Suppression of TNF-α and IL-1 Signaling Identifies a Mechanism of Homeostatic Regulation of by IL-27

This information is current as George D. Kalliolias, Rachael A. Gordon and Lionel B. of September 28, 2021. Ivashkiv J Immunol 2010; 185:7047-7056; Prepublished online 22 October 2010; doi: 10.4049/jimmunol.1001290

http://www.jimmunol.org/content/185/11/7047 Downloaded from

References This article cites 55 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/185/11/7047.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on September 28, 2021

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Suppression of TNF-a and IL-1 Signaling Identifies a Mechanism of Homeostatic Regulation of Macrophages by IL-27

George D. Kalliolias,* Rachael A. Gordon,* and Lionel B. Ivashkiv*,†

IL-27 is a pleiotropic with both activating and inhibitory functions on innate and acquired immunity. IL-27 is expressed at sites of inflammation in cytokine-driven autoimmune/inflammatory diseases, such as rheumatoid arthritis, psoriasis, inflammatory bowel disease, and sarcoidosis. However, its role in modulating disease pathogenesis is still unknown. In this study, we found that IL-27 production is induced by TNF-a in human macrophages (Mf) and investigated the effects of IL-27 on the responses of primary human Mf to the endogenous inflammatory TNF-a and IL-1. In striking contrast to IL-27–mediated aug- mentation of TLR-induced cytokine production, we found that IL-27 suppressed Mf responses to TNF-a and IL-1b, thus Downloaded from identifying an anti-inflammatory function of IL-27. IL-27 blocked the proximal steps of TNF-a signaling by downregulating cell-surface expression of the signaling receptors p55 and p75. The mechanism of inhibition of IL-1 signaling was downregulation of the ligand-binding IL-1RI concomitant with increased expression of the receptor antagonist IL-1Ra and the decoy receptor IL- 1RII. These findings provide a mechanism for suppressive effects of IL-27 on innate immune cells and suggest that IL-27 regulates inflammation by limiting activation of Mf by inflammatory cytokines while preserving initial steps in host defense by augmenting responses to microbial products. The Journal of Immunology, 2010, 185: 7047–7056. http://www.jimmunol.org/

nterleukin-27 is a heterodimeric cytokine composed of EBI3 pathogen recognition by TLRs triggers the synthesis of IL-27. and p28 subunits that share similarity with the p40 and p35 Downstream of TLRs, MyD88-dependent activation of NF-kB I subunits of IL-12 (1). The IL-27R is a heterodimer composed and TRIF-dependent activation of IFN regulatory factor (IRF) 3 of a WSX-1 subunit, which confers ligand specificity, and the drive the induction of EBI3 and p28 subunits of IL-27 (5). Type I gp130 signaling subunit, which is also used by the IL-6 family of and II IFNs are also involved in the induction of p28 by activating cytokines (2). WSX-1 bears a STAT1-binding site, and gp130 has several members of the IRF family including IRF1, IRF3, IRF7, four STAT3-docking sites, two of which can also recruit STAT1. and IRF9 (6–9). Recently, it has been reported that type I IFNs In lymphocytes, IL-27 activates STAT1, STAT3, STAT5, and low trigger production of IL-27 by downregulating the expression of by guest on September 28, 2021 amounts of STAT4 (3). In myeloid cells, IL-27–induced phos- the intracellular isoform of in murine DCs (10). phorylation of STAT1 and STAT3 has been observed (2). Our In humans, IL-27 is expressed at sites of chronic sterile in- group has found that in human monocytes (Mo), IL-27 induces flammation, such as the pannus (inflammatory synovial tissue) of sustained activation of STAT1, resulting in potent induction of rheumatoid arthritis (RA) (11), psoriatic skin lesions (12), in- inflammatory STAT1 target genes and augmentation of TLR re- flamed intestine in Crohn’s disease, and sarcoid granulomas (13). sponses. In contrast, STAT3-mediated gene induction and sup- IL-27 is a pleiotropic cytokine, and its role in these diseases pressive functions were not readily apparent in IL-27–stimulated remains enigmatic and elusive (14–17). Initially, IL-27 was dis- primary human Mo (4). covered as a cytokine that promotes the early stages of Th1 dif- The major cellular source of IL-27 is APC: Mo, macrophages ferentiation by inducing expression of IL-12Rb2 and rendering (Mf), and dendritic cells (DCs) (1). In the setting of infections, T cells responsive to the Th1-promoting effects of IL-12 (3, 18, 19). More recently, the preponderance of evidence suggests that the dominant in vivo function of IL-27 is immunoregulatory by *Arthritis and Tissue Degeneration Program, Department of Medicine, Hospital for suppressing Th1, Th2, and Th17 cells (5). IL-27 also has both Special Surgery and †Immunology and Microbial Pathogenesis Program, Weill Cor- nell Graduate School of Medical Sciences, New York, NY 10021 activating and suppressive effects on innate immune cells (16). Received for publication April 20, 2010. Accepted for publication September 22, Our group has described STAT1-mediated proinflammatory ef- 2010. fects of IL-27 in human Mo including: 1) induction of chemo- This work was supported by the Stavros S. Niarchos International Fellowship Ex- kines, such as CXCL9 and CXCL10, that are well-known for change Program (to G.D.K.) and by a National Institutes of Health grant (to L.B.I.). recruiting inflammatory cells; and 2) augmented production of Address correspondence and reprint requests to Dr. Lionel B. Ivashkiv, Department proinflammatory cytokines in response to TLR ligands (4). In of Medicine, Hospital for Special Surgery, 535 East 70th Street, Research Building 4th Floor, New York, NY 10021. E-mail address: [email protected] contrast, our laboratory and others (20, 21) observed that IL-27 has tissue-protective capacities by inhibiting osteoclastogenesis. Abbreviations used in this paper: Control, control stained for p55; Con- trol Isotype, control macrophage stained with isotype control; Control Stained, con- Other reports suggest that IL-27 can suppress inflammatory trol macrophage stained for IL-1RI; DC, ; h, human; IL-1AcP, IL-1 cytokine production in Mf and DCs (22, 23), although these accessory protein; IL-1Ra, IL-1R antagonist; IL-27, IL-27–treated macrophage stain- ed for p55; IL-27 Isotype, IL-27–treated macrophage stained with isotype control; suppressive effects in murine cells appeared modest relative to IL-27 Stained, IL-27–treated macrophage stained for IL-1RI; IRF, IFN regulatory IL-10, the potent Mf-deactivating cytokine that strongly activates factor; Mf, macrophage; Mo, monocyte; qPCR, quantitative PCR; RA, rheumatoid STAT3 (24). Overall, evidence from animal models supports the arthritis. concept that IL-27, depending on the context, can be either pro- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 or anti-inflammatory (25). Along these lines, in adjuvant-induced www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001290 7048 IL-27 INHIBITS RESPONSES OF MACROPHAGES TO TNF-a AND IL-1 arthritis, proteoglycan-induced arthritis, and animal models of dia- with specific Abs. ECL was used for detection. Abs to ERK, p-ERK, betes mellitus, IL-27 is pathogenic (26–28), whereas in experi- p-p38, p-IkBa,IkBa, and cleaved IL-1b were purchased from Cell Sig- mental autoimmune encephalomyelitis and in collagen-induced naling Technology (Beverly, MA), and the Abs to p38 and c-Fos were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). For sandwich arthritis, IL-27 is protective and may represent a potential treat- ELISA, paired capture and detection Abs to IL-27 (R&D Systems), ment (11, 29). IL-8 (R&D Systems), IL-6 (BD Pharmingen, San Diego, CA), and In our current study, we wished to investigate the potential role IL-1Ra (R&D Systems) were used according to the instructions of the of IL-27 in the process of chronic sterile inflammation. We first manufacturer. a For flow cytometry, goat PE-conjugated Ab to human IL-1RI, goat PE- observed that TNF- , a key player in the synovial inflammation of conjugated control Ab, mouse PE-conjugated Ab to human TNFRSF1A RA, skin inflammation of psoriasis, and intestinal inflammation (p55), mouse PE-conjugated control Ab, mouse fluorescein-conjugated Ab of inflammatory bowel disease, triggers the production of large to human TNFRSF1B (p75), and mouse fluorescein-conjugated control Ab amounts of IL-27 by human Mf (synovial fluid Mf of patients (R&D Systems) were used according to the instructions of the manufac- with RA or Mf generated from peripheral blood CD14+ Mo). In turer. FcRs on cell surface were blocked by human FcR Blocking Reagent (Miltenyi Biotec). striking contrast to augmentation of inflammatory TLR responses (4), we found that IL-27 suppressed responses of human Mf to Real-time quantitative RT-PCR endogenous inflammatory factors TNF-a and IL-1b, thus identi- Total RNA was extracted using an RNeasy mini kit (Qiagen, Valencia, CA), fying a potent anti-inflammatory function of IL-27. In contrast to and 1 mg total RNA was reverse transcribed using a First Strand cDNA IL-10, which suppresses both TLR and TNF-a/IL-1 responses by Synthesis kit (Fermentas, Glen Burnie, MD). Quantitative PCR (qPCR) STAT3-mediated suppression of downstream cytokine gene tran- was performed using iQ SYBR Green Supermix and iCycler iQ thermal scription (24, 30), IL-27 worked by suppressing proximal TNF-a cycler (Bio-Rad, Hercules, CA) following the manufacturer’s protocols, Downloaded from and IL-1 signaling. IL-27 suppressed TNF-a–mediated proin- and triplicate reactions were run for each sample. The oligonucleotide primers used were: hGAPDH:59-ATCAAGAAGGTGGTGAAGCA-39 and flammatory functions by downregulating cell-surface expression 59-GTCGCTGTTGAAGTCAGAGGA-39; hIL-8:59-TCTGTTAAATCTG- of the signaling receptors TNFRSF1A (p55) and TNFRSF1B GCAACCC-39 and 59-TAAAGGAGAAACCAAGGCAC-39; hIL-1b:59- (p75). IL-27 inhibited IL-1b–induced signaling in human Mf by TTCTTCGACACATTGGATAACG-39 and 59-TGGAGAACACCACTTG- downregulating the expression of the signaling receptor IL-1RI, TTGCT-39; hIL-6:59-TAATGGGCATTCCTTCTTCT-39 and 59-TGTCC- TAACGCTCATACTTTT-39; hTNFRSF1A:59-CCCCCGGTGACTGTCC- inducing expression of the receptor antagonist IL-1Ra, and by CAACTTT-39 and 59-GGCGCTGTCCTCCCACTTCTGA-39; hTNFRSF- http://www.jimmunol.org/ upregulating the expression of the decoy receptor IL-1RII. These 1B:59-TGAGCTTGGCAGCTGAACTA-39 and 59-AGCCAGCCAGTCT- results identify a mechanism by which IL-27 suppresses Mf ac- GACATCT-39; hIL-1RI:59-AGCTGGCTGGGTGGTTCAT-39 and 59-CG- tivation by endogenous inflammatory factors and provide insights ATTCTGGCATTTTCTCATAGTC-39; hIL-1RII:59-GGGGGAAATGAT- 9 9 9 about the basis of context-dependent activating versus suppressive CACAGGAATGGT-3 and 5 -CCCATGAAGGCCAGCAATACAACA-3 ; hIL-1Ra:59-CGCTCAGGTCAGTGATGTTAA-395 and 9-GAAGATGTG- functions of IL-27 in innate immune and chronic inflammatory CCTGTCCTGTGT-39; and hIL-1AcP:59-CCTCTCGGGGCAACATCA- responses. AC-39 and 59-GACCGCCTGGGACTTTTCTTC-39.

