IFN-Dependent Antiviral Immune Responses in Domain-Like

The Nucleotide Oligomerization Binding Domain-Like Receptor NLRC5 Is Involved in IFN-Dependent Antiviral Immune Responses

This information is current as Sven Kuenzel, Andreas Till, Michael Winkler, Robert of September 28, 2021. Häsler, Simone Lipinski, Sascha Jung, Joachim Grötzinger, Helmut Fickenscher, Stefan Schreiber and Philip Rosenstiel J Immunol published online 8 January 2010 http://www.jimmunol.org/content/early/2010/01/08/jimmun

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 8, 2010, doi:10.4049/jimmunol.0900557 The Journal of Immunology

The Nucleotide Oligomerization Binding 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*

Nucleotide oligomerization binding 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 Downloaded from 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 http://www.jimmunol.org/ 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: 000–000.

he nucleotide oligomerization binding domain (Nod)-like lecular patterns (PAMPs) and other danger signals are directly or receptor (NLR) family represents a group of intracellular indirectly recognized by the LRRs. This recognition process in- T sensors of the innate immune system. At least 22 members duces NLR oligomerization and activation of NLR-dependent sig- have been identified within the human genome (1, 2). The family nal transduction pathways via induced-proximity signaling (3). In by guest on September 28, 2021 members are characterized by three distinct functional domains: an particular, activation of NF-kB, MAPKs, and proinflammatory N-terminal effector-binding domain, a centrally located NAIP, caspases have been implicated as downstream events of NLR acti- CIITA, HET-E, and TP1 domain (NACHT) and a carboxy-terminal vation (4–6). Interestingly, several NLR family members (e.g., leucine-rich repeat (LRR) domain. The effector-binding domain is NOD1, NOD2, NLRP3/NALP3, NLRP1/NALP1, NLRA/MHC usually composed of caspase-recruitment domain(s) (CARD), class II transcription activator) have been shown to contribute to a Pyrin domain, or a baculovirus inhibitor of apoptosis protein do- susceptibility for chronic inflammatory diseases (7–15). Thus, it is main. Current knowledge suggests that pathogen-associated mo- assumed that NLR proteins represent pivotal components of the innate immune system and that impaired NLR function is closely linked to pathological conditions. *Institute of Clinical Molecular Biology and ‡Biochemical Institute, Christian- Albrechts University; and †Institute for Infection Medicine and xFirst Department In this report, we describe the genomic organization, regulation, for General Internal Medicine, University Hospital Schleswig-Holstein, Campus and cellular function of NLRC5 (also referred to as NOD27). Kiel, Kiel, Germany 1S.K. and A.T. contributed equally to this work. Materials and Methods Received for publication February 17, 2009. Accepted for publication December 5, Cell lines and transfection 2009. This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 617-A20 Human cervical carcinoma HeLaS3 cells (ACC161), human acute (to P.R.) and Grant SFB 617-A24 (to H.F.) and the Clusters of Excellence “Inflam- monocytic cell line THP-1 (ACC16), human colonic carcinoma cell lines mation at Interfaces” and “The Future Ocean” (to A.T., S.S., H.F., and P.R.). Work on CaCo2 (ACC169), and HT-29 (ACC299) were purchased from the Ger- CMV is supported by Deutsche Forschungsgemeinschaft Grant Wi1725 (to M.W.). man Collection of Microorganisms and Cell Cultures (DSMZ, Address correspondence and reprint requests to Dr. Philip Rosenstiel, Institute of Braunschweig, Germany). Human foreskin fibroblasts (HFFs) were iso- Clinical Molecular Biology, Christian-Albrechts University, Schittenhelmstr. 12, D- lated from human foreskin and cultivated up to passage number 25 in 24105 Kiel, Germany. E-mail address: [email protected] 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 The online version of this paper contains supplemental material. (Paschberg, Austria). Media were supplemented with 10% FCS and Abbreviations used in this paper: CARD, caspase-recruitment domain; FKBP, FK506 penicillin/streptomycin (each at 50 mg/ml); all cell lines were grown in binding protein; GAS, IFN-g activation sequence; ISRE, IFN-specific response ele- 5% CO2 at 37˚C. One day before transfection, cells were typically seeded ment; LRR, leucine-rich repeat; NACHT, NAIP, CIITA, HET-E, and TP1 domain; at a density of 5 3 105 cells/2 ml on six-well plates or at 2 3 104/100 ml HFF, human foreskin fibroblast; NLR, nucleotide oligomerization binding domain- like receptor; Nod, nucleotide oligomerization binding domain; PAMP, pathogen- on a 96-well plate. Transfection of plasmids was performed using Fu- associated molecular pattern; poly I:C, polyinosinic-polycytidylic acid; siRNA, small gene6 (Roche, Basel, Switzerland), small interfering RNA (siRNA) interfering RNA. transfection was performed using Lipofectamine2000 (Invitrogen, Carlsbad, CA). For all procedures, standard protocols were used ac- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 cording to the manufacturers’ manuals.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900557 2 CHARACTERIZATION OF NLRC5 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

