon addition of exogenous dimerizer AP20187.Native protein complexes were blotted onto polyvinylidene difluoridemembranes (Millipore, Billerica, MA) as described below.

SDS-PAGE and immunoblotting

Cells (53 105 cells/well) were grown in a six-well plate overnight, followedby transfection and stimulation as indicated. After washing in ice-cold PBS,cells were pelleted and lysed in buffer containing 1% SDS, 10 mM Tris (pH7.6), 1mMNa3VO4, andmixtures for inhibitionofphosphatases andproteases(Sigma-Aldrich) followed by boiling for 5 min and sonification. Lysed cellswere cleared from debris by centrifugation, and supernatants were collected.A total of 15 mg protein extract was separated by denaturing SDS-PAGE and

FIGURE 2. A, Subcellular localization of NLRC5.

GFP-NLRC5 was overexpressed in HeLaS3 cells.

Nuclear DNA was counterstained using DAPI. Fluo-

rescence microscopy reveals a spot-like cytosolic

localization. B, Characterization of an Ab raised

against NLRC5. Overexpression of GFP-NLRC5 fu-

sion 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 anti-

NLRC5 Ab, whereas anti-GFP Ab exhibited strong

signals as expected. C, Using the evaluated Ab, cy-

tosolic localization of endogenous NLRC5 protein

could be confirmed. Irrelevant primary Ab was used

as negative control.

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transferred onto polyvinylidene difluoride membranes (Millipore). Afterblocking, membranes were probed with specific primary Abs, washed, andincubated with HRP-conjugated IgG as secondary Ab. Proteins were visual-ized by chemiluminescence (Amersham Biosciences, Piscataway, NJ). Todetermine even transfer and equal loading, membranes were stripped andreprobed with Ab specific for b-actin (Sigma-Aldrich).

Fluorescence microscopy

HeLaS3cellswere seededon sterile coverslips at 23105 cells/well in six-wellplates and grown overnight. The next day, cells were transiently transfectedwith overexpression plasmid encodingGFP-NLRC5.After 24 h, the cellswerewashed 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 stainedwith anti-NRLC5 Ab (1:250 in PBS/BSA) (or irrelevant primary Ab as neg-

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

RNA isolation and RT-PCR

Total RNAwas isolated from human cell lines using the RNeasy kit (Qiagen,Valencia,CA),and250ngtotalRNAwasreverse-transcribedtocDNAusingtheAdvantage RT-for-PCR kit (Clontech). Resulting cDNAwas analyzed usingstandard semiquantitative RT-PCR procedures and the following primers: 59-TGACTTCCTTCTGCGTCTGCACAG-39 (NLRC5-for), 59-CATTGTCGG-CAAGCTCCTGGAG-39 (NLRC5-rev), 59-ATTCAGCTCTCTGGGCTGTG-ATC-39 (IFN-a-for), 59-CATCACACAGGACTCCAGGTCATT-39 (IFN-a-rev),59-ATGCCAGAAGTGGGTGGAGAAC-39 (PKRIR-for), 59-AGCCTCATG-TCCATCCAGAGGTAT-39 (PKRIR-rev), 59-TGGCTCCTCAGGCAAGG-GC-39 (OAS1-for), 59-TGGTACCAGTGCTTGACTAGGCGG-39 (OAS1-

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

mRNAwas 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.

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

Real-time PCR

cDNA derived from stimulation and infection experiments was analyzed forNLRC5 and IL-8 mRNA levels using TaqMan Gene Expression Assays(NLRC5; Hs00260008_m1, IL-8; Hs00174103_m1; housekeeping geneACTB; Hs99999903_m1; Applied Biosystems, Foster City, CA) on the ABIPrism 7900HT Fast Sequence Detection System (Applied Biosystems). Allother target genes were quantified using the primer sets given above and theSYBR Green PCR Master Mix System (Applied Biosystems). Transcriptamounts were normalized to those of the housekeeping gene b-actin usingthe following primer pair: ACTB-F 59-GATGGTGGGCATGGGTCAG-39and ACTB-R 59-CTTAATGTCACGCACGATTTCC-39. Acquired real-timePCR data were analyzed using the 22DDCt method (20). Fold changes werecalculated based on the ratio between treated sample and untreated control;p values were generated using the Mann-Whitney U test. Transcripts wereconsidered differentially expressed when the p value was #0.05.

