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

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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 tol- erance represented a more general phenomenon. Preincubation with a high TNF dose induces both absolute and induction toler- ance, 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 phos- phorylation 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 paral- ysis. 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
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