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. The Journal of Immunology 3

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 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 anti- NLRC5 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. Downloaded from

Bacterial and viral infection could be detected using the preimmune serum (data not shown). The an-

tagonistic IFN-g Ab IP-500 was purchased from Genzyme (Cambridge, http://www.jimmunol.org/ Infection of different epithelial cell lines with Listeria monocytogenes MA). MitoTracker and LysoTracker staining solutions were purchased from serotype 1/2a strain EGD was performed as described before (16). HFF Invitrogen. DAPI was from Sigma-Aldrich (St. Louis, MO). JAK inhibitor 1 cells were infected with human CMV AD169-BAC as described (17, 18). was purchased from Calbiochem (San Diego, CA) and was compared with its To inactivate and remove extracellular viral particles, HFF cells were diluent DMSO as negative control. The oligomerizer AP20187 was from additionally washed with citrate buffer (pH 3.0). Ariad. Polyinosinic-polycytidylic acid (poly I:C) that serves as surrogate Plasmid construction stimulus for CMV infection was provided by Calbiochem. Luciferase assay Expression plasmid for NLRC5 was generated by amplifying full-length cDNA coding for NLRC5 from pancreas cDNA and inserting cDNA coding For quantification of promotor transcactivation and pathway-specific for NLRC5 into destination vectors by Gateway cloning (Invitrogen). The reportergen activity, transient transfection was used. HeLaS3 cells were by guest on September 28, 2021 following primers were used for full-length amplification: forward 59- seeded in a 96-well plate at a density of 1.5 3 104 per well. The next day, cells ATGGACCCCGTTGGCCTCCA-39 and reverse 59-TCAAGTACCC- were transfected with 40 ng wild-type promoter construct (pGL3-NLRC5) or CAAGGGGCCTGG-39. The N-terminal region of NLRC5 coding region the deletion constructs (pGL3-DkB/STAT, pGL3-DSTAT) in combination containing the predicted CARD domain (bases 1–663) was cloned into with 10 ng reference plasmid pRL-TK (Promega). After 24 h, cells were pC4M-Fv2E vector (Ariad, Cambridge, MA) to obtain an FK506 binding stimulated with epidermal growth factor (100 ng/ml), TNF-a (10 ng/ml), protein (FKBP) fusion construct (CARD-FKBP) by using restriction sites LPS (10 mg/ml), flagellin (500 ng/ml), TGF-b (25 ng/ml), muramyl di- EcoRI and XbaI. Primers were as follows: forward 59-ATGGACCCC- peptide (50 mg/ml), peptidoglycan of Staphylococcus aureus (10 mg/ml), GTTGGCCTCCAGCT-39 and reverse 59-CGGGCCCTTGTTAACCC- IFN-g (1000 U/ml), IFN-a (subtype A/D hybrid; 1000 U/ml), or IFN-b TGGT-39. Expression constructs for NLRC5 are illustrated in the (1000 U/ml) or infected with Listeria monocytogenes (multiplicity of in- Supplemental Fig. 2. The control plasmids encoding GFP and GFP-NOD2 fection = 100 bacteria/cell). For analysis of signaling events downstream of have been described before (16). The promoter region of NLRC5 spanning NLRC5, pathway-specific cis-reporter constructs pAP1-LUC, pNF-kB- basepairs 21to21673 (relative to