Materials and Methods Results by guest on September 28, 2021 Cell culture Human Mf derived from synovial fluid of patients with RA and PBMCs from whole blood of healthy volunteers and mononuclear cells from peripheral blood of healthy donors produce IL-27 in from synovial fluids of 13 patients with RA (fulfilling the American College response to TNF-a of Rheumatology criteria [31]) were isolated by density gradient centri- fugation using Ficoll (Invitrogen Life Technologies, Carlsbad, CA). CD14+ Recently, it has been reported that IL-27 is expressed in the pannus cells were purified from fresh PBMCs and synovial fluid-derived of patients with RA (11), but the stimulus for IL-27 production mononuclear cells using anti-CD14 magnetic beads (Miltenyi Biotec, during synovial inflammation has not yet been identified. TLR- + Auburn, CA) as recommended by the manufacturer. Purity of CD14 cells mediated stimulation of APCs is a well-characterized inducer of was .97% as verified by FACS. Cells were cultured in RPMI 1640 me- dium (Invitrogen Life Technologies) supplemented with 10% FBS IL-27 (5–9). Although TLR ligands have been implicated in sy- (HyClone, Logan, UT) and 100 U/ml penicillin and streptomycin in the novial inflammation (32), we wished to investigate whether en- presence or absence of 10 ng/ml human M-CSF (hM-CSF) (PeproTech, dogenous inflammatory factors, such as TNF-a, which has a more Rocky Hill, NJ). The following human cytokines were used to stimulate established role in RA pathogenesis and is abundant in the cells as indicated: human (h)IL-27 (3–100 ng/ml; R&D Systems, Minne- a b inflamed synovium (33), can trigger production of IL-27. In this apolis, MN), hTNF- (10 ng/ml; PeproTech), hIL-1 (10 ng/ml; R&D f + Systems), and hIL-10 (100 ng/ml; R&D Systems). In some experiments, context, we isolated M (CD14 cells) from synovial fluid of 13 etanercept (10 mg/ml; Amgen, Thousand Oaks, CA) was used to block the patients with active RA and cultured them overnight (12 h) in the effects of TNF-a; hIgG1 (10 mg/ml; R&D Systems) was used as an isotype presence or absence of TNF-a (10 ng/ml). Interestingly, we ob- control. The following TLR ligands were used to stimulate cells as in- served that TNF-a stimulation of patients’ Mf led to a significant dicated: Pam3CysSer(Lys)4 (EMC Microcollections, Tu¨bingen, Germany), , LPS (InvivoGen, San Diego, CA), and CL097 (InvivoGen). In all of the production of IL-27 protein (n = 13; p 0.001, paired Student t experiments, the culture medium was not replenished, and cells were not test) (Fig. 1A). It is of note that the amount of the TNF-a–induced washed before cytokine or TLR ligand stimulation. The cell density used IL-27 was substantial; the mean concentration of IL-27 in culture in our cultures for immunoblotting, IL-27, IL-8, and IL-6 ELISA was 2 3 6 supernatants was 0.5 ng/ml (ranging from 0.18–1.11 ng/ml), and 10 cells/ml, whereas for FACS, IL-1R antagonist (IL-1Ra) ELISA, and a 3 6 induction of IL-27 by TNF- was observed in all 13 independent RNA extraction was 4.5 10 cells/ml. Experiments with human cells a were approved by the Hospital for Special Surgery Institutional Review experiments (Fig. 1A). When TNF- function was blocked using Board. the soluble TNFR etanercept, TNF-a–induced production of IL-27 by patients’ Mf was abrogated (n =5;p , 0.05, paired Student t Immunoblotting, ELISA, and FACS test) (Fig. 1B), supporting the conclusion that, in our experimental Whole-cell extracts were prepared by lysis of cells in buffer containing 20 system, TNF-a triggered the production of IL-27 by RA patient- mM HEPES (pH 7), 300 mM NaCl, 10 mM KCl, 1 mM MgCl2, 0.1% Triton derived synovial Mf. 3 X-100, 0.5 mM DTT, 20% glycerol, and 1 proteinase inhibitor mixture a (Roche, Basel, Switzerland). A total of 5 or 10 mg whole-cell lysates were We next wished to explore whether TNF- stimulation induces fractioned on polyacrylamide gels using SDS-PAGE, transferred to poly- IL-27 production by human Mf and Mo obtained from peripheral vinylidene fluoride membranes (Millipore, Bedford, MA), and incubated blood of healthy individuals. CD14+ cells from PBMCs of healthy The Journal of Immunology 7049 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. Human Mf produce IL-27 in response to TNF-a. A and B, CD14+ cells isolated from synovial fluids of patients with active RA were cultured overnight (12 h) in the presence or absence of hTNF-a (10 ng/ml). For B, the effect of TNF-a was blocked by etanercept (10 mg/ml); human IgG1 (10 mg/ml) was used as an isotype control. C and D, CD14+ cells isolated from PBMCs of healthy donors were differentiated for 2 d in the presence of M-CSF (10 ng/ml) and then were cultured overnight as in A and B. E, Fresh Mo (CD14+ cells isolated from PBMCs of healthy donors) were cultured overnight (12 h) in the presence or absence of TNF-a or LPS. For F, Mo were cultured as described in B. Production of IL-27 (A–E) and IL-8 (F) in response to TNF-a was measured in culture supernatant with ELISA. Paired Student t test was used for statistical analysis. donors cultured for 2 d with M-CSF to differentiate them into Mf- IL-27 suppresses TNF-a responses by downregulating the like cells were stimulated overnight with TNF-a. In all eight in- expression of the signaling receptors p55 and p75 and by dependent experiments, there was a robust (0.26–1.85 ng/ml) and inhibiting proximal TNF-a–induced signaling in human Mf significant (p = 0.001, paired Student t test) induction of IL-27 We have recently reported that IL-27 enhances responses of a protein post stimulation of cells with 10 ng/ml TNF- (Fig. 1C) human Mo/Mf to TLR ligands (4). Cells exposed to IL-27 pro- that was prevented by etanercept (Fig. 1D). Freshly isolated pe- duce significantly higher amounts of protein and mRNA of the ripheral blood Mo produced, as expected, high amounts of IL-27 proinflammatory cytokines IL-6, IL-1b, and TNF-a in response to in response to overnight stimulation with 10 ng/ml LPS (Fig. 1E). various TLR ligands including Pam3CysSer(Lys)4 (TLR2 ligand), TNF-a stimulation of Mo induced a robust production of IL-8 LPS (TLR4 ligand), and CL097 (TLR7/8 ligand) (Fig. 2A,2B) (4). (Fig. 1F), but surprisingly failed to induce IL-27 production even In our current study, we wished to explore whether IL-27 also when TNF-a was used at the high concentration of 50 ng/ml modulates responsiveness of human Mf to mediators that drive (n =5;p . 0.05, paired Student t test) (Fig. 1E). Overall, these synovial inflammation including TNF-a and IL-1b. CD14+ cells, results indicate that human Mf (derived from synovial fluid of derived from healthy donors’ PBMCs, were cultured for 2 d with RA patients or peripheral blood of healthy donors), but not Mo, M-CSF in the presence or absence of IL-27 and were then produce substantial amounts of IL-27 in response to TNF-a sti- stimulated with TNF-a. As expected, M-CSF–generated Mf mulation; the amounts of TNF-a–induced IL-27 in Mf were responded to TNF-a stimulation with a robust induction of IL-8 comparable to those induced in Mo by LPS. mRNA (Fig. 2C, black bar). Surprisingly, in cells exposed to 100 7050 IL-27 INHIBITS RESPONSES OF MACROPHAGES TO TNF-a AND IL-1 Downloaded from http://www.jimmunol.org/