siRNA-mediated knockdown of NLRC5

Synthetic siRNAs targeting NLRC5 were purchased from Applied Bio-systems/Ambion. Target sequences were as follows: siRNA1 (ID 141058),F: 59-CGGGUGGAAGAAUAUGUGAtt-39, R: 59-UCACAUAUUCUUC-CACCCGtg-39; siRNA2 (ID 141060), F: 59-CCAACAUCCUAGAGCAC-AGtt-39, R: 59-CUGUGCUCUAGGAUGUUGGtc-39; siRNA3 (ID 141059),F: 59-GCAGACAGGCUAUGCUUUCtt-39, R: 59-GAAAGCAUAGCCU-GUCUGCtg-39. As control, unspecific siRNANegative Control #2 (AppliedBiosystems/Ambion) was used.

ResultsGenomic organization and expression of NLRC5

In the human genome, NLRC5 is located at the locus 16q13, span-ning a region of ∼96 kbp. The gene contains 39 exons, and thetranslation start site is located in the third exon. The gene structure isconserved among many vertebrates and seems to have occurred by

a single gene duplication event early in mammalian evolution(Supplemental Fig. 1). The predicted protein consists of 1866 aasand shows the typical tripartite domain structure characterizingNLR family members (21). The N terminus comprises a predictedCARD domain spanning amino acids 1–91. The predicted NACHTdomain is located between position 220 and 383. The predicted C-terminal LRR region consists of 713 aa residues and shows an un-usual architecture compared with other NLRs, because the 20 LRRunits 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 tissuepanels for RT-PCR analysis. NLRC5 mRNA is expressed ubiqui-tously, with highest expression levels in the brain, lung, and prostate(Fig. 1C) and medium to high expression in a variety of tissues in-cluding the heart, digestive tract, and thymus. The lowest expressionwas detected in spleen, lymph nodes, and leukocytes. To assess thesubcellular localization of the NLRC5 protein, we generated a GFP-NLRC5 fusion construct for transient transfection of HeLaS3 cells.Fluorescence microscopy revealed a spot-like expression of theGFP-NLRC5 fusion protein in the cytosol (Fig. 2A). Using Lyso-Tracker and MitoTracker staining (Invitrogen), we found no evi-dence for lysosomal or mitochondrial localization of NLRC5(Supplemental Fig. 3 and data not shown). Next, we used peptideimmunization to raise an Ab against the NLRC5 protein. Validationof the Ab using control plasmids (Fig. 2B) showed that neither GFPnor GFP-NOD2 were recognized by the NLRC5 Ab, but a specificsignal was detectable that corresponds to the predicted 230-kDa sizeof GFP-NRLC5. Interestingly, all experiments revealed the exis-tence of a double-band pattern that might be caused by translationalmodifications of yet unknown nature. Using this validated Ab, wecould demonstrate that NLRC5 protein expression is restricted tothe cytosolic compartment (Fig. 2C).

NLRC5 expression is specifically regulated by IFN-g

For characterization of the transcriptional regulation of NLRC5, thepromoter region was cloned into the pGL3B luciferase reportergene vector. The resulting reporter gene construct (pGL3-NLRC5)

FIGURE 4. Regulatory elements

controlling NLRC5 promoter activ-

ity. A, The NLRC5 promoter region

contains two predicted STAT con-

sensus binding sites (one of which is

overlapping with a predicted NF-kB

consensus site). Individual and

combined deletion of these two reg-

ulatory elements significantly im-

pairs the IFN-g response of the

promoter construct. pp , 0.05. Note

that the empty vector backbone ac-

tivity (vector control) accounts for

,10% of the NLRC5 promoter

construct. B, Preincubation with

pharmacological JAK inhibitor re-

duces the regulatory influence of

IFN-g on NLRC5 promoter activity.

pp , 0.05. All experiments were

performed in triplicate.

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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.