the first exon) was amplified from LUC, pISRE-LUC, and pGAS-LUC (Clontech, Palo Alto, CA) were used. human genomic DNA and inserted into pGL3-Basic vector (Promega, The pathway-specific reporter gene constructs (22.5 ng) were cotransfected Madison, WI) by using restriction sites XhoI and HindIII and the following with either GFP-NLRC5 fusion construct, NLRC5-FKBP fusion construct, primers: forward 59-GAGGAGGTCACTCTTCCAGAACC-39 and reverse or the appropriate empty control vectors (22.5 ng) in combination with pRL- 59-GCTCTGCACGAAACTGAAAGTAGAAG-39. For bioinformatic analysis TK (5 ng). After 24 h, the oligomerizer AP20187 was added at 0.1 mM. of the NLRC5 promoter sequence, the MatInspector software (Genomatix, Transfection of siRNAwas performed 24 h prior to transfection of luciferase Mu¨nchen, Germany; http://www.genomatix.de/) was used. For specific vectors. Intracellular delivery of poly I:C was performed as previously de- mutagenesis of the constructs, the QuickChange site-directed mutagenesis scribed (19). Dual luciferase assay was performed using the Dual-Luciferase kit (Stratagene, La Jolla, CA) was used. For deletion of the combined kB/ Reporter Assay System (Promega) and a MicroLumatPlus Luminometer STAT binding site, the forward primer 59-GGACTTCATGCACAGTG- (Berthold Technologies, Bad Wildbad, Germany). GGACTTCATGTA-39 and reverse primer 59-TACATGAAGTCCCACT- GTGCATGAAGTCC-39 were used; for deletion of the single STAT binding Native gel electrophoresis site, forward primer 59-TCAGCCAGGGGTAGGAGCGAAC-39 and reverse primer 59-GTTCGCTCCTACCCCTGGCTGA-39 were used. The resulting Native glycine polyacrylamide gels were used for analysis of NLRC5- deletion constructs were termed pGL3-DkB/STAT and pGL3-DSTAT, re- FKBP oligomerization upon addition of exogenous dimerizer AP20187. spectively. An additional construct harboring both deletions was termed Native protein complexes were blotted onto polyvinylidene difluoride pGL3-DSTAT/STAT. membranes (Millipore, Billerica, MA) as described below. Abs and reagents SDS-PAGE and immunoblotting Antiserum against NLRC5 was generated by a commercial supplier (Euro- Cells (5 3 105 cells/well) were grown in a six-well plate overnight, followed gentec, Seraing, Belgium). In short, the NLRC5-specific peptide by transfection and stimulation as indicated. After washing in ice-cold PBS, CGQIENLSFKSRK was used for immunization of rabbit host animals to cells were pelleted and lysed in buffer containing 1% SDS, 10 mM Tris (pH generateanmAbdirectedagainstNLRC5protein.Specificityoftheantiserum 7.6), 1 mM Na3VO4, and mixtures for inhibitionof phosphatases and proteases was tested by immunoblotting of lysates containing overexpressed fusion (Sigma-Aldrich) followed by boiling for 5 min and sonification. Lysed cells proteins GFP-NLRC5, GFP-NOD2, or GFP alone. Only GFP-NLRC5– were cleared from debris by centrifugation, and supernatants were collected. containing lysates revealed a specific band using anti-NLRC5 Ab. No signal A total of 15 mg protein extract was separated by denaturing SDS-PAGE and 4 CHARACTERIZATION OF NLRC5 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