FIGURE 2. IL-27 primes human Mf for enhanced TLR responses and suppresses responses of human Mf to TNF-a. A and B, CD14+ cells isolated from healthy donors’ peripheral blood were cultured with M-CSF (10 ng/ml) in the presence or absence of IL-27 (100 ng/ml) for 24 h and then were stimulated with various TLR ligands including Pam3CysSer(Lys)4 (TLR2 ligand), LPS (TLR4 ligand), and CL097 (TLR7/8 ligand). For A, ELISA was used to measure production of IL-6 protein in culture supernatants following 6 h stimulation with Pam3CysSer(Lys)4 (10 ng/ml), LPS (1 ng/ml), or CL097 (1 mg/ml). For B, qPCR was used to measure the mRNA expression of IL-6 and IL-1b mRNA following 3 h stimulation with Pam3CysSer(Lys)4 (10 ng/ml), and results are depicted as expression relative to GAPDH. For A and B, paired Student t test was used for statistical analysis. CD14+ cells isolated from healthy donor PBMCs were cultured for 2 d with M-CSF (10 ng/ml) (C) or without M-CSF (D) in the presence or absence of IL-27 (100 ng/ml) and then were stimulated by guest on September 28, 2021 with TNF-a (10 ng/ml) for 3 h. Induction of IL-8 and IL-1b mRNA was measured by qPCR, and the results of the independent experiments were pooled (n = 5 for C and n = 3 for D). The mean levels of TNF-a–induced IL-8 mRNA were 238% relative to GAPDH mRNA levels (C) and 240% of GAPDH (D); IL-1b mRNA was induced to 22% of GAPDH levels (D). For comparison of pooled data from different donors, the induction of IL-8 and IL-1b mRNA in response to TNF-a in control cells (cells cultured without IL-27 pretreatment) was set as 100. Induction of IL-8 and IL-1b mRNA in cells pretreated with IL-27 is depicted as percentage relative to induction in control cells. For statistical analysis, paired Student t test was used. ng/ml IL-27, the induction of IL-8 mRNA following TNF-a performed. Interestingly, at both time points tested (3 h and 24 h stimulation was attenuated (Fig. 2C, gray bar; n =5;p , 0.0001, pretreatment with 100 ng/ml IL-27 or 100 ng/ml IL-10), IL-27 paired Student t test). A significant suppressive effect of IL-27 displayed a suppressive effect comparable to that of IL-10 on on TNF-a–mediated induction of IL-8 and IL-1b mRNA was also the TNF-a–mediated induction of IL-8 mRNA expression in observed in the absence of M-CSF in our experimental system human Mf (Fig. 3C; n = 3; no statistically significant difference (Fig. 2D; n =3;p = 0.001 for the attenuation of IL-8 mRNA was found by paired Student t test). expression and p = 0.014 for the suppression of IL-1b mRNA The induction of TNF-a target genes, including induction of expression, paired Student t test). IL-8, is dependent upon TNF-a–induced activation of NF-kB and The above-mentioned suppressive effects of IL-27 on hu- MAPK pathways (34), and thus we investigated whether IL-27 man Mf were dose dependent. Whereas 3 ng/ml IL-27 was only inhibited TNF-a signaling. In agreement with the literature, we moderately suppressive, pre-exposure of cells to higher doses found that stimulation of human Mf with TNF-a (10 ng/ml) in- of IL-27 (10, 30, and 100 ng/ml) significantly suppressed the duced rapid phosphorylation of IkBa (Fig. 4A, upper panel, lanes mRNA expression of the TNF-a target IL-8 (Fig. 3A; n =4;p , 2–4) and rapid phosphorylation of ERK and p38 MAPKs (Fig. 4B, 0.01) in a dose-dependent manner. Interestingly, the inhibitory first and third panel, lanes 2–4). Notably, we observed strong effects of 100 ng/ml IL-27 on the TNF-a–mediated expression of inhibition of TNF-a–induced IkBa, ERK, and p38 phosphoryla- IL-8 mRNA were significant even when the cells were pre- tion by IL-27 (Fig. 4A,4B, lanes 6–8). In summary, these results exposed to IL-27 for only 1 h (Fig. 3B, first gray bar on the demonstrate that IL-27 attenuates responses of human Mf to left; n =3;p , 0.05, paired Student t test). The potency of in- TNF-a by inhibiting TNF-a–induced signaling. hibition increased with increased duration of IL-27 exposure, with We next investigated the effects of IL-27 on the expression of the an average inhibition of 46.1% after 1 h (Fig. 3B) increasing to two signaling receptors for TNF-a. In the presence of IL-27 (100 91.7% after 48 h of IL-27 pretreatment (Fig. 3B, last gray bar on ng/ml) for 48 h, we observed a significant downregulation of the the right; n =3;p , 0.01, paired Student t test). To further ad- p55 receptor on the cell surface of human Mf, measured by FACS dress the potency of the anti-inflammatory effects of IL-27 on (Fig. 5A; n = 5; average percentage of inhibition, 50; p = 0.001, human Mf at the earlier time points, a head-to-head comparison paired Student t test). Interestingly, exposure of Mf to IL-27 for 1, of IL-27 with the powerful anti-inflammatory cytokine IL-10 was 3, 6, 24, and 48 h had no effect on the expression level of p55 The Journal of Immunology 7051 Downloaded from

