PP1-Associated Signaling and − B/AP-1 Κ Inhibition of NF- Tolerance

Identification of Two Forms of TNF Tolerance in Human Monocytes: Differential Inhibition of NF-κB/AP-1− and PP1-Associated Signaling This information is current as of October 3, 2021. Johannes Günther, Nico Vogt, Katharina Hampel, Rolf Bikker, Sharon Page, Benjamin Müller, Judith Kandemir, Michael Kracht, Oliver Dittrich-Breiholz, René Huber and Korbinian Brand

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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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 26, 2014, doi:10.4049/jimmunol.1301610 The Journal of Immunology

Identiﬁcation of Two Forms of TNF Tolerance in Human Monocytes: Differential Inhibition of NF-kB/AP-1– and PP1-Associated Signaling

Johannes Gunther,*€ ,1 Nico Vogt,*,1 Katharina Hampel,*,1 Rolf Bikker,* Sharon Page,* Benjamin Muller,*€ Judith Kandemir,* Michael Kracht,† Oliver Dittrich-Breiholz,‡ Rene´ Huber,* and Korbinian Brand*

The molecular basis of TNF tolerance is poorly understood. In human monocytes we detected two forms of TNF refractoriness, as follows: absolute tolerance was selective, dose dependently affecting a small group of powerful effector molecules; induction tolerance represented a more general phenomenon. Preincubation with a high TNF dose induces both absolute and induction tolerance, whereas low-dose preincubation predominantly mediates absolute tolerance. In cells preincubated with the high TNF dose, we Downloaded from observed blockade of IkBa phosphorylation/proteolysis and nuclear p65 translocation. More prominent in cells preincubated with the high dose, reduced basal IkBa levels were found, accompanied by increased IkBa degradation, suggesting an increased IkBa turnover. In addition, a nuclear elevation of p50 was detected in tolerant cells, which was more visible following high-dose preincubation. TNF-induced phosphorylation of p65-Ser536, p38, and c-jun was inhibited, and basal inhibitory p65-Ser468 phosphorylation was increased in tolerant cells. TNF tolerance induced by the low preincubation dose is mediated by glycogen synthesis kinase-3, whereas high-dose preincubation-mediated tolerance is regulated by A20/glycogen synthesis kinase-3 and http://www.jimmunol.org/ protein phosphatase 1–dependent mechanisms. To our knowledge, we present the ﬁrst genome-wide analysis of TNF tolerance in monocytic cells, which differentially inhibits NF-kB/AP-1–associated signaling and shifts the kinase/phosphatase balance. These forms of refractoriness may provide a cellular paradigm for resolution of inﬂammation and may be involved in immune paralysis. The Journal of Immunology, 2014, 192: 000–000.

umor necrosis factor is a master cytokine involved in involved in inflammation, for example, sepsis (4) or chronic in- inflammation and immunity (1, 2). The rapid induction of flammatory disease (5), but also in malignant processes (6). The cytokines such as TNF, chemokines, and other antimi- balance between protection against excessive immune response and

T by guest on October 3, 2021 crobial effector molecules is fundamental for orchestrating a immune paralysis determines the patients’ fate, for example, in se- coordinated immune response. TNF tolerance means that pre- vere sepsis. exposure to TNF reduces sensitivity to subsequent stimulation Animal research reveals that TNF-mediated effects, such as with this cytokine (3). This form of refractoriness is involved in fever, gastrointestinal toxicity, liver injury, and anorexia, are af- the modulation of TNF signaling and may represent a protective fected by TNF tolerance (7–11). Moreover, several forms of cross- mechanism preventing the cell and organism from excessive and/ tolerance between TNF and LPS have been described (7, 12, 13). or prolonged cytokine stimulation (4). In contrast, TNF tolerance Because TNF tolerance appears more slowly than that of LPS, may be a paradigm for processes resulting in immune paralysis and different mechanisms seem to be responsible for the two phe- shutdown of the immune response (4). TNF tolerance is presumably nomena (14). Only a few results from cell culture studies char- acterizing the molecular basis of TNF tolerance exist to date (9, 15, 16). At the beginning of this study, it was unclear whether the *Institute of Clinical Chemistry, Hannover Medical School, D-30625 Hannover, phenomenon of TNF tolerance exists in primary monocytes as ma- Germany; †Rudolf-Buchheim-Institute of Pharmacology, Justus-Liebig-Universita¨t Giessen, D-35392 Giessen, Germany; and ‡Institute of Physiological Chemistry, jor producers of TNF coordinating innate and adaptive immunity Hannover Medical School, D-30625 Hannover, Germany (17). An 18-h preincubation of monocytic THP-1 cells with a high 1J.G., N.V., and K.H. contributed equally to this work. TNF dose, IL-1b or LPS induces tolerance against stimulation with Received for publication June 20, 2013. Accepted for publication January 24, 2014. the same agonist and several forms of cross-tolerance, accompanied k k a This work was supported by the Deutsche Forschungsgemeinschaft (SFB 566) and by reduced degradation of NF- B inhibitor protein I B and at- the Vereinte Deutsche Gesellschaft fur€ Klinische Chemie und Laboratoriumsmedizin tenuated phosphorylation of JNK and ERK (18). In contrast, when € (Stiftung fur Pathobiochemie und Molekulare Diagnostik). THP-1 cells were preincubated for 72 h with a low TNF dose, no The microarray data presented in this article have been submitted to the Gene Expres- inhibition of IkBa proteolysis and NF-kB DNA-binding activity sion Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE45371. was found (19). Under this condition the transcription factor Address correspondence and reprint requests to Prof. Korbinian Brand, Institute b k of Clinical Chemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, D-30625 C/EBP interacts with NF- B-p65 and inhibits its phosphoryla- Hannover, Germany. E-mail address: [email protected] tion, thereby blocking the expression of NF-kB–dependent target The online version of this article contains supplemental material. genes, for example, IL-8 (3). A recent report demonstrates that Abbreviations used in this article: GSK3, glycogen synthesis kinase-3; IKK, inhibitor TNF induces glycogen synthesis kinase-3 (GSK3)–mediated cross- of kb kinase; M, medium; PP1, protein phosphatase 1; qPCR, quantitative PCR; tolerance to endotoxin in macrophages (20). The hitherto available rhAb, human rAb; siRNA, small interfering RNA; T, TNF. studies show that the basic mechanism and functional consequences Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 of TNF tolerance have not yet been satisfactorily elucidated.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301610 2 TNF TOLERANCE IN HUMAN MONOCYTES

The present study uses human monocytes as the gold standard Quantitative PCR to investigate the phenomenon of TNF tolerance on a genome- Cultured cells were lysed, and total RNA was isolated using the RNeasy wide level. We demonstrate that TNF tolerance is a prominent Mini Kit or Micro Kit (Qiagen). To remove contaminating DNA, treatment phenomenon in primary monocytes of healthy individuals. We with RNase-free DNase I (Qiagen) was performed. RNA concentrations established two forms of TNF refractoriness, as follows: absolute were assessed using a Nanodrop ND-1000. Total RNA was reverse tran- tolerance, mediated by low and high TNF doses, is a very specific scribed (SuperScript-II; Invitrogen), and quantitative PCR (qPCR) was performed using platinum SYBR-Green qPCR SuperMix UDG (Invitrogen) mechanism inhibiting a small, albeit powerful group of effector and a LightCycler 480 (Roche). The amplification protocol included enzy- molecules, whereas induction tolerance, predominantly induced matic degradation of contaminating uracil-containing DNA (50˚C, 2 min) and by high doses, represents a more general phenomenon. TNF tol- activation of the DNA polymerase (95˚C, 2 min), followed by 45 amplifi- erance differentially modulates NF-kB/AP-1–associated signaling. cation cycles (95˚C, 10 s; 59˚C, 15 s; 72˚C, 20 s). The following primers were applied: IL8 (59-TCCTGTTTCTGCAGCTCTGG-39,59-GGCCACT- Low-dose TNF-induced tolerance is regulated by GSK3, whereas CTCAATCACTCTC-39), IL6 (59-ACAGCCACTCACCTCTTCAG-39,59- high-dose TNF-mediated tolerance is controlled by A20/GSK3 GTGCCTCTTTGCTGCTTTCAC-39), IL1A (59-TGACTGCCCAAGATG- and protein phosphatase 1 (PP1)–dependent mechanisms. Abso- AAGAC-39,59-CCAAGCACACCCAGTAGTC-39), CCL20 (59-GAAGGC- lute and induction TNF tolerance dose dependently affect the TGTGACATCAATGC-39,59-GGGCTATGTCCAATTCCATTC-39), PTGS2 9 9 9 kinase/phosphatase balance and may represent different cellu- (5 -GGGCCAGCTTTCACCAAC-3 ,5-ATCTTTGACTGTGGGAGGATAC- 39), and IkBa (59-CGAGCAGATGGTCAAGGAGC-39,59-CAGCCAAGT- lar strategies to protect against excessive TNF stimulation and GGAGTGGAGTC-39). IL-8 mRNA expression was alternatively assessed resolve inflammation. using universal probe library probe 72 (Roche) and IL-8–specific primers (59- AGACAGCAGAGCACACAAGC-39,59-CACAGTGAGATGGTTCCTTCC- 39), according to the standard protocol (21). Target gene expression levels were