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 ative control) followed by staining with FITC-conjugated secondary anti- blocking, membranes were probed with specific primary Abs, washed, and rabbit Ab. Slips were transferred onto glass slides and examined using an incubated with HRP-conjugated IgG as secondary Ab. Proteins were visual- apotome-equipped Axio Imager Z1 microscope (Zeiss, Jena, Germany). ized by chemiluminescence (Amersham Biosciences, Piscataway, NJ). To determine even transfer and equal loading, membranes were stripped and RNA isolation and RT-PCR reprobed with Ab specific for b-actin (Sigma-Aldrich). Total RNA was isolated from human cell lines using the RNeasy kit (Qiagen, Fluorescence microscopy Valencia,CA),and250ngtotalRNAwasreverse-transcribedtocDNAusingthe Advantage RT-for-PCR kit (Clontech). Resulting cDNA was analyzed using HeLa S3 cells were seeded on sterile coverslips at 2 3 105 cells/well in six-well standard semiquantitative RT-PCR procedures and the following primers: 59- plates and grown overnight. The next day, cells were transiently transfected TGACTTCCTTCTGCGTCTGCACAG-39 (NLRC5-for), 59-CATTGTCGG- with overexpression plasmid encoding GFP-NLRC5. After 24 h, the cells were CAAGCTCCTGGAG-39 (NLRC5-rev), 59-ATTCAGCTCTCTGGGCTGTG- washed twice in ice-cold PBS, fixed for 20 min at room temperature with 4% ATC-39 (IFN-a-for), 59-CATCACACAGGACTCCAGGTCATT-39 (IFN-a-rev), paraformaldehyde (Carl Roth, Karlsruhe, Germany), and stained with DAPI 59-ATGCCAGAAGTGGGTGGAGAAC-39 (PKRIR-for), 59-AGCCTCATG- (Roche) for 15 min. Alternatively, untransfected cells were fixed and stained TCCATCCAGAGGTAT-39 (PKRIR-rev), 59-TGGCTCCTCAGGCAAGG- with anti-NRLC5 Ab (1:250 in PBS/BSA) (or irrelevant primary Ab as neg- GC-39 (OAS1-for), 59-TGGTACCAGTGCTTGACTAGGCGG-39 (OAS1- The Journal of Immunology 5