FIGURE 4. IL-27 blocks TNF-a–induced activation of signaling path- ways in human Mf. CD14+ cells isolated from healthy donor PBMCs were cultured for 2 d with M-CSF (10 ng/ml) in the presence or absence of IL- 27 (100 ng/ml) and then were stimulated with TNF-a (10 ng/ml) for 5, 10, and 15 min. Immunoblotting was used to measure Ser32 phosphorylation of 202 204 180 182 IkBa (A), Thr /Tyr phosphorylation of Erk1/Erk2, and Thr /Tyr http://www.jimmunol.org/ phoshorylation of p38 (B). Representative results of at least three in- dependent experiments are shown.

IL-27 abrogates responses of human Mf to IL-1b IL-1b is an important inflammatory cytokine that utilizes MyD88- dependent signaling pathways similar to TLR-induced MyD88- dependent pathways (35). We then investigated whether IL-27 enhances (similar to LPS and other TLR ligands) or suppresses FIGURE 3. IL-27 suppresses responses of human Mf to TNF-a in a (similar to TNF-a) responses of human Mf to IL-1b. In agree- by guest on September 28, 2021 dose- and time-dependent manner, and these suppressive effects of IL-27 f b + ment with the literature, human M stimulated with IL-1 pro- are comparable to the effects of IL-10. CD14 cells isolated from healthy duced mature IL-1b protein (cleaved form with molecular mass of donor PBMCs were cultured for 48 h with M-CSF (10 ng/ml) in the 17 kDa) (Fig. 6A, upper panel, lanes 3 and 4). In the presence of presence or absence of IL-27 (A–C) or IL-10 (C) and then were stimulated b with TNF-a (10 ng/ml) for 3 h. Induction of IL-8 mRNA was measured by IL-27, production of the cleaved form of IL-1 protein in response b qPCR and the results of the independent experiments were pooled. A, Cells to IL-1 stimulation was strongly attenuated (Fig. 6A, upper were pretreated for 48 h with different doses of IL-27 (3–100 ng/ml) and panel, lanes 6–8). We also used qPCR to measure expression of then were stimulated with TNF-a. B, Cells were pretreated with 100 ng/ml IL-1b target genes. IL-27 significantly suppressed the expression IL-27 for 1, 3, 6, 24, or 48 h and then were stimulated with TNF-a. The of IL-1b, IL-6, and IL-8 mRNA in response to IL-1b stimulation induction of IL-8 mRNA in response to TNF-a in control cells (cells (data not shown and Fig. 6B,6C; n =3;p , 0.05, paired Student cultured without IL-27 pretreatment) was set as 100. Induction of IL-8 t test). Interestingly, in the absence of M-CSF in our system, the mRNA in cells pretreated with IL-27 is depicted as percentage relative to IL-1b–mediated induction of IL-1b, IL-8, and IL-6 mRNA was induction in control cells (A, B). C, Cells were pretreated for 3 or 24 h with almost completely inhibited (average inhibition .97%) by IL-27 (100 ng/ml) or IL-10 (100 ng/ml) and then stimulated with TNF-a. pre-exposure of cells to 100 ng/ml IL-27 (Fig. 6D; n =3;p , Results are depicted as percentage of inhibition relative to control cells (cells stimulated with TNF-a without IL-27 or IL-10 pretreatment). For 0.001, paired Student t test). b statistical analysis, paired Student t test was used. *p . 0.05; **p , 0.05; We next investigated whether IL-27 suppresses IL-1 responses ***p , 0.01. by inhibiting IL-1b–induced signaling. After stimulating human Mf with 10 ng/ml IL-1b, we observed the expected activation of mRNA (Fig. 5B). Similarly, cell-surface expression of the second the classical NF-kB pathway as manifested by rapid degradation receptor for TNF-a, p75, was also significantly reduced by of the IkBa protein (Fig. 7A, upper panel, lanes 1–4). We also IL-27 (Fig. 5C; n = 5; average percentage of inhibition, 29.5; p = observed rapid phosphorylation of ERK and p38 (Fig. 7B, first and 0.019, paired Student t test), whereas the mRNA expression of p75 third panel, lanes 1–4), indicating activation of MAPK pathways. was not inhibited (Fig. 5D). Culture of Mf with IL-27 resulted in IL-1b–mediated degradation of IkBa and phosphorylation of shifts in isotype control staining, most likely secondary to changes ERK and p38 were prevented in the presence of IL-27 (Fig. 7A, in autofluorescence as FcRs were blocked as described in Mate- 7B, lanes 5–8), suggesting that IL-27 suppresses the effects of IL- rials and Methods, and in each experimental condition, the ap- 1b on human Mf by blocking IL-1b–induced signaling. propriate background staining was subtracted to calculate IL-27– induced changes specific receptor staining. These results suggest IL-27 downregulates the expression of the signaling receptor that IL-27–mediated downregulation of cell-surface expression IL-1RI and induces IL-1Ra and the decoy receptor IL-1RII of TNFRs may contribute to suppression of downstream signal IL-1b first binds to the IL-1RI receptor subunit on the surface transduction. of target cells, and subsequently, IL-1 accessory protein (IL-1AcP) 7052 IL-27 INHIBITS RESPONSES OF MACROPHAGES TO TNF-a AND IL-1 Downloaded from