Materials and Methods normalized to hGAPDH (59-AGGTCGGAGTCAACGGAT-39,59-TCCTGG- Downloaded from Isolation and culture of primary human monocytes AAGATGGTGATG-39)orb2-microglobulin (59-TGTGCTCGCGCTACTC- TCTCTT-39,59-CGGATGGATGAAACCCAGACA-39). Generation of ex- Blood samples from healthy donors were provided by the Institute of ternal standard curves and normalization of cDNA amount were performed, Transfusion Medicine, Hannover Medical School. Informed patient consent as previously described (21). Relative expression values and fold changes was obtained, and the experiments were approved by the Hannover Medical were calculated, as published (22). Statistical analyses were performed using School ethics committee in accordance with the Declaration of Helsinki. GraphPad Prism 5.0 (GraphPad Prism software). Monocytes were isolated by Biocoll (Biochrom) density gradient centri- fugation using LeucoSeptubes (Greiner Bio-One), followed by negative Microarray experiments http://www.jimmunol.org/ selection with magnetic antibiotin microbeads (Monocyte Isolation Kit II; Miltenyi Biotec), according to manufacturer’s instructions. Purified cells The Whole Human Genome Oligo Microarray 4x44K (G4112F, design ID were cultured in 12- or 24-well plates (Thermo Scientific) at a density of 014850; Agilent) was used in this study. The microarray contains 45,015 4 3 106 or 2 3 106 cells in a final volume of 2 ml or 1 ml endotoxin-free oligonucleotide probes covering ∼31,000 human transcripts. Total RNA RPMI 1640 supplemented with 7% FCS, 100 U/ml penicillin, 100 mg/ml was applied to prepare Cy3- or Cy5-labeled cRNA (Amino Allyl Mes- streptomycin (Biochrom), 1.8% OPI media supplement (Sigma-Aldrich), sageAmp II Kit; Ambion), according to the manufacturer’s instructions. and 0.8% Life Technologies MEM nonessential amino acids solution cRNA fragmentation, hybridization, and washing steps were carried out (Invitrogen). Following an adherence step for 1 h, cells were washed according to Agilent’s Two-Color Microarray-Based Gene Expression three times with supplemented medium. Endotoxin contamination was Analysis Protocol V5.7. A total of 300 ng of each labeled cRNA sample excluded using the Limulus amebocyte lysate assay (Lonza) (,10 pg was used for cohybridization. Slides were scanned on the Agilent Micro endotoxin/ml). Array Scanner G2505C (pixel resolution 5 mm, bit depth 20). Data ex- by guest on October 3, 2021 traction was performed with Feature Extraction Software V10.7.3.1 using Flow cytometry the recommended default extraction protocol file (GE2_107_Sep09.xml). To correct for systematic bias at high-end fluorescence intensity mea- Purity of isolated monocytes was assessed by dual cell labeling (30 min, surements, the highest 2% of processed signals in the green channel were 4˚C, dark) using Alexa Fluor405-CD45 (Invitrogen) and allophycocyanin- further normalized according to an intensity-dependent, nonlinear strategy, CD14 (BD Biosciences) human rAbs (rhAb). Contaminations with other to optimize fitting to their respective counterparts in the red channel. Heat cell types were excluded using allophycocyanin-CD3, PE-CD19, FITC- maps were created using the program Mayday (23). CD56 (BD Biosciences), and FITC-CD15 (Invitrogen) rhAbs. For detec- The complete microarray data have been deposited in National Center for tion of TNFR1 and 2, cells were labeled with PE-TNFR1 and FITC-TNFR2 Biotechnology Information’s Gene Expression Omnibus and are accessible rhAbs (Santa Cruz). Cells were then fixed in 1% paraformaldehyde (Sigma- through GEO series accession number GSE45371 (http://www.ncbi.nlm. Aldrich) and stored at 4˚C until detection using the LSR II flow cytometer nih.gov/geo/query/acc.cgi?acc=GSE45371). and FACSDiVa software (BD Biosciences). Western blot analysis Culture of THP-1 and HeLa cells Whole-cell extracts were prepared by incubating cells in hot lysis buffer Human monocytic THP-1 and HeLa cells (DSMZ) were maintained in (75 mM Tris[hydroxymethyl]-aminomethan [Tris]-HCl, 0.5% NaDodSO m 4 RPMI 1640 supplemented with 7.5% FCS, 100 U/ml penicillin, and 100 g/ [SDS], 50 mM DTT, 1 mg/100 ml bromphenol blue [Applichem], 15% 3 5 ml streptomycin (Biochrom). THP-1 were cultured at 5 10 cells/well in glycerol [Merck]; 5 min, 96˚C). Alternatively, sonication (10 s) in ex- 12-well plates, and HeLa were plated at 2 3 105 cells/well in 6-well plates traction buffer (150 mM NaCl [Merck], 25 mM MgCl2 [Roth], 50 mM (Sarstedt). Tris-HCl, 10% glycerol, 1% Nonidet P-40, and three tablets protease inhibitor [Roche] per 50 ml) was performed. Cytosolic extracts were ob- Tolerance experiments tained using cytosolic extraction buffer (10 mM HEPES [Sigma-Aldrich], Unless otherwise indicated, cells were pretreated with 40 or 400 U TNF/ml 10 mM KCl [Roth], 300 mM Saccharose [Roth], 1.5 mM MgCl2, 0.1% (Sigma-Aldrich) for 48 h, followed by short exposure with 400 U TNF/ml Nonidet P-40, and three tablets protease inhibitor per 50 ml; for 5 min on for 2 h (mRNA expression), 4–6 h (protein secretion), 15 min (protein ice). Protein concentrations were determined by Bradford assay (Bio-Rad). phosphorylation), or 30 min (EMSA). Electrophoresis was performed with 12% Tris/glycin SDS-polyacrylamide gels (Biostep), and proteins were transferred to nitrocellulose membranes ELISA (0.45 mm; Bio-Rad) using the Western blot technique. After blocking with 5% skim milk (Merck) or 5% BSA (Roche) in TBS (140 mM NaCl, Culture supernatants were harvested and subsequently analyzed using the 20 mM Tris-HCl)/0.1% Tween 20 (Sigma-Aldrich), membranes were Quantikine Human CXCL8/IL-8 Immunoassay (rel="tag">R&D Systems) and the incubated (4˚C, overnight) with primary Abs against the following ELx808 Absorbance Microplate Reader (BioTek Instruments), according proteins: A20 (D13H3), AKT (67E7), b-catenin (6B3), c-jun (60A8), GSK3b to the manufacturers’ instructions. Protein levels were normalized to the (27C10), IkBa (44D4), p38 (D13E1), p65 (22B4), p-AKT (Ser473) (D9E), respective DNA levels. Total DNA was isolated using the QIAamp DNA p-c-jun (Ser63)-II, p-GSK3b (Ser9) (D85E12), p-IkBa (Ser32) (14D4), Mini Kit (Qiagen), and the concentration was assessed using a Nanodrop p-IKKa/b (Ser176/180) (16A6), p-JNK1/2 (Thr183/Tyr185) (81E11), p-p38 ND-1000 (PeqLab). (Thr180/Tyr182) (D3F9), p-p65 (Ser536), p-p65 (Ser468) (Cell Signaling), actin The Journal of Immunology 3

(11C; Sigma-Aldrich), or PPP1R14C (Novus Biologicals). Next, mem- for 4 h. Afterward, 6 ml RMPI/20% FCS were added for 24 h. For tol- branes were incubated (1 h, room temperature) with HRP-conjugated sec- erance experiments, transfected cells were seeded in 12-well plates (1 ml/ ondary Ab (Alexis Biochemicals). Proteins were visualized, as previously well) and pretreated with TNF. described (21). Densitometric analysis was performed with TotalLab TL100 software. Results EMSA Preincubation time course and dose-response experiments Nuclear extracts were prepared and analyzed by EMSA, as previously Human monocytes were isolated from blood of healthy donors in described (19). a multiple step procedure (purity .97%) (Fig. 1A). Isolated mono- Inhibitor experiments cytes were incubated with increasing doses of TNF, and IL-8 mRNA/protein was determined. These experiments showed a Addition of GSK3-inhibitor SB216763 (Sigma-Aldrich) and TNF pretreatment were performed simultaneously. Calyculin A (Cell Signaling Tech- continuous increase in IL-8 mRNA/protein (TNF, 0.4–1000 U/ml). nology) and okadaic acid (Merck) were added 30 min before TNF short The expression of IL-8 mRNA was used as an indicator for the exposure. Cycloheximide (Merck) was added for the indicated time in- majority of our experiments. In initial preincubation time course tervals following TNF preincubation for 48 h. experiments, monocytes were preincubated in medium or with Transfection experiments 400 U/ml TNF up to 48 h and then stimulated with 400 U/ml TNF for 2 h. Following TNF preincubation, a high IL-8 mRNA Small interfering RNA (siRNA) transfection of HeLa cells was performed concentration was observed at 6 h, whereas an intermediate level using A20 siRNA, Alexa Fluor 488–coupled AllStars negative control siRNA, and FlexiTube GeneSolution or HiPerFect Transfection Reagent was found at 24 h and a low level after 48 h (Fig. 1B). When we (Qiagen), according to manufacturer’s instructions. For plasmid transfec- restimulated the TNF-preincubated cells, we found a complete Downloaded from tion of THP-1 cells, PPP1R14C human cDNA open reading frame clone inhibition of IL-8 mRNA expression at 48 h, representing (OriGene) and X-treme gene HP transfection reagent (Qiagen) were used. an optimal condition to further study tolerance. To evaluate the Freshly grown THP-1 were seeded at 3 3 105 cells/ml in Opti-MEM (Life Technologies) and incubated at 37˚C for 2 h. Following preincubation of minimal dose required to induce TNF tolerance, monocytes were 6 mg plasmid and 21 ml transfection reagent for 30 min in 600 ml Opti- preincubated with decreasing doses of TNF for 48 h and then short MEM, 6 ml cell suspension was transfected and subsequently incubated exposed to high TNF doses. This showed that TNF preincubation http://www.jimmunol.org/ by guest on October 3, 2021 FIGURE 1. Conditions to induce TNF tolerance in human monocytes. (A) Primary human monocytes were isolated up to a purity of . 97% determined by flow cytometry (inset). The cells were incubated with increasing doses of TNF, and the levels of IL-8 mRNA and protein (supernatant) were determined after 2 or 4 h, respectively. Data are normalized to the unstimulated control. (B) Monocytes were preincubated (Pre) in control medium (2) or in the presence of 400 U/ml TNF (+) up to 48 h and then stimulated with 400 U TNF for 2 h (SE). (C) Cells were preincubated with decreasing doses of TNF for 48 h and then exposed to the high TNF dose for 2 h. (D) Schematic scheme of the conditions to induce TNF tolerance in human monocytes. Monocytes were preincubated for 48 h with medium (naive cells) or TNF (40 or 400 U/ml) to make tolerant cells, which was followed by short exposure to medium or TNF (400 U/ml). The symbols to describe these conditions were used throughout the manuscript. (A–C) Representative experiments (n $ 5) are shown measured in duplicates (mean 6 SD). 4 TNF TOLERANCE IN HUMAN MONOCYTES as low as 25 U/ml was sufficient to induce tolerance (Fig. 1C). Following preincubation with TNF for 48 h, no downregulation of TNFR1/R2 was observed using flow cytometry (data not shown), showing that TNF tolerance is not due to receptor downregulation. Our incubation conditions are summarized, as follows: monocytes were preincubated with 400 (high dose) or 40 U/ml (low dose) to induce tolerance (Fig. 1D). TNF tolerance in healthy individuals Next, we investigated whether healthy individuals differ in their ability to develop TNF tolerance. Monocytes from 18 healthy donors were preincubated with 400 U/ml TNF for 48 h and then restimulated. This revealed that these individuals differ strongly in their sensitivity to TNF (Fig. 2A); IL-8 mRNA fold-induction values varied from 5-fold up to .110-fold. Initially, TNF tolerance was defined by the following formula: 400 + TNF (T) divided by medium (M) + T # 0.25 (inhibition $ 75%). Fifteen of 18 individuals showed the picture of TNF tolerance, and a partial tolerance was observed in 3 individuals (Fig. 2B). Similar results were obtained when lower preincubation doses (6.25–200 U/ml, Downloaded from 20 donors) were used (Fig. 2C). Absolute tolerance in human monocytes Microarrays were performed to characterize TNF tolerance on a genome-wide basis. Human monocytes from five different donors (Fig. 2, asterisks) were TNF preincubated for 48 h with 400 U/ml http://www.jimmunol.org/ (n = 3) or 40 U/ml (n = 2). The cells were then short exposed to TNF (400 U/ml) for 2 h. To determine all the genes responding to TNF stimulation, we filtered the obtained data with regard to at least 2-fold induction following TNF short-term exposure of naive monocytes (M + T/M + M $ 2) identifying 360 genes (Supplemental Table I). First, TNF tolerance was defined by the following formula: 400 + T or 40 + T divided by M + T # 0.25 (see Fig. 2B). Because under this condition the 400 + T or 40 + T by guest on October 3, 2021 value is ,M + T, we defined this form of refractoriness as absolute tolerance. Following preincubation with 400 U/ml TNF, we detected 51 genes showing absolute tolerance (Fig. 3A, Supplemental Table II). Eleven of these genes belonged to a class of highly TNF-inducible genes (M + T/M + M $ 10) (Fig. 3B). Under 40 2 T conditions, we identified 24 genes affected by absolute tolerance (Fig. 3C, Supplemental Table II). Applying less stringent criteria (40 + T/M + T # 0.5), we identified 99 genes showing tolerance, including our indicator IL-8 (Supplemental Table III). Our data demonstrate that, under absolute tolerance, a selective number of TNF-inducible genes is inhibited, predominantly involved in inflammation, growth/differentiation, and chemotaxis/migration (Fig. 3D). Thirty-one (61%) of the 51 genes affected by high-dose absolute tolerance were regulated by NF-kB and/or AP-1 transcription factors, with similar results using low preincubation doses FIGURE 2. TNF tolerance in different donors. (A) Isolated monocytes (Supplemental Table II) (24, 25). Microarrays were confirmed in from 18 healthy donors were preincubated with control medium or 400 primary monocytes by qPCR, for which several highly inducible U/ml TNF for 48 h and then subjected to TNF (400 U/ml for 2 h). The genes were selected (IL-8, IL-6, IL-1A, CCL20, PTGS2) (Fig. expression of IL-8 mRNA (duplicates) was measured as a readout. The 4A). mRNA data were confirmed on protein levels in primary data were normalized to the M + M condition. The M + T and the 400 + T values are depicted. (B) The data shown in (A) were further analyzed. monocytes using IL-8 as a readout (Fig. 4B). Absolute TNF tol- Tolerance was defined by the following formula: 400 + T/M + T # 0.25. erance was also demonstrated on both levels in monocytic THP-1 The cutoff for TNF tolerance is indicated by the dashed line. (C)Cells cells, another independent monocytic model, regardless of whether were treated as in (A). Different TNF concentrations were used for pre- low (Fig. 4C) or high (data not shown) preincubation doses were incubation (6.25–200 U/ml). The asterisks in (A–C) indicate the donors used. that were further investigated by microarrays. Induction tolerance in human monocytes Second, TNF tolerance was defined by a different formula; the short-term stimulation of tolerant cells leading to the name in- degree of induction was calculated within the same condition duction tolerance. When cells were preincubated with 400 U/ml (naive, tolerant) for the 360 TNF-inducible genes: 400 + T/400 + M TNF, we identified 227 tolerant genes (63%), also visualized by or 40 + T/40 + M # 1.25. This means no induction following TNF the heat map (white or pale red bands) (Fig. 5A, Supplemental The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 3. Absolute tolerance in human monocytes identified by microarray. Primary human monocytes derived from five different donors (1–5) were preincubated for 48 h in medium or with a high (400 U/ml, 1–3) or a low (40 U/ml, 4–5) TNF concentration. The cells were then stimulated with TNF (400 U/ml) for 2 h, which was followed by microarrays. The data were normalized by dividing the gene expression levels by the respective value in native unstimulated cells (M + M) for each experiment. (A) TNF tolerance was defined by the equation 400 + T/M + T # 0.25. (B) Eleven of the genes shown in (A) belong to a class of highly TNF-inducible genes (M + T/M + M $ 10). These genes are also indicated by boxes in (A). (C) TNF tolerance was defined by the equation 40 + T/M + T # 0.25. Again, the highly inducible tolerant genes are indicated by boxes. (D) The gene groups that were affected by absolute tolerance (400 U/ml preincubation) were analyzed in terms of their major functional importance for the cell using genecards (http://www.genecards.org). Shown are five major gene groups that are affected by this form of tolerance (%calculation). The genes not belonging to these major groups are summarized under “other.” The absolute number of genes is indicated in brackets.

