Variant L1007fsinsc in the Crohn's Disease-Associated NOD2 An

The Journal of Immunology

Genome-Wide Expression Proﬁling Identiﬁes an Impairment of Negative Feedback Signals in the Crohn’s Disease-Associated NOD2 Variant L1007fsinsC

Susanne Billmann-Born,* Andreas Till,* Alexander Arlt,† Simone Lipinski,* Christian Sina,*,† Anna Latiano,‡ Vito Annese,‡ Robert Ha¨sler,* Martin Kerick,* Thomas Manke,x Dirk Seegert,‖ Adedayo Hanidu,# Heiner Scha¨fer,† David van Heel,** Jun Li,# Stefan Schreiber,*,†,1 and Philip Rosenstiel*,1

NOD2 is an intracellular receptor for the bacterial cell wall component muramyl dipeptide (MDP), and variants of NOD2 are associated with chronic inflammatory diseases of barrier organs (e.g., Crohn’s disease, asthma, and atopic eczema). It is known that activation of NOD2 induces a variety of inflammatory and antibacterial factors. The exact transcriptomal signatures that define the cellular programs downstream of NOD2 activation and the influence of the Crohn-associated variant L1007fsinsC are yet to be defined. To describe the MDP-induced activation program, we analyzed the transcriptomal reactions of isogenic HEK293 cells expressing NOD2wt or NOD2L1007fsinsC to stimulation with MDP. Importantly, a clear loss of function could be observed in the cells carrying the Crohn-associated variant L1007fsinsC, whereas the NOD2wt cells showed differential regulation of growth factors, chemokines, and several antagonists of NF-kB (e.g., TNFAIP3 [A20] and IER3). This genotype- dependent regulation pattern was confirmed in primary human myelomonocytic cells. The influence of TNFAIP3 and IER3 in the context of NOD2 signaling was characterized, and we could validate the predicted role as inhibitors of NOD2-induced NF-kB activation. We show that IER3 impairs the protective effect of NOD2wt against bacterial cytoinvasion. These results further our understanding of NOD2 as a first-line defense molecule and emphasize the importance of simultaneous upregulation of counterregulatory anti-inflammatory factors as an integral part of the NOD2-induced cellular program. Lack of these regulatory events due to the L1007fsinsC variant may pivotally contribute to the induction and perpetuation of chronic inflammation. The Journal of Immunology, 2011, 186: 4027–4038.

rohn’s disease (CD) is a human chronic relapsing-remitting with an increased risk for the manifestation of CD (4–6). Rec- inflammatory bowel disease. CD is presumed to result ognition of MDP via the LRRs of NOD2 leads to an activation of C from the combined effects of genetic predisposition and NF-kB pathway by recruitment and induced proximity signaling environmental factors that precipitate into an aberrant inflam- of the receptor interacting protein kinase 2, the NF-kB activating matory response. A significant progress in the understanding of kinase complex, IKKa,-b, and NEMO (7). The CD-associated disease etiopathogenesis was achieved by identifying the associ- frameshift mutation L1007fsinsC in the LRR of NOD2, which ation of polymorphisms in the NOD2/CARD15 gene with an in- leads to a partial truncation of the protein, and other single nu- creased risk for CD in white populations. The NOD2/CARD15 cleotide polymorphisms causing amino acid exchanges in or close gene, a member of the NOD-like receptor (NLR) family (1), to the LRR (R702W, G908R), result in decreased MDP respon- encodes for an intracellular protein implicated as a pattern rec- siveness and MDP-mediated NF-kB activation (8). Several re- ognition receptor for the bacterial cell wall component muramyl ports have described putative target genes of NOD2 activation dipeptide (MurNAc-L-Ala-D-isoGln, or MDP) (2, 3). Mutations in [e.g., IL-8 (9), b-defensin 2 (10), IL-1b (11), and DMBT1 (12)]. A the leucine-rich repeats (LRRs) of NOD2, which are homologous microarray analysis of intestinal specimens of Nod22/2 mice has to the LRRs in TLRs and plant resistance genes, are associated revealed a complex dysregulation of the expression levels of genes