FIGURE 5. IL-27 downregulates the cell-surface protein levels of the TNF-a signaling receptors p55 and p75 on human Mf. CD14+ cells isolated from healthy donors’ peripheral blood were cultured with M-CSF (10 ng/ml) in the presence or absence of IL-27 (100 ng/ml) for 48 h. A, Expression level of p55 http://www.jimmunol.org/ protein on the cell surface was measured by FACS. B, qPCR was used to measure the mRNA expression of p55, and results are depicted relative to GAPDH. C, Expression level of p75 protein on the cell surface was measured by FACS. Panel labels same as defined in A. D, qPCR was used to measure the mRNA expression of p75, and results are depicted relative to GAPDH. For A and C, representative results of five independent experiments are shown. For B and D, pooled results of three independent experiments are shown. Control, control Mf stained for p55; IL-27, IL-27–treated Mf stained for p55; Control Isotype, control Mf stained with isotype control; IL-27 Isotype, IL-27–treated Mf stained with isotype control. is recruited, thus forming a trimolecular signaling complex that binds and sequesters IL-1b but does not elicit any signal and by activates NF-kB and MAPK signaling pathways (35). Because IL- IL-1Ra that competes with IL-1b for binding with IL-1RI (35). In

1b has potent proinflammatory and tissue-destructive effects, its this context, we wished to investigate whether the inhibitory effect by guest on September 28, 2021 function is tightly regulated by the decoy receptor IL-1RII that of IL-27 on IL-1b signaling and function was due to an effect on

FIGURE 6. IL-27 suppresses responses of human Mf to IL-1b. CD14+ cells isolated from healthy donor PBMCs were cultured for 1 d with M-CSF (10 ng/ml) (A–C) or without M-CSF (D) in the presence or absence of IL-27 (100 ng/ml) and then were stimulated with IL-1b (10 ng/ml) for 1, 3, or 6 h. For A, immunoblotting was used to measure production of the cleaved mature form of IL-1b protein, and representative results of three independent experiments are shown. For B–D, qPCR was used to measure induction of IL-1b, IL-6, and IL-8 mRNA, and the results of three independent experiments were pooled. In B, the mean levels of IL-1b–induced IL-1b mRNA relative to GAPDH mRNA levels were 670%, 144%, and 176% at the 1 h, 3 h, and 6 h time points, respectively. In C, the mean levels of IL-1b–induced IL-6 mRNA were 1.0% of GAPDH. In D, the mean levels of IL-1b–induced mRNA relative to GAPDH were 510% (IL-1b), 470% (IL-8), and 4.5% (IL-6). For comparison of pooled data from different donors, induction of IL-1b, IL-6, and IL-8 mRNA in response to IL-1b in control cells (cells cultured without IL-27 pretreatment) was set as 100. Induction of IL-1b, IL-6, and IL-8 mRNA in cells pretreated with IL-27 was depicted as percentage relative to induction in control cells. For statistical analysis, paired Student t test was used. The Journal of Immunology 7053

IL-27 significantly suppressed the mRNA expression of the sig- naling receptor IL-1RI (Fig. 8A; n =9;p , 0.01, paired Student t test). This suppressive effect on IL-1RI mRNA expression was readily observed within 3 h following stimulation of cells with IL-27 and was sustained for at least 48 h (Fig. 8B). A similarly significant suppressive effect of IL-27 on the IL-1RI mRNA ex- pression was observed in our system in the absence of M-CSF (average inhibition of IL-1RI mRNA, 77.5%; n =5;p , 0.001, paired Student t test, data not shown). In agreement with our observations at the mRNA level, the expression of IL-1RI protein on the cell surface was also significantly reduced by IL-27 in all four donors tested (Fig. 8C, average percentage of inhibition, 69.2). IL-1RI expression has recently been shown to depend on basal expression of the transcription factor c-Fos (36). IL-27 downregulated the expression of c-Fos protein in human Mf (Fig. 8D), suggesting that IL-27 suppresses IL-1RI expression by tar- geting its transcriptional regulator c-Fos. In contrast to the suppression of IL-1RI expression, IL-27

significantly increased the production of the receptor antag- Downloaded from onist IL-1Ra by human Mf (Fig. 9A). Interestingly, the induction b k FIGURE 7. IL-27 inhibits IL-1 –induced activation of NF- B and of IL-1Ra protein by IL-27 was more robust in the absence f + MAPK signaling pathways in human M . CD14 cells isolated from of M-CSF in our system. In supernatants collected from 24-h healthy donors’ PBMCs were cultured for 1 d with M-CSF (10 ng/ml) in cultures, the average amount of the IL-27–induced IL-1Ra pro- the presence or absence of IL-27 (100 ng/ml) and then were stimulated for 5, 10, and 15 min with IL-1b (10 ng/ml). Immunoblotting was used to tein measured by ELISA was 4.87 ng/ml and 1.64 ng/ml, in the measure total IkBa (A), Thr202/Tyr204 phosphorylation of Erk1/Erk2, and absence and presence of M-CSF, respectively (Fig. 9A). The http://www.jimmunol.org/ Thr180/Tyr182 phoshorylation of p38 (B). Representative results of at least decoy receptor IL-1RII was also induced in human Mf by IL-27 three independent experiments are shown. in the presence of M-CSF (Fig. 9B; n =7;p , 0.01, paired Student t test). Finally, the expression of IL-1AcP was not the above-mentioned molecules. Interestingly, we observed that in modulated by IL-27 (Fig. 9C). Overall, IL-27 coordinately sup- human CD14+ cells purified from PBMCs of healthy individuals, pressed expression of an activating IL-1R component while by guest on September 28, 2021

FIGURE 8. IL-27 downregulates IL-1RI and suppresses the expression of c-Fos. CD14+ cells isolated from healthy donors’ peripheral blood were cultured with M-CSF (10 ng/ml) in the presence or absence of IL-27 (100 ng/ml) for 24h (A, C, D) or for 3, 24, and 48 h (B). A and B, qPCR was used to measure the mRNA expression of IL-1RI, and results are depicted relative to GAPDH. Paired Student t test was used for statistical analysis. For B, representative results of three independent experiments are shown. C, Expression level of IL-1RI protein on the cell surface was measured by FACS. Left panel shows representative results of four independent experiments, and right panel depicts the percentage of inhibition of IL-1RI expression by IL-27 in four donors that were tested. D, Immunoblotting was used to measure the expression of the transcription factor c-Fos (representative results of at least five independent experiments are shown). Control Stained, control Mf stained for IL-1RI; Control Isotype, control Mf stained with isotype control; IL-27 Stained, IL-27–treated Mf stained for IL-1RI; IL-27 Isotype, IL-27–treated Mf stained with isotype control. 7054 IL-27 INHIBITS RESPONSES OF MACROPHAGES TO TNF-a AND IL-1