Table IV), and 10 genes when the lower dose was used (data not levels in 400 + M than in M + T cells (400 + M/M + T , 0.5) (Fig. shown). These 227 genes showing induction tolerance could be 5B), and 155 genes showed comparable/higher 400 + M levels further subdivided, as follows: 72 genes displayed lower expression (400+M/M+T$ 0.5) (Fig. 5C). These genes, again mostly 6 TNF TOLERANCE IN HUMAN MONOCYTES

observed. In the following, cells were treated with a proteasome inhibitor briefly before short exposure because it is difficult to determine IkBa phosphorylation under the condition of stimulus- induced proteolysis. A modestly elevated level of IkBa phosphorylation was detected in unstimulated tolerant cells especially preincubated with high doses (Fig. 6D). As expected, we observed a dramatic increase in IkBa phosphorylation when naive cells were short-term TNF exposed, peaking at 5 min. A similar increase in IkBa phosphorylation was observed in 40 + T cells, albeit to a less degree and delayed. In contrast, in high-dose pretreated cells, no further TNF-induced phosphorylation of IkBa was observed. This was supported by gel shifts demonstrating a TNF- stimulated increase in NF-kB activity in 40 + T but not 400 + T cells (Fig. 6E). This demonstrates that TNF tolerance affects the NF-kB pathway in 400 + T cells by inhibiting restimulation- induced IkBa phosphorylation/proteolysis. In addition, mRNA expression and protein stability of IkBa were examined under tolerance-inducing conditions. Following preincubation with both the low and the high TNF dose, modestly increased IkBa mRNA levels were measured (Fig. 6F). As mentioned above, more promi- Downloaded from nent in cells preincubated with the high dose, reduced basal levels of IkBa protein were detected that were accompanied by increased IkBa protein degradation compared with naive cells (Fig. 6G). Taken together, our results suggest an increased IkBa turnover in tolerant cells. http://www.jimmunol.org/ Effect of low- and high-dose preincubation on nuclear translocation of NF-kB proteins as well as p65, p38, and c-jun phosphorylation The nuclear levels of NF-kB proteins were also assessed in tolerant cells. In response to TNF restimulation, a marked increase in FIGURE 4. Confirmation of array data on mRNA and protein levels in nuclear p65 in nontolerant and low-dose preincubated cells, but monocytes and THP-1 cells. (A) Primary monocytes were incubated under not in high-dose preincubated cells, was observed (Fig. 7A). standard tolerance conditions using the low and high TNF preincubation Preincubation with the low dose induced a modest increase in by guest on October 3, 2021 dose, respectively, and then short-term exposed to the high TNF dose. nuclear p50 (see TNF SE, 0 h; Fig. 7A), whereas a marked in- After 2 h, the mRNA expression of the indicated genes was measured. The crease was observed following preincubation with the high dose, data were normalized to the M + M condition, and depicted are the M + T consistent with earlier results (18). When cells were restimulated, B and 40 + T or 400 + T fold-induction values. ( ) Monocytes were incu- we found a marked increase in nuclear p50 in naive cells and a bated under standard conditions using low or high preincubation doses. further increase in low-dose pretreated cells. No additional elevation Following preincubation, cells were washed with PBS and short-term ex- of p50 in the nucleus was detected when high-dose preincubated posed to the high TNF dose for 6 h. The level of IL-8 protein was measured in the supernatant by immunoassay and normalized to the amount of cells were restimulated. This suggests a regular translocation of p65/ DNA/well. (C) Monocytic THP-1 cells were incubated as described in (A) p50 heterodimers into the nucleus following restimulation of naive or (B) (40 U/ml TNF preincubation), and the expression of mRNA or and low-dose preincubated cells as well as a contribution of ele- protein was determined. Representative experiments (n $ 5), which are vated p50 levels to the inhibition of NF-kB–dependent gene ex- shown throughout this figure, were performed in triplicates (mRNA) or pression in tolerant cells (26). Short-term exposure of naive cells duplicates (protein). to TNF led to a marked p65 phosphorylation on Ser536 (Fig. 7B). In unstimulated 400 + M cells, a modest increase in p65 (Ser536) phosphorylation was found. Remarkably, TNF short-term–induced 536 controlled by NF-kB/AP-1, can be attributed to six prominent p65 (Ser ) phosphorylation was inhibited in all tolerant cells, groups involved in signaling/transcription, inflammation, metab- regardless of whether low or high preincubation doses were ap- olism, growth/differentiation, chemotaxis/migration, and transport plied. Furthermore, in unstimulated tolerant cells, an increased 468 (Fig. 5D). phosphorylation of p65 (Ser ), negatively regulating basal p65 activity (27), was found (Fig. 7B). We also investigated whether k a Effect of low- and high-dose TNF preincubation on I B the MAPK/AP-1 pathway (25) is affected in tolerant cells. As ex- phosphorylation and proteolysis pected, TNF short-term exposure lead to phosphorylation of p38, Next, we monitored how TNF tolerance affects the NF-kB system JNK, and c-jun (Fig. 7C, data not shown). Remarkably, phos- (24). As expected, TNF (40 or 400 U/ml) led to initial IkBa phorylation of these proteins was strongly inhibited in tolerant cells proteolysis at 0.25 h and recovery within 1 h (Fig. 6A). Interest- under both low- and high-dose preincubation conditions. These ingly, following exposure up to 48 h, we observed a continuous data suggest that the NF-kB system is inhibited in both 40 + T and decrease in IkBa predominantly in cells pretreated with the high 400 + T tolerant cells by inhibition of p65 phosphorylation. The TNF dose. When low-dose preincubated cells were re-exposed to increase in p65 (Ser468) phosphorylation may contribute to tolerance- TNF up to 120 min, we observed a regular, somewhat attenuated mediated inhibition of gene expression. In addition, the MAPK IkBa proteolysis/recovery (Fig. 6B, 6C). Remarkably, in high- signaling pathway is inhibited in TNF-tolerant cells, presumably dose pretreated cells, no TNF-inducible IkBa proteolysis was leading to inhibition of AP-1–dependent gene expression. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 5. Induction tolerance in human monocytes. The same set of microarray data described in Fig. 3 was further analyzed. (A) Tolerance was deﬁned by the equation 400 + T/400 + M # 1.25. (B and C) The set of genes identiﬁed by the strategy described under (A) was further characterized by the equations 400 + T/M + T , 0.5 and 400 + M/M + T $ 0.5. (D) Shown are six major gene groups (% calculation) that are affected by induction tolerance (as extracted from http://www.genecards.org). The absolute number of genes is indicated in brackets.

Low-dose TNF-induced tolerance is mediated by the GSK3 inhibitor SB216763, the effect of low- dose TNF-induced GSK3, whereas high-dose preincubation involves tolerance was completely reversed in a dose-dependent manner A20/GSK3-dependent mechanisms (Fig. 8A, left). Preincubation with SB216763 increased the level It has been shown that GSK3/A20 mediates TNF cross-tolerance to of b-catenin, an established substrate for GSK3 (28). Under high- endotoxin (20). Most important, when cells were incubated with dose TNF preincubation, the expression of IL-8 was modestly 8 TNF TOLERANCE IN HUMAN MONOCYTES

FIGURE 6. Low-dose and high-dose TNF preincubation affects IkBa phosphorylation and proteolysis. (A) THP-1 monocytic cells were incubated with 40 or 400 U/ml TNF up to 48 h. The level of IkBa was measured by Western blot at the indicated time points. Actin was used as a loading control. (B) Naive and tolerant cells preincubated (Pre) with medium (M) or TNF (40 or 400 U/ml) were short- term exposed to 400 U/ml TNF (SE) up to 120 min, and IkBa levels were measured. (C) The experiments shown in (B) were analyzed by densitometric analysis. The respective SE values at 0 min were defined as the 100% controls (dashed line). (D) Cells were preincubated (48 h) under standard conditions (M, 40 and 400 U/ml TNF). The proteasome inhibitor MG-132 was added 30 min prior to restimulation (SE). The levels of p-IkBa Downloaded from and total IkBa were determined by West- ern blot. (E) Cells were preincubated with the low-dose or high-dose TNF and then short-term exposed to the high TNF dose. The activation of NF-kB was determined by gel shift assay, and NF-kB is indi- http://www.jimmunol.org/ cated by the bracket. Oct-1 was used as a loading control. (F) Following preincubation of cells with medium or the low/ high TNF dose, the expression of IkBa mRNAwasdeterminedbyqPCR.A representative experiment is shown that was determined in triplicates (mean 6 SD). The level of mRNA in naive cells was defined as 1. (G) Cells were preincubated with medium (M) or TNF for by guest on October 3, 2021 48 h, and then 10 mg/ml cycloheximide was added. The levels of IkBa were determined by Western blot at the indicated time points. (A–G) Representative examples of at least three independent experiments. elevated in inhibitor-treated cells, regardless of whether they were induced tolerance. Our results also demonstrate that A20 plays restimulated or not (Fig. 8A, right). Remarkably, in low-dose pre- a major role in TNF tolerance forms that are induced by the high exposed cells, the inhibition of p65 (Ser536) phosphorylation was dose but is not involved in low-dose–dependent TNF tolerance. completely reversed in the presence of SB216763 (Fig. 8B). Un- der high-dose conditions, only a modest increase in p65 phos- High-dose–induced TNF tolerance affects the PP1 phorylation was detected in inhibitor-treated cells. In addition, we phosphatase system detected an upregulation of c-jun expression in the presence of The inhibition of several kinase cascades in TNF-tolerant cells SB216763 only in tolerant cells (Fig. 8C). Inhibition of GSK3 prompted us to look at the phosphatase system as counteracting phosphorylation leads to the activation of this kinase (28, 29). principle (31, 32). Microarrays identified an increased expression Our experiments showed an inhibition of GSK3-b phosphoryla- of the PP1 phosphatase catalytic subunit PPP1CB and a down- tion on Ser9 in TNF-tolerant cells (Fig. 8D). Our arrays also dem- regulation of the PP1 phosphatase inhibitory subunit PPP1R14C onstrated a marked upregulation of A20 (30) in TNF-tolerant cells (33) following TNF preincubation (Fig. 9A, 9B). Remarkably, (400 + M: 8.9 6 3.2-fold, n = 3, GEO series accession number preincubation with the PP1 phosphatase inhibitor calyculin A led GSE45371), and we showed that A20 levels were significantly to a strong upregulation of IL-8 expression predominantly in those increased following TNF exposure (Fig. 8E). Most important, tolerant cells that were pretreated with the high TNF dose (Fig. 9C). using an A20 siRNA approach, we demonstrated a strong upreg- No effect on IL-8 was observed using the inhibitor okadaic acid ulation of IL-8 only in those cells in which TNF tolerance was in a dose selectively reducing PP2A activity (34). Furthermore, induced by high-dose TNF (Fig. 8F). Finally, high TNF dose– overexpression of the inhibitory subunit PPP1R14C increased the induced inhibition of inhibitor of kb kinase (IKK) phosphoryla- expression of IL-8 solely in tolerant cells, again more dramatic tion was almost completely reversed by A20 siRNA (Fig. 8G). when the high preincubation dose was used (Fig. 9D). Finally, Our data suggest that GSK3 plays a key role in mediating low- calyculin A treatment elevated the levels of phosphorylated IKK, dose–dependent TNF tolerance and also participates in high-dose– IkBa, p65, and JNK (Fig. 9E), demonstrating a connection be- The Journal of Immunology 9