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 Downloaded from IFN-g on NLRC5 promoter activity. pp , 0.05. All experiments were performed in triplicate. http://www.jimmunol.org/ rev), 59-ACCGGAAGGAACCATCTCACT-39 (IL-8-for), 59-AAACTTCTC- a single gene duplication event early in mammalian evolution CACAACCCTCTGC-39 (IL-8-rev), 59-CCCTGATAATCCTGACGAGG-39 (Supplemental Fig. 1). The predicted protein consists of 1866 aas (CMV-IE2-for), and 59-GGTTCTCGTTGCAATCCTCGG-39 (CMV-IE2- and shows the typical tripartite domain structure characterizing rev). Housekeeping gene GAPDH was used as control and amplified using the following primers: 59-CCAGCCGAGCCACATCGC-39 (GAPDH-for) NLR family members (21). The N terminus comprises a predicted and 59-ATGAGCCCCAGCCTTCTCCAT-39 (GAPDH-rev). To analyze the CARD domain spanning amino acids 1–91. The predicted NACHT tissue-specific expression pattern of NLRC5, we used commercially avail- domain is located between position 220 and 383. The predicted C- able human cDNA tissue panels (Clontech) and RT-PCR. Relative expres- terminal LRR region consists of 713 aa residues and shows an un- sion was determined by normalization to housekeeping gene GAPDH using by guest on September 28, 2021 densitometry (ImageJ software, http://rsb.info.nih.gov/ij/). usual architecture compared with other NLRs, because the 20 LRR units are scattered irregularly along the domain (Fig. 1A,1B). Real-time PCR To describe the tissue-specific expression pattern of NLRC5,we cDNA derived from stimulation and infection experiments was analyzed for designed exon-spanning primer pairs and used human cDNA tissue NLRC5 and IL-8 mRNA levels using TaqMan Gene Expression Assays panels for RT-PCR analysis. NLRC5 mRNA is expressed ubiqui- (NLRC5; Hs00260008_m1, IL-8; Hs00174103_m1; housekeeping gene tously, with highest expression levels in the brain, lung, and prostate ACTB; Hs99999903_m1; Applied Biosystems, Foster City, CA) on the ABI Prism 7900HT Fast Sequence Detection System (Applied Bio- (Fig. 1C) and medium to high expression in a variety of tissues in- systems). All other target genes were quantified using the primer sets given cluding the heart, digestive tract, and thymus. The lowest expression above and the SYBR Green PCR Master Mix System (Applied Bio- was detected in spleen, lymph nodes, and leukocytes. To assess the systems). Transcript amounts were normalized to those of the house- subcellular localization of the NLRC5 protein, we generated a GFP- keeping gene b-actin using the following primer pair: ACTB-F NLRC5 fusion construct for transient transfection of HeLaS3 cells. GATGGTGGGCATGGGTCAG and ACTB-R CTTAATGTCACGCAC- GATTTCC. Acquired real-time PCR data were analyzed using the 22DDCt Fluorescence microscopy revealed a spot-like expression of the method (20). Fold changes were calculated based on the ratio between GFP-NLRC5 fusion protein in the cytosol (Fig. 2A). Using Lyso- treated sample and untreated control; p values were generated using the Tracker and MitoTracker staining (Invitrogen), we found no evi- Mann-Whitney U test. Transcripts were considered differentially expressed dence for lysosomal or mitochondrial localization of NLRC5 when the p value was #0.05. (Supplemental Fig. 3 and data not shown). Next, we used peptide siRNA-mediated knockdown of NLRC5 immunization to raise an Ab against the NLRC5 protein. Validation Synthetic siRNAs targeting NLRC5 were purchased from Applied Bio- of the Ab using control plasmids (Fig. 2B) showed that neither GFP systems/Ambion. Target sequences were as follows: siRNA1 (ID 141058), F: nor GFP-NOD2 were recognized by the NLRC5 Ab, but a specific CGGGUGGAAGAAUAUGUGAtt, R: UCACAUAUUCUUCCACCCGtg; signal was detectable that corresponds to the predicted 230-kDa size siRNA2 (ID 141060), F: CCAACAUCCUAGAGCACAGtt, R: CUGUG- of GFP-NRLC5. Interestingly, all experiments revealed the exis- CUCUAGGAUGUUGGtc; siRNA3 (ID 141059), F: GCAGACAGGCUA- UGCUUUCtt, R: GAAAGCAUAGCCUGUCUGCtg. As control, unspecific tence of a double-band pattern that might be caused by translational siRNA Negative Control #2 (Applied Biosystems/Ambion) was used. modifications of yet unknown nature. Using this validated Ab, we could demonstrate that NLRC5 protein expression is restricted to Results the cytosolic compartment (Fig. 2C). Genomic organization and expression of NLRC5 In the human genome, NLRC5 is located at the locus 16q13, span- NLRC5 expression is specifically regulated by IFN-g ning a region of ∼96 kbp. The gene contains 39 exons, and the For characterization of the transcriptional regulation of NLRC5, the translation start site is located in the third exon. The gene structure is promoter region was cloned into the pGL3B luciferase reporter conserved among many vertebrates and seems to have occurred by gene vector. The resulting reporter gene construct (pGL3-NLRC5) 6 CHARACTERIZATION OF NLRC5 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

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. The Journal of Immunology 7