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was transfected into HeLaS3. Baseline reporter gene expressionby the empty vector backbone accounted for,10% of the NLRC5promoter construct (data not shown). Cells were stimulated with10 different stimuli, including cytokines and a panel of PAMPs.Alternatively, cells were infected with cytoinvasive Listeriamonocytogenes to check for direct response to bacterial infection.As shown in Fig. 3A and 3B, only stimulation with IFN-g resultedin a significant increase in luciferase activity, whereas none of theother stimuli, including IFN-a and IFN-b, affected NLRC5 pro-moter transactivation. These findings were supported by quanti-tative real-time PCR data showing that NLRC5 mRNA levels weresignificantly 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, thiseffect was dose-dependent, as shown by RT-PCR and quantitativereal-time PCR (Fig. 3D). As control, quantification of IL-8 mRNAlevels was used to demonstrate responsiveness of the cells to thevarious stimuli (Supplemental Fig. 5A–C). Using the validatedanti-NLRC5 Ab, we could demonstrate that NLRC5 protein ex-

pression exhibits the same response to IFN-g stimulation becauseNLRC5 protein levels were remarkably increased 6 to 12 h afterstimulation with IFN-g (Fig. 3E).Next, we aimed to understand the regulatory mechanisms un-

derlying the IFN-g–dependent effect on NLRC5 expression. Usingbioinformatical prediction tools, the NLRC5 promoter region wasanalyzed for the presence of potential cis-regulatory elements. Thecomputational analysis revealed the presence of two STAT consensusbinding sites. The STAT family of transcription factors representsa central component of multiple signal transduction cascades, in-cluding the IFN and IL-6 pathways (22, 23). The NLRC5 promoterregion contains two individual potential STATbinding sites located atpositions21336 and2452, respectively. Interestingly, the STAT siteat 21336 partially overlaps with a predicted NF-kB consensusbinding site. To further analyze a possible involvement of thesebinding sites in NLRC5 promoter activation, we created deletionconstructs by site-directed mutagenesis. Individual or combined de-letion of the predicted regulatory elements resulted in significantdecrease of IFN-g–dependent promoter transactivation (Fig. 4A).

FIGURE 6. Signal transduction events

downstreamofNLRC5.Pathway-specific

cis-reporter gene constructs (A–D) were

cotransfected in combination with either

GFP-tagged NLRC5, CARD-FKBP fu-

sion construct, or the appropriate control

vectors. In addition, FKBP-expressing

cells were stimulated with exogenous

oligomerizer AP20187 to enforce oligo-

merization of the (FKBP)2 domain. Al-

though 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 en-

forced oligomerization of the CARD do-

main of NLRC5 (C and D). Values

represent mean of relative luciferase ac-

tivity. pp , 0.05; ppp , 0.01. All ex-

periments were performed in triplicate.

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Moreover, pretreatment with a specific inhibitor of JAK activity priorto IFN-g stimulation decreased promoter transactivation almostcompletely (Fig. 4B). Because JAK activity is essential for phos-phorylation and nuclear translocation of STATs (24), these data fur-ther emphasize the role of IFN-g–dependent JAK/STAT signaling forNLRC5 transcriptional regulation .

Viral infection contributes to transcriptional regulation ofNLRC5 via autocrine IFN-g

The secretion of the cytokine IFN-g is a pivotal cellular response toviral infection (25). In order to elucidate whether the expression ofNLRC5 is directly linked toviral infection,we used awell-establishedviral infection model that is based on the infection of HFFs withhuman CMV (17, 18). To differentiate between effects that originatefrom direct viral infection and effects depending on sole presence ofinactive virus particles, heat-inactivated virus particles were used inaparallel experimental setup. Six, 12, or 24hpostinfectionwithCMV,expression levels of NLRC5 mRNA were checked by quantitativereal-time PCR. Efficiency of the viral infection was checked by

concomitant detection of CMV-IE2. As shown in Fig. 5A, a strongincrease inNLRC5 expressionwas detectable thatwas peaking at 12 hpostinfection. Remarkably, only intact virus particleswere capable ofinducing this effect, whereas heat-inactivated CMV did not alterNLRC5 mRNA levels within 24 h (Fig. 5A). Thus, the ability to ef-ficiently infect human cells appears to be a prerequisite for regulationof NLRC5 expression.To investigate if autocrine JAK/STAT signaling in response to in-

fection is involved in these regulatory events, we infected cells inpresence of JAK inhibitor to block JAK/STAT signaling. Blockade ofJAK-dependent pathways resulted in almost complete abrogation ofNLRC5 upregulation (Fig. 5B). These results were confirmed by thefact that the supernatant of infected cells was able to upregulateNLRC5 mRNA in cells that were not infected (Fig. 5C). To analyzewhether autocrine IFN-g secretion initiated by viral infection is re-sponsible for regulation ofNLRC5, we blocked endogenous IFN-g byaddition of an inhibitory Ab. As shown in Fig. 5D, supernatant ofinfected cells preincubated with anti–IFN-g Ab resulted in impairedinduction 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.