immune and inflammatory responses by attenuating Mf activation in response to endogenous inflammatory cytokines. Our findings that IL-27 inhibits TNF and IL-1 responses stand in striking contrast to IL-27–mediated augmentation of TLR-induced inflammatory cytokine production in human Mf (Fig. 2A,2B) (4). This dual regulation suggests that IL-27 has a different function in the context of an infection due to TLR stimulation than its func- tion during chronic sterile inflammation that is driven primarily by endogenous cytokines, such as TNF-a and IL-1. In the first case, IL-27 augments innate immunity to eliminate pathogens by increasing responses of Mf to pathogen-associated molecular patterns (TLR ligands) (4). In the latter case, IL-27 functions as a homeostatic cytokine by suppressing responses of Mf to TNF-a and IL-1b. A similar pleiotropic and context-dependent function of IL-27 has been described in adaptive immunity (5, 14–17). In the early phase of the adaptive immune response, IL-27 promotes Th1 polarization of naive lymphocytes to promote a robust re- sponse to weak infectious stimuli (37). When an immune response

is established, the function of IL-27 transitions to immunosup- Downloaded from pressive aiming to restrict inflammation by inhibiting polarization of T cells (5). IL-27 is expressed at sites of TNF-a–driven inflammation, such as the RA synovium (11), the psoriatic skin (12), and the intestine of patients with Crohn’s disease (13). Our finding that TNF-a

induces production of IL-27 by synovial fluid Mf supports the http://www.jimmunol.org/ hypothesis that TNF-a stimulation of Mf in the joint microen- vironment may contribute to the observed expression IL-27 in the pannus of RA patients. It is not yet clear whether IL-27 expressed in TNF-a–driven diseases is predominantly pathogenic or repre- sents a homeostatic attempt to control inflammation and restrain tissue destruction. In a recent report, we observed that IL-27 is tissue protective by inhibiting osteoclastogenesis (20). Our cur- rent study further supports a homeostatic function of IL-27 in cytokine-driven inflammation by suppressing Mf activation. An- by guest on September 28, 2021 other group of investigators that described the expression of IL-27 FIGURE 9. IL-27 induces the expression of IL-1Ra and upregulates in psoriatic skin lesions also observed anti-inflammatory effects + the decoy receptor IL-1RII. CD14 cells isolated from healthy donors’ on human keratinocytes (12). In addition, several studies indicate peripheral blood were cultured with M-CSF (10 ng/ml) (A–C) or without that IL-27 suppresses Th1 and Th17 cells (10, 29, 38–41), two M-CSF (A, C) in the presence or absence of IL-27 (100 ng/ml) for 24 h. A, subsets of T cells that have been implicated in the pathogenesis of Production of IL-1Ra protein was measured in culture supernatants with RA (42), psoriasis, and Crohn’s disease (43, 44). These results are ELISA. qPCR was used to measure the mRNA expression of IL-1RII (B) and IL-1AcP (C). Results are depicted relative to GAPDH, and paired consistent with reports of suppressive properties of IL-27 in sev- Student t test was used for statistical analysis. eral mouse models of autoimmune and inflammatory diseases (10, 11, 22, 29, 38, 40, 41, 45–51). However, the contribution of IL-27–mediated suppression of Mf function, as described in inducing inhibitors of the IL-1R and thereby suppressed human this study, to suppressing inflammation in vivo is difficult to assess Mf responses to IL-1b. in animal models, as murine Mf are minimally responsive to IL-27 (4). The alternative approach of ex vivo analysis of human Discussion arthritic Mf suggests that the in vivo functions of IL-27 are IL-27 is a highly pleiotropic cytokine that can promote or suppress regulated by the synovial microenvironment (20), and definitive immune and inflammatory responses depending on context (15–17, resolution of this question would require a clinical trial of IL-27 in 25). One of the challenges in understanding IL-27 function has human diseases with concomitant measurement of Mf function been to elucidate molecular mechanisms that underlie its acti- and cytokine production. vating versus suppressive functions. In this study, we have iden- The anti-inflammatory effects of IL-27 on human Mf are more tified a new suppressive function of IL-27, namely, inhibition of circumscribed than the effects of the prototypic Mf-deactivating Mf responses to the key endogenous inflammatory cytokines cytokine IL-10. IL-10 potently blocks inflammatory cytokine TNF-a and IL-1b. IL-27 suppressed Mf responses to TNF-a and production in response to multiple Mf-activating stimuli, inclu- IL-1b by blocking proximal steps in signaling by TNFRs and ding TNF, IL-1, and multiple TLRs (24). The broad suppressive IL-1Rs. IL-27 downregulated the protein expression of the sig- activity of IL-10 is explained by its targeting of inflammatory naling receptors p55 and p75 on the surface of human Mf. The cytokine gene transcription; inhibition of transcription appears to mechanism of inhibition of IL-1 signaling was downregulation of occur via STAT3-mediated induction of as yet unknown tran- the ligand-binding IL-1RI concomitant with increased expression scriptional repressors (30). In contrast, IL-27 exhibits receptor- of the IL-1Ra protein and the decoy receptor IL-1RII. These specific inhibitory effects that are explained by selective target- findings provide a mechanism for suppressive effects of IL-27 on ing of IL-1 and TNFRs and proximal signaling components, innate immune cells and suggest that IL-27 regulates innate whereas TLR-induced cytokine responses are actually augmented The Journal of Immunology 7055