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 Downloaded from cells were stimulated with exogenous oligomerizer AP20187 to enforce oligomerization of the (FKBP)2 domain. Al- though NLRC5 does not influence AP-1 or NF-kBactivity(A and B), our data demonstrate a significant enhancement of ISRE- and GAS-dependent reporter gene http://www.jimmunol.org/ 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. by guest on September 28, 2021

was transfected into HeLaS3. Baseline reporter gene expression pression exhibits the same response to IFN-g stimulation because by the empty vector backbone accounted for ,10% of the NLRC5 NLRC5 protein levels were remarkably increased 6 to 12 h after promoter construct (data not shown). Cells were stimulated with stimulation with IFN-g (Fig. 3E). 10 different stimuli, including cytokines and a panel of PAMPs. Next, we aimed to understand the regulatory mechanisms un- Alternatively, cells were infected with cytoinvasive Listeria derlying the IFN-g–dependent effect on NLRC5 expression. Using monocytogenes to check for direct response to bacterial infection. bioinformatical prediction tools, the NLRC5 promoter region was As shown in Fig. 3A and 3B, only stimulation with IFN-g resulted analyzed for the presence of potential cis-regulatory elements. The in a significant increase in luciferase activity, whereas none of the computational analysis revealed the presence of two STATconsensus other stimuli, including IFN-a and IFN-b, affected NLRC5 pro- binding sites. The STAT family of transcription factors represents moter transactivation. These findings were supported by quanti- a central component of multiple signal transduction cascades, in- tative real-time PCR data showing that NLRC5 mRNA levels were cluding the IFN and IL-6 pathways (22, 23). The NLRC5 promoter significantly increased at 8 h after stimulation with IFN-g (Fig. region contains two individual potential STATbinding sites located at 3C). Similar findings were obtained in a variety of other cell lines positions 21336 and 2452, respectively. Interestingly, the STAT site (THP-1, CaCo2, and HT-29; Supplemental Fig. 4). Moreover, this at 21336 partially overlaps with a predicted NF-kB consensus effect was dose-dependent, as shown by RT-PCR and quantitative binding site. To further analyze a possible involvement of these real-time PCR (Fig. 3D). As control, quantification of IL-8 mRNA binding sites in NLRC5 promoter activation, we created deletion levels was used to demonstrate responsiveness of the cells to the constructs by site-directed mutagenesis. Individual or combined de- various stimuli (Supplemental Fig. 5A–C). Using the validated letion of the predicted regulatory elements resulted in significant anti-NLRC5 Ab, we could demonstrate that NLRC5 protein ex- decrease of IFN-g–dependent promoter transactivation (Fig. 4A). 8 CHARACTERIZATION OF NLRC5 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

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.

Moreover, pretreatment with a specific inhibitor of JAK activity prior concomitant detection of CMV-IE2. As shown in Fig. 5A, a strong to IFN-g stimulation decreased promoter transactivation almost increase in NLRC5 expression was detectable that was peaking at 12 h completely (Fig. 4B). Because JAK activity is essential for phos- postinfection. Remarkably, only intact virus particles were capable of phorylation and nuclear translocation of STATs (24), these data fur- inducing this effect, whereas heat-inactivated CMV did not alter ther emphasize the role of IFN-g–dependent JAK/STAT signaling for NLRC5 mRNA levels within 24 h (Fig. 5A). Thus, the ability to ef- NLRC5 transcriptional regulation . ficiently infect human cells appears to be a prerequisite for regulation of NLRC5 expression. Viral infection contributes to transcriptional regulation of To investigate if autocrine JAK/STAT signaling in response to in- NLRC5 via autocrine IFN-g fection is involved in these regulatory events, we infected cells in The secretion of the cytokine IFN-g is a pivotal cellular response to presence of JAK inhibitor to block JAK/STAT signaling. Blockade of viral infection (25). In order to elucidate whether the expression of JAK-dependent pathways resulted in almost complete abrogation of NLRC5 is directly linked to viral infection, we used a well-established NLRC5 upregulation (Fig. 5B). These results were confirmed by the viral infection model that is based on the infection of HFFs with fact that the supernatant of infected cells was able to upregulate human CMV (17, 18). To differentiate between effects that originate NLRC5 mRNA in cells that were not infected (Fig. 5C). To analyze from direct viral infection and effects depending on sole presence of whether autocrine IFN-g secretion initiated by viral infection is re- inactive virus particles, heat-inactivated virus particles were used in sponsible for regulation of NLRC5, we blocked endogenous IFN-g by a parallel experimental setup. Six, 12, or 24 h postinfection with CMV, addition of an inhibitory Ab. As shown in Fig. 5D,supernatantof expression levels of NLRC5 mRNA were checked by quantitative infected cells preincubated with anti–IFN-g Ab resulted in impaired real-time PCR. Efficiency of the viral infection was checked by induction of NLRC5. The Journal of Immunology 9

