Tolerance in Human Monocytes: Differential Inhibition of NF-Kb/AP-1– and PP1-Associated Signaling

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 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 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 T cytokines such as TNF, chemokines, and other antimi- balance between protection against excessive immune response and 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- 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 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 extraction 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 (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 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

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, 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 (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 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

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 prominent 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 Ik Ba turnover in tolerant cells. 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 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

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 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 indicated 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 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 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 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 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

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 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 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.

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). 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