*Institute of Clinical Molecular Biology, Christian-Albrechts-University, 24105 Kiel, Network, an unrestricted grant from Boehringer Ingelheim, and through a Compe- Germany; †Department of General Internal Medicine, University Hospital Schleswig- tence Network from the German Ministry for Education and Research. Holstein, Campus Kiel, D-24105 Kiel, Germany; ‡UU.OO. Gastroenterologia, Endo- The sequences presented in this article have been submitted to the Gene Expression scopia Digestiva e Laboratorio di Ricerca, Ospedale Istituto di Ricovero e Cura Omnibus under accession Nos. GSM560855–GSM560881. a Carattere Scientifico Casa Sollievo della Sofferenza, 71013 San Giovanni Rotondo, Italy; xMax Planck Institute of Molecular Genetics, 14195 Berlin, Germany; ‖Conaris Address correspondence and reprint requests to Dr. Philip Rosenstiel, Institute of Research Institute AG, 24118 Kiel, Germany; #R&D Center, Boehringer Ingelheim Clinical Molecular Biology, University Hospital Schleswig-Holstein, Campus Kiel, Pharmaceuticals, Ridgefield, CT 06877; and **Blizard Institute of Cell and Molec- Schittenhelmstraße 12, D-24105 Kiel, Germany. E-mail address: p.rosenstiel@ ular Science, Barts and The London School of Medicine and Dentistry, Queen Mary mucosa.de University of London, London E1 4NS, United Kingdom The online version of this article contains supplemental material. 1S.S. and P.R. share senior authorship of this work. Abbreviations used in this article: BMDM, bone marrow-derived macrophage; CD, Received for publication January 12, 2010. Accepted for publication January 13, Crohn’s disease; DD-MDP, MurNAc-D-Ala-D-isoGln; FRT, FLP recombinase target; 2011. LRR, leucine-rich repeat; MDP, muramyl dipeptide; MOI, multiplicity of infection; NLR, NOD-like receptor; RLU, relative light unit; siRNA, small interfering RNA; This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB TF, transcription factor. 415/617), the clusters of excellence (Future Ocean and Inflammation at Interfaces), an internal grant from the University of Kiel (to P.R.), the National Genome Research Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000085 4028 TRANSCRIPTOMAL PROFILES OF NOD2 ACTIVATION relevant for immune function and barrier integrity (13). A pro- culture medium (DMEM, 10% FCS) and treated with 6 pmol/well either teomics approach using two-dimensional gel electrophoresis and Stealth negative control siRNA (GC high; Invitrogen) or Stealth siRNA mass spectrometry in NOD2-overexpressing HEK 293 cells for targeting human IEX-1 (HSS 189629; Invitrogen) dissolved in 12 ml/well HiperFect reagent (Qiagen). After 48 h incubation at standard culture the identification of proteins differentially regulated upon MDP conditions, siRNA-treated cells were subject to further treatment. stimulation revealed the existence of complex regulation patterns To check the efficacy of the IEX-1 knockdown, siRNA-transfected mock- of abundant proteins such as peroxiredoxin 4 (14). Due to limi- or NOD2-HEK293 cells were washed with PBS, and total RNAwas isolated tation in the method, it can be hypothesized that the observed using the Qiashredder/RNeasy kits (Qiagen) following the manufacturer’s protocol. A total of 1 mg total RNA was reverse transcribed (37˚C, 1 h) changes only represent a small section of the cellular changes using M-MLV RT (Promega) and 1 mg dT15 primer (Promega). For induced by NOD2 signaling. quantification of IEX-1 mRNA, 2 ml cDNA reaction was submitted to real- To improve our understanding of the protective cellular pro- time PCR as described (16). gram initiated by NOD2 activation, we undertook a detailed com- parative examination of the transcriptional responses of NOD2wt- Bacterial strains L1007fsinsC and NOD2 -expressing cells by using genome-wide Salmonella typhimurium (strain SL1344) was used to infect HEK293 cells microarray analysis. Microarrays are powerful tools employed for in a gentamicin protection assay. THP-1 cells were infected with Listeria. deciphering dynamic signatures of activated genes in response to Listeria monocytogenes (serotype 1/2a strain EGD) were used as model defined stimuli. This experimental approach allowed us to com- organisms for bacterial cytoinvasion; the noninvasive strain L. innocua (serotype 6b strain ATCC 51742) was used as control. Overnight cultures prehend the mechanisms that define the physiological innate im- of bacteria were diluted and grown to midlogarithmic phase. Bacteria were mune responses downstream of NOD2 activation. We verified harvested by centrifugation, washed, and resuspended in cell-culture me- selected parts of the activation program by independent methods dium without antibiotics. For stimulation experiments and gentamicin (quantitative RT-PCR and Western blot) in independent experi- protection assays, a multiplicity of infection (MOI) of 100 bacteria per cell was added. ments using primary monocytic cells from NOD2wt or L1007fsinsC NOD2 individuals. Functional experiments identify the Abs and reagents ubiquitin ligase A20 (TNFAIP3) and IER3 as crucial negative regulators for NOD2-induced NF-kB activation and contribute to the Anti-NOD2 Ab was purchased from Cayman Chemicals (Ann Arbor, L1007fsinsC MI) and Novus Biologicals (clone 2D9; Littleton, CO). Anti-A20 Ab understanding of the disturbed responses of the NOD2 was purchased from Stressgen (Victoria, Canada). Recombinant human variant associated with the development of several barrier dis- TNF-a was purchased from R&D Systems. MDP and its inactive stereo- orders. isomer MurNAc-D-Ala-D-isoGln (DD-MDP) were obtained from Bachem (Bubendorf, Switzerland) and applied at a concentration of 1–10 Materials and Methods mg/ml. Cell culture and transfection Plasmid constructs HEK293 epithelial cells (ACC305), HeLa cells (ACC161), and THP-1 myelomonocytic cells (ACC16) were purchased from the German Col- The constructs pcDNA3.1-NOD2wt, pcDNA3.1-NOD2L1007fsinsC, and lection of Microorganisms and Cell Cultures (Deutsche Sammlung pFlag-IER3 for overexpression of NOD2 variants or IER3, respectively, von Mikroorganismen und Zellkulturen, Braunschweig, Germany). All have been described before (9, 17). The luciferase reporter construct IER3- LUC(-493) containing the proximal promoter of IER3 has been described cells were cultivated at standard conditions of 5% CO2 and 37˚C. Isogenic stable cell lines HEK293-mock, HEK293-NOD2wt, and HEK293- (17). The overexpression construct for A20 (pUNO-hTNFAIP3) was pur- NOD2L1007fsinsC were generated with the FLP-IN system (Invitrogen, chased from InvivoGen (San Diego, CA). Carlsbad, CA) as described (14). In brief, HEK293 cells with a single genomic FLP recombinase target (FRT) cassette were purchased from Microarray experiments Invitrogen. These host cells were transfected with an expression vector wt L1007fsinsC mock FLP-IN HEK293-NOD2 , HEK293-NOD2 , and HEK293 containing the respective open reading frame under the control of a CMV cells were cultivated in medium without FCS for 7 h before stimulation promoter with flanking FRT sites and a hygromycin-resistance sequence. with 10 mg/ml MDP for 0, 2, and 6 h. Cells were harvested simultaneously, Coexpressed FLP recombinase was then employed to catalyze a homolo- and total RNA was isolated using the RNeasy Kit (Qiagen). RNA was gous recombination event between the FRT sites in the host cells and the quality checked with Agilent RNA 6000 nanochips and either transcribed expression vector. to cDNA (M-MLV Reverse Transcriptase; Promega) for real-time PCR Isolation and cultivation of PBMCs from patients homozygous either for experiments or used for microarray hybridization. The experimental setup NOD2wt (n = 4) or the NOD2L1007fsinsC variant (n = 3) were performed as was repeated independently three times. Five micrograms total RNA was previously described (15). 2 2 used to synthesize cRNA using the Affymetrix expression protocol (ex- Nod2 / knockout mice were purchased from The Jackson Laboratory pression analysis technical manual; Affymetrix, Santa Clara, CA). Ten (Bar Harbor, ME) strain B6.129S1-Nod2tm1Flv/J. Murine bone marrow- micrograms labeled and fragmented cRNA was hybridized to HG- derived macrophages (BMDMs) were isolated as follows: femurs and U133Plus2.0 arrays (Affymetrix, Santa Clara, CA), and signal detection Nod2 tibiae of homozygote knockout mice were perfused with ice-cold was performed following the manufacturer’s instructions. GeneChips Macrophage Medium Murin (PAA Laboratories, Pasching Austria) con- Human Genome HG-U133Plus2.0 detects .54,600 probe sets, which taining penicillin 10 mg/ml, streptomycin 10 mg/ml, amphotericin 25 mg/ allows analysis of .47,000 transcripts, and has been widely employed to ml, and recombinant murine M-CSF (ImmunoTools, Friesoythe, Germany) study genome-wide expression signatures (18). 20 ng/ml. The eluted cell suspension was filtered (Cell Strainer, 70 mm Nylon Filter; BD Biosciences, Franklin Lakes, NJ) and cultured at standard conditions for 7–10 d. Adherent cells were seeded to six-well tissue culture Data deposition plates at a density of 800,000 cells/well for experiments. The cel-files of the 27 microarray experiments are deposited at the Gene Transfections of HEK293 and HeLa cells were performed with Fugene Expression Omnibus public database (http://www.ncbi.nlm.nih.gov/geo; 6 (Roche, Basel, Switzerland) using indicated amounts of the respective accession numbers GSM560855–GSM560881). plasmids. Transfection of A20 small interfering RNA (siRNA) was performed Data analysis using siPORT Amine (Ambion, Austin, TX) according to the manufacturer’s instructions. A20 targeting siRNA was purchased from Qiagen Image analysis, absent/present calls, and interarray normalization have been (Hilden, Germany). The target sequence for siA20-1 was 59-AAAGA- performed using the default settings of Gene Chip Operating Software 1.1 CACACGCAACTTTAAA-39, and the target sequence for siA20-2 was 59- (Affymetrix). The intensity distributions of the normalized data were CCGAGCTGTTCCACTTGTTAA-39. Efficiency of the knockdown was checked and regarded as sufficiently normalized. Spotfire DecisionSite 8.0 analyzed by Western blot. (TIBCO, Palo Alto, CA) was used for IL-8 similarity search based on IER3 siRNA transfection was performed as follows: NOD2- or mock- Pearson’s correlation. Cluster analysis was performed by Cluster 3.0 from HEK293 cells grown on 12-well plates were supplied with 1 ml fresh M. Eisen (19). The Journal of Immunology 4029

