IL-27, a , and IFN-λ1, a Type III IFN, Are Coordinated To Regulate Virus Replication through Type I IFN

This information is current as Yanhua Cao, Rui Zhang, Wei Zhang, Chengliang Zhu, Yi of September 23, 2021. Yu, Yu Song, Qing Wang, Lan Bai, Yingle Liu, Kailang Wu and Jianguo Wu J Immunol 2014; 192:691-703; Prepublished online 11 December 2013;

doi: 10.4049/jimmunol.1300252 Downloaded from http://www.jimmunol.org/content/192/2/691

Supplementary http://www.jimmunol.org/content/suppl/2013/12/11/jimmunol.130025 Material 2.DC1 http://www.jimmunol.org/ References This article cites 56 articles, 26 of which you can access for free at: http://www.jimmunol.org/content/192/2/691.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IL-27, a Cytokine, and IFN-l1, a Type III IFN, Are Coordinated To Regulate Virus Replication through Type I IFN

Yanhua Cao,* Rui Zhang,* Wei Zhang,* Chengliang Zhu,* Yi Yu,* Yu Song,* Qing Wang,* Lan Bai,* Yingle Liu,*,† Kailang Wu,*,† and Jianguo Wu*,†,‡

IL-27, a member of the IL-12 family, plays a critical role in the control of innate and adaptive immune responses. IFN-l1, a member of the type III IFN family, shows antiviral abilities. In this study, we investigated the effects of IL-27 and IFN-l1 on the replication of hepatitis B virus (HBV), a major pathogen associated with a high risk for cirrhosis, liver failure, and hepatocellular carcinoma. We revealed that HBV infection activates IL-27 expression and IFN-l1 production and demonstrated that viral-activated IL-27

and IFN-l1 are coordinated to inhibit HBV replication. Initially, HBV infection upregulates IL-27 expression, which, in turn, Downloaded from stimulates IFN-l1 production through regulating ERK1/2 signaling and by enhancing NF-kB nuclear translocation to bind to the IFN-l1 promoter. Moreover, IL-27–activated IFN-l1 upregulates IFN-l1 receptor (IL-28R1 and IL-10Rb) activity, resulting in the activation of the STAT1/2 pathway, which, in turn, induces the expression of IFN-stimulated , including IFN-inducible dsRNA-activated kinase, oligoadenylate synthetase 1, and IFN-induced GTP-binding protein 1 and, finally, inhibits HBV protein expression and viral capsid–associated DNA replication. More interestingly, we also revealed that type I IFN (IFN-a)is also involved in the downregulation of HBV replication mediated by IL-27. Thus, we identified a previously unknown mechanism http://www.jimmunol.org/ by which IL-27 and IFN-l1 are coordinated to regulate virus replication through type I IFN. The Journal of Immunology, 2014, 192: 691–703.