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NLRC5 oligomerization activates IFN-specific responseelement- and IFN-g activation sequence-dependent signaltransduction pathways

Next we asked which signaling pathways are activated downstreamofNLRC5 activation. Because it is a common phenomenon ofNLRsthat ectopic overexpression leads to ligand-independent autoacti-vation of downstream signaling events (3), we used overexpressionof GFP-NLRC5 to screen for potential signaling cascades initiatedby NLRC5 activity. In addition, we overexpressed a fusion proteinconsisting of the N-terminal CARD of NLRC5 fused (FKBP)2,whose oligomerization could be exogenously triggered by additionof the synthetic oligomerizer AP20187 (for illustration, see Sup-plemental Fig. 6). As shown by native glycine gel analyses, additionof the exogenous oligomerizer resulted in enforced oligomerizationof CARD-FKBPmonomers (Supplemental Fig. 7). Next, bothGFP-NLRC5 and CARD-FKBP constructs were expressed in parallelwith a number of pathway-specific reporter gene constructs thatcould monitor the activation of specific regulatory promoter ele-ments (Fig. 6). No activation of AP-1 or NF-kB signaling pathwayswas detectable (Fig. 6A, 6B). This was surprising because severalmembers of the NLR family have been shown to directly activateNF-kB and/or MAPK signaling cascades (26–28). In contrast, ourdata point to a specific role of NLRC5 for the activation of signalingpathways that uses the consensus binding sites IFN-specific re-sponse element (ISRE) and IFN-g activation sequence (GAS) (Fig.6C, 6D). These regulatory elements represent the major bindingsites for STAT family members. Supporting these data, we dem-onstrate increased and prolonged phosphorylation of STAT1downstream of IFN-g in the presence of overexpressed NLRC5(Supplemental Figs. 8, 9).

In order to analyze whether this activation profile was alsodetectable on the target gene level, we overexpressed GFP-NLRC5in epithelial cells and used RT-PCR and real-time PCR to char-acterize potential NLRC5-dependent downstream target genes. Asshown in Fig. 7A, the overexpression resulted in remarkable in-crease in mRNA levels of IFN-a, PRKRIR, and OAS1 24 h aftertransfection, whereas expression of IL-8 was not affected. Next,we checked the influence of siRNA-mediated knockdown ofNLRC5 on the target gene level. As demonstrated in Fig. 7B, ef-ficient knockdown of NLRC5 by three individual siRNAs results inreduction of endogenous mRNA levels of the investigated targetgenes as compared with control. To analyze whether this effect isalso relevant in the context of viral infection, we next transfectedHFF cells with siRNA and quantified expression of IFN-a in re-sponse to CMV. siRNA-mediated knockdown of NLRC5 signifi-cantly reduces induction of IFN-a after CMV infection (Fig. 8A),further emphasizing the role of NLRC5 as an essential componentof antiviral signaling pathways. Supporting this view, NLRC5knockdown also significantly reduces induction of GAS-drivenluciferase 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 pathwayscontributing to antiviral defense mechanisms.

DiscussionIn this report, we describe the genomic organization, regulation,and aspects of cellular function of the NLR family memberNLRC5. This gene family has attracted much attention becausemutations in several NLR genes have been linked to a variety ofinflammatory 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.