ANOVA analysis either pNF-kB-Luc (Clontech, Mountain View, CA) or IER3-Luc(-493) in combination with reference plasmid pRL-TK (Promega) and expres- Genes that have not been flagged as present in at least 2 out of 27 microarray sion plasmids for A20 or IER3 as indicated. Twenty-four hours post- experiments were regarded as not present and excluded from further transfection, cells were stimulated with MDP (5 or 10 mg/ml) for indicated analysis. Normalized data were analyzed using R (version 2.0) software in time periods. Cells were washed, lysed in passive lysis buffer (Promega), conjunction with the bioconductor-package limma (version 1.8.10) (20, 21). and assayed for reporter gene activity using Dual Glow Luciferase reporter Our analysis focused on variation in gene expression attributed to three assay system (Promega) and the Tecan Genios Pro microplate luminometer different genotypes and the effect of stimulation with MDP in the different (Tecan Trading). Each experiment was repeated independently (n = 3). genotypes. Therefore, we analyzed our data using an ANOVA approach. Firefly luciferase expression was normalized to constitutive Renilla lu- As proposed by Kerr et al. (22), a fixed-effects model was used to estimate ciferase expression and indicated as relative light units (RLU). the differences in expression concerning the questions of interest. For each individual gene on the array, the model can be described as:

n Y ¼ + Cn þ e; n¼1 in which Y represents the normalized intensity, Cn represents the 1..n factor combinations, and e is an independent error term with E(e) = 0 and Var(e)= s2. Questions of interest have been addressed by formulating contrasts. The p values of the genewise F-statistics generated by bioconductor-package limma have been adjusted for multiple testing by the method of Benjamini and Hochberg (23) to control the false-discovery rate at a level of a = 0.05. Gene ontology analysis was performed as previously published (24) by comparing significantly upregulated and downregulated transcripts. Bi- ological processes associated to the transcripts were retrieved from the Gene Ontology Consortium (http://www.geneontology.org). Significantly enriched transcription factor (TF) binding motifs were detected using the statistical models introduced by Manke et al. (25). Only motifs with known TFs were chosen. Statistical significance of other experiments was tested by performing Student’s t test as indicated by t test in the figure legends. Settings for the t test were unpaired, two tailed if not mentioned otherwise in the figure legends (26).

TaqMan real-time PCR For confirmation of the results derived from Affymetrix-based gene expression profiling, we performed real-time PCR for a selected panel of differentially regulated genes. FLP-IN HEK293 cells and PBMCs from donors with known NOD2 genotype were treated as described and used for RNA isolation (see above). The corresponding cDNA was arrayed on 384-well plates for real-time PCR quantitation using Assays-on-Demand Gene Expression Assays fom Applied Biosystems (Foster City, CA) for A20/TNFAIP3 (Hs00234712_m1), MAP3K8 (Hs00178297_m1), BMP2 (Hs00154192_m1), 4-1BB/TNFRSF9 (Hs00155512_m1), and IER3 (Hs00277234_s1) on the ABI Prism 7900HT Sequence Detection System (Applied Biosystems) according to the manufacturer’s instructions. Rela- tive transcript levels were determined using the standard curve quantitation method and b-actin as the endogenous control gene. In an additional experimental setup, THP-1 were infected with L. monocytogenes or L. innocua (MOI = 100, 1 h), followed by killing of extracellular bacteria by gentamicin (10 mg/ml), cultivation of cells for the times indicated, and isolation of RNA. A20 expression was detected as described above. For detection of A20 in Nod2 knockout mouse BMDMs, cells were isolated as described above. Murine BMDMs were stimulated with MDP (10 mg/ml), and mRNA expression was quantified as described above. Murine TaqMan assays were IL-1b (Mm01336189_m1), IL-6 (Mm99999064_m1), and Tnfaip3 (Mm00437121_m1).

ELISA Stably transfected HEK293 cells were cultivated overnight in FCS-free medium and stimulated with MDP (10 mg/ml) for 24 h. Supernatants were collected, cleared by centrifugation, and assayed for IL-8 release using ELISA kits (Biosource International). Cell viability was determined in parallel using a commercial MTT assay (Promega).

Western blots FIGURE 1. Characterization of NOD2 stable cell lines used in the study. Cells were lysed in Laemmli buffer in the presence of protease inhibitors. wt L1007fsinsC Cleared protein extracts were subjected to SDS-PAGE, and Western blotting FLP-IN HEK293 cells stably expressing NOD2 or NOD2 or was performed as previously described (27). A20 protein levels were transfected with the empty FRT cassette (FLP-IN HEK293 mock) were detected using speciﬁc Ab (see above) and compared with b-actin by stimulated with MDP (10 mg/ml) for 0, 2, and 6 h. A, Supernatants were densitometry, applying ImageJ software from the National Center for collected and assayed for human IL-8 by ELISA (mean 6 SD, n = 3 in- Biotechnology Information (http://rsb.info.nih.gov/ij/index.html). dependent experiments; t test, **p , 0.01). B, mRNA was analyzed for NOD2 expression by TaqMan real-time PCR (mean 6 SD, n = 3). C, Luciferase assay Protein expression was detected by Western blot with anti–NOD2-Ab Quantiﬁcation of NF-kB activity and IER3 promotor activity was per- (Novus). Note the size difference of NOD2wt and NOD2L1007fsinsC due to formed as previously described (28). In short, cells were transfected with the premature stop codon caused by the frameshift mutation. 4030 TRANSCRIPTOMAL PROFILES OF NOD2 ACTIVATION

Gentamicin protection assay Results wt To determine the inﬂuence of IER3 expression on bacterial cytoinvasion, we MDP induces a distinct activation program in NOD2 , but not L1007fsinsC performed gentamicin protection assays with S. typhimurium. HEK293 NOD2 cells cells were transfected with pcDNA3.1-NOD2, pFlag-IER3, a combination of both plasmids, or empty vector (mock transfection). The cells were To elucidate the MDP-induced activation program, we generated infected with Salmonella using an MOI of 100 bacteria/cell. After 1 h isogenic HEK293 cells stably expressing wild-type NOD2 or infection time at 37˚C, extracellular bacteria were killed by replacing the NOD2L1007fsinsC using an FRT recombinase-based approach. Cells medium by DMEM containing gentamicin (10 mg/ml) and 10% FCS for 3 h. Infected cells were washed with PBS, lysed in 1% Triton X-100/PBS, carrying the inserted vector cassette were used as controls (mock and plated on solid media for counting of CFU. transfectant). HEK293 cells represent an accepted model system

FIGURE 2. NOD2 activation results in specific changes in transcriptomal profile patterns. FLP-IN HEK293 stably expressing NOD2wt or NOD2L1007fsinsC and control cells (FLP-IN HEK293mock) were stimulated with MDP (10 mg/ml) for 0, 2, and 6 h. Cells were harvested simultaneously, and RNA was isolated. Resulting cDNA of three independent experiments was used for hybridization on Affymetrix HG-U133Plus2.0 microarrays. Cluster analysis of 158 MDP-regulated transcripts is shown, with transcription levels in linear color range: green = low expression, black = medium expression, and red = high expression level. Inclusion criteria were present call; ANOVA, p , 0.05, fold-change .1.4. The full list of included transcripts is given in Supplemental Table I. Only wild-type NOD2 but not the CD-associated variant L1007fsinsC exhibited a specific gene expression pattern when compared with NOD2-negative cells. The Journal of Immunology 4031 for exploration of NOD2 signaling pathways, as the cells normally NOD2L1007fsinsC cells was not able to identify MDP-dependent do not express NOD2 at detectable levels (29). To demonstrate transcriptomal profiles (data not shown). the validity of the system, we analyzed the secretion of IL-8 as The transcripts showing differential regulation were clustered a marker of NOD2 activation by MDP as described previously (9, into six different classes (i.e., up/downregulated at 2, 2, and 6 h or 30). Only in NOD2wt cells was a significant IL-8 induction upon up/downregulated at 6 h) reflecting the kinetics of regulation to MDP challenge detectable, whereas the control cells and the further differentiate transient versus sustained responses (Fig. 2, NOD2L1007fsinsC cells did not show a significant IL-8 release (Fig. Supplemental Table I). Gene ontology analysis of the clustered 1A). NOD2 mRNA levels were similar in the HEK-NOD2wt and genes revealed enrichment of several immunity, differentiation, HEK-NOD2L1007fsinsC cells at baseline and after 2 h and 6 h of MDP and signaling terms (Fig. 3). The individual genes of the gene on- stimulation (Fig. 1B). NOD2wt and NOD2L1007fsinsC showed com- tology results are shown in Supplemental Table II. Transcripts in- parable protein levels in the Western blot (Fig. 1C), whereas mock- duced by MDP stimulation included expected cytokines, growth transfected cells and unaltered HEK293 cells do not express NOD2. factors (IL-8, BMP2, amphiregulin), and chemokines (CXCL2 To comprehensively investigate NOD2-mediated innate im- and CXCL3). The transcriptional response is also defined by mune responses, we analyzed transcriptomal signature patterns using genes that are involved in the interaction between cells and their genome-wide cDNA microarrays. Samples were harvested from environment (e.g., the ion channel SCN3A, the solute carriers HEK293-mock, HEK293-NOD2wt, and HEK293-NOD2L1007fsinsC SLC24A1/SLC1A2, molecules important for biosynthesis and cell cultures at 0, 2, and 6 h of MDP stimulation. ANOVA analysis modification of extracellular matrix [SDC4, CSS3, TFPI2, and was performed to specify significantly differential transcript lev- HS3ST3A1]). Most upregulated genes, however, belong to the els after MDP stimulation in at least one genotype. This analysis group of molecules involved in negative modulation of signal accounted formal statistical support for 294 probe sets (represent- transduction (e.g., TNFAIP3, IER3, TFs JUN, ATF3, FOSL2, and ing 0.96% of the interrogated transcripts), which was extended REL, adaptor molecules NFKBIA and NFKBIZ, and kinases/ by 26 significant probe sets using a correlation-based similarity phosphatases DUSP1/2, IKBKB, and MAP3K8). Downregulated search for an IL-8–like expression profile. Interestingly, the tran- molecules included TFs (ETV7, KLF12, and EBF2) and mole- scripts significantly regulated by MDP were exclusively found cules involved in cytoskeletal dynamics and vesicle transport in the NOD2wt genotype. Neither in the control nor in the (SSH1, COPA, and KIF22). We termed this whole collection of NOD2L1007fsinsC cells was a panel of MDP-induced transcripts ∼100 genes the NOD2-induced activation program, as it defines detectable. Even a refined search only using the data from the signaling pathways and induced functions that are triggered upon

FIGURE 3. Gene Ontology: biological functions, associated to significantly regulated transcripts. The bar length represents the significance [-logP], orange indicates a depleted function, green indicates an enriched function, and light green/orange indicates not significant enrichment/depletion. 4032 TRANSCRIPTOMAL PROFILES OF NOD2 ACTIVATION activation of the pathogen-associated molecular pattern receptor homozygous for the NOD2L1007fsinsC mutation. Four NOD2 wild- NOD2 by addition of its minimal elicitor structure MDP. type healthy volunteers served as controls. Concording with the To predict signaling pathways underlying each of the tran- data from the HEK293 cells, we found significant MDP-dependent scriptomal change classes, we first calculated the binding affinities changes for the investigated transcripts only in the NOD2wt group, of 202 vertebrate TFs for the human promoters corresponding to whereas carriers of the NOD2L1007fsinsC variant showed unaltered the uniquely mapped transcripts of the NOD2-induced activation transcript levels after stimulation (Fig. 5, right panels). program. Binding models for TF were retrieved from the TRANSFAC database. From the promoters, we only retained those The ubiquitin ligase TNFAIP3/A20 is upregulated by NOD2 k sequence regions that are conserved between human and mouse activation and downregulates MDP-induced NF- B activation or more distant vertebrates. To compare the conserved affinities As our results demonstrated a substantial upregulation of A20 of different factors, they were converted into p values using the transcript levels in isogenic HEK293-NOD2wt cells and also in statistical model introduced by Manke et al. (25). In this way, we primary NOD2 wild-type monocytes by stimulation with purified obtained a p value for each TF and every promoter. Finally, we MDP, we conducted a set of experiments to characterize the re- scored the enrichment of motifs in the different classes using gulation and function of the A20 protein in the context of NOD2 Fisher’s method for combining p values, under the assumption that activation, which has been described as a modulator of NLR ac- all promoters in a class are independent of each other. Fig. 4 tivity (31). Myelomonocytic THP1 cells express endogenous shows the significantly enriched TFs sorted by class and p value, NOD2 and thus represent an appropriate model to validate re- according to which the motifs were ranked. Interestingly, in group sults from our overexpression model (29). In THP-1 cells, as well 1 and group 2 (early transient and sustained upregulation), NF-kB as in the isogenic HEK293-NOD2wt cells, but not in HEK293- is enriched, which reflects the known canonical signaling pathway NOD2L1007fsinsC, an upregulation of A20 protein could be detected of NOD2. It will be interesting to demonstrate whether and how upon stimulation with the NOD2 ligand LD-MDP by Western blot the predicted additional TFs are directly or indirectly related (Fig. 6A,6B). These results confirmed the observations regarding with the known signaling events. A20 mRNA induction in primary monocytes of patients (Fig. 5, To confirm the MDP-induced transcriptomal signature indicated right panels). To exclude an unspecific effect, the DD-MDP ste- by the microarray analysis, we used the independent method of reoisomer was employed, which is not recognized by NOD2. real-time PCR to assess the transcript levels of five genes that Stimulation with this compound did not result in upregulation of showed an upregulation after MDP stimulation. All target genes A20 in any of the employed cell types. Further, we confirmed showed regulation patterns that were consistent with the microarray the NOD2 dependence of the observed A20 induction in murine data (Fig. 5, left and middle panels). Nod2-deficient BMDM cells. To characterize the cell system, we To further demonstrate the biological significance of the cellu- tested induction of IL-1b and IL-6 mRNA by MDP stimulation, lar system employed for the microarray analysis, we isolated both well known to be induced downstream of NOD2 activation. monocytic cells from the peripheral blood of three CD patients Murine BMDMs stimulated with MDP showed induction of A20, IL-1b, and IL-6 mRNA only in wild-type cells, whereas Nod2 knockout cells were unresponsive (Fig. 6C). This result mimics the failure of A20 induction in L1007fsinsC homozygote patients. Summarizing the observations in primary monocytes, THP-1 cells, and murine BMDMs, we can conclude that functional NOD2 is essential for MDP-induced A20 induction. To assess the ability of A20 to interfere with NOD2-induced signaling pathways, we coexpressed increasing amounts of A20 together with NOD2wt in HeLa cells. Western blots of HeLa cell lysates did not show endogenous NOD2, but showed low amounts of endogenous A20 in Western blot and rising amounts of A20 corresponding to rising expression plasmid charge. Overex- pression of A20 did not affect expression levels of the NOD2wt plasmid (Fig. 6D, left panel). In this coexpression setup, we determined NF-kB activation after MDP stimulation by reporter gene assay. Expression of A20 dose-dependently inhibited NOD2- dependent NF-kB transactivation (Fig. 6D, right panel). A robust NF-kB activation after MDP stimulation (10 mg/ml) was seen exclusively in the NOD2wt-transfected cells with no or low amounts of coexpressed A20. Thus, the NOD2-mediated induction of A20 could be part of a negative-feedback loop modulating NF- kB activation downstream of NOD2. To confer evidence to the specificity of this effect, we performed an A20 knockdown with two different siRNAs in HeLa cells. The efficiency of the knockdown was controlled by West- ern blot, and then the same siRNA setup was conducted to an NF-kB reporter gene assay. Indeed, we could observe the reversed effect of A20 overexpression by applying A20 siRNA: FIGURE 4. Significantly enriched TFs sorted by class and p value, NF-kB induction was significantly stronger in A20 compro- according to which the motifs were ranked. In group 1 and group 2 (early mised cells compared with cells expressing normal endogenous transient and sustained upregulation), NF-kB is enriched, which reflects A20 levels (control siRNA transfected cells) downstream of the known canonical signaling pathway of NOD2. MDP stimulation (Fig. 6E). The Journal of Immunology 4033

FIGURE 5. Verification of genotype-dependent transcript regulation by NOD2 activation in primary PBMCs. Left and center panels, FLP-IN HEK293 stably expressing NOD2wt or NOD2L1007fsinsC and control cells (FLP-IN HEK293mock) cells were stimulated with MDP (10 mg/ml) for 0, 2, and 6 h. RNA was isolated and subjected to Affymetrix HG-U133Plus2.0 microarray (left panels) or to TaqMan real-time PCR (center panels) for selected genes (mean 6 SD, n = 3 independent experiments; t test, *p , 0.05, **p , 0.01, ***p , 0.001). Right panels, PBMCs homozygous for NOD2wt or NOD2L1007fsinsC were isolated from donor blood and stimulated with MDP (10 mg/ml) for 0, 2, and 6 h. RNA was isolated and subjected to TaqMan real-time PCR for selected genes (mean 6 SD, n [wild-type] = 4 results of four volunteers and n [L1007fsinsC] = 3 results of three patients; t test, *p , 0.05, **p , 0.01). The data confirmed the initial screening experiment based on microarray analysis showing that NOD2 wild-type but not the insertion variant exhibits a specific gene induction profile. In addition, primary monocytes from donors carrying NOD2 wild-type allele show a comparable regulation, whereas donors homozygous for L1007fsinsC allele exert a minor response. 4034 TRANSCRIPTOMAL PROFILES OF NOD2 ACTIVATION

FIGURE 6. TNFAIP3/A20 is upregulated by NOD2 activation and contributes to negative regulation of MDP-dependent NF-kB activation. THP-1 cells (A) and FLP-IN HEK293 (B) stably expressing NOD2wt or NOD2L1007fsinsC were stimulated with either MDP (LD-MDP) (10 mg/ml) or the biological inactive stereoisomer DD-MDP (10 mg/ml) for indicated times (0, 2, 6, and 24 h). Cells were lysed and proteins blotted with anti-A20 (Stressgen) or anti- actin as loading control. OD of A20 protein bands was quantiﬁed by ImageJ, divided by the corresponding actin density value, and expressed as arbitrary units (representative result of three independent experiments). C, BMDMs of Nod2 knockout (Nod22/2) mice (n = 10) and Nod2wt mice (n = 10) were stimulated with MDP (10 mg/ml). TaqMan real-time PCR revealed A20 induction only in Nod2 wild-type cells. The well known Nod2-dependent induction of IL-1b and IL-6 mRNA serves as positive control (mean 6 SD, n = 10 independent experiments; t test, **p , 0.01, ***p , 0.001). D, A20 overexpression inhibits MDP-induced NF-kB activity. Interference of NOD2 and A20 coexpression was excluded by blotting lysates of HeLa cells expressing NOD2wt (500 ng pcDNA3.1-NOD2wt) and rising amounts of A20 (0, 62, 125, 250, 500, and 1000 ng pUNO-hTNFAIP3). HeLa cells were transfected in 96-well plates with 2 ng plasmid encoding for NOD2wt (pcDNA3.1-NOD2wt) and rising amounts of plasmid expressing A20 (0, 2, 4, 8, 16, and 32 ng pUNO-hTNFAIP3), whereas total amount of DNA was adjusted to 35 ng with control plasmid (pcDNA3.1mock). Cells were stimulated with MDP (10 mg/ ml) at different time points (24, 8, 4, and 0 h) before harvesting. Induction of NF-kB activity was determined by using a dual luciferase reporter assay The Journal of Immunology 4035

In addition, we found a strong upregulation of A20 expression The general finding that stimulation of NOD2wt but not the CD- postinfection of THP-1 monocytes with cytoinvasive L. mono- associated variant NOD2L1007fsinsC by MDP leads to a highly cytogenes, whereas the noninvasive strain L. innocua resulted in specific gene expression profile is in good accordance with pre- significantly lower A20 induction (Fig. 6F). This finding, although vious data from our group and others claiming that the truncation not specific for NOD2 function (32, 33), may contribute to our of the NOD2 protein by frameshift mutation in the LRR region understanding of A20 expression as a reaction to cytoinvasive results in a loss-of-function phenotype. In contrast, Maeda and bacterial infection. colleagues (36) have shown by a transgenic mouse model that a genetic variant representing the human NOD2L1007fsinsC allele NOD2-induced upregulation of IER3 modulates the NOD2- does not diminish NOD2 function but results in increased NF-kB dependent NF-kB response activation and IL-1b release. This discrepancy might reflect di- As a second NOD2 target gene, we addressed the function of IER3 vergent regulatory mechanisms on the background of functional in the context of cellular MDP sensing. To further confirm the di- differences between human and murine NOD2 variants. The lack rect involvement of NOD2 in regulation of IER3, we transfected of specific gene induction profiles by the disease-associated NOD2 a luciferase reporter gene construct driven by the IER3 promoter, variant and the confirmation in PBMCs of patients carrying the IER3-Luc (-493), into HEK293 cells. Determination of luciferase risk variant as reported in this study favor the concept that the high wt levels demonstrated NOD2 -dependent transactivation of the levels of NF-kB activation observed in CD (37, 38) represent IER3 promoter upon MDP stimulation (Fig. 7A). This effect could a secondary inflammatory reaction caused by functional defects of be suppressed by cotransfection of a vector encoding a mutant the intestinal immunological barrier induced by lack of NOD2 IkBa, which serves as a superrepressor of NF-kB activation (data signals and are not evoked by a distinct genetically determined not shown). gain-of-function program directly downstream of NOD2. We further analyzed the influence of IER3 expression on The identification of various transcriptomal profile clusters NOD2-induced NF-kB activity by luciferase assay. Direct physi- with distinct kinetics and regulatory trends reflects the complexity cal interaction of IER3 with the p65/RelA subunit leads to a neg- of gene regulation networks. For example, cluster 1, which covers ative interference with NF-kB signals (34). We could show that genes that are transiently induced after 2 h, contains well-known expression of IER3 dose-dependently attenuated the MDP-induced immediately early response genes regulated in an NF-kB–depen- NF-kB activity (Fig. 7B). This effect could be reversed by ap- dent manner (e.g., NFKBIA, IER3, REL, and TNFAIP3). The bona plying IER3 targeting siRNA, which resulted in enhanced MDP- fide presence of IL-8, one of the first genes described to be induced induced NF-kB activity (Fig. 7C; for analysis of siIER3 efficiency, by NOD2 activation (9, 11), in this cluster also emphasizes the see Supplemental Fig. 1), which argues for an IER3-specific ef- validity of our experimental setup. It seems likely that a specific set fect. of genes with contribution to regulatory signal transduction mech- To analyze the functional contribution of IER3 to other NOD2- anisms (TFs, adaptors, and kinases/phosphatases) is regulated in dependent downstream events, we asked if the presence of IER3 a complex manner by a multifactorial cellular signaling network. wt protein interferes with the bactericidal properties of NOD2 (30, The comparison of the architecture of 59 upstream regulatory re- wt 35) using gentamicin-protection assays. As expected, NOD2 gions of the different gene clusters revealed a highly significant overexpression resulted in a reduced relative infection of HEK293 enrichment of NF-kB sites in the two clusters 1 and 2 (transient and cells with invasive S. typhimurium, emphasizing the role of NOD2 sustained upregulation). Several other TF consensus sites are also as cytosolic surveillance protein and mediator of antimicrobial significantly enriched, suggesting complex regulatory events be- defense mechanisms. Interestingly, overexpression of IER3 com- yond canonical NF-kB activation. Interestingly, the motif for pletely reversed the antibacterial effect exerted by NOD2 ex- POU1F1 (also known as PIT-1) is significantly enriched in the pression (Fig. 7D). An opposite effect was not observable when promoters regulating transcripts that are downregulated early IER3 expression levels were silenced by IER3-specific siRNAs during NOD2 activation. The transactivation potential of this (Supplemental Fig. 2). Also, no significant difference in cytoin- homeodomain-containing protein is enhanced via IkBa through vasion was observed between wild-type and Ier3-deficient murine an NF-kB–independent association with histone deacetylase 1 and BMDMs postinfection with S. typhimurium. 3 (39). Taken together, we identified the early response gene IER3 as Whether these signaling events are directly triggered by NOD2 a novel downstream target of NOD2 and show further that IER3 or are secondary events elicited by regulation of NOD2 target comprises a negative regulatory capacity on NOD2-dependent genes will be an interesting topic of future investigations. NF-kB signaling and potentially antibacterial effects. Regarding functional impact of newly identified NOD2 target genes on subcellular processes triggered by pathogen-associated Discussion molecular pattern recognition, it is important to note that MAP3K8 In this study, we analyzed the complex transcriptomal program (Cot/Tpl2) kinase is crucially involved in cellular signaling events induced by NOD2wt and the NOD2L1007fsinsC variant in isogenic activated upon LPS detection by TLR4 (40, 41). Moreover, deg- HEK293 cell populations to understand the multifactorial contri- radation of MAP3K8 protein after LPS treatment represents a bution of NOD2 dysfunction in CD. major mechanism to control TLR-specific activation of NF-kB

(pNFkB-luc and pRL-TK) as described in Materials and Methods. Data are expressed as relative luciferase activity (RLU) (mean 6 SD of quadruplicate cultures; t test, **p , 0.01, representative of three independent experiments). E, A20 knockdown enhances MDP-induced NF-kB activity. Two different siRNAs targeting A20 mRNA were transfected to HeLa cells (20 nM). Efﬁciency of the knockdown was evaluated by Western blot, indicating an ∼40% knockdown of A20 protein. Upon stimulation with MDP (10 mg/ml), both siRNAs induced higher NF-kB activity in a dual luciferase assay compared with irrelevant siControl (mean 6 SD of ﬁve repeated cultures, representative result of three independent experiments; t test, **p , 0.01). F, A20 expression is induced by intracellular bacterial challenge. THP-1 cells were infected with L. monocytogenes or noncytoinvasive L. innocua (MOI = 100, 1 h) followed by killing of extracellular bacteria by gentamicin (10 mg/ml), cultivation of cells for the times indicated, and isolation of RNA. A20 expression was detected by TaqMan real-time PCR (mean 6 SD, n = 3 independent experiments; t test, **p , 0.01). 4036 TRANSCRIPTOMAL PROFILES OF NOD2 ACTIVATION

FIGURE 7. Upregulation of IER3 by NOD2 downregulates MDP-dependent activation of NF-kB. A, MDP administration activates IER3 promotor activity in NOD2 cells. HEK293 cells expressing NOD2wt or NOD2L1007fsinsC were transfected with IER3 promoter-luciferase reporter gene construct (IER- Luc-493). Upon stimulation with MDP (10 mg/ml) luciferase activity was detected (mean 6 SD, n =3;t test, *p , 0.05). B, IER3 dose-dependently attenuates the MDP-induced NF-kB activity in NOD2-expressing cells. HEK293 cells were transfected with 2 ng pcDNA3.1-NOD2wt or pcDNA3.1mock expression vector and pFlag-IER3 in rising concentrations (0, 10, 20, and 30 ng). Following stimulation with MDP (1 and 10 mg/ml), NF-kB induction was determined by using a dual luciferase reporter assay (pNF-kB and pRL-TK) as described in Materials and Methods. Data are expressed as relative luciferase activity (RLU) (mean 6 SD, n =3;t test, ***p , 0.001). C, Knockdown of IER3 enhances NOD2-dependent NF-kB response. HEK293-NOD2wt cells grown on 12-well plates were treated with 6 pmol/well either Stealth negative control siRNA (GC high; Invitrogen) or Stealth siRNA targeting human IER3 (HSS 189629; Invitrogen). After 48 h incubation at standard culture conditions, siRNA-treated cells were transfected with dual luciferase assay plasmids (pNF-kB and pRL-TK). After treatment with MDP (2 mg/ml) for 6 h, cells were lysed and used for the dual luciferase assay procedure using Dual- Glow luciferase assay system (Promega). Data are expressed as fold change of MDP-stimulated over unstimulated relative luciferase activity (RLU) (mean 6 SEM, n = 4; paired t test, *p , 0.05). D, Overexpression of IER3 results in increased intracellular bacterial infection. HEK293 cells expressing NOD2wt or IER3 or both were infected with S. typhimurium at an MOI of 100. After 1 h incubation at 37˚C, extracellular bacteria were killed with gentamicin (10 mg/ml), and intracellular bacteria were quantified as CFU (mean 6 SD, n = 3 independent experiments; t test, **p , 0.01). and MAPKs (42). Thus, our data further corroborate the hypoth- Interestingly, we identified two transcripts, IER3 and A20, that esis that regulation of MAPK-mediated signal transduction is of have been implicated as critical negative regulators of NF-kB ac- substantial importance for the cross-talk between TLR and NLR tivity. The early response gene IER3 has been shown to directly proteins. contribute to negative feedback regulation of TNF-a–induced NF- The Journal of Immunology 4037 kB signaling in HEK293 cells. The molecular mechanisms in- Recent studies emphasize the role of NOD2-dependent type I volved in this inhibitory effect are related to both specific in- and II IFN production in antiviral and antiparasitic responses activation of proteasomal degradation processes by the 26S pro- (51–53). NOD2 was shown to interact with REL (c-Rel) and is teasome (43) and direct interaction with the p65 subunit of NF- required for optimal nuclear accumulation of c-Rel, which in turn kB (34). Overexpression of IER3 protein also negatively influ- is relevant for IL-2 transcription, Th1/Th2 polarization, and IFN-g ences NOD2-mediated NF-kB activity, pointing to a putative role release. In our study, we show also transcriptional upregulation of of IER3 in a negative-feedback loop downstream of NOD2. In c-Rel following MDP stimulation in NOD2wt cells, which may contrast, high IER3 levels introduced by overexpression also re- contribute to the role of NOD2 in the cross-talk of innate and duce the direct antibacterial activity exerted by NOD2, suggesting adaptive immunity. a bipotential role of IER3 in inflammatory propagation and tissue In summary, our study identifies a cellular transcriptomal sig- damage. Whereas silencing of endogenous IER3 transcript levels nature program elicited by MDP-dependent activation of NOD2, in HEK293 cells do not affect NOD2 antibacterial activity, the which dissects a complex cellular regulatory network and sheds data from the Ier3-deficient BMDMs argue against a general a new light on the role of NOD2 variants associated with CD. effect of the gene on cytoinvasion. Although we are aware of the inherent problems of overexpression results, the findings could Acknowledgments be explained by a threshold level of the modulating IER3 func- We thank Tanja Kaacksteen, Yasmin Brodtmann, Alina Gra¨ff, Dorina O¨ ls- tion. As HEK293 cells are known to express low endogenous ner, and Melanie Schlapkohl for expert technical assistance and Olga IER3 protein levels (43), knocking down these intrinsically low Gavrilova for excellent mouse services. levels may not affect antibacterial activity in the 1 h time win- dow of infection, whereas higher levels as induced by over- Disclosures expression or longer stimulation may still exert a NOD2-specific A.H. and J.L. are employees of Boehringer Ingelheim. effect. In the same line, we identified another gene implicated in negative regulation of NF-kB signaling. The cytosolic zinc finger References protein TNFAIP3/A20 has been described to be involved in NF- 1. Rosenstiel, P., G. Jacobs, A. Till, and S. Schreiber. 2008. 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