ytokines play a critical role in controlling the innate and IL-27 is produced mainly by activated macrophages and den- adaptive immune responses. IL-27, a member of the IL-12 dritic cells (DCs) and is a cytokine that regulates Th function dur- C family, is a heterodimeric anti-inflammatory cytokine ing autoimmune and pathogen-induced immune responses (3, 4). In composed of EBV-induced protein 3 (EBI3) and IL-27p28 protein early studies, IL-27 was initially described as a proinflammatory (1). IL-27p28 is a four-helical cytokine requiring association with cytokine that promoted Th1 responses (1, 5). However, subsequent by guest on September 23, 2021 EBI3 to secrete efficiently and activate functionally. The signal- studies in multiple models of infectious and autoimmune diseases transduction receptor complex for IL-27 is composed of the cy- confirmed anti-inflammatory roles for IL-27 in Th1, Th2, and tokine receptor IL-27Ra (WSX-1 or alternatively cytokine Th17 responses (6). Recent work showed that IL-27 can induce receptor [TCCR]) and the gp130 protein (2). T cells to produce the anti-inflammatory cytokine IL-10 (7–9). Overexpressing IL-27 decreases regulatory T cell frequencies, induces spontaneous inflammation, and activates proliferation of + *State Key Laboratory of Virology, College of Life Sciences, Wuhan University, naive human B cells and CD4 T cells (10, 11). IL-27 has ther- Wuhan 430072, People’s Republic of China; †Wuhan Institute of Biotechnology, apeutic potential through various mechanisms: inhibiting repli- Wuhan East Lake High Technology Development Zone, Wuhan 430075, People’s cation of HIV (12) and hepatitis C virus (HCV) (13), enhancing Republic of China; and ‡Chinese-French Liver Disease Research Institute, Zhongnan Hospital, Wuhan University, Wuhan 430072, People’s Republic of China antitumor immune responses (14), directing antiproliferative effects Received for publication January 25, 2013. Accepted for publication November 7, on tumors (15–17), and reducing the inflammation associated with 2013. experimental autoimmune arthritis (18, 19). We recently demon- This work was supported by research grants from the Major State Basic Research strated that during influenza A virus (IAV) infection, IL-27 is Development Program (973 Program) (2012CB518900), the National Natural Sci- strongly induced through cyclooxygenase-2 (COX-2)– and protein ence Foundation of China (31230005, 812111146, and 81171525), the National Mega Project on Major Infectious Disease Prevention (2012ZX10002006-003 and kinase A (PKA)-signaling pathways (20). 2012ZX10004-207), and the National Mega Project on Major Drug Development Upon detecting viral components, host cells activate antiviral (2011ZX09401-302). responsestopreventviralinfectionbyactivatingTLRsandIFN Address correspondence and reprint requests to Prof. Jianguo Wu, State Key Labo- cascades. Type I IFNs (IFN-a/b) play an essential role in both the ratory of Virology and College of Life Sciences, Wuhan University, Wuhan 430072, P.R. China. E-mail address: [email protected] innate immune response and the induction of adaptive immunity The online version of this article contains supplemental material. against viral infections. Viral infections trigger the production of IFN-a/b (21), which leads to the activation of several hundred IFN- Abbreviations used in this article: CHB, chronic hepatitis B virus; COX-2, cyclo- oxygenase-2; DC, dendritic cell; EBI3, EBV-induced protein 3; HBV, hepatitis B stimulated genes (ISGs). These genes encode a variety of antiviral virus; HCV, hepatitis C virus; IAV, influenza A virus; ISG, IFN-stimulated ; and , leading to host protection from further viral ISRE, IFN stimulation response element; mERK, mutant ERK; Mx1, IFN-induced GTP-binding protein; NC, negative control; OAS1, oligoadenylate synthetase 1; infections (22). PKA, protein kinase A; PKR, IFN-inducible dsRNA-activated protein kinase; rh, IFN-l1 (also called IL-29) belongs to the type III IFN (IFN-l) recombinant human; sh, short hairpin; si, small interfering; siRNA, small interfering family, which is composed of three members: IFN-l1 (IL-29), RNA; TCCR, T cell cytokine receptor. IFN-l2 (IL-28A), and IFN-l3 (IL-28B) (23, 24). PBMCs and Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 DCs are the major producers of IFN-l when infected by viruses or www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300252 692 IL-27 INHIBITS VIRUS REPLICATION BY ACTIVATING IFN-l1 stimulated by polyinosinic-polycytidylic acid or LPS (25). It was and 20 ng/ml IL-4 (PeproTech, Rocky Hill, NJ) for 7 d. Medium was reported that NF-kB and IRF3/7 are required for induction of changed2,4,and6dafterculturing. IFN-l1 expression (26). Although IFN-l binds to distinct receptors, Reagents IL-28R1 and IL-10Rb (24), it activates signal transduction path- ways by a manner similar to that activated by type I IFN (23, 24) Recombinant human (rh)IL-27 and rhIFN-l1 protein