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of NOD2 are associated with susceptibility for Crohn’s disease orBlau syndrome (7–10, 30), whereas NLRP3/NALP3 has beenlinked to rare hereditary fever syndromes and Crohn’s disease(11–14, 31, 32). These genetic findings underscore the importanceof NLRs as surveillance proteins for host defense and immunityand point to their potential as novel therapeutic targets (33).Among the NLR family members identified and characterizedso far, NLRC5 displays an unusual architecture of its carboxy-terminal LRR because its repeat units are irregularly scatteredalong the domain. The LRR domain of NLR is generally con-sidered to represent the sensor region of the proteins. The mo-lecular structures that are recognized by NLR proteins representeither conserved molecular signatures of bacteria (PAMPs) orendogenous molecules that arise from conditions associated withcellular danger (danger-associated molecular patterns) (34). In-terestingly, some NLRs are described to constitute highly selectivesensor platforms that only respond to a specific molecular elicitor,whereas others exhibit a promiscuous selectivity by responding toa variety of stimuli. For example, NLRC4/IPAF responds to cy-tosolic presence of the bacterial protein flagellin, whereas NLRP3/NALP3 is activated by various endogenous danger-associatedmolecular patterns like monosodium urate crystals or low potas-sium ion concentration (35). Nevertheless, it is still unclearwhether this response is caused by direct physical interactions ofthe respective NLR with its elicitor or indirectly by mediators ofunknown nature. The identification of physiological elicitors ofNLR signaling and the dissection of the exact interaction mode ofputative ligand and cognate receptor still remain important tasksfor future studies. Because both the identity of a potential physi-ological elicitor of NLRC5 and the downstream signaling eventswere unknown, we used enforced oligomerization of an FKBPfusion protein and overexpression to simulate cellular events thattrigger oligomerization of NLRC5. Our data demonstrate thatNLRC5 oligomerization can efficiently trigger GAS- and ISRE-dependent promoter activity. In subsequent experiments, a panelof known PAMPs was used to test stimulus specificity of NLRC5-dependent GAS/ISRE activation. However, from the tested com-pounds (e.g., LPS, flagellin, single-stranded RNA, muramyl di-peptide), the biochemical identity of a potential endogenousNLRC5 elicitor could not be elucidated (data not shown).Our data demonstrate a direct link between signaling events

triggered by viral infection and transcriptional regulation ofNLRC5 and argue for autocrine IFN-g activating JAK/STAT sig-naling as a key player of this autoregulatory loop. Moreover, weshow that activation of NLRC5 implies specific activation of ISREand GAS elements and upregulation of IFN-a, PRKRIR, andOAS1 without affecting transcription of chemokine IL-8. Thesefindings are underscored by our data demonstrating that knock-down of NLRC5 significantly impairs induction of these targetgenes in the context of CMV infection. IFN-a transcriptionalregulation has been shown to be dependent on JAK/STAT sig-naling via the IFN-stimulated gene factor 3 transcription factorcomplex (36). PRKRIR encodes for a protein that was first iden-tified as a regulator of P58(IPK), a cellular inhibitor of the RNA-dependent protein kinase (37). In addition, OAS proteins representa group of IFN-inducible enzymes contributing to activation ofRNAseL that degrade viral RNAs and thus inhibit viral replica-tion. Conversely, IL-8 transcriptional regulation, which was usedas a control, is mainly dependent on NF-kB and AP-1 trans-activation activity, thus supporting our data on a specific inductionof GAS/ISRE by NLRC5 activation. Given the regulatory role ofIFN-g on NLRC5 gene regulation, these data argue for a specificrole of NLRC5 as an endogenous amplifier of antiviral signalingpathways.

By now, only few members of the NLR family have been as-sociated with antiviral immune responses, whereas the majority ofNLRs have been shown to contribute to innate defense mechanismsagainst pathogenic bacteria (38–40). The recently characterizedNLR family member NLRX1 interacts with mitochondrial anti-viral signaling adaptor in the outer membrane of mitochondria andrepresents a checkpoint modulating mitochondrial antiviral re-sponses (41). Furthermore, NLRX1 is capable of amplifying NF-kB and JNK pathways activated by different proinflammatorystimuli via production of reactive oxygen species (42). A recentstudy reports on the specific detection of influenza virus bycomponents of the ASC/NLRP3 inflammasome, but the directrequirement for NLRP3 in the sensing process is still under debate(43). Our data add NLRC5 to the list of NLR family membersinvolved in mechanisms that respond to viral infection and em-phasize the role of NLRs as general cytosolic surveillance proteinsinvolved in both bacterial and viral infection and endogenousdanger response.In summary, we have characterized the genomic organization of

NLRC5 and describe complex regulatory mechanisms identifyingNLRC5 as target gene of IFN-g and mediator of IFN-mediatedantiviral signaling pathways. Further analyses on NLRC5 regula-tion and function will help us to fully understand the impact ofNLR family members on molecular defense strategies againstviral pathogens.

AcknowledgmentsWe thank Tanja Kaacksteen, Melanie Schlapkohl, Dorina Oelsner, Yasmin

Brodtmann, and Alina Graff for their technical assistance.

DisclosuresThe authors have no financial conflicts of interest.

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