Using microarrays, we detected two forms of TNF refractoriness, that is, absolute and induction tolerance. Absolute tolerance was defined by the equation 400 + T or 40 + T divided by M + T # 0.25 (abbreviations: Fig. 1D) with the 400 + T or 40 + T values , M + T values ($75% inhibition). Under high-dose preincubation, we detected 51 of 360 TNF-inducible genes affected by absolute tolerance, and, when we applied the 40 + T condition, we found 24 inhibited genes. The arrays were confirmed by RT-PCR and on protein levels in monocytes and monocytic THP-1 cells. This proves that the inhibition of mRNA expression observed under tolerance results in a functional protein output, and the experiments with pure cell lines demonstrate that TNF tolerance is not dependent on the potential presence of small amounts of contaminating other blood cells. Next, induction tolerance was defined by calculating the degree of induction within the same condition (naive, tolerant) for the identified TNF-inducible genes (400 + T/400 + M or 40 + T/40 + M # 1.25), which means basically no induction following TNF short exposure. More than half (63%) of the TNF-inducible genes displayed the induction tolerance almost exclusively when the high preincubation dose Downloaded from was used. TNF tolerance affected six major groups of genes predominantly regulated by NF-kB/AP-1 transcription factors (24, 25). Under absolute tolerance, an inhibition of genes playing a role in inflammation, growth/differentiation, and chemotaxis/migration FIGURE 7. Low-dose and high-dose TNF preincubation affects nuclear was found, whereas induction tolerance inhibits genes involved http://www.jimmunol.org/ p50 and p65 levels as well as phosphorylation of p65, p38, and c-jun. (A) in signaling/transcription, inflammation, metabolism, growth/dif- Cells were preincubated for 48 h with medium (M) or the low/high TNF ferentiation, chemotaxis/migration, and transport. One interest- dose and then short exposed to TNF for the indicated time intervals. The ing candidate not represented by these groups is tissue factor, a levels of p50 and p65 were determined in nuclear extracts by Western blot key procoagulatory molecule (41), which was also completely (loading control: TBP). (B) Cells were incubated under standard conditions, and the levels of phospho-p65 (Ser536, Ser468) and total p65 were inhibited under absolute tolerance. Taken together, absolute tol- determined by Western blot (loading control: actin). (C) Cells were incu- erance appears to represent a selective negative-regulatory mecha- bated under standard conditions. The phosphorylation of p38 and c-jun nism that dose dependently affects a relatively small, albeit ex- was determined by phospho-Western blot. In addition, total levels of these tremely powerful group of molecules basically involved in each by guest on October 3, 2021 kinases were measured. (A–C) Representative examples of at least three major monocytic function (17). This is different from LPS toler- independent experiments. ance characterized by a more global nature of unresponsiveness of macrophages to restimulation (42, 43). TNF tolerance is not induced by direct contact with bacterial products such as LPS tol- tween NF-kB/MAPK and PP1 phosphatases. Our data show that, erance but is dependent on a specific cytokine. Therefore, TNF under TNF tolerance induced by the high dose, the PP1 phosphatase tolerance can be less manipulated by excessive production of certain system is upregulated, which may be at least partially caused by a bacterial products and may thus represent a more precise second reduced expression of the inhibitory PPP1R14C subunit and finally protection line. A relatively large group of TNF-inducible genes contributes to the inhibition of NF-kB/AP-1–associated kinases. is inhibited by induction tolerance, suggesting that this form of tolerance is a more general phenomenon to organize cellular func- Discussion tions in the presence of high TNF. It is striking to speculate that Despite the fact that TNF is considered to be one of the master this form of tolerance represents a protective principle, leaving the cytokines involved in regulation of inflammation and immune basal mRNA supply open, but does not allow a further increase. response (1, 24) the phenomenon of TNF tolerance is poorly un- There are little and varying data as to how TNF tolerance derstood to date. When this study was initiated, it was unclear regulates the NF-kB system. Under low dose–mediated tolerance, whether this form of tolerance existed in monocytes at all. To our we found in THP-1 monocytic cells that TNF short-term–induced knowledge, this study represents the first genome-wide analysis of IkBa proteolysis and NF-kB activation (EMSA) were not affected TNF tolerance using primary human monocytes. TNF tolerance (19). In contrast, another paper identified reduced IkBa degra- was observed in monocytes preincubated for 48 h with a TNF dose dation and NF-kB–binding activity in THP-1 cells preincubated as low as 25 U/ml. To systematically characterize TNF toler- for 18 h with high TNF doses (18). In addition, it was shown ance, we selected high and low TNF doses (400 and 40 U/ml) for that TNF short-term pretreatment of fibroblasts negatively affects preincubation to induce tolerance according to the literature (35) IkBa resynthesis (44). Our present results resolve these discrep- and our own dose-response experiments. Similar concentrations ancies, as follows. First, preincubation of THP-1 cells with TNF may be locally achieved in vivo for a certain time period in the for 48 h led to reduced basal IkBa levels, which were more presence of monocytic cells, for example, in an acute or chronic in- prominent when the high dose was used. This is in contrast to LPS flammatory environment (36–40). Monocytes of 83% of the healthy tolerance characterized by elevated IkBa levels (43). In addition, donors showed complete tolerance (400 U/ml preincubation), we measured a modestly increased mRNA expression of IkBa whereas 17% exhibited a partial tolerance (with similar results in tolerant cells accompanied by increased protein degradation, using lower preincubation doses), which demonstrates that TNF suggesting an increased IkBa turnover. Second, under low-dose tolerance is a prominent phenomenon in this cell type. preincubation-dependent tolerance, restimulation with TNF induced 10 TNF TOLERANCE IN HUMAN MONOCYTES Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 8. Low-dose TNF-induced tolerance is mediated by GSK3, whereas high-dose TNF-induced tolerance is dependent on A20/GSK3-dependent mechanisms. (A) THP-1 monocytic cells were incubated under standard conditions (low [left] or high [right] TNF dose for preincubation) in the presence of increasing concentrations of SB216763, and IL-8 mRNA expression (mean 6 SD, n = 3) was used as a readout (qPCR). The IL-8 value in M + T cells was defined as the 100% control for each condition. The inset shows the effect of SB216763 on b-catenin levels. (B) Cells were incubated under standard conditions in the absence or presence of 20 mM SB216763. The phosphorylation of p65 on Ser536 was monitored by Western blot analysis. (C) Cells were incubated under standard conditions in the presence of SB216763, and the level of c-jun was determined. (D) Cells were incubated under standard conditions, and the phosphorylation of GSK3-b (Ser9) was monitored. (E) Cells were incubated under standard conditions, and the expression of A20 was examined by Western blot. (F) Cells were preincubated under standard conditions in the presence of control (scrambled) or specific (against A20) siRNA, and the expression of IL-8 (mean 6 SD, n = 3) was determined. The IL-8 value in M + T cells was defined as the 100% control for each condition. The inset shows the effect on A20 protein expression of control or specific siRNA, respectively. (G) Cells were incubated under standard conditions using the high TNF dose for preincubation in the presence of control or specific A20 siRNA, and the phosphorylation of IKK was monitored by Western blot. Each panel of Fig. 7 depicts representative data of at least three independent experiments. a regular modestly impaired IkBa phosphorylation/proteolysis and transcription. Consistent with results reported earlier (18), we translocation of NF-kB subunits p65 and p50 in monocytic cells. detected increased levels of p50 in the nucleus of tolerant cells Third, in high-dose preincubated cells, no TNF-induced IkBa that were more pronounced in high-dose preincubated cells and phosphorylation/proteolysis and increase in NF-kB activity could be accompanied by a change of the cytosolic p50/p65 ratio toward detected. p50 (data not shown). As a consequence, the p50 increase may lead The TNF-induced phosphorylation of p65 (Ser536) was inhibited to the formation of transcriptionally inactive p50/p50 homodimers in tolerant cells in our experiments, regardless of whether low or (24), thereby contributing to the inhibition of NF-kB–dependent high preincubation doses were used. We have shown that low-dose genes in the same way as found for LPS tolerance (26, 45). Taken TNF-induced tolerance is associated with an inhibition of p65 together, we demonstrate that when cells were preincubated with (Ser536) phosphorylation (3), leading to inhibited NF-kB–dependent high TNF doses, NF-kB signaling was inhibited at two regulatory The Journal of Immunology 11

FIGURE 9. TNF tolerance mediated by the high TNF dose affects the PP1 phosphatase system. (A and B) The data were obtained from the microarray experiments shown in Figs. 3 and 5. Depicted are the relative expression levels of two PP1 phosphatase catalytic subunits PPP1CB and PPP3CC and several PP1 phosphatase regulatory/inhibitory subunits, PPP1R14C, PPP1R3E, and PPP1R12B (mean 6 SD). The M + M value was deﬁned in (A) as 1 and in (B) as 100%. (C) THP-1 monocytic cells were incubated under standard conditions. The phosphatase inhibitor calyculin A or the PP2A inhibitor ocadaic acid was added 30 min before short exposure to TNF. The expression of IL-8 was determined by qPCR (mean 6 SD, n = 2). The M + T value in the absence or presence of the inhibitors was deﬁned as 100% and is indicated by a dashed line. (D) Cells were transfected with either a Downloaded from PPP1R14C (R14C) expression vector or the empty control vector (GFP). Cells were then preincubated for 48 h in medium or with the low or the high TNF dose, and the expression of IL-8 mRNA was monitored. The values obtained using the GFP control vector were subtracted from the values obtained http://www.jimmunol.org/ with the PPP1R14C expression vector (mean 6 SD, n = 3). (E) Cells were incubated with calyculin A or okadaic acid for 45 min, and the phosphorylation of IKK, IkBa, p65, and JNK was determined by phospho-Western blot in whole-cell extracts. (C–E) Representative examples of at least three independent experiments. by guest on October 3, 2021

levels, that is, IkBa phosphorylation/proteolysis and p65 phos- proach. This suggests that a GSK3-dependent mechanism plays phorylation, whereas in low-dose TNF-treated tolerant cells only a key role in mediating low-dose–induced TNF refractoriness. an inhibition of p65 phosphorylation was observed. This is a likely GSK3 has been shown to be involved in the inhibition of TNF- explanation as to why high-dose TNF preincubation is more ef- induced NF-kB–dependent gene expression on a transcriptional fective in inhibiting NF-kB–dependent genes. The increase in level (47), and it has been shown that GSK3 regulates chromatin transcriptionally inactive p50 presumably contributes to the inhi- accessibility (48), which may negatively affect p65 phosphoryla- bition of the NF-kB system potentially to a greater extent in cells tion (49, 50). Previously, we have shown that C/EPBb is involved preincubated with the high dose. Our data also suggest that there in the inhibition of p65 (Ser536) phosphorylation mediated by low- are two different groups of NF-kB–regulated genes showing a dose TNF-dependent tolerance (3). Another group has found that differing sensitivity to the degree of inhibition. GSK3-b–dependent phosphorylation of C/EBPb is associated with Database analysis revealed that a major group of genes affected inhibitory effects on gene expression (51), and our experiments by absolute/induction tolerance is regulated by AP-1, which is demonstrate an inhibition of CEBPb phosphorylation using a activated by MAPK pathways (46). Our data showed that the TNF- GSK3 inhibitor (data not shown). In unstimulated tolerant cells, induced phosphorylation of p38 and JNK was completely inhib- we also observed an upregulation of p65 phosphorylation on Ser468, ited in tolerant cells regardless of whether high or low doses were a site phosphorylated by GSK3 and involved in negative regula- used for preincubation. An impaired activation of JNK under TNF tion of basal p65 transcriptional activity (27). GSK3 is inactivated tolerance has been found earlier (18). Our data also demonstrated by p38 and AKT-dependent phosphorylation (52, 53). Under TNF that the phosphorylation of c-jun was significantly inhibited in tolerance we observed an inhibition of p38, JNK, and AKT (data tolerant cells. It has been shown that JNK and p38 cooperate to not shown) phosphorylation accompanied by reduced GSK3-b regulate inflammatory gene expression, and a variety of genes is phosphorylation presumably associated with activation of this cooperatively controlled by AP-1 and NF-kB (24, 25, 46). kinase. It has also been shown that GSK3 inhibits the activation Innate and adapted immunity can be regulated by GSK3 (28, 29), of JNK and p38 (54) and is also involved in the inhibition of and TNF-induced LPS cross-tolerance is reversed by GSK3 in- c-jun–mediated transcription (28). For example, it has been sug- hibitory strategies (20). Remarkably, our data showed that inhi- gested that AP-1, CREB, and NF-kB form an integrated tran- bition of GSK3 led to a complete reversal of low-dose TNF- scriptional network largely responsible for maintaining repression induced tolerance. In addition, low-dose TNF-mediated inhibition of genes downstream of GSK3 signaling (55). It should also be of p65 (Ser536) phosphorylation could be reversed by this ap- mentioned that GSK3 phosphorylates c-jun targeting this protein 12 TNF TOLERANCE IN HUMAN MONOCYTES for degradation (56). In our experiments, we observed an up- TNF tolerance dose dependently modulates NF-kB/AP-1–associated regulation of c-jun in the presence of a GSK3 inhibitor solely in signaling. Low-dose–induced TNF tolerance is mediated by GSK3, tolerant cells, suggesting an increased c-jun turnover under tol- whereas high-dose–induced tolerance is regulated by A20/GSK3- erance. and PP1 phosphatase–dependent mechanisms. Absolute and in- GSK3 inhibition only partially affected high-dose–induced TNF duction TNF tolerance affects the kinase/phosphatase balance tolerance and inhibition of p65 phosphorylation, suggesting that representing different cellular means to protect against excessive additional mechanism contributes to this form of refractoriness. TNF stimulation and orchestrate/resolve inflammation. A patho- For example, the upregulation of PP1 phosphatase systems under physiological scenario may arise from a situation in which one has high-dose–dependent tolerance conditions also found in this study not enough or vice versa too much tolerance, resulting in either may prevent a complete reversal of TNF tolerance by GSK in- excessive inflammation or immune paralysis. hibitory strategies. GSK3 is also involved in the upregulation of A20 expression (30). Remarkably, when we used a siRNA approach against A20, a significant upregulation of gene expression Acknowledgments was observed only in high-dose but not in low-dose TNF-induced We are very thankful to Prof. Dr. H. W. L. Ziegler-Heitbrock, Dr. C. Cappello, tolerant cells. We also detected a dose-dependent upregulation of Dr. F. Bollig, J. Mages, and R. Lang for numerous critical discussions throughout the development of this study. We thank I. Rudnick for technical A20 in tolerant cells. In this study, it should also be mentioned that assistance. In addition, we are very thankful to Corne´lia La Fouge`re-Brand for in those cells, in which tolerance was induced by the high dose, typing and proofreading the manuscript as well as for critical discussions. our array data showed an upregulation of TNIP1-3 (data not shown) involved in the regulation of A20 (30). These data suggest Disclosures Downloaded from that A20 plays a major role in mediating TNF tolerance induced The authors have no financial conflicts of interest. by high TNF doses and also indicate that GSK3-b–independent mechanisms contribute to A20 upregulation. A20 has been shown to negatively affect NF-kB signaling upstream of the IKK com- References plex (57). Our data show an almost complete reversal of the high- 1. Locksley, R. M., N. Killeen, and M. J. Lenardo. 2001. The TNF and TNF re- dose TNF-induced inhibition of IKK phosphorylation in the ceptor superfamilies: integrating mammalian biology. Cell 104: 487–501.

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TNF-inducible genes (360) identified by the microarrays are listed. These genes were identified by the formula M+T/M+M ≥2, and shown is the log2 induction (mean±SD). The Table contains the gene (alphabetical order), the systematic name, and the probe name.

Gene Systematic Name Probe Name log2 induction (mean±SD) 7A5 NM_182762 A_32_P131031 3,06 ± 0,68 ABTB2 NM_145804 A_23_P356616 2,65 ± 0,53 ACSL1 NM_001995 A_23_P110212 2,95 ± 0,36 ACSL5 NM_203380 A_24_P201360 1,97 ± 0,61 ACVR2A NM_001616 A_23_P153930 2,80 ± 0,20 ADA NM_000022 A_23_P210482 1,63 ± 0,30 ADM NM_001124 A_23_P127948 3,15 ± 0,31 ADORA2A NM_000675 A_23_P109436 3,89 ± 0,22 AK3L1 NM_001005353 A_32_P108655 3,81 ± 1,22 AK3L2 NM_001002921 A_23_P200524 3,27 ± 1,40 ALS2CR4 NM_152388 A_23_P370097 1,69 ± 0,40 AMPD3 NM_000480 A_23_P116286 1,77 ± 0,32 AMZ1 NM_133463 A_24_P383649 3,08 ± 0,64 ANKRD15 NM_153186 A_32_P64570 4,34 ± 0,48 ANKRD22 NM_144590 A_23_P161428 1,56 ± 0,65 APOL3 NM_145641 A_24_P416997 2,36 ± 0,69 AQP9 NM_020980 A_23_P106362 2,84 ± 0,94 AREG NM_001657 A_23_P259071 2,62 ± 0,63 ARHGAP20 NM_020809 A_23_P422933 1,69 ± 0,44 ARHGEF7 NM_145735 A_23_P417891 2,01 ± 0,41 ARL5B NM_178815 A_23_P378588 3,64 ± 0,73 ARNTL2 NM_020183 A_23_P53345 2,07 ± 0,40 ARRDC3 NM_020801 A_24_P274615 1,84 ± 0,38 ARRDC4 NM_183376 A_23_P339818 1,89 ± 0,25 ATF5 NM_012068 A_23_P119337 1,66 ± 0,28 ATP2B1 NM_001682 A_23_P128319 2,36 ± 0,20 AXUD1 NM_033027 A_23_P121011 2,03 ± 0,46 B4GALT1 NM_001497 A_24_P103803 2,04 ± 0,20 B4GALT5 NM_004776 A_24_P239731 2,10 ± 0,60 BAZ1A NM_013448 A_23_P76799 1,95 ± 0,33 BCAR3 NM_003567 A_23_P97394 1,35 ± 0,17 BCL2A1 NM_004049 A_23_P152002 3,08 ± 1,08 BCMO1 NM_017429 A_23_P124300 1,37 ± 0,13 BIC NR_001458 A_32_P108156 4,69 ± 1,07 BID NM_197966 A_23_P154929 1,59 ± 0,23 BIRC3 NM_001165 A_23_P98350 2,80 ± 0,55 BLR1 NM_032966 A_24_P252945 2,47 ± 0,83 BRPF3 NM_015695 A_24_P414712 1,31 ± 0,26 BTBD4 NM_025224 A_32_P518489 1,95 ± 0,72

I Supplementary Table I, continued

Gene Systematic Name Probe Name log2 induction (mean±SD) BTG1 NM_001731 A_23_P87560 1,88 ± 0,47 BTG2 NM_006763 A_23_P62901 1,64 ± 0,48 BTG3 NM_006806 A_23_P80068 2,92 ± 0,14 BTNL8 NM_024850 A_23_P7412 1,39 ± 0,20 CCL1 NM_002981 A_23_P49759 1,88 ± 0,65 CCL19 NM_006274 A_23_P123853 1,42 ± 0,48 CCL20 NM_004591 A_23_P17065 7,76 ± 0,50 CCL22 NM_002990 A_24_P313418 1,67 ± 0,26 CCL23 NM_005064 A_24_P319088 4,23 ± 0,33 CCL3 D00044 A_23_P373017 3,11 ± 0,18 CCL3L3 NM_001001437 A_23_P321920 3,33 ± 0,86 CCL4 NM_002984 A_23_P207564 5,22 ± 0,30 CCL5 NM_002985 A_23_P152838 4,59 ± 0,34 CCL8 NM_005623 A_23_P207456 3,55 ± 0,48 CCR7 NM_001838 A_23_P343398 2,91 ± 1,22 CCRL2 NM_003965 A_23_P69310 1,84 ± 0,37 CCRN4L NM_012118 A_24_P213794 2,99 ± 0,85 CD274 NM_014143 A_23_P338479 2,70 ± 0,75 CD40 NM_001250 A_23_P57036 3,00 ± 0,46 CD44 NM_000610 A_23_P24870 1,78 ± 0,25 CD58 NM_001779 A_23_P138308 1,59 ± 0,30 CD6 NM_006725 A_23_P311875 3,17 ± 0,46 CD69 NM_001781 A_23_P87879 4,03 ± 1,04 CD80 NM_005191 A_24_P320033 3,88 ± 0,36 CD83 NM_004233 A_23_P70670 2,80 ± 0,58 CDC42EP2 NM_006779 A_23_P1602 3,91 ± 0,42 CDGAP NM_020754 A_24_P349039 1,88 ± 0,17 CDKN2B NM_078487 A_24_P360674 3,93 ± 0,40 CENTD1 NM_015230 A_32_P83784 1,38 ± 0,22 CGN NM_020770 A_24_P45728 2,10 ± 0,54 CH25H NM_003956 A_23_P86470 3,38 ± 1,54 CHML NM_001821 A_23_P46118 2,36 ± 0,52 CHST2 NM_004267 A_23_P40847 3,24 ± 0,48 CIAS1 NM_004895 A_23_P9883 1,83 ± 0,27 CLCF1 NM_013246 A_23_P138760 2,25 ± 0,62 CLEC4D NM_080387 A_23_P25235 2,56 ± 1,00 CLEC4E NM_014358 A_24_P78531 3,20 ± 0,55 CLIC4 NM_013943 A_23_P135494 2,50 ± 0,22 CRIM1 NM_016441 A_23_P51105 3,06 ± 0,77 CSF2 NM_000758 A_23_P133408 4,45 ± 0,52 CSF3 NM_000759 A_23_P501754 4,03 ± 0,46 CSRP2 NM_001321 A_23_P44724 3,06 ± 0,61 CTNND2 NM_001332 A_24_P380196 1,84 ± 0,34 CXCL1 NM_001511 A_23_P7144 6,09 ± 0,70 CXCL2 NM_002089 A_23_P315364 3,58 ± 0,81 CXCL3 NM_002090 A_24_P251764 3,29 ± 0,40 CXCL5 NM_002994 A_23_P110204 3,81 ± 0,82 CXCL6 NM_002993 A_23_P155755 4,29 ± 1,28

II Supplementary Table I, continued

Gene Systematic Name Probe Name log2 induction (mean±SD) CYP7B1 NM_004820 A_23_P169092 3,00 ± 1,01 DC-UbP NM_152277 A_23_P19061 1,89 ± 0,37 DDIT4 NM_019058 A_23_P104318 2,21 ± 0,42 DGKH NM_152910 A_23_P502980 1,90 ± 0,73 DLC1 NM_182643 A_24_P940115 2,00 ± 0,20 DNAJB5 NM_012266 A_23_P112241 2,56 ± 0,22 DOC1 NM_182909 A_23_P252052 2,41 ± 0,27 DOT1L NM_032482 A_23_P408768 2,21 ± 0,33 DSU NM_018000 A_23_P108948 1,51 ± 0,44 DUSP1 NM_004417 A_23_P110712 2,11 ± 0,40 DUSP2 NM_004418 A_24_P37409 2,41 ± 0,61 DUSP5 NM_004419 A_23_P150018 3,44 ± 1,03 DUSP6 NM_001946 A_23_P139704 1,87 ± 0,58 DYRK3 NM_001004023 A_24_P345209 2,45 ± 1,05 E2F7 NM_203394 A_32_P210202 3,16 ± 1,29 EBI3 NM_005755 A_24_P370201 3,20 ± 0,26 ECE1 NM_001397 A_24_P154080 1,72 ± 0,50 EDEM1 NM_014674 A_24_P285768 1,53 ± 0,22 EDN1 NM_001955 A_23_P214821 3,58 ± 0,34 EGR3 NM_004430 A_23_P216225 2,86 ± 1,09 EHD1 NM_006795 A_23_P52647 3,52 ± 0,57 EID3 NM_001008394 A_23_P65068 2,02 ± 0,42 EMP2 NM_001424 A_23_P106682 1,46 ± 0,16 EREG NM_001432 A_23_P41344 5,53 ± 0,71 ESPL1 NM_012291 A_23_P32707 1,84 ± 0,23 ETV3 NM_005240 A_23_P400945 1,96 ± 0,37 EYA3 NM_001990 A_23_P74737 1,60 ± 0,35 EZH2 NM_004456 A_23_P259641 1,73 ± 0,13 F3 NM_001993 A_23_P126782 5,57 ± 1,02 FAM107B NM_031453 A_23_P149975 1,19 ± 0,14 FAM49A NM_030797 A_23_P21560 1,49 ± 0,20 FAM57A NM_024792 A_23_P50000 1,63 ± 0,38 FCAR NM_133280 A_24_P348265 2,15 ± 0,67 FEZ1 NM_005103 A_24_P201552 4,94 ± 0,85 FFAR2 NM_005306 A_23_P397391 1,58 ± 0,20 FJX1 NM_014344 A_23_P150693 1,80 ± 0,49 FMNL3 NM_175736 A_23_P379200 1,73 ± 0,38 FOSL1 NM_005438 A_23_P322519 2,85 ± 0,81 FOSL2 NM_005253 A_23_P348121 2,24 ± 0,59 FPRL1 NM_001462 A_23_P55649 1,46 ± 0,21 FSCN1 NM_003088 A_23_P168532 3,67 ± 0,79 FUT4 NM_002033 A_23_P12965 1,48 ± 0,29 FZD7 NM_003507 A_23_P209449 1,36 ± 0,23 G0S2 NM_015714 A_23_P74609 5,83 ± 0,38 GBP1 NM_002053 A_23_P62890 3,67 ± 0,58 GBP2 NM_004120 A_23_P85693 2,92 ± 0,26 GCH1 NM_000161 A_23_P163079 4,79 ± 0,42 GJB2 NM_004004 A_23_P204947 4,45 ± 0,48