(4). Consistent with this, we found that although STAT3 is acti- 11. Niedbala, W., B. Cai, X. Wei, A. Patakas, B. P. Leung, I. B. McInnes, and vated by IL-27, IL-27 is a weak inducer of STAT3 target genes F. Y. Liew. 2008. 27 attenuates collagen-induced arthritis. Ann. Rheum. Dis. 67: 1474–1479. relative to IL-10 (4). This difference in downstream gene ex- 12. Shibata, S., Y. Tada, N. Kanda, K. Nashiro, M. Kamata, M. Karakawa, pression and mechanism of inhibition between IL-27 and IL-10 T. Miyagaki, H. Kai, H. Saeki, Y. Shirakata, et al. 2010. Possible roles of IL-27 in the pathogenesis of psoriasis. J. Invest. Dermatol. 130: 1034–1039. suggests that, in contrast to IL-10, IL-27–mediated suppressive 13. Larousserie, F., S. Pflanz, A. Coulomb-L’Hermine´, N. Brousse, R. Kastelein, and effects may not be predominantly STAT3 dependent. Indeed, O. Devergne. 2004. Expression of IL-27 in human Th1-associated granuloma- IL-27 inhibits IL-1 responses at least in part by downregulating tous diseases. J. Pathol. 202: 164–171. 14. Kastelein, R. A., C. A. Hunter, and D. J. Cua. 2007. Discovery and biology of Fos expression and thus expression of IL-1RI. As Fos is also IL-23 and IL-27: related but functionally distinct regulators of inflammation. downregulated by IFNs acting via STAT1 (52, 53), it is possible Annu. Rev. Immunol. 25: 221–242. that STAT1 contributes to the homeostatic functions of IL-27 that 15. Yoshida, H., M. Nakaya, and Y. Miyazaki. 2009. Interleukin 27: a double-edged sword for offense and defense. J. Leukoc. Biol. 86: 1295–1303. we have described. 16. Yoshida, H., Y. Miyazaki, and M. Yoshiyuki. 2008. Regulation of immune TLR ligands, TNF-a, and IL-1b are potent activators of Mf, responses by interleukin-27. [Published erratum appears in 2009 Immunol. Rev. inducing production of proinflammatory mediators that are crucial 227: 283.] Immunol. Rev. 226: 234–247. f 17. Batten, M., and N. Ghilardi. 2007. The biology and therapeutic potential of for an effective immune response to pathogens. Activators of M interleukin 27. J. Mol. Med. 85: 661–672. also trigger homeostatic mechanisms that restrain inflammation 18. Chen, Q., N. Ghilardi, H. Wang, T. Baker, M. H. Xie, A. Gurney, I. S. Grewal, and limit associated tissue damage. One key homeostatic mech- and F. J. de Sauvage. 2000. Development of Th1-type immune responses requires the TCCR. Nature 407: 916–920. anism that TLR ligands activate is the production of IL-10 (54), 19. Takeda, A., S. Hamano, A. Yamanaka, T. Hanada, T. Ishibashi, T. W. Mak, a potent anti-inflammatory cytokine that limits inflammatory cy- A. Yoshimura, and H. Yoshida. 2003. Cutting edge: role of IL-27/WSX-1 sig- tokine production and controls the duration and intensity of im- naling for induction of T-bet through activation of STAT1 during initial Downloaded from Th1 commitment. J. Immunol. 170: 4886–4890. mune responses (24). Interestingly, TNF-a does not induce IL-10 20. Kalliolias, G. D., B. Zhao, A. Triantafyllopoulou, K. H. Park-Min, and production in Mf (55), but according to our findings, induces the L. B. Ivashkiv. 2010. Interleukin-27 inhibits human osteoclastogenesis by ab- synthesis of IL-27 that suppresses responses of Mf to TNF-a and rogating RANKL-mediated induction of nuclear factor of activated T cells c1 and suppressing proximal RANK signaling. Arthritis Rheum. 62: 402–413. IL-1b. In this context, our observations suggest that TNF-a– 21. Furukawa, M., H. Takaishi, J. Takito, M. Yoda, S. Sakai, T. Hikata, A. Hakozaki, stimulated production of IL-27 may represent a selective homeo- S. Uchikawa, M. Matsumoto, K. Chiba, et al. 2009. IL-27 abrogates recep- tor activator of NF-kappa B ligand-mediated osteoclastogenesis of human static mechanism that limits activation of Mf by inflammatory http://www.jimmunol.org/ granulocyte-macrophage colony-forming unit cells through STAT1-dependent cytokines, but preserves responses to microbial products that are inhibition of c-Fos. J. Immunol. 183: 2397–2406. sensed by TLRs. 22. Ho¨lscher, C., A. Ho¨lscher, D. Ru¨ckerl, T. Yoshimoto, H. Yoshida, T. Mak, C. Saris, and S. Ehlers. 2005. The IL-27 receptor chain WSX-1 differentially regulates antibacterial immunity and survival during experimental tuberculosis. Acknowledgments J. Immunol. 174: 3534–3544. We thank the physicians of the Hospital for Special Surgery (Drs. Sergio 23. Wang, S., Y. Miyazaki, Y. Shinozaki, and H. Yoshida. 2007. Augmentation of antigen-presenting and Th1-promoting functions of dendritic cells by WSX-1 Schwartzman, Susan Goodman, Joseph Markenson, Theodore Fields, (IL-27R) deficiency. J. Immunol. 179: 6421–6428. Ronald MacKenzie, and Dalit Ashany) for providing synovial fluids from 24. Moore, K. W., R. de Waal Malefyt, R. L. Coffman, and A. O’Garra. 2001. patients with RA. We also thank Drs. Baohong Zhao and Kyung-Hyun Park- Interleukin-10 and the interleukin-10 receptor. Annu. Rev. Immunol. 19: 683–

Min for critical review of the manuscript. 765. by guest on September 28, 2021 25. Villarino, A. V., E. Huang, and C. A. Hunter. 2004. Understanding the pro- and anti-inflammatory properties of IL-27. J. Immunol. 173: 715–720. Disclosures 26. Goldberg, R., G. Wildbaum, Y. Zohar, G. Maor, and N. Karin. 2004. Suppression of ongoing adjuvant-induced arthritis by neutralizing the function of the p28 The authors have no financial conflicts of interest. subunit of IL-27. J. Immunol. 173: 1171–1178. 27. Cao, Y., P. D. Doodes, T. T. Glant, and A. Finnegan. 2008. IL-27 induces a Th1 immune response and susceptibility to experimental arthritis. J. Immunol. 180: References 922–930. 1. Pflanz, S., J. C. Timans, J. Cheung, R. Rosales, H. Kanzler, J. Gilbert, L. Hibbert, 28. Wang, R., G. Han, J. Wang, G. Chen, R. Xu, L. Wang, X. Li, B. Shen, and Y. Li. T. Churakova, M. Travis, E. Vaisberg, et al. 2002. IL-27, a heterodimeric cy- 2008. The pathogenic role of interleukin-27 in autoimmune diabetes. Cell. Mol. tokine composed of EBI3 and p28 protein, induces proliferation of naive CD4(+) Life Sci. 65: 3851–3860. T cells. Immunity 16: 779–790. 29. Batten, M., J. Li, S. Yi, N. M. Kljavin, D. M. Danilenko, S. Lucas, J. Lee, F. J. de 2. Pflanz, S., L. Hibbert, J. Mattson, R. Rosales, E. Vaisberg, J. F. Bazan, Sauvage, and N. Ghilardi. 2006. Interleukin 27 limits autoimmune encephalo- J. H. Phillips, T. K. McClanahan, R. de Waal Malefyt, and R. A. Kastelein. 2004. myelitis by suppressing the development of -producing T cells. Nat. Immunol. 7: 929–936. WSX-1 and constitute a signal-transducing receptor for IL-27. 30. El Kasmi, K. C., A. M. Smith, L. Williams, G. Neale, A. D. Panopoulos, J. Immunol. 172: 2225–2231. A. Panopolous, S. S. Watowich, H. Ha¨cker, B. M. Foxwell, and P. J. Murray. 3. Lucas, S., N. Ghilardi, J. Li, and F. J. de Sauvage. 2003. IL-27 regulates IL-12 2007. Cutting edge: A transcriptional repressor and corepressor induced by the responsiveness of naive CD4+ T cells through Stat1-dependent and -independent STAT3-regulated anti-inflammatory signaling pathway. [Published erratum mechanisms. Proc. Natl. Acad. Sci. USA 100: 15047–15052. appears in 2008. J. Immunol. 180: 3612.] J. Immunol. 179: 7215–7219. 4. Kalliolias, G. D., and L. B. Ivashkiv. 2008. IL-27 activates human monocytes via 31. Arnett, F. C., S. M. Edworthy, D. A. Bloch, D. J. McShane, J. F. Fries, STAT1 and suppresses IL-10 production but the inflammatory functions of IL-27 N. S. Cooper, L. A. Healey, S. R. Kaplan, M. H. Liang, H. S. Luthra, et al. 1988. are abrogated by TLRs and p38. J. Immunol. 180: 6325–6333. The American Rheumatism Association 1987 revised criteria for the classifi- 5. Stumhofer, J. S., and C. A. Hunter. 2008. Advances in understanding the anti- cation of rheumatoid arthritis. Arthritis Rheum. 31: 315–324. inflammatory properties of IL-27. Immunol. Lett. 117: 123–130. 32. O’Neill, L. A. 2008. Primer: Toll-like receptor signaling pathways—what do 6. Liu, J., X. Guan, and X. Ma. 2007. Regulation of IL-27 p28 gene expression in rheumatologists need to know? Nat. Clin. Pract. Rheumatol. 4: 319–327. macrophages through MyD88- and -gamma-mediated pathways. J. 33. Feldmann, M., and S. R. Maini. 2008. Role of cytokines in rheumatoid arthritis: Exp. Med. 204: 141–152. an education in pathophysiology and therapeutics. Immunol. Rev. 223: 7–19. 7. Pirhonen, J., J. Sire´n, I. Julkunen, and S. Matikainen. 2007. IFN-alpha regulates 34. Aggarwal, B. B. 2003. Signalling pathways of the TNF superfamily: a double- Toll-like receptor-mediated IL-27 gene expression in human macrophages. J. edged sword. Nat. Rev. Immunol. 3: 745–756. Leukoc. Biol. 82: 1185–1192. 35. Dinarello, C. A. 2009. Immunological and inflammatory functions of the 8. Molle, C., M. Nguyen, V. Flamand, J. Renneson, F. Trottein, D. De Wit, interleukin-1 family. Annu. Rev. Immunol. 27: 519–550. F. Willems, M. Goldman, and S. Goriely. 2007. IL-27 synthesis induced by TLR 36. Kim, J. H., H. M. Jin, K. Kim, I. Song, B. U. Youn, K. Matsuo, and N. Kim. ligation critically depends on IFN regulatory factor 3. J. Immunol. 178: 7607– 2009. The mechanism of osteoclast differentiation induced by IL-1. J. Immunol. 7615. 183: 1862–1870. 9. Molle, C., M. Goldman, and S. Goriely. 2010. Critical role of the IFN-stimulated 37. Hunter, C. A., A. Villarino, D. Artis, and P. Scott. 2004. The role of IL-27 in the gene factor 3 complex in TLR-mediated IL-27p28 gene expression revealing development of T-cell responses during parasitic infections. Immunol. Rev. 202: a two-step activation process. J. Immunol. 184: 1784–1792. 106–114. 10. Shinohara, M. L., J. H. Kim, V. A. Garcia, and H. Cantor. 2008. Engagement of 38. Villarino, A., L. Hibbert, L. Lieberman, E. Wilson, T. Mak, H. Yoshida, the type I interferon receptor on dendritic cells inhibits T helper 17 cell de- R. A. Kastelein, C. Saris, and C. A. Hunter. 2003. The IL-27R (WSX-1) is re- velopment: role of intracellular osteopontin. Immunity 29: 68–78. quired to suppress hyperactivity during infection. Immunity 19: 645–655. 7056 IL-27 INHIBITS RESPONSES OF MACROPHAGES TO TNF-a AND IL-1