NLRC5 oligomerization activates IFN-specific response In order to analyze whether this activation profile was also element- and IFN-g activation sequence-dependent signal detectable on the target gene level, we overexpressed GFP-NLRC5 transduction pathways in epithelial cells and used RT-PCR and real-time PCR to char- Next we asked which signaling pathways are activated downstream acterize potential NLRC5-dependent downstream target genes. As of NLRC5 activation. Because it is a common phenomenon of NLRs shown in Fig. 7A, the overexpression resulted in remarkable in- that ectopic overexpression leads to ligand-independent autoacti- crease in mRNA levels of IFN-a, PRKRIR, and OAS1 24 h after vation of downstream signaling events (3), we used overexpression transfection, whereas expression of IL-8 was not affected. Next, of GFP-NLRC5 to screen for potential signaling cascades initiated we checked the influence of siRNA-mediated knockdown of by NLRC5 activity. In addition, we overexpressed a fusion protein NLRC5 on the target gene level. As demonstrated in Fig. 7B, ef- ficient knockdown of NLRC5 by three individual siRNAs results in consisting of the N-terminal CARD of NLRC5 fused (FKBP)2, whose oligomerization could be exogenously triggered by addition reduction of endogenous mRNA levels of the investigated target of the synthetic oligomerizer AP20187 (for illustration, see Sup- genes as compared with control. To analyze whether this effect is plemental Fig. 6). As shown by native glycine gel analyses, addition also relevant in the context of viral infection, we next transfected of the exogenous oligomerizer resulted in enforced oligomerization HFF cells with siRNA and quantified expression of IFN-a in re- of CARD-FKBP monomers (Supplemental Fig. 7). Next, both GFP- sponse to CMV. siRNA-mediated knockdown of NLRC5 signifi- NLRC5 and CARD-FKBP constructs were expressed in parallel cantly reduces induction of IFN-a after CMV infection (Fig. 8A), with a number of pathway-specific reporter gene constructs that further emphasizing the role of NLRC5 as an essential component could monitor the activation of specific regulatory promoter ele- of antiviral signaling pathways. Supporting this view, NLRC5 ments (Fig. 6). No activation of AP-1 or NF-kB signaling pathways knockdown also significantly reduces induction of GAS-driven Downloaded from was detectable (Fig. 6A,6B). This was surprising because several luciferase activity in response to poly I:C, as shown in Fig. 8B. members of the NLR family have been shown to directly activate Taken together, our data argue for the NLR family member NF-kB and/or MAPK signaling cascades (26–28). In contrast, our NLRC5 as being both target and activator of signaling pathways data point to a specific role of NLRC5 for the activation of signaling contributing to antiviral defense mechanisms. pathways that uses the consensus binding sites IFN-specific response element (ISRE) and IFN-g activation sequence (GAS) (Fig. Discussion http://www.jimmunol.org/ 6C,6D). These regulatory elements represent the major binding In this report, we describe the genomic organization, regulation, sites for STAT family members. Supporting these data, we dem- and aspects of cellular function of the NLR family member onstrate increased and prolonged phosphorylation of STAT1 NLRC5. This gene family has attracted much attention because downstream of IFN-g in the presence of overexpressed NLRC5 mutations in several NLR genes have been linked to a variety of (Supplemental Figs. 8, 9). inflammatory diseases (29). For example, different genetic variants by guest on September 28, 2021