III Supplementary Table I, continued

Gene Systematic Name Probe Name log2 induction (mean±SD) GPR109A NM_177551 A_23_P329924 2,15 ± 0,54 GPR109B NM_006018 A_23_P64721 4,33 ± 0,72 GPR132 NM_013345 A_24_P201994 2,66 ± 0,51 GPR35 NM_005301 A_23_P154245 1,51 ± 0,34 GPR84 NM_020370 A_23_P25155 2,76 ± 0,20 GRAMD1A NM_020895 A_23_P56213 2,97 ± 0,54 GRAMD3 NM_023927 A_23_P22350 2,55 ± 0,07 HEY1 NM_012258 A_32_P83845 5,81 ± 1,20 HIVEP1 NM_002114 A_23_P19619 2,38 ± 0,30 HIVEP2 NM_006734 A_23_P214766 3,45 ± 0,34 HSA251708 AJ251708 A_24_P932084 2,80 ± 0,30 HSD11B1 NM_181755 A_23_P63209 2,27 ± 0,77 IBRDC2 NM_182757 A_24_P406060 3,87 ± 0,31 IBRDC3 NM_153341 A_23_P321388 2,54 ± 0,51 ICAM1 NM_000201 A_23_P153320 3,34 ± 0,56 IER3 NM_003897 A_23_P42257 3,30 ± 0,39 IFNB1 NM_002176 A_23_P71774 2,54 ± 0,76 IFNGR2 NM_005534 A_23_P29036 1,69 ± 0,28 IGSF21 NM_032880 A_32_P78101 2,14 ± 0,54 IL12B NM_002187 A_23_P7560 4,53 ± 0,97 IL15RA NM_172200 A_23_P138680 2,90 ± 0,48 IL18 NM_001562 A_23_P104798 2,75 ± 0,16 IL18R1 NM_003855 A_24_P208567 2,58 ± 1,29 IL1A NM_000575 A_23_P72096 8,19 ± 0,63 IL1B NM_000576 A_23_P79518 6,16 ± 0,53 IL1F9 NM_019618 A_23_P17053 4,12 ± 0,90 IL20 NM_018724 A_23_P46482 2,58 ± 1,63 IL23A NM_016584 A_23_P76078 3,91 ± 0,76 IL28A NM_172138 A_23_P409438 1,72 ± 0,25 IL2RA NM_000417 A_23_P127288 4,22 ± 1,10 IL32 NM_001012631 A_23_P15146 1,82 ± 0,19 IL4I1 NM_172374 A_23_P502520 1,59 ± 0,27 IL6 NM_000600 A_23_P71037 6,54 ± 0,50 IL7R NM_002185 A_23_P404494 4,47 ± 0,97 IL8 NM_000584 A_32_P87013 5,80 ± 1,08 INHBA NM_002192 A_23_P122924 2,65 ± 1,38 INSIG1 NM_198336 A_23_P22027 3,08 ± 0,40 IRAK2 NM_001570 A_23_P80635 2,79 ± 0,88 IRAK3 NM_007199 A_23_P162300 2,00 ± 0,20 ITGB8 NM_002214 A_24_P273599 2,93 ± 0,73 JAG1 NM_000214 A_23_P210763 5,20 ± 0,38 JUNB NM_002229 A_23_P4821 1,72 ± 0,25 KCNA3 NM_002232 A_23_P201138 3,68 ± 0,34 KCNJ2 NM_000891 A_23_P329261 3,45 ± 0,45 KCNN4 NM_002250 A_23_P67529 2,14 ± 0,40 KMO NM_003679 A_23_P200838 2,17 ± 0,47 KRT23 NM_015515 A_23_P78248 2,50 ± 0,66 KYNU NM_003937 A_24_P11506 2,24 ± 0,31

IV Supplementary Table I, continued

Gene Systematic Name Probe Name log2 induction (mean±SD) LAD1 NM_005558 A_23_P415510 2,98 ± 0,88 LAMB3 NM_001017402 A_23_P86012 3,36 ± 0,52 LAMP3 NM_014398 A_23_P29773 2,38 ± 1,03 LCT NM_002299 A_23_P79217 1,86 ± 0,29 LENG9 NM_198988 A_32_P493225 3,08 ± 0,55 LIMS3 NM_033514 A_23_P365685 1,69 ± 0,50 LINCR NM_001080535 A_23_P328740 3,15 ± 0,61 LITAF NM_004862 A_23_P3532 1,45 ± 0,47 LONRF1 NM_152271 A_23_P94216 1,90 ± 0,39 LRFN5 NM_152447 A_23_P163195 3,11 ± 0,78 LRP12 NM_013437 A_23_P8906 1,92 ± 0,62 LRRC32 NM_005512 A_24_P389916 4,24 ± 1,02 LSS NM_002340 A_24_P110799 1,88 ± 0,29 MAFF NM_012323 A_23_P103110 2,27 ± 0,56 MAP2K3 NM_145109 A_23_P118427 2,28 ± 0,40 MAP3K8 NM_005204 A_23_P23947 3,09 ± 0,46 MARCH3 NM_178450 A_23_P321511 1,92 ± 0,32 MCL1 NM_021960 A_24_P319635 1,92 ± 0,53 MCOLN2 NM_153259 A_23_P23639 2,75 ± 0,55 MCTP1 NM_024717 A_24_P212481 1,79 ± 0,47 MESDC1 NM_022566 A_23_P99891 1,42 ± 0,25 MET NM_000245 A_23_P359245 3,66 ± 1,39 MFSD2 NM_032793 A_23_P43820 2,81 ± 0,33 MN1 NM_002430 A_23_P6381 2,27 ± 0,73 MOBKL2C NM_145279 A_24_P225339 1,87 ± 0,31 MSC NM_005098 A_23_P256948 4,29 ± 0,31 MTF1 NM_005955 A_23_P74241 2,36 ± 0,18 MYC NM_002467 A_23_P215956 2,70 ± 0,87 MYO1G NM_033054 A_23_P257542 1,37 ± 0,37 N4BP3 NM_015111 A_23_P58747 2,03 ± 0,38 NAB1 NM_005966 A_23_P209805 1,80 ± 0,31 NBN NM_002485 A_23_P251480 2,75 ± 0,33 NFE2L3 NM_004289 A_23_P42718 2,20 ± 0,32 NFKB1 NM_003998 A_23_P30024 3,00 ± 0,27 NFKB2 NM_002502 A_23_P202156 2,56 ± 0,25 NFKBIA NM_020529 A_23_P106002 2,45 ± 0,28 NFKBIE NM_004556 A_23_P30655 1,26 ± 0,12 NFKBIZ NM_031419 A_23_P212089 3,19 ± 0,82 NUMB NM_001005743 A_23_P88381 1,50 ± 0,16 OASL NM_003733 A_23_P139786 2,62 ± 0,56 OGFRL1 NM_024576 A_23_P7791 1,89 ± 0,30 OLR1 NM_002543 A_24_P124624 1,99 ± 0,61 OPTN NM_001008211 A_23_P1461 1,45 ± 0,28 OR11H12 NM_001013354 A_23_P128868 2,06 ± 0,63 OSM NM_020530 A_23_P166408 2,38 ± 0,23 OTUD1 AB188491 A_32_P60459 1,55 ± 0,39 P2RX7 NM_002562 A_23_P25354 1,66 ± 0,17 PBEF1 NM_005746 A_24_P408772 2,87 ± 0,40

V Supplementary Table I, continued

Gene Systematic Name Probe Name log2 induction (mean±SD) PBX4 NM_025245 A_23_P90419 2,76 ± 0,61 PDE4B NM_002600 A_24_P325333 4,44 ± 0,47 PDE4DIP NM_022359 A_23_P149153 1,60 ± 0,28 PDGFA NM_002607 A_23_P113701 2,84 ± 0,82 PDGFB NM_002608 A_24_P339944 2,32 ± 0,44 PDSS1 NM_014317 A_23_P161152 1,97 ± 0,47 PELI1 NM_020651 A_23_P120345 1,85 ± 0,46 PHLDA2 NM_003311 A_23_P47614 3,00 ± 1,07 PHLDB1 NM_015157 A_23_P24555 2,80 ± 0,49 PHLPPL NM_015020 A_23_P418234 1,36 ± 0,16 PIK3AP1 NM_152309 A_23_P104445 1,38 ± 0,22 PIM2 NM_006875 A_24_P379104 2,64 ± 0,29 PIM3 NM_001001852 A_23_P61398 1,75 ± 0,39 PLAGL2 NM_002657 A_23_P6151 1,89 ± 0,20 PLAUR NM_001005377 A_23_P16469 1,56 ± 0,52 PLCB4 NM_000933 A_23_P28898 1,77 ± 0,52 PLEK NM_002664 A_23_P209678 2,69 ± 0,36 PLEKHC1 NM_006832 A_23_P88347 3,32 ± 1,51 PLK3 NM_004073 A_23_P51646 2,13 ± 0,39 PMAIP1 NM_021127 A_23_P207999 2,97 ± 0,28 PNPLA1 NM_173676 A_32_P526255 3,04 ± 0,95 PNRC1 NM_006813 A_23_P145074 2,57 ± 0,26 PPP1R15B NM_032833 A_23_P160449 2,68 ± 0,47 PPP3CC NM_005605 A_23_P157495 1,71 ± 0,09 PRDM11 NM_020229 A_23_P13057 2,09 ± 0,42 PRPF3 NM_004698 A_23_P97573 1,33 ± 0,26 PSTPIP2 NM_024430 A_23_P208119 2,74 ± 0,51 PTGER2 NM_000956 A_23_P151710 2,64 ± 0,45 PTGER4 NM_000958 A_23_P435394 2,91 ± 0,46 PTGIR NM_000960 A_23_P340848 1,87 ± 0,47 PTGS2 NM_000963 A_24_P250922 6,05 ± 0,61 PTPN1 NM_002827 A_23_P338890 1,93 ± 0,27 PTPN12 NM_002835 A_23_P8763 1,32 ± 0,11 PTPRJ NM_002843 A_24_P282434 1,55 ± 0,25 PTX3 NM_002852 A_23_P121064 4,46 ± 1,13 PVR NM_006505 A_24_P65616 1,94 ± 0,44 RAB6IP1 NM_015213 A_23_P321201 1,78 ± 0,27 RABGEF1 NM_014504 A_23_P250825 1,83 ± 0,43 RAP2C NM_021183 A_24_P374319 1,57 ± 0,42 RAPGEF2 XM_944412 A_24_P912587 3,18 ± 0,68 RASGEF1B NM_152545 A_23_P331186 1,79 ± 0,44 RASL11A NM_206827 A_23_P14124 1,96 ± 0,43 REL NM_002908 A_23_P56938 2,48 ± 0,66 RELB NM_006509 A_23_P55706 1,94 ± 0,42 RHOF NM_019034 A_24_P104115 2,06 ± 0,37 RIPK2 NM_003821 A_24_P124032 1,79 ± 0,28 RND1 NM_014470 A_23_P53370 2,37 ± 0,54 RRAD NM_004165 A_24_P262127 3,81 ± 0,38

VI Supplementary Table I, continued

Gene Systematic Name Probe Name log2 induction (mean±SD) SAMD14 NM_174920 A_24_P185186 2,99 ± 0,48 SDC4 NM_002999 A_24_P373976 2,56 ± 0,55 SEC24A BC019341 A_23_P329593 1,53 ± 0,26 SERPINB2 NM_002575 A_24_P245379 3,24 ± 0,45 SERPINB8 NM_198833 A_23_P4561 2,02 ± 0,27 SERPINB9 NM_004155 A_23_P30687 4,31 ± 0,48 SERPINE1 NM_000602 A_24_P158089 1,56 ± 0,46 SESN3 NM_144665 A_23_P361448 1,58 ± 0,35 SGPP2 NM_152386 A_23_P153971 4,92 ± 0,21 SH2D2A NM_003975 A_23_P160618 1,67 ± 0,35 SKIL NM_005414 A_23_P351215 1,71 ± 0,33 SLAMF7 NM_021181 A_24_P353638 2,43 ± 0,64 SLC1A2 NM_004171 A_23_P162068 2,68 ± 1,02 SLC2A6 NM_017585 A_23_P169249 2,87 ± 0,43 SLC35F2 NM_017515 A_23_P339098 1,69 ± 0,30 SLC39A8 NM_022154 A_23_P41424 3,18 ± 0,39 SLC43A3 NM_199329 A_23_P358548 1,62 ± 0,35 SLC7A5 NM_003486 A_24_P335620 2,86 ± 0,38 SLCO4A1 NM_016354 A_23_P5903 3,17 ± 0,57 SNFT NM_018664 A_23_P160720 2,28 ± 0,72 SNX10 NM_013322 A_24_P98109 1,46 ± 0,32 SOCS1 NM_003745 A_24_P48014 1,53 ± 0,60 SOCS3 NM_003955 A_23_P207058 4,30 ± 0,62 SOD2 NM_000636 A_23_P134176 2,70 ± 0,21 SPHK1 NM_021972 A_23_P38106 1,61 ± 0,56 SPSB1 NM_025106 A_23_P200096 2,04 ± 0,48 SQLE NM_003129 A_23_P146284 1,70 ± 0,21 SRC NM_005417 A_23_P308603 2,60 ± 0,20 SRrp35 NM_080743 A_23_P110903 4,01 ± 1,88 SRXN1 NM_080725 A_23_P320113 1,86 ± 0,16 STAT4 NM_003151 A_23_P305198 3,30 ± 0,84 STX11 NM_003764 A_23_P156788 2,06 ± 0,31 SYNPO2 AL833294 A_23_P310094 3,14 ± 1,04 TACR1 NM_015727 A_24_P148590 2,23 ± 0,41 TANK NM_133484 A_24_P257108 1,76 ± 0,31 TARP NM_001003799 A_23_P59543 3,72 ± 1,46 TBC1D9 NM_015130 A_23_P41487 1,78 ± 0,36 TCEB3 NM_003198 A_23_P74097 1,17 ± 0,09 THBS1 NM_003246 A_23_P206212 3,41 ± 0,44 THRAP1 NM_005121 A_23_P27247 1,50 ± 0,23 TICAM1 NM_182919 A_23_P90311 1,70 ± 0,27 TIFA NM_052864 A_24_P350686 2,45 ± 0,42 TINAG NM_014464 A_23_P168329 1,63 ± 0,66 TJP2 NM_201629 A_24_P201153 1,91 ± 0,41 TLR2 NM_003264 A_23_P92499 2,20 ± 0,10 TMEM88 NM_203411 A_23_P77859 2,52 ± 0,18 TNF NM_000594 A_24_P50759 3,61 ± 0,32 TNFAIP2 NM_006291 A_23_P421423 2,76 ± 0,46

VII Supplementary Table I, continued

Gene Systematic Name Probe Name log2 induction (mean±SD) TNFAIP3 NM_006290 A_24_P166527 3,73 ± 0,79 TNFAIP6 NM_007115 A_23_P165624 5,69 ± 0,55 TNFAIP8 NM_014350 A_32_P219520 2,79 ± 0,29 TNFRSF10B NM_003842 A_23_P169030 1,78 ± 0,35 TNFRSF18 NM_148901 A_24_P411121 2,66 ± 0,29 TNFRSF4 NM_003327 A_32_P26092 2,57 ± 0,45 TNFRSF9 NM_001561 A_23_P51936 3,74 ± 0,47 TNFSF15 NM_005118 A_24_P167012 1,99 ± 0,41 TNFSF9 NM_003811 A_23_P67224 1,95 ± 0,44 TNIP1 NM_006058 A_23_P30435 2,48 ± 0,39 TNIP2 NM_024309 A_23_P258418 2,30 ± 0,48 TNIP3 NM_024873 A_23_P386478 7,63 ± 0,90 TP53BP2 NM_005426 A_23_P12526 1,75 ± 0,37 TP53INP2 NM_021202 A_24_P357465 2,16 ± 0,47 TRAF1 NM_005658 A_24_P89891 5,71 ± 0,33 TRAF3 NM_145725 A_23_P37068 1,35 ± 0,17 TRIM36 NM_018700 A_23_P110569 2,32 ± 0,11 TRIP10 NM_004240 A_23_P50349 2,51 ± 0,38 UAP1 NM_003115 A_23_P160460 1,52 ± 0,44 UPB1 NM_016327 A_23_P120822 2,53 ± 0,29 USP12 NM_182488 A_24_P237613 3,29 ± 0,51 USP13 NM_003940 A_23_P40989 1,33 ± 0,31 UXS1 NM_025076 A_23_P67829 1,53 ± 0,21 VCAM1 NM_001078 A_23_P34345 3,11 ± 0,42 WNT5A NM_003392 A_23_P211926 3,29 ± 1,08 WTAP NM_152858 A_23_P215037 3,55 ± 0,23 YRDC NM_024640 A_23_P62840 1,93 ± 0,19 ZBTB10 NM_023929 A_24_P64071 3,79 ± 0,95 ZC3H12A NM_025079 A_23_P326160 3,63 ± 0,53 ZHX2 NM_014943 A_23_P168951 2,07 ± 0,30 ZNF140 NM_003440 A_23_P150841 1,23 ± 0,09 ZNF697 NM_001080470 A_23_P322076 2,10 ± 0,51 ZNFX1 NM_021035 A_24_P23034 1,63 ± 0,37

VIII Supplementary Table II

Summary of genes affected by absolute tolerance in human monocytes. The 51 genes induced by preincubation with the high TNF dose (Figure 3A; 400+T/M+T ≤0.25) and the 24 genes induced by the low TNF preincubation dose (Figure 3C; 40+T/M+T ≤0.25) are summarized according to their function. A regulation by NF-ĸB and/or AP-1 is indicated (extracted from http://www.genecards.org). Genes affected by the high dose (normal font), by the low dose (italic), and under both conditions (bold) are indicated accordingly.

Category Gene Gene Name Regulation

Inflammation IL18R1 interleukin 18 receptor 1 NF-κB

IL1A interleukin 1 alpha AP-1

IL6 interleukin 6 NF-κB

IL8 interleukin 8 NF-κB

MAP3K8 mitogen-activated protein kinase kinase kinase 8 NF-κB

OLR1 oxidized low density lipoprotein receptor 1 AP-1

PTGS2 prostaglandin-endoperoxide synthase 2 AP-1

PTX3 pentraxin 3 NF-κB

TNF tumor necrosis factor NF-κB, AP-1

TNFSF15 tumor necrosis factor superfamily member 15 NF-κB

Growth/ IL12B interleukin 12B Differentiation

AREG amphiregulin NF-κB, AP-1

CSF2 colony stimulating factor 2

CSF3 colony stimulating factor 3 AP-1

STAT4 signal transducer and activator of transcription 4

HEY1 hairy/enhancer-of-split related with YRPW motif 1 NF-κB, AP-1

EGR3 early growth response 3 AP-1

PDGFA platelet-derived growth factor alpha AP-1

PDGFB platelet-derived growth factor beta pleckstrin homology-like domain family A member PHLDA2 2

IX Supplementary Table II, continued

Category Gene Gene Name Regulation

Growth/ TMEM88 transmembrane protein 88 AP-1 Differentiation Chemotaxis/ CCL20 C-C motif chemokine ligand 20 Migration

CCL4 C-C motif chemokine ligand 4

CXCL1 C-X-C motif chemokine ligand 1 NF-κB

CXCL2 C-X-C motif chemokine ligand 2 NF-κB

CXCL3 C-X-C motif chemokine ligand 3

CXCL6 C-X-C motif chemokine ligand 6 NF-κB

FEZ1 fasciculation and elongation protein zeta 1

THBS1 thrombospondin 1 pleckstrin homology-domain containing protein family C PLEKHC1 member 1 CD6 CD6 molecule NF-κB, AP-1

Signaling/ GPR35 G protein-coupled receptor 35 AP-1 Transcription ARL5B ADP-ribosylation factor-like 5B AP-1

ARRDC3 arrestin domain containing 3

DOT1L DOT1-like histone H3 methyltransferase AP-1

RASGEF1B RasGEF domain family member 1B

Metabolism CHML Rab escort protein 2 NF-κB, AP-1

INSIG1 insulin induced gene 1 major facilitator superfamily domain-containing MFSD2A protein 2A UAP1 UDP-N-acteylglucosamine pyrophosphorylase 1 AP-1

potassium voltage-gated channel shaker-related Transport KCNA3 subfamily member 3 solute carrier organic anion transporter family SLCO4A1 member 4A1 Cell Cycle/ ESPL1 extra spindle pole bodies homolog 1 NF-κB, AP-1 Proliferation TRIM36 tripartite motif containing 36

IL20 interleukin 20 AP-1

CD69 CD69 molecule NF-κB, AP-1

X Supplementary Table II, continued

Category Gene Gene Name Regulation

Cell Cycle/ INHBA inhibin beta A AP-1 Proliferation

Procoagulation F3 Tissue factor NF-κB, AP-1

SERPINB2 serpin peptidase inhibitor member 2 NF-κB, AP-1

Vasoconstrictive EDN1 endothelin 1 NF-κB

v-maf musculoaponeurotic fibrosarcoma oncogene Oncogene MAFF AP-1 homolog F

Unknown ANKRD15 KN motif and ankyrin repeat domains 1

LENG9 leukocyte receptor cluster member 9 AP-1

LIMS3 LIM and senescent cell antigen-like domains 3

BIC MIR155 host gene

FAM57A family with sequence similarity 57 member A NF-κB

XI Supplementary Table III

Summary of 99 genes affected by low-dose-induced tolerance applying less stringent criteria. Genes were selected using the formula: 40+T/M+T ≤0.5.

CSF2 EDN1 DNAJB5 CSF3 DUSP2

F3 IL12B MAP2K3 CXCL3 IFNB1

IL6 LENG9 MYC CXCL2 BTBD4

PTGS2 ARL5B PPP1R15B KCNA3 LRP12

CCL20 IL23A REL TNF PHLDB1

IL1A USP12 ATP2B1 CD69 DUSP6

HEY1 IL1F9 EID3 SERPINB2 STX11

SERPINB9 IL18R1 LRFN5 RAPGEF2 SDC4

ANKRD15 MFSD2 P2RX7 PMAIP1 PIM2

IL8 INHBA EDEM1 THBS1 OGFRL1

CXCL1 TNFSF15 TICAM1 ARRDC3 BIC

JAG1 PHLDA2 CD83 ESPL1 STAT4

IL1B EGR3 RASGEF1B DOT1L UAP1

EREG CXCL6 CHML OLR1 FAM57A

INSIG1 PDGFB TRIM36 TMEM88 DDIT4

MAP3K8 SPSB1 AREG PLEKHC1 CCRN4L

CLCF1 LCT

XII Supplementary Table IV

List of genes affected by induction tolerance in human monocytes. The table shows the list of 227 genes inhibited by TNF induction tolerance (400+T/400+M ≤1.25) depicted in Figure 5A. Again, the genes are summarized into functional groups, and NF-ĸB- and/or AP-1-dependent genes were identified by monitoring online database genecards.