39. Amadi-Obi, A., C. R. Yu, X. Liu, R. M. Mahdi, G. L. Clarke, R. B. Nussenblatt, receptor) gene-deficient mice display enhanced resistance to leishmania donovani I. Gery, Y. S. Lee, and C. E. Egwuagu. 2007. TH17 cells contribute to uveitis and infection but develop severe liver immunopathology. Am.J.Pathol.168: 158–169. scleritis and are expanded by IL-2 and inhibited by IL-27/STAT1. Nat. Med. 13: 48. Fitzgerald, D. C., B. Ciric, T. Touil, H. Harle, J. Grammatikopolou, J. Das 711–718. Sarma, B. Gran, G. X. Zhang, and A. Rostami. 2007. Suppressive effect of IL-27 40. Stumhofer, J. S., A. Laurence, E. H. Wilson, E. Huang, C. M. Tato, on encephalitogenic Th17 cells and the effector phase of experimental autoim- L. M. Johnson, A. V. Villarino, Q. Huang, A. Yoshimura, D. Sehy, et al. 2006. mune encephalomyelitis. J. Immunol. 179: 3268–3275. Interleukin 27 negatively regulates the development of interleukin 17-producing 49. Fitzgerald, D. C., G. X. Zhang, M. El-Behi, Z. Fonseca-Kelly, H. Li, S. Yu, T helper cells during chronic inflammation of the central nervous system. Nat. C. J. Saris, B. Gran, B. Ciric, and A. Rostami. 2007. Suppression of autoimmune Immunol. 7: 937–945. inflammation of the central nervous system by secreted by in- 41. Guo, B., E. Y. Chang, and G. Cheng. 2008. The type I IFN induction pathway terleukin 27-stimulated T cells. Nat. Immunol. 8: 1372–1379. constrains Th17-mediated autoimmune inflammation in mice. J. Clin. Invest. 50. Awasthi, A., Y. Carrier, J. P. Peron, E. Bettelli, M. Kamanaka, R. A. Flavell, V. K. Kuchroo, M. Oukka, and H. L. Weiner. 2007. A dominant function for 118: 1680–1690. interleukin 27 in generating interleukin 10-producing anti-inflammatory T cells. 42. Lubberts, E. 2010. Th17 cytokines and arthritis. Semin. Immunopathol. 32: 43– Nat. Immunol. 8: 1380–1389. 53. 51. Stumhofer, J. S., J. S. Silver, A. Laurence, P. M. Porrett, T. H. Harris, 43. Brand, S. 2009. Crohn’s disease: Th1, Th17 or both? The change of a paradigm: L. A. Turka, M. Ernst, C. J. Saris, J. J. O’Shea, and C. A. Hunter. 2007. Inter- new immunological and genetic insights implicate Th17 cells in the pathogenesis leukins 27 and 6 induce STAT3-mediated T cell production of interleukin 10. of Crohn’s disease. Gut 58: 1152–1167. Nat. Immunol. 8: 1363–1371. 44. Tesmer, L. A., S. K. Lundy, S. Sarkar, and D. A. Fox. 2008. Th17 cells in human 52. Hu, X., J. Chen, L. Wang, and L. B. Ivashkiv. 2007. Crosstalk among Jak-STAT, disease. Immunol. Rev. 223: 87–113. Toll-like receptor, and ITAM-dependent pathways in macrophage activation. J. 45. Artis, D., A. Villarino, M. Silverman, W. He, E. M. Thornton, S. Mu, S. Summer, Leukoc. Biol. 82: 237–243. T. M. Covey, E. Huang, H. Yoshida, et al. 2004. The IL-27 receptor (WSX-1) is 53. Takayanagi, H., S. Kim, K. Matsuo, H. Suzuki, T. Suzuki, K. Sato, T. Yokochi, an inhibitor of innate and adaptive elements of type 2 immunity. J. Immunol. H. Oda, K. Nakamura, N. Ida, et al. 2002. RANKL maintains bone homeostasis 173: 5626–5634. through c-Fos-dependent induction of interferon-beta. Nature 416: 744–749. 46. Miyazaki, Y., H. Inoue, M. Matsumura, K. Matsumoto, T. Nakano, M. Tsuda, 54. Berg, D. J., R. Ku¨hn, K. Rajewsky, W. Mu¨ller, S. Menon, N. Davidson, S. Hamano, A. Yoshimura, and H. Yoshida. 2005. Exacerbation of experimental G. Gru¨nig, and D. Rennick. 1995. Interleukin-10 is a central regulator of the Downloaded from allergic asthma by augmented Th2 responses in WSX-1-deficient mice. J. response to LPS in murine models of endotoxic shock and the Shwartzman re- Immunol. 175: 2401–2407. action but not endotoxin tolerance. J. Clin. Invest. 96: 2339–2347. 47. Rosas, L. E., A. A. Satoskar, K. M. Roth, T. L. Keiser, J. Barbi, C. Hunter, F. J. de 55. Saraiva, M., and A. O’Garra. 2010. The regulation of IL-10 production Sauvage, and A. R. Satoskar. 2006. Interleukin-27R (WSX-1/T-cell cytokine by immune cells. Nat. Rev. Immunol. 10: 170–181. http://www.jimmunol.org/ by guest on September 28, 2021