were purchased from R&D Systems (Minneapolis, MN). Protein A/G was from Santa Cruz through inducing tyrosine phosphorylation of STAT proteins (27). Biotechnology (Santa Cruz, CA). Abs specific for p-STAT1, p-STAT2, IFN-l also shows antiviral abilities, similar to those of type I IFN STAT2, PKR, OAS1, p-ERK, ERK1/2, IL-28R1, IL-10Rb, NF-kB p65, (28), by inhibiting the replication of a number of viruses, including NF-kB p50, IRF3, and IRF7 were purchased from Santa Cruz Biotech- HCV, hepatitis B virus (HBV), encephalomyocarditis virus, IAV, nology. Ab specific for STAT1 was purchased from Cell Signaling Tech- nology (Beverly, MA). Ab specific for Mx1 was purchased from Proteintech and vesicular stomatitis virus (23, 24, 28–31). Group (Chicago, IL). Ab specific for lamin A/C was purchased from HBV is a major human pathogen chronically infecting .350 EPITOMICS (Burlingame, CA). Abs specific for GAPDH and b-actin were million individuals worldwide and is associated with a high risk purchased from CWBIO (Beijing, China). Flow cytometry Abs specific for for developing cirrhosis, liver failure, and hepatocellular carci- IL-27Ra (WSX-1), IFN-a/b R1, IFN-a/b R2, IL-28R1, and IL-10Rb were noma (32, 33). HBV replication is noncytopathically inhibited by purchased from R&D Systems. Neutralizing type I IFN receptor (IFNAR) chain 2 Ab (CD118) and neutralizing IFN-l1 Ab were purchased from R&D type I IFN (IFN-a/b) (34), type II IFN (IFN-g) (35), and type III Systems. IFN (IFN-l) (30). Our previous study (36) showed that HBV en- The inhibitors used in this study were purchased from Sigma-Aldrich hances IL-27 expression both in vivo and in vitro. We also dem- (St. Louis, MO): U0126 (iERK; 10 mM), H-89 (iPKA; 10 mM), LY294002 onstrated that HBV induces a novel inflammation network involving (iPI3K; 10 mM), SP600125 (iJNK; 10 mM), and SB203580 (iP38; 10 mM). All inhibitors were dissolved in DMSO. three inflammatory factors: IFN-l1, IL-8, and COX-2 (37). Al- Downloaded from though IFN-a is administered to HBV-infected patients (38), some Cell culture patients fail to respond to the treatment (39, 40). Thus, a new or Human hepatocytes, HepG2 and Huh7 cells, were grown in DMEM sup- improved immunotherapy is needed to treat HBV infection. plemented with 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 In this study, we investigated the effects of IL-27 and IFN-l1on mg/ml streptomycin sulfate at 37˚C with 5% CO2. HepG2.2.15 cells that HBV replication. We revealed that HBV infection upregulates IL-27 stably express HBV (ayw, one of HBV subtypes) were derived from HepG2 cells and were maintained in DMEM containing 400 mg/ml G418. expression, which, in turn, stimulates IFN-l1 production through http://www.jimmunol.org/ regulating ERK1/2 signaling and by enhancing NF-kBnuclear Plasmid construction translocation to bind to the IFN-l1 promoter. Moreover, we dem- The DNA fragment carrying HBV genomic DNA (ayw subtype) was am- onstrated that IL-27–activated IFN-l1 regulates IFN-l1 receptors plified from HepG2.2.15 cells and inserted into pBluescript II (Invitrogen, (IL-28R1 and IL-10Rb), resulting in activation of the STAT1/2 Carlsbad, CA) to generate plasmid pHBV1.3. The IFN stimulation re- pathway, which, in turn, induces the expression of ISGs, including sponse element (ISRE) luciferase reporter plasmid (pGL3-ISRE-Luc) was IFN-inducible dsRNA-activated protein kinase (PKR), oligoadeny- a gift from Dr. Hongbing Shu (Wuhan University). Mutant Erk (mERK) late synthetase 1 (OAS1), and IFN-induced GTP-binding protein 1 plasmids, which express ERK1 and ERK2 mutants that block ERK kinase activities by competing with endogenous kinases, were kindly provided (Mx1), in a manner similar to type I IFN and, finally, inhibits HBV by Dr. Melanie H. Cobb (University of Texas Southwestern Medical Center, protein expression and viral capsid–associated DNA replication. Our Dallas, TX). by guest on September 23, 2021 study revealed a novel mechanism by which IL-27 and IFN-l1are coordinated to regulate virus replication through type I IFN. Luciferase assay HepG2 cells were transfected with reporter plasmids and then treated with rhIL-27 protein. Cells were lysed with luciferase cell culture lysis reagent Materials and Methods (Promega, Madison, WI). Cell lysates and luciferase assay substrate (Promega) Clinical samples were mixed, and the light intensity was detected by a luminometer (Turner T20/ 20). Assays were performed in triplicate. Peripheral blood samples were obtained from 30 patients with chronic HBV (CHB; 22 males and 8 females; mean age 47 6 14.6 y) who were ELISA admitted to Rennin Hospital of Wuhan University. All patients were confirmed HBV positive (but negative for HCV, hepatitis D virus, and IFN-l1 released in culture supernatants was quantified using a Human HIV), had no evidence of concomitant illness, and showed no sero- IFN-l1 Platinum ELISA kit (eBioscience, San Diego, CA), according to logical markers suggