Transcriptome Assessment Reveals a Dominant Role for TLR4 in the Activation of Human Monocytes by the Alarmin MRP8

This information is current as Selina K. Fassl, Judith Austermann, Olympia of September 28, 2021. Papantonopoulou, Mona Riemenschneider, Jia Xue, Damien Bertheloot, Nicole Freise, Christoph Spiekermann, Anika Witten, Dorothee Viemann, Susanne Kirschnek, Monika Stoll, Eicke Latz, Joachim L. Schultze, Johannes Roth and Thomas Vogl Downloaded from J Immunol 2015; 194:575-583; Prepublished online 10 December 2014; doi: 10.4049/jimmunol.1401085 http://www.jimmunol.org/content/194/2/575 http://www.jimmunol.org/

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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 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Transcriptome Assessment Reveals a Dominant Role for TLR4 in the Activation of Human Monocytes by the Alarmin MRP8

Selina K. Fassl,* Judith Austermann,*,† Olympia Papantonopoulou,‡ Mona Riemenschneider,x Jia Xue,‡ Damien Bertheloot,{ Nicole Freise,* Christoph Spiekermann,* Anika Witten,x Dorothee Viemann,‖ Susanne Kirschnek,# Monika Stoll,x Eicke Latz,{,**,†† Joachim L. Schultze,‡ Johannes Roth,*,† and Thomas Vogl*,†

The alarmins myeloid-related (MRP)8 and MRP14 are the most prevalent cytoplasmic in phagocytes. When re- Downloaded from leased from activated or necrotic phagocytes, extracellular MRP8/MRP14 promote inflammation in many diseases, including infec- tions, allergies, autoimmune diseases, rheumatoid arthritis, and inflammatory bowel disease. The involvement of TLR4 and the multiligand for advanced glycation end products as receptors during MRP8-mediated effects on inflammation remains controversial. By comparative bioinformatic analysis of genome-wide response patterns of human monocytes to MRP8, endotoxins, and various cytokines, we have developed a model in which TLR4 is the dominant receptor for MRP8-mediated phagocyte acti-

vation. The relevance of the TLR4 signaling pathway was experimentally validated using human and murine models of TLR4- and http://www.jimmunol.org/ receptor for advanced glycation end products–dependent signaling. Furthermore, our systems biology approach has uncovered an antiapoptotic role for MRP8 in monocytes, which was corroborated by independent functional experiments. Our data confirm the primary importance of the TLR4/MRP8 axis in the activation of human monocytes, representing a novel and attractive target for modulation of the overwhelming innate immune response. The Journal of Immunology, 2015, 194: 575–583.

nnate immune mechanisms have a key role in inflammatory inflammatory activation of the innate immune system by patho- diseases such as infections, cardiovascular diseases, inflam- gens, the release of cytokines and chemokines triggers the immune matory bowel diseases, and autoimmune diseases. During response, often leading to chronic conditions. During the past

I by guest on September 28, 2021 decade it has become increasingly evident that the immune re- sponse is also triggered by endogenous ligands known as damage- *Institute of Immunology, University of Muenster, 48149 Muenster, Germany; †Interdisciplinary Center for Clinical Research, University of Muenster, 48149 Muen- associated molecular patterns (DAMPs) or alarmins (1). DAMPs ster, Germany; ‡Genomics and Immunoregulation, Life and Medical Sciences Insti- are intracellular molecules primarily involved in cell homeostasis, tute, University of Bonn, 53115 Bonn, Germany; xGenetic Epidemiology of Vascular Disorders, University of Muenster, 48149 Muenster, Germany; {Institute of Innate but they can also act as extracellular danger signals when released Immunity, University Hospital, University of Bonn, 53127 Bonn, Germany; ‖Clinic by damaged or activated cells. DAMPs are recognized by specific for Pediatric Pneumology and Neonatology, Hannover Medical School, 30625 Hann- members of the TLR family as well as the multiligand receptor for over, Germany; #Institute for Medical Microbiology and Hygiene, University Med- ical Center, 79104 Freiburg, Germany; **Department of Infectious Diseases and advanced glycation end products (RAGE) (2, 3). Alarmins such as Immunology, University of Massachusetts Medical School, Worcester, MA 01605; high-mobility group protein B1 and heat shock proteins orches- †† and German Center for Neurodegenerative Diseases, 53117 Bonn, Germany trate key events in the inflammatory response not only in infec- Received for publication April 28, 2014. Accepted for publication November 13, tious diseases, but also, more importantly, in sterile inflammation 2014. (4–6). This work was supported by Interdisciplinary Center of Clinical Research at the University of Muenster Grant Vo2/014/09 (to T.V.), German Research Foundation We have previously characterized the phagocyte-specific Grant CRC 1009 B8 and B9 (to T.V. and J.R.), Federal Ministry of Education and myeloid-related proteins (MRPs) MRP8 (S100A8) and MRP14 Research Project AID-NET (to J.R.), and by a grant from the German Research (S100A9) as DAMPs (7). Both proteins belong to the - Foundation (Sonderforschungsbereich 704 and Excellence Cluster ImmunoSensa- tion) (to J.L.S.). The research leading to these results has received funding from binding S100 protein family and are known to form homodimeric the People Programme (Marie Curie Actions) of the European Union Seventh Frame- as well as heterodimeric MRP8/MRP14 complexes. MRP8 work Programme FP7/2077-2013 under Research Executive Agency Grant 317445. homodimers activate cells of the innate immune system and in- Address correspondence and reprint requests to Dr. Thomas Vogl, Institute of Immu- duce proinflammatory cytokine and adhesion molecule expression nology, University of Muenster, Ro¨ntgenstrasse 21, 48149 Muenster, Germany. E-mail address: [email protected] in phagocytes and endothelial cells, resulting in leukocyte re- The online version of this article contains supplemental material. cruitment and activation (8–10). Studies involving mice deficient for MRP14 have uncovered a pivotal role for MRP8/MRP14 in the Abbreviations used in this article: cIAP1, cellular inhibitor of 1; CRA, coregulation analysis; DAMP, damage-associated molecular pattern; FDR, false dis- promotion of inflammation in many clinically relevant conditions covery rate; GO, ontology; GOEA, enrichment analysis; HC, (7, 9, 11–15). hierarchical clustering; HEK, human embryonic kidney; IP-10, IFN-g–inducible pro- tein 10; MIG, monokine induced by IFN-g; MRP, myeloid-related protein; PCA, However, the identity of the receptor responsible for MRP8- principal component analysis; PCCM, Pearson correlation coefficient matrix; qRT- mediated effects on inflammation is still a matter of debate. PCR, quantitative real-time PCR; RAGE, receptor for advanced glycation end prod- RAGE has been described as the receptor for several S100 proteins, ucts; RPL, ribosomal protein L; WT, wild-type. including MRP8 and MRP14 (16, 17). In vitro studies have Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 demonstrated that MRP8/MRP14 binds to immobilized RAGE www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401085 576 ACTIVATION OF MONOCYTES BY MRP8 IS TLR4-DEPENDENT

(18, 19), and that RAGE but not TLR4 associates with MRP8/ Steinheim, Germany), and 20 ng/ml GM-CSF or 1% culture supernatant MRP14 in colon tumor cells (20). However, studies using TLR4 or from a GM-CSF–producing cell line (B16 melanoma cells). Macrophage RAGE knockout mouse strains indicate that TLR4 is the main differentiation was induced by removal of . ER-Hoxb8 macro- phages were used on day 5 of differentiation and were stimulated (2.5 3 receptor that functions during activation of murine phagocytes (9). 106) with 5 mg/ml murine MRP8 for 4 h. Similarly, conflicting data also exist in humans. MRP8/MRP14 colocalizes with RAGE in LNCaP cells (21), and a functional Reagents and Abs role for MRP8/MRP14–RAGE interaction has been described in LPS from Escherichia coli 055:B5, polymyxin B, and anti–b-actin (AC15) inflammation-associated cancer (22). MRP8/MRP14 also pro- Ab were obtained from Sigma-Aldrich. Annexin V–allophycocyanin motes tumor cell growth and mediates endotoxin-induced car- (31490016X2) and recombinant murine GM-CSF were purchased from diomyocyte dysfunction in a RAGE-dependent manner (23, 24). ImmunoTools (Friesoythe, Germany) and staurosporine from Alexis Bio- chemicals (San Diego, CA). Anti–Bcl-xL (610212) Ab was obtained from In contrast to this, MRP8/MRP14 also induces RAGE-independent BD Biosciences (San Diego, CA). Abs against cleaved caspase-3 (9661), cell death in several tumor cell lines (25) and displays a TLR4- cellular inhibitor of apoptosis 1 (cIAP-1; 4952), IkBa (4812), phospho-p38 dependent catabolic effect on human chondrocytes (26). (9211), total p38 (9212), phospho-MEK (9121), phospho-ERK1/2 (9101), Recent systematic comparisons of the inflammatory processes of and total ERK (9102) were purchased from Cell Signaling Technology mice and humans have suggested that inflammatory responses in (Danvers, MA). human diseases correlate poorly with the corresponding mouse Protein expression and purification of MRP8 models. These data clearly underline the need for translational medical research to generate reliable conclusions on human MRP8 was expressed in E. coli BL21(DE3) cells and purified as previously described (9, 30). Briefly, pET11/20 expression vector containing human inflammatory diseases (27). However, experimental systems or murine MRP8 cDNA was used to transform E. coli BL21(DE3) bacteria. Downloaded from involving primary human phagocytes are limited, as human Bacteria were grown at 37˚C in 23 YT for 24 h. Afterward, bacteria were knockout models are not available and efficiencies of small in- harvested, lysed, and the inclusion bodies were prepared. The inclusion terfering RNA studies are limited by low transfection and high cell bodies pellet was dissolved in 8 M urea buffer, and to establish proper damage/mortality rates. We therefore took a systems biology ap- refolding samples were adjusted to pH 2.0–2.5 first by adding hydrochloric acid. After 60 min incubation at room temperature, samples were stepwise proach to defining the response of human monocytes to MRP8 and dialyzed to get adapted to pH 7.4 for refolding in the presence of 2 mM subsequent downstream signaling pathways. We have confirmed DTT. After centrifugation (10 min, 60,000 3 g, 4˚C) to pellet-aggregated http://www.jimmunol.org/ the previously elucidated proinflammatory functions of murine as material, samples were further dialyzed and applied to anion exchange well as human MRP8 at the genome-wide level, and have also column and gel filtration chromatography. The MRP8 protein concen- trations were determined by UV absorption at 280 nm using a specific uncovered a novel antiapoptotic role for MRP8 in monocytes. absorption coefficient of 1.02 (human MRP8) or 0.40 (murine MRP8) Additional bioinformatic analyses clearly demonstrate a dominant ml/mg21/cm21, respectively. Possible endotoxin contaminations were de- role for human TLR4 during MRP8-mediated phagocyte activa- termined by a Limulus amebocyte lysate assay (BioWhitaker, Walkersville, tion. Taken together, our data have identified the TLR4/MRP8 axis MD) and could either not be detected or were ,1 pg LPS/mgMRP8inthe as a potent and novel target in humans for future investigations different batches. into anti-inflammatory approaches. Determination of cytokine concentrations by guest on September 28, 2021 Materials and Methods Human TNF-a, IL-1b, and IL-6 were analyzed in culture supernatants 6 Cell culture conditions from human monocytes (1.6 3 10 cells/ml) using commercial ELISA kits (BD Biosciences). Mouse TNF-a and IL-1b were analyzed in culture Approval was obtained from the Ethics Committee of the Medical Faculty supernatants of Hoxb8 cells (2.5 3 106 cells/ml) using commercial ELISA of Muenster for these studies. Human monocytes were isolated from blood kits (mouse TNF-a from BD Biosciences; mouse IL-1b from eBioscience, samples after leukapheresis according to Roth et al. (28). Purity was always San Diego, CA). .90% as ascertained by flow cytometry. Cells were cultivated overnight in Teflon bags in McCoy’s 5a medium (Biochrom, Berlin, Germany) sup- Gene array expression studies and bioinformatic analysis of plemented with 15% FCS (Life Technologies, Eggenstein, Germany) prior microarray data to use for stimulation or in functional assays. Human embryonic kidney (HEK)293 cells stably transfected with human Total RNA was isolated using the NucleoSpin RNA II kit (Macherey- TLR4-CD14-MD2 or TLR2 were purchased from InvivoGen (San Nagel, Duren,€ Germany), and hybridization to Affymetrix (Santa Clara, CA) Diego, CA) together with control cells transfected with empty vectors. 133 Plus 2.0 Gene Chip arrays was performed as previ- HEK293 cells stably transfected with human RAGE were provided by M. E. ously described (31). Data were imported into the Partek Genomics Suite Bianchi (Division of Genetics and Cell Biology and Division of Regen- v6.6 (Partek, St. Louis, MO) using robust multiarray average prior to batch erative Medicine, Stem Cells and Gene Therapy, San Raffaele University correction. Robust multiarray average is an algorithm used for background and Scientific Institute, Milan, Italy) and used as described for TLR4- correction, log2 transformation, and quantile normalization of Affymetrix expressing HEK293 cells (9). Cells were grown in DMEM (Biochrom) expression data. A one-way ANOVA test was performed to calculate the supplemented with 4.5 g/l glucose and 10% FCS at 37˚C in 5% CO2. 1000 most variable and differentially expressed genes across the different Human monocytes (1.6 3 106/ml) or HEK293 cells (5 3 105/ml) were stimulation groups. Differentially expressed genes were defined by a fold stimulated with 5 mg/ml human MRP8, 1 ng/ml LPS, 10 ng/ml TNF-a,or change .2.5 or ,22.5 and a false discovery rate (FDR)–corrected p value 1 ng/ml IL-1b for 4 h. of ,0.05. To visualize the structure within the data, we performed hier- HEK293 cells stably expressing TLR9 were transiently transfected with archical clustering (HC) and principal component analysis (PCA) on the an NF-kB–driven luciferase reporter and wild-type (WT) RAGE or 1000 most variable genes, with default settings in the Partek Genomics mCherry (control) using GeneJuice (Novagen) as transfection reagent. Suite, on FDR-corrected p values according to the expression values of the Cells were incubated at 37˚C for 24 h to allow protein expression. Cells samples across the conditions. In addition, we performed co-regulation were subsequently stimulated with a dose gradient of CpG2006 (Metabion) analysis (CRA) based on Pearson correlation coefficients using BioLay- from 63 to 250 nM either alone or together with 5 mg/ml MRP8. As out Express 3D (32), as well as Pearson correlation coefficient matrix controls, cells were also stimulated with MRP8 alone (5 mg/ml), LPS (200 (PCCM) analysis, which was plotted as a heat map (33). To identify the ng/ml), or human TNF-a (1 ng/ml). Luciferase assay readout was done by differences and similarities between MRP8- and LPS-stimulated con- incubating cell supernatants with coelentrazine (Promega) and subsequent ditions, differentially expressed genes were visualized with BioVenn (34) measurement of luminescence with a SpectraMax i3 plate reader (Mo- and SigmaPlot version 10.0 (Systat Software, San Jose, CA) as a ratio- lecular Devices). ranked (log2) plot. To determine enhanced or inhibited biological functions ER-Hoxb8 macrophage progenitors derived from WT, TLR42/2, of MRP8-stimulated monocytes, we used the 200 most strongly upregu- RAGE2/2, and MyD882/2 C57BL/6 mice were generated as previously lated and downregulated genes to generate and visualize networks based described (29). Cells were maintained in RPMI 1640 medium (Biochrom) on gene ontology (GO) enrichment analysis (GOEA) using BiNGO, supplemented with 10% FCS, 1 mM estradiol (E-2758; Sigma-Aldrich, EnrichmentMap, and Word Clouding in Cytoscape (33). Data are provided The Journal of Immunology 577 in Omnibus (GSE56681; http://www.ncbi.nlm.nih.gov/ identified novel aspects of MRP8 biology, such as the induction of geo/query/acc.cgi?acc=GSE56681). genes involved in apoptotic processes in leukocytes (Fig. 1D). In Quantitative real-time PCR fact, when deconvoluting the genes from the GO terms, it was found that a large number of antiapoptotic genes were induced in Total RNA was isolated using a NucleoSpin RNA II kit (Macherey-Nagel). One microgram of total RNA was reverse-transcribed to cDNA using response to MRP8 stimulation (Supplemental Table III). To ad- RevertAid H Minus transcriptase (Fermentas, St. Leon-Roth, Germany). dress whether the MRP8-induced effects on cell survival identified Primers used are listed in Supplemental Table I. in silico were indeed functionally relevant, we confirmed the Quantitative real-time PCR (qRT-PCR) was performed using SYBR upregulation of antiapoptotic genes by qRT-PCR (Supplemental Green (PeqLab, Erlangen, Germany), and data were acquired with the CFX Table III) and demonstrate that expression of antiapoptotic pro- 384 system and CFX Manager software version 3.0 (Bio-Rad, Munich, Germany) as previously described (35). Each assay was set up in duplicate teins cIAP-1 and Bcl-xL were significantly upregulated in mono- and the relative expression was calculated using 2DCT, and the house- cytes after treatment with MRP8 compared with unstimulated keeping genes ribosomal protein L (RPL) and GAPDH were used as cells. We also observed decreased activation of caspase-3 during controls. staurosporine-induced apoptosis after MRP8 stimulation (Fig. 1E). Induction and analysis of apoptosis We quantified apoptosis rates by staining for annexin V and in addition necrosis with propidium iodide (Fig. 1F). MRP8- Monocytes were treated as described previously (36). Monocytes presti- mulated with MRP8 were treated with 400 nM staurosporine for an ad- stimulated monocytes displayed higher spontaneous survival ditional 6 h or were left untreated. The percentage of apoptotic and rates compared with untreated cells, and they were also signifi- necrotic cells was determined by staining with allophycocyanin-conjugated cantly protected from staurosporine-induced apoptosis as indi- annexin V and propidium iodide and analyzed by flow cytometry using cated by GOEA. These antiapoptotic effects are known for TLR Downloaded from a FACSCalibur flow cytometer (BD Biosciences) and FlowJo software version 7.6.5 (Tree Star, Ashland, OR). agonists as LPS, and the MRP8 stimulation induces similar effects (Fig. 1E, 1F). Western blot analysis To rule out endotoxin contamination in protein preparations, we Cells were lysed in high-salt buffer containing a protease inhibitor mixture strictly screened all protein batches for potential contamination by or a nuclear extraction kit (Epigentek, Farmingdale, NY) was used for cell LPS (9). Our recombinant-prepared protein was shown to be en-

fractionation, and equal amounts of protein were separated on SDS- tirely free of any contaminating E. coli proteins (Fig. 2A). En- http://www.jimmunol.org/ polyacrylamide gels and transferred to nitrocellulose membranes. Mem- dotoxin was not detectable in our protein preparations by the branes were probed with primary Abs overnight at 4˚C. Primary Abs were detected with HRP-conjugated secondary Abs and developed with ECL. Limulus amebocyte assay (detection limit 1 pg/mg protein). This indicates that the maximal possible endotoxin contamination of Statistical analysis MRP8 protein preparations is ,5 pg LPS in the experiments, All values given throughout are expressed as mean 6 SEM or mean 6 SD which alone did not induce any cytokine induction (data not from at least three independent experiments. Statistical analyses were shown). Furthermore, MRP8-induced effects could not be inhib- performed using t tests and Mann–Whitney U tests. A p value ,0.05 was ited by addition of polymyxin B (50 mg/ml) at concentrations that judged to be significant. efficiently blocked the action of 1 ng/ml LPS. Finally, heat- inactivated MRP8 failed to stimulate monocytes, whereas LPS by guest on September 28, 2021 Results was still fully active at these conditions (Fig. 2B). MRP8-regulated gene expression in human monocytes We performed a global gene expression analysis to define the MRP8 and LPS stimulation induce the most closely related cellular response of human monocytes to MRP8 treatment. response patterns in human monocytes Monocytes from individual donors were stimulated with MRP8 or Which of the receptors present on human monocytes is respon- left untreated to assess changes in gene expression patterns. Using sible for MRP8-mediated effects during inflammation is still hierarchical clustering of the most variable genes (p , 0.05) within a matter for debate. To define the relevant MRP8 receptor the dataset, we demonstrate that MRP8 induces a strong tran- in human monocytes, we compared the genome-wide tran- scriptional response (Fig. 1A). This was further corroborated by scriptomes of MRP8-stimulated monocytes with human mono- PCA showing that MRP8-stimulated monocytes were distinct cytes activated by LPS, TNF-a,orIL-1b. Following previously from unstimulated cells (Fig. 1B). We analyzed 22,277 transcripts developed approaches (33), we performed several bioinfor- using Affymetrix U133 arrays and identified 1,512 differentially maticanalyses,includingCRA(Fig.3A),PCA(Fig.3B),HC expressed genes (ANOVA test; fold change .2.5 or ,22.5, FDR- (Fig. 3C), and PCCM analyses (Fig. 3D), to determine the re- corrected p , 0.05), 516 of which were upregulated and 996 lationship of MRP8-induced transcriptional reprogramming with downregulated by MRP8 (Supplemental Table II). Differential the other stimuli. CRA revealed a clear group-based network regulation of 13 selected genes identified by microarray analysis structure with control samples separated from stimulated sam- was validated by quantitative real-time PCR (qRT-PCR; ples. Moreover, the MRP8-stimulated samples were most closely Supplemental Table III). Additionally, we confirmed our find- related to LPS stimulation, whereas TNF- and IL-1b–stimulated ings at the protein level for three secreted cytokines (IL-6, TNF-a, sampleswereplacedinasubcluster(Fig.3A).Thesefindings and IL-1b) by ELISA of cell culture supernatants (Fig. 1C). No- were corroborated by PCA, again showing that MRP8 stimula- tably, RNA expression of both potential MRP8 receptors, TLR4 tion is most closely related to LPS stimulation (Fig. 3B). Using and RAGE, showed no alterations in MRP8-treated monocytes. the most variable genes within the dataset, we found using HC To elucidate the biological processes induced by MRP8, we that most gene clusters were similarly regulated by MRP8 and performed GOEA based on the 200 most differentially expressed LPS (Fig. 3C). Finally, PCCM analysis also confirmed that genes followed by network visualization of enriched GO terms MRP8 stimulation was most closely related to LPS stimulation using BiNGO and EnrichmentMap (Fig. 1D). As expected, the of monocytes (Fig. 3D). We further visualized the groups of overwhelming majority of genes were related to immune cell genes differentially expressed after MRP8 or LPS stimulation activation, including categories such as NF-kB signaling, che- using a Venn diagram (Fig. 3E) and a ratio-ranked (log2)plot motaxis, cell migration, and inflammatory response, as well as (Fig.3F).Mostgenesdifferentially regulated by LPS were also leukocyte activation and signal transduction. However, we also regulated by MRP8 (purple area), although MRP8 induced an 578 ACTIVATION OF MONOCYTES BY MRP8 IS TLR4-DEPENDENT Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. Transcriptional regulation in MRP8-stimulated human monocytes. Human monocytes were left untreated or stimulated with 5 mg/ml MRP8 or 1 ng/ml LPS for 4 h. (A) Hierarchical clustering of the 1000 most variable genes. Data were z-score normalized and ranked according to change in expression upon stimulus. (B) PCA of the 1000 most variable genes within the dataset (p , 0.05, one-way ANOVA test). (C) Secretion of IL-6, TNF-a, and IL-1b in the supernatants was analyzed by ELISA. Data represent mean values 6 SEM of eight independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. (D) Network visualization of GOEA of the top 200 upregulated and 200 downregulated genes using BiNGO and EnrichmentMap. Red nodes represent enriched GO terms, whereas node size represents corresponding FDR-adjusted enrichment p value (q value). Edge thickness shows overlap of genes between neighbor nodes. (E and F) Spontaneous and staurosporine-induced apoptosis were analyzed after incubation with staurosporine for 6 h.

Antiapoptotic proteins cIAP-1 and Bcl-xL and cleavage of caspase-3 were analyzed by Western blot. One representative blot is shown from the four independent experiments performed (E). Percentage of apoptotic and necrotic cells was evaluated by parallel staining with annexin V (x-axis) and pro- pidium iodide (y-axis), respectively, and quantified by flow cytometry. Data represent mean values 6 SEM of six independent experiments. **p , 0.01. One representative dot plot is shown (F). The Journal of Immunology 579

controls as demonstrated by qRT-PCR, which is in accordance with results derived from human monocytes. In contrast, HEK293RAGE, HEK293TLR2, and HEK293MOCK cells showed no response, neither to MRP8 nor to LPS stimulation. HEK293TLR2 cells responded well to lipoprotein stimulation, providing a posi- tive control for TLR2 activation (Fig. 5A, 5B). As a positive control for RAGE-dependent signaling we used the HEK293RAGE/TLR9 model recently described (37). Although RAGE nicely increased CpG2006-induced TLR9 response, MRP8 neither antagonized nor amplified this effect. Of note, MRP8 alone did not activate RAGE in this system (Fig. 5C). FIGURE 2. MRP8 preparation is free of any endotoxin contamination. To prove that signaling pathways are identical in the human (A) Purity of MRP8 preparation by staining. (B) and murine systems, we stimulated estrogen-regulated Hoxb8 Human monocytes were stimulated with 5 mg/ml MRP8 or 1 ng/ml LPS in progenitor cell–derived macrophages from WT, TLR42/2, the presence or absence of polymyxin B for 4 h, or stimulated with MRP8 RAGE2/2, and MyD882/2 mice on day 5 of macrophage differ- and LPS that was preincubated for 30 min at 80˚C prior to stimulation. 2 2 entiation. RAGE / cells responded to MRP8 stimulation in a TNF-a levels in the supernatants were determined by ELISA. *p , 0.05, ***p , 0.001. manner similar to WT controls, as demonstrated by induction of TNF-a, IL-1b, and IL-6 shown by qRT-PCR at the RNA level

(Fig. 5D) and TNF-a and IL-1b at the protein level (Fig. 5E). In Downloaded from even stronger response (red area; Fig. 3E). Using the ratio- contrast, removal of TLR4 or the downstream signaling adaptor molecule MyD88 abolished activation by MRP8. ranked (log2) plot, we confirmed that the large number of genes significantly induced or repressed by MRP8 stimulation Moreover, blocking the TLR4 receptor with the mAb HTA125 that were not significantly altered in response to LPS (red dots) or TLR4 antagonist Rhodobacter sphaeroides LPS in human did, however, show the same regulation trends. This provides monocytes confirmed our findings. Both agents efficiently further support for the notion that MRP8 and LPS trigger a very inhibited the MRP8- as well as the LPS-induced secretion of http://www.jimmunol.org/ similar transcriptional reprogramming of monocytes. Among the TNF-a (Fig. 5F). These results confirm TLR4 as the dominant genes most significantly induced were IL-6, CCL20, and IL-1a, receptor of MRP8-mediated activation of monocytes and mac- all of which are well known to be induced by LPS via TLR4 rophages. signaling. The similarity of gene expression patterns, together with the induction of common cytokines in MPR8- and LPS- Discussion stimulated human monocytes, has provided us with evidence DAMPs are a heterogeneous group of proteins that are released that MRP8 is a specific of human TLR4. upon cell stress and tissue damage and activate the innate and adaptive immune systems. Recent evidence has indicated that MyD88-dependent and -independent TLR4 signaling pathways uncontrolled release of DAMPs is pivotal in many inflammatory by guest on September 28, 2021 are activated by MRP8 stimulation and autoimmune diseases (1). However, the underlying molec- To further confirm the role of TLR4 in MRP8-mediated stimulation ular mechanisms mediating DAMP-induced inflammation are of human monocytes, we analyzed pathway members downstream currently not well defined. The contribution of various DAMP of TLR4 signaling. Several members of the MAPK family are receptors such as TLR4 and RAGE has also been a controversial activated by phosphorylation during TLR4 signaling. Accordingly, topic of discussion (17). Especially in the case of MRP8 and we detected activation of MEK, ERK1/2, and p38 in MRP8- MRP14, numerous studies have described various contributions stimulated monocytes (Fig. 4A). We also observed an induction of both receptors to several different aspects of inflammation. of IkBa degradation (Fig. 4A), resulting in NF-kB activation and Both proteins belong to the DAMP family, and they promote translocation (Fig. 4B). Furthermore, qRT-PCR demonstrated that inflammatory processes during infections, allergies, and auto- inflammatory cytokines such as TNF-a, IL-6, and IL-8, which are immune diseases (1). However, the initial events during protein known to be induced via MyD88- and IkBa/NF-kB–dependent binding to its receptor are not yet clear. Several studies have pathways, are upregulated after MRP8 treatment. This is in ad- demonstrated interactions of MRP8/MRP14 with RAGE, fo- dition to cytokines such as CD80, IFN-g–inducible protein 10 cusing mainly on signaling pathways involving activation of (IP-10), and monokine induced by IFN-g (MIG), which are in- MAPK p38 or the transcription factor NF-kB, including cellular volved in the MyD88-independent, Toll/IL-1R domain–containing mechanisms downstream of the MRP8/MRP14–RAGE interac- adapter inducing IFN-b/IFN regulatory factor 3–dependent TLR4 tion. However, no structural data regarding the ligand-receptor signaling pathway (Fig. 4C). Overall, this clearly demonstrates recognition sites have been described so far (21, 22, 24, 38). that both known TLR4 signaling pathways are activated by MRP8 Similar MRP8/MRP14–RAGE interactions were observed in in a similar manner to LPS. a study done by Boyd et al. (23) on purified cardiomyocytes, during which RAGE was coimmunoprecipitated with MRP8/ MRP8 induces cytokines via TLR4 but not RAGE signaling in MRP14. Binding of MRP8 and/or MRP14 to both TLR4 (9, various cell models 19) and RAGE (18, 19) has been demonstrated in BIAcore Because we could not detect substantial amounts of RAGE ex- studies, and strong binding constants in the low nanomolar pression on the human monocytes by flow cytometry and Western range could be calculated for both receptors. Although MRP8 blot analyses (data not shown), we used HEK293 cells stably has been unequivocally demonstrated to be an active component transfected with TLR4/CD14/MD2 or RAGE as well as TLR2 as for phagocyte stimulation in vitro (9), the biologically relevant controls for specificity to verify that TLR4 is the primary receptor complex forms of MRP8/MRP14 in vivo, however, are still to be for human MRP8. HEK293TLR4/CD14/MD2 cells showed a strong defined. Furthermore, it is not clear whether MRP8/MRP14 induction of proinflammatory cytokines, including TNF-a, IL-8, interacts directly with certain amino acids within the RAGE and MCP-1, on stimulation with MRP8 and LPS as positive receptor or to carboxylated glycans covalently attached to 580 ACTIVATION OF MONOCYTES BY MRP8 IS TLR4-DEPENDENT Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. MRP8 and LPS stimulation induce a similar expression pattern in human monocytes. Human monocytes were left untreated or stimulated with 5 mg/ml MRP8, 1 ng/ml LPS, 1 ng/ml IL-1b, or 10 ng/ml TNF-a for 4 h. RNA was isolated and used for gene expression arrays. For (A)–(D), the 1000 most variable genes (FDR-corrected p , 0.05) within the dataset were used. (A) Correlation networks with Pearson correlation coefficient of 0.90. (B) PCA. (C) HC. Data were z-score normalized and ranked according to change in expression upon stimulus. (D) PCCM. (E) Venn diagram of genes with differential expression (MRP8 and LPS versus control; fold-change limit 2.5, FDR-corrected p , 0.05, one-way ANOVA test) as a result of MRP8 (red) or LPS stimulation (blue), as well as genes that are coregulated by both treatments (purple). Numbers of upregulated and downregulated genes are given. (F) The same gene sets in (E) visualized as a ratio-ranked (log2) plot. The Journal of Immunology 581