Category Gene Gene Name Regulation

Signaling/ RRAD Ras-related associated with diabetes NF-κB, AP-1 Transcription PLCB4 phospholipase C beta 4

GPR109B hydroxycarboxylic acid receptor 3

ADORA2A adenosine A2a receptor NF-κB

OASL 2'-5'-oligoadenylate synthetase-like

PDE4B phosphodiesterase 4B human immunodeficiency virus type I HIVEP1 NF-κB, AP-1 enhancer binding protein 1 human immunodeficiency virus type I HIVEP2 AP-1 enhancer binding protein 2 PTGER4 prostaglandin E receptor 4

ARRDC4 arrestin domain containing 4

OPTN optineurin NF-κB

GPR132 G protein-coupled receptor 132 AP-1

FZD7 frizzled family receptor

TJP2 tight junction protein 2 AP-1

PTGER2 prostaglandin E receptor 2

MCOLN2 mucolipin 2

GJB2 gap junction protein, beta 2 AP-1

RABGEF1 RAB guanine nucleotide exchange factor 1 NF-κB

RAPGEF2 Rap guanine nucleotide exchange factor 2 AP-1

TICAM1 toll-like receptor adaptor molecule 1 NF-κB

GPR35 G protein-coupled receptor 35 AP-1

CD83 CD83 molecule NF-κB

XIII Supplementary Table IV, continued

Category Gene Gene Name Regulation

Signaling/ SOCS3 suppressor of cytokine signaling 3 Transcription protein phosphatase 3 catalytic subunit PPP3CC gamma MSC musculin NF-κB, AP-1

ZNF140 zinc finger protein 140

DOT1L DOT1-like histone H3 methyltransferase AP-1

NAB1 EGR1 binding protein 1

EZH2 enhancer of zeste homolog 2

ZHX2 zinc fingers and homeoboxes 2 bromodomain adjacent to zinc finger BAZ1A domain 1A bromodomain and PHD finger containing BRPF3 protein 3 ATF5 activating transcription factor 5 NF-κB, AP-1 protein phosphatase 1 regulatory PPP1R15B subunit 15B transcription elongation factor B TCEB3 polypeptide 3 PLAGL2 pleiomorphic adenoma gene-like 2

NFE2L3 nuclear factor (erythroid-derived 2)-like 3 NF-κB

THRAP1 mediator complex subunit 13

CTNND2 catenin delta 2

EYA3 eyes absent homolog 3

tumor necrosis factor alpha-induced Inflammation TNFAIP2 protein 2 TLR2 toll-like receptor 2 NF-κB, AP-1 CIAS1 NLR family pyrin domain containing protein 3 (NLRP3) NFKB2 NF-κB 2 NF-κB receptor-interacting serine-threonine RIPK2 NF-κB kinase 2 FCAR Fc fragment of IgA receptor NF-κB

LITAF lipopolysaccharide-induced TNF factor

IL18 interleukin 18 NF-κB, AP-1

XIV Supplementary Table IV, continued

Category Gene Gene Name Regulation

Inflammation MAP2K3 mitogen-activated protein kinase kinase 3 NF-κB, AP-1 mitogen-activated protein kinase kinase MAP3K8 NF-κB kinase 8 OLR1 oxidized low density lipoproteinreceptor 1 AP-1

IL18R1 interleukin 18 receptor 1 NF-κB

EBI3 Epstein-Barr virus induced 3 NF-κB v-rel reticuloendotheliosis viral oncogene RELB NF-κB, AP-1 homolog B FPRL1 formyl peptide receptor 2

IL32 interleukin 32 NF-κB

NFKBIA NF-κB inhibitor alpha NF-κB, AP-1 TRAF family member-associated NFKB TANK NF-κB activator NFKB1 NF-κB 1 NF-κB

TRAF1 TNF receptor-associated factor 1 NF-κB, AP-1

TRAF3 TNF receptor-associated factor 3 NF-κB, AP-1

PLEK pleckstrin NF-κB

CLCF1 cardiotrophin-like cytokine factor 1

IL2RA interleukin 2 receptor alpha NF-κB

CLEC4D C-type lectin domain family 4 member D

LAMP3 lysosomal-associated membrane protein 3

MYO1G myosin IG

CD80 CD80 molecule AP-1 PIK3AP1 phosphoinositide-3-kinase adaptor PIK3AP1 NF-κB protein 1 SH2D2A SH2 domain containing protein 2A NF-κB, AP-1 tumor necrosis factor receptor superfamily TNFRSF9 NF-κB member 9 DNAJB5 DnaJ homolog subfamily B member 5

SRXN1 sulfiredoxin 1

MTF1 metal-regulatory transcription factor 1 NF-κB, AP-1

XV Supplementary Table IV, continued

Category Gene Gene Name Regulation

Inflammation DUSP1 dual specificity phosphatase 1

GBP1 guanylate binding protein 1

IL28A interleukin 28A AP-1

IFNGR2 interferon gamma receptor 2

Metabolism PBEF1 nicotinamide phosphoribosyltransferase acyl-CoA synthetase long-chain family ACSL1 AP-1 member 1 acyl-CoA synthetase long-chain family ACSL5 member 5 AMZ1 AMZ1 archaelysin family metallopeptidase 1 AP-1

PDSS1 prenyl diphosphate synthase subunit 1

GCH1 GTP cyclohydrolase 1 AP-1 UDP-Gal:betaGlcNAc beta 1,4- B4GALT1 galactosyltransferase polypeptide 1 KMO kynurenine 3-monooxygenase UDP-Gal:betaGlcNAc beta 1,4- B4GALT5 galactosyltransferase polypeptide 5 cytochrome P450 family 7 subfamily B CYP7B1 polypeptide 1 KYNU kynureninase

AK3L1 Adenylate kinase 3 alpha-like 1

MARCH3 membrane-associated ring-CH finger 3

UPB1 ureidopropionase beta

OTUD1 OTU domain containing protein 1

ADA adenosine deaminase NF-κB, AP-1

FUT4 fucosyltransferase 4 AP-1

LSS lanosterol synthase

NBN nibrin NF-κB, AP-1

GBP2 guanylate binding protein 2

ECE1 endothelin converting enzyme 1 AP-1

LCT lactase NF-κB, AP-1

XVI Supplementary Table IV, continued

Category Gene Gene Name Regulation

UDP-N-acteylglucosamine Metabolism UAP1 AP-1 pyrophosphorylase 1 major facilitator superfamily domain MFSD2A containing protein 2A splA/ryanodine receptor domain and SOCS SPSB1 box containing 1 ER degradation enhancer, mannosidase EDEM1 alpha-like 1 SQLE squalene epoxidase

CHST2 carbohydrate sulfotransferase 2

HSD11B1 hydroxysteroid 11-beta dehydrogenase 1

INSIG1 insulin induced gene 1 IBRDC2 ring finger protein 144B (RNF144B) IBRDC3 ring finger protein 19B (RNF19B) Growth/ signal transducer and activator of STAT4 Differentiation transcription 4 CSRP2 cysteine and glycine-rich protein 2 AP-1 cysteine rich transmembrane BMP CRIM1 AP-1 regulator 1 ACVR2A activin A receptor type IIA protein tyrosine phosphatase non-receptor PTPN1 type 1 JUNB jun B proto-oncogene NF-κB, AP-1

DUSP5 dual specificity phosphatase 5

FOSL2 FOS-like antigen 2 NF-κB, AP-1

DSU MREG melanoregulin

PDGFA platelet-derived growth factor alpha AP-1

PDGFB platelet-derived growth factor beta

IL20 interleukin 20 AP-1

CSF2 colony stimulating factor 2

INHBA inhibin, beta A AP-1

BTG1 B-cell translocation gene 1 NF-κB, AP-1

BTG3 B-cell translocation gene 3 NF-κB

XVII Supplementary Table IV, continued

Category Gene Gene Name Regulation

Growth/ NUMB numb homolog Differentiation

WTAP Wilms tumor 1 associated protein 7A5 metastasis associated in colon cancer 1 (MACC1) PIM3 pim-3 oncogene

BTG2 BTG family member 2 NF-κB, AP-1

PTPRJ protein tyrosine phosphatase receptor type J v-src sarcoma (Schmidt-Ruppin A-2) viral SRC AP-1 oncogene homolog v-myc myelocytomatosis viral oncogene MYC homolog EID3 EP300 interacting inhibitor of differentiation 3 NF-κB

PBX4 pre-B-cell leukemia homeobox 4

TMEM88 transmembrane protein 88 AP-1

Chemotaxis/ RHOF ras homolog gene family member F Migration

RAP2C member of RAS oncogene family NF-κB

CCRL2 C-C motif chemokine receptor-like 2 NF-κB CDGAP (ARHGAP3 Rho GTPase activating protein 31 1) RND1 Rho family GTPase 1 NF-κB

ITGB8 integrin, beta 8

FMNL3 formin-like 3

CD58 CD58 molecule AP-1

CD44 CD44 molecule NF-κB protein tyrosine phosphatase non-receptor PTPN12 type 12 CCR7 C-C motif chemokine receptor 7 NF-κB

CCL22 C-C motif chemokine ligand 22 NF-κB, AP-1

ICAM1 intercellular adhesion molecule 1 NF-κB

PLEKHC1 fermitin family member 2 ArfGAP with RhoGAP domain, ankyrin CENTD1 repeat, and PH domain 2

XVIII Supplementary Table IV, continued

Category Gene Gene Name Regulation

Chemotaxis/ LAMB3 laminin beta 3 NF-κB Migration

BLR1 C-X-C motif chemokine receptor 5

PVR poliovirus receptor NF-κB

FEZ1 fasciculation and elongation protein zeta 1

CCL19 C-C motif chemokine ligand 19 NF-κB

potassium inwardly-rectifying channel Transport KCNJ2 AP-1 subfamily J member 2 potassium intermediate/small conductance KCNN4 calcium-activated channel subfamily N AP-1 member 4 SLC7A5 solute carrier family 7 member 5 AP-1

ATP2B1 plasma membrane Ca(2+) ATPase

SEC24A SEC24 family member A AP-1 solute carrier organic anion transporter SLCO4A1 family member 4A1 SLC39A8 solute carrier family 39 member 8

APOL3 apolipoprotein L 3 AP-1

AQP9 aquaporin 9

AMPD3 adenosine monophosphate deaminase 3

SLC43A3 solute carrier family 43 member 3 AP-1

SLC1A2 solute carrier family 1 NF-κB, AP-1

CLIC4 chloride intracellular channel 4

tumor necrosis factor alpha-induced Apoptosis TNFAIP8 protein 8 MCL1 myeloid cell leukemia sequence 1 NF-κB, AP-1

BID BH3 interacting domain death agonist NF-κB, AP-1

IL15RA interleukin 15 receptor alpha AP-1

BIRC3 baculoviral IAP repeat containing 3 NF-κB, AP-1 TNFRSF10 tumor necrosis factor receptor superfamily NF-κB, AP-1 B member 10b TP53BP2 tumor protein p53 binding proteim 2 NF-κB

Antiinflammatory IRAK3 interleukin-1 receptor-associated kinase 3 NF-κB, AP-1

XIX Supplementary Table IV, continued

Category Gene Gene Name Regulation

Antiinflammatory P2RX7 purinergic receptor P2X NF-κB

NFKBIE NF-κB inhibitor epsilon NF-κB, AP-1 tumor necrosis factor alpha-induced TNFAIP3 protein 3 ETV3 ets variant 3 NF-κB

Oncogene BCAR3 breast cancer anti-estrogen resistance 3

PIM2 pim-2 oncogene NF-κB v-rel reticuloendotheliosis viral oncogene REL AP-1 homolog MET met proto-oncogene AP-1 v-maf musculoaponeurotic fibrosarcoma MAFF AP-1 oncogene homolog F

Coagulation SERPINB9 serpin peptidase inhibitor clade B member 9

SERPINB2 serpin peptidase inhibitor member 2 NF-κB, AP-1 aryl hydrocarbon receptor nuclear ARNTL2 AP-1 translocator-like 2 PTGIR prostaglandin I2 receptor NF-κB

Cell Cycle/ PH domain and leucine rich repeat protein PHLPPL Proliferation phosphatase 2 ESPL1 extra spindle pole bodies homolog 1 NF-κB, AP-1 mRNA processing SRrp35 serine/arginine-rich splicing factor 12 PRP3 pre-mRNA processing factor 3 PRPF3 homolog

Unknown SYNPO2 synaptopodin 2 AP-1

LAD1 ladinin 1 AP-1

USP13 ubiquitin specific peptidase 13

TARP TCR gamma alternate reading frame protein

GRAMD3 GRAM domain containing 3

ALS2CR4 transmembrane protein 237

TP53INP2 tumor protein p53 inducible nuclear protein 2 AP-1

FJX1 four jointed box 1

MESDC1 mesoderm development candidate 1 AP-1

XX Supplementary Table IV, continued

Category Gene Gene Name Regulation

ankyrin repeat and BTB domain containing Unknown ABTB2 NF-κB protein 2 LON peptidase N-terminal domain and ring LONRF1 AP-1 finger 1 CGN cingulin NF-κB multiple C2 domains transmembrane MCTP1 protein 1 FAM49A family with sequence similarity 49 member A

ZNFX1 zinc finger NFX1-type containing protein 1 DOC1 filamin A interacting protein 1-like (FILIP1L) RAB6IP1 DENN/MADD domain containing 5A (DENND5A) proline-serine-threonine phosphatase PSTPIP2 NF-κB interacting protein 2 PDE4DIP phosphodiesterase 4D interacting protein AP-1

EMP2 epithelial membrane protein 2

SLC35F2 solute carrier family 35 member F2

IL4I1 interleukin 4 induced 1 NF-κB, AP-1 LIM and senescent cell antigen-like LIMS3 domains 3 leucine rich repeat and fibronectin type III LRFN5 domain containing protein 5 OGFRL1 opioid growth factor receptor-like 1

FAM57A family with sequence similarity 57 member A NF-κB

BIC MIR155 host gene family with sequence similarity 107 FAM107B member B GRAMD1A GRAM domain containing 1A NF-κB, AP-1

BTNL8 butyrophilin-like 8

MN1 meningioma 1

ARRDC3 arrestin domain containing 3

XXI