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 (24h) 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. 10 CHARACTERIZATION OF NLRC5 of NOD2 are associated with susceptibility for Crohn’s disease or By now, only few members of the NLR family have been as- Blau syndrome (7–10, 30), whereas NLRP3/NALP3 has been sociated with antiviral immune responses, whereas the majority of linked to rare hereditary fever syndromes and Crohn’s disease NLRs have been shown to contribute to innate defense mechanisms (11–14, 31, 32). These genetic findings underscore the importance against pathogenic bacteria (38–40). The recently characterized of NLRs as surveillance proteins for host defense and immunity NLR family member NLRX1 interacts with mitochondrial anti- and point to their potential as novel therapeutic targets (33). viral signaling adaptor in the outer membrane of mitochondria and Among the NLR family members identified and characterized represents a checkpoint modulating mitochondrial antiviral re- so far, NLRC5 displays an unusual architecture of its carboxy- sponses (41). Furthermore, NLRX1 is capable of amplifying NF- terminal LRR because its repeat units are irregularly scattered kB and JNK pathways activated by different proinflammatory along the domain. The LRR domain of NLR is generally con- stimuli via production of reactive oxygen species (42). A recent sidered to represent the sensor region of the proteins. The mo- study reports on the specific detection of influenza virus by lecular structures that are recognized by NLR proteins represent components of the ASC/NLRP3 inflammasome, but the direct either conserved molecular signatures of bacteria (PAMPs) or requirement for NLRP3 in the sensing process is still under debate endogenous molecules that arise from conditions associated with (43). Our data add NLRC5 to the list of NLR family members cellular danger (danger-associated molecular patterns) (34). In- involved in mechanisms that respond to viral infection and em- terestingly, some NLRs are described to constitute highly selective phasize the role of NLRs as general cytosolic surveillance proteins sensor platforms that only respond to a specific molecular elicitor, involved in both bacterial and viral infection and endogenous whereas others exhibit a promiscuous selectivity by responding to danger response. a variety of stimuli. For example, NLRC4/IPAF responds to cy- In summary, we have characterized the genomic organization of Downloaded from tosolic presence of the bacterial protein flagellin, whereas NLRP3/ NLRC5 and describe complex regulatory mechanisms identifying NALP3 is activated by various endogenous danger-associated NLRC5 as target gene of IFN-g and mediator of IFN-mediated molecular patterns like monosodium urate crystals or low potas- antiviral signaling pathways. Further analyses on NLRC5 regula- sium ion concentration (35). Nevertheless, it is still unclear tion and function will help us to fully understand the impact of whether this response is caused by direct physical interactions of NLR family members on molecular defense strategies against

the respective NLR with its elicitor or indirectly by mediators of viral pathogens. http://www.jimmunol.org/ 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 Acknowledgments for future studies. Because both the identity of a potential physi- We thank Tanja Kaacksteen, Melanie Schlapkohl, Dorina Oelsner, Yasmin ological elicitor of NLRC5 and the downstream signaling events Brodtmann, and Alina Gra¨ff for their technical assistance. were unknown, we used enforced oligomerization of an FKBP fusion protein and overexpression to simulate cellular events that Disclosures trigger oligomerization of NLRC5. Our data demonstrate that The authors have no financial conflicts of interest. NLRC5 oligomerization can efficiently trigger GAS- and ISRE- by guest on September 28, 2021 dependent promoter activity. 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