which in turn suppress adaptive immunity and thereby facilitate tumor growth. However, the relevance of either RAGE or TLR4 to any of these studies has not been investigated so far (40, 41). Whether murine data are always transferable to the human system has been controversial. Recent systematic comparisons of inflammatory processes between mice and humans have suggested that inflammatory responses in human diseases correlate only poorly with the corresponding mouse models (27). To better un- derstand the underlying molecular mechanisms of inflammation in humans, we have in the present study taken a systems biology approach utilizing the transcriptome of phagocytes to generate MRP8-receptor interaction models. We focused on MRP8 in this study because this molecule has been unequivocally identified as a biologically active component of MRP8/MRP14 complexes (9). We have used microarray technology to define the MRP8-induced transcriptome in human monocytes and determined a dominant proinflammatory response to MRP8 at the genome-wide level in human monocytes. Linking our data to prior knowledge by GOEA

combined with GO term network construction corroborated the Downloaded from strong inflammatory signal induced by MRP8. Additionally, this approach has revealed an antiapoptotic role for MRP8 in monocytes as indicated by the network visualization of GOEA. We could experimentally confirm that MRP8 modulates the mitochondria-dependent apoptotic pathway in monocytes. Ex-

pression of genes involved in this antiapoptotic mechanism was http://www.jimmunol.org/ significantly upregulated at the mRNA and protein levels. MRP8- stimulated monocytes were likewise significantly protected from staurosporine-induced apoptosis. These findings are in contrast to the proapoptotic effect of MRP8/MRP14 described in endothelial cells and several tumor cell lines (25, 42). The molecular path- ways responsible for these cell-specific differences are, however, not yet clear. FIGURE 4. MRP8-induced phosphorylation of MEK, ERK, and To elucidate whether TLR4 is the main receptor for MRP8 on p38, degradation of IkBa and NF-kB translocation, as well as induction human monocytes, we have performed a comparative genomic by guest on September 28, 2021 a of TNF- , IL-6, CD80, IP-10, and MIG in human monocytes. Human analysis, again utilizing the transcriptome to determine overlaps or monocytes were left untreated or stimulated with MRP8 for 4 h, and cell differences between various stimuli in comparison with MRP8. We lysates were prepared for Western blot (A and B) or RNA was isolated (C). (A and B) Cell lysates of three independent experiments were analyzed by postulated that transcriptional changes induced via TLR4 should Western blot with Abs indicated in the figure (one representative Western closely mimic those induced by LPS, whereas downstream effector blot is shown). (C) qRT-PCR analysis of the expression of TNF-a, IL-6, cytokines such as IL-1b or TNF-a should induce alternative CD80, IP-10, and MIG. Results are presented relative to baseline ex- responses (Fig. 3). Utilizing a set of previously developed tech- pression in unstimulated cells, and RPL was used as a housekeeping niques, including CRA, PCA, HC, and PCCM, we have demon- control gene. Data are shown as mean values 6 SEM from four inde- strated on a global level that MRP8 stimulation is most closely pendent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. related to LPS stimulation, strongly indicating that TLR4 is a prime candidate for the MRP8 receptor. Single-transcript anal- ysis using ratio plots further corroborated these findings. Our data RAGE (16, 19). For isolated MRP14, similar findings were did show some quantitative differences between MRP8 and LPS found. Although it could be shown that MRP14 binds to both stimulation, which are most likely explained by different dosing receptors TLR4 as well as RAGE (19), cytokine induction could effects. Owing to the low expression level of RAGE in human only be observed for MRP14-TLR4–dependent activation in monocytes, we cannot completely rule out the possibility of an a cell line culturing model (39). Studies on phagocytes from additional MRP8/RAGE axis, which might be relevant in other TLR4 mutant mice clearly indicate that TLR4 is mainly re- cell types. However, using a HEK293 cell line–based system sponsible for MRP8-dependent activation in these cells (9). where RAGE was overexpressed to a high level, we neither could Further evidence of this TLR4 dependency comes from two detect direct nor costimulatory or antagonistic effects of MRP8 on studies that show that in MRP14-deficient mice, or when TLR4 these cells, indicating that RAGE has an inferior relevance for is blocked with TAK-242 (a small molecule TLR4 inhibitor), MRP- mediated effects. the cellular response to MRP8/MRP14 is almost completely To experimentally validate this computationally derived model abrogated (12, 26). In one of these studies, blocking of RAGE indicating a role for TLR4 in MRP8 stimulation in human or carboxylated glycans by specific Abs did not change the monocytes, we analyzed downstream elements of TLR4 signaling MRP14-mediated expression of matrix metalloproteinases, type in MRP8-treated monocytes. We observed that expression of II collagen, or cytokines (26). MyD88-dependent genes such as TNF-a, IL-6, and IL-8 as well as There are also no consistent data suggesting that either RAGE MyD88-independent genes such as CD80, IP-10, and MIG is in- or TLR4 is the dominant receptor during MRP8/MRP14-mediated duced by MRP8, indicating that both TLR4 signaling pathways tumor genesis (18, 20, 40). MRP8/MRP14 promotes tumor are activated on MRP8 stimulation. Analysis of HEK293 cells growth by inducing so-called myeloid-derived suppressor cells, stably transfected with either TLR4/CD14/MD2 or RAGE con- 582 ACTIVATION OF MONOCYTES BY MRP8 IS TLR4-DEPENDENT Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. MRP8 stimulation of transfected HEK293 cells is TRL4/CD14/MD2-dependent. (A and B) Stably transfected HEK293 cells (TLR4/CD14/ MD2, RAGE, or empty vector) were left untreated or stimulated with 5 mg/ml MRP8 or 1 ng/ml LPS (A) and TLR2-expressing HEK293 cells were stimulated with 5 mg/ml MRP8 or lipoprotein (LP) (B). Activation of cells was investigated by qRT-PCR analysis of TNF-a, IL-8, and MCP-1 mRNA expression. Results are shown as relative to baseline expression in unstimulated cells, and RPL was used as a housekeeping control gene. (C) HEK293 cells stably expressing TLR9 were transiently transfected with an NF-kB–driven luciferase reporter and RAGE or mCherry (control). Cells were stimulated as indicated with CpG2006 (nM), MRP8 (5 mg/ml), LPS (200 ng/ml), or TNF-a (1 ng/ml). Data are shown as mean 6 SD for triplicate samples and are representative of two experiments. (D and E) WT, TLR42/2,RAGE2/2, or MyD882/2 Hoxb8 cells were differentiated for 5 d down the macrophage lineage and stimulated with 5 mg/ml MRP8 for 4 h. (D) qRT-PCR analysis of expression levels of TNF-a, IL-1b, and IL-6. RPL was used as a housekeeping control gene, and data are presented as copies per 10,000 copies of RPL. (E) TNF-a and IL-1b protein levels in supernatants of stimulated cells were determined by ELISA. Results are mean values 6 SEM from three independent experiments. (F) Human monocytes were preincubated for 30 min with 2 mg/ml Ab to human TLR4 (clone HTA125) or isotype-matched control Ab (IgG2a) or 1 mg/ml TLR4 antagonist Rhodobacter sphaeroides LPS, followed by 4 h stimulation with LPS or MRP8 as indicated. *p , 0.05, **p , 0.01, ***p , 0.001.

firmed signaling of MRP8 through TLR4, and blocking of this therefore be attractive targets for novel therapeutic approaches in- receptor in human monocytes abolishes TNF-a secretion. Finally, volving the prevention of the binding of these molecules to their we confirmed that these signaling pathways are identical in receptor. Taken together, our results clearly indicate that data obtained humans and mice. Collectively, these data unequivocally confirm in murine inflammatory models can be transferred to the human TLR4 as the primary receptor for MRP8 in human monocytes system in the case of MRP8-dependent signaling in monocytes. under inflammatory conditions as previously described (9). Because the MRP8/MRP14 heterodimer is one of the most Acknowledgments significantly upregulated molecules in many inflammatory diseases We thank H. Berheide, H. Hater, D. Lagemann, and U. Nordhues for (7, 14), the mechanism described in the present study is likely to excellent technical support. be highly clinically relevant. Targeted deletion of these genes in mice results in a significant phenotype under inflammatory Disclosures conditions such as autoimmune diseases, arthritis, infections, LPS- The authors have no financial conflicts of interest. induced shock, and allergies (12, 43). Expression of these pro- teins in humans correlates very well with disease activity, espe- cially in predicting the response to therapy for arthritis and References 1. Chan, J. K., J. Roth, J. J. Oppenheim, K. J. Tracey, T. Vogl, M. Feldmann, inflammatory bowel disease, as well as the risk of flares in re- N. Horwood, and J. Nanchahal. 2012. Alarmins: awaiting a clinical response. J. mitting–relapsing disease courses (7, 8, 11). These molecules may Clin. Invest. 122: 2711–2719. The Journal of Immunology 583

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Supplementary Table I. Sequences of primer pairs used for qRT‐PCR.

Human primers Gene Forward primer Reverse primer IL‐6 5´‐AGAGGCACTGGCAGAAAACAAC‐3´ 5´‐AGGCAAGTCTCCTCATTAATCC‐3´ IL‐19 5´‐TCTGCGGCAATGTCAGGAA‐3´ 5´‐GGATTTAATGGCAGCAGCGT‐3´ SOCS1 5´‐AGCTCCTTCCCCTTCCAGATTT‐3´ 5´‐CCACATGGTTCCAGGCAAGTAATA‐3´ G‐CSF 5´‐ACAAGCAGAGGTGGCCAGAG‐3´ 5´‐CAAACCATGTCCCAAAAGTCTTAAG‐3´ SOCS3 5´‐CCTCAAGACCTTCAGCTCCAA‐3´ 5´‐ACTGCGCTCCAGTAGAAGCC‐3´ TNFAIP3 5´‐CGTCAGAAGCTGGCTGATTTG‐3´ 5´‐CCTTGTCGGTCTCCTCCATTTC‐3´ IL‐10 5´‐GCTGAGAACCAAGACCCAGACA‐3´ 5´‐CGGCCTTGCTCTTGTTTTCA‐3´ IL‐1α 5´‐AGACCTACGCCTGGTTTTCC‐3´ 5´‐GCTGAAGGAGATGCCTGAGAT‐3´ CD80 5´‐ CTGCTTTGCCCCAAGATGC‐3´ 5´‐CAGATCTTTTCAGCCCCTTGC‐3´ SOCS2 5´‐ CTGACCAAACCGCTCTACACGT‐3´ 5´‐ TTGTTGGTAAAGGCAGTCCCC‐3´ NFκB5´‐TTATGTATGTGAAGGCCCATCC‐3´ 5´‐TGGTCCCACATAGTTGCAGATT‐3´ TNF‐α 5´‐CTTCTCGAACCCCGAGTGAC‐3´ 5´‐TGAGGTACAGGCCCTCTGATG‐3´ Bcl‐XL 5´‐TGCGTGGAAAGCGTAGACAAG‐3´ 5´‐TCCACAAAAGTATCCCAGCCG‐3´ cIAP1 5´‐CAAGTGGTTTCCAAGGTGTGAGTA‐3´ 5´‐GATGTGGATAGCAGCTGTTCAAGTAG‐3´ cIAP2 5´‐GAAAAGCGCCAACACGTTTG‐3´ 5´‐AGGGACCCCAGCAGGAAAA‐3´ IP‐10 5´‐AGACATCTCTTCTCACCCTTC‐3´ 5´‐GGAACCTCCAGTCTCAGCACCA‐3´ MIG 5´‐TTTGGCTGACCTGTTTCTCC‐3´ 5´‐TTCCTCTTGGGCATCATCTT‐3´ RPL 5´‐AGGTATGCTGCCCCACAAAAC‐3´ 5´‐TGTAGGCTTCAGACGCACGAC‐3´ MCP‐15´‐TCGCCTCCAGCATGAAAGTC‐3´ 5´‐TTGCATCTGGCTGAGCGAG‐3´ GAPDH 5´‐CAAATTCCATGGCACCGTCA‐3´ 5´‐TCGCCCCACTTGATTTTGG‐3´ Murine primers TNF‐α 5´‐AGAAACACAAGATGCTGGGACAGT‐3´ 5´‐CCTTTGCAGAACTCAGGAATGG‐3´ IL‐1β 5´‐TGGGCTGGACTGTTTCTAATGC‐3´ 5´‐ TGTCTTGGCCGAGGACTAAGG‐3´ IL‐65´‐TGAGATCTACTCGGCAAACCTAGTG‐3´ 5´‐CTTCGTAGAGAACAACATAAGTCAGATACC‐3´ RPL 5´‐TGGTCCCTGCTGCTCTCAAG‐3´ 5´‐GGCCTTTTCCTTCCGTTTCTC‐3´ GAPDH 5´‐CCCACTCTTCCACCTTCGATG‐3´ 5´‐GTCCACCACCCTGTTGCTGTAG‐3´

Supplementary Table I FASSL et al. ACTIVATION OF MONOCYTES BY MRP8 IS TLR4-DEPENDENT

Supplementary Table III. Selected MRP8‐induced genes. Regulation was confirmed by qRT‐PCR and involvement of genes in apoptosis‐related GO‐Terms are indicated. Apoptosis‐ Gene array, n = 3 qRT‐PCR, n = 6 related GO‐ Terms Gene Gene ID N‐fold P N‐fold P G‐CSF (CSF3) 1440 18.14 1.05E‐10 133.8 ± 29.21 0.0011 SOCS1 8651 10.64 4.36E‐10 17.1 ± 4.8 0.0076 IL‐19 29949 6.12 0.0004 32.6 ± 4.4 0.0004 CD80 941 4.17 1.06E‐07 5.8 ± 1.0 0.0001 IL‐6 3569 X 171.70 1.20E‐10 228.4 ± 67.6 0.0072 IL‐1A 3552 X 123.40 2.00E‐12 61.8 ± 41.8 0.0037 PTGS2 5743 X 51.86 1.33E‐11 IL2RA 3559 X 24,44 1.86E‐12 IL‐12B 3593 X 26.44 6.20E‐12 IL‐1B 3552 X 20.43 1.41E‐10 SERPINB9 5272 X 16.90 1.06E‐08 WNT5A 7474 X 16.76 2.21E‐08 TNFRSF4 7293 X 13.66 1.70E‐09 BCL2A1 597 X 13.16 6.46E‐12 TRAF1 7185 X 13.13 1.31E‐09 F3 2152 X 12.28 7.16E‐10 PMAIP1 5366 X 8.85 2.71E‐10 AMIGO2 347902 X 7.87 1.47E‐12 MST4 51765 X 7.87 6.65E‐10 IL‐10 3586 X 7.28 4.75E‐06 15.3 ± 2.7 0.0004 CASP5 838 X 7.02 1.15E‐09 TNF 7124 X 6.80 1.11E‐07 2.8 ± .42 0.0017 TNFAIP3 7128 X 6.55 5.39E‐11 3.4 ± 1.3 0.037 NFKB1 4790 X 6.54 5.06E‐11 6.0 ± 1.9 0.0277 RIPK2 8767 X 5.90 6.46E‐08 CCL2 6347 X 4.94 1.99E‐05 ARNT2 9915 X 4.76 8.27E‐06 PIM2 11040 X 4.71 9.37E‐12 PIM1 5292 X 4.64 4.14E‐07 IDO1 3620 X 4.57 2.25E‐06 SLC11A2 4891 X 4.40 3.05E‐09 BCL6 604 X 4.35 3.88E‐07 CD44 960 X 2.94 1.22E‐08 CFLAR 8837 X 4.01 2.44E‐06 SERPINB2 5055 X 3.96 4.71E‐06 PPIF 10105 X 3.82 1.94E‐06 TCF7L2 6934 X 3.69 2.59E‐08 SOCS2 8835 X 3.67 0.0002 5.0 ± 1.4 0.0128 IL‐7 3574 X 3.62 3.65E‐06 BCL2L14 79370 X 3.43 0.0002 P2RX4 5025 X 3.36 1.05E‐07 RELA 5970 X 3.34 1.37E‐07 CDH1 999 X 3.31 0.002

Supplementary Table III FASSL et al. ACTIVATION OF MONOCYTES BY MRP8 IS TLR4-DEPENDENT

PSEN1 5663 X 3.17 5.76E‐06 SPHK1 8877 X 3.13 1.35E‐07 IL‐18 3606 X 3.08 1.32E‐05 CASP4 837 X 3.01 8.90E‐08 ADA 100 X 2.99 6.17E‐10 ZAK 51776 X 2.96 1.43E‐06 B4GALT1 2683 X 2.78 5.50E‐05 PML 5371 X 2.78 0.002 NME1 4830 X 2.70 1.52E‐05 ELK1 2002 X 2.62 5.81E‐08 VEGFA 7422 X 2.56 4.09E‐06 SOCS3 9021 X 3.19 5.06E‐06 8.5 ± 3.2 0.0428 DNAJB6 10049 X 3.05 3.93E‐07 NLRP3 114548 X 3.35 5.15E‐06 BCL2L1 598 X 2.81 1.14E‐05 3.1 ± .48 0.0014 DAPK1 1612 X 3.09 0.001 SOD2 6648 X 13.18 2.95E‐07

Overrepresented genes for apoptosis related GO‐IDs

GO‐ID Description n N p x 12501 programmed cell death 720 14941 8.67 27 regulation of programmed cell 43067 death 641 14941 7.72 24 10941 regulation of cell death 649 14941 7.82 24 8219 cell death 823 14941 9.91 27 16265 death 827 14941 9.96 27 6915 apoptotic process 706 14941 8.51 26 42981 regulation of apoptotic process 633 14941 7.63 24 cell‐type specific apoptotic 97285 process 30 14941 0.36 5 71887 leukocyte apoptotic process 11 14941 0.13 3 8637 apoptotic mitochondrial changes 29 14941 0.35 4 70227 lymphocyte apoptotic process 8 14941 0.10 2 n= Total number of genes that are included in the GO‐ID N= Total number of genes that are included in the GO annotation p= Number of input genes expected to be included in the GO‐ID (null hypothesis) x= Number of input genes that are included in the GO‐ID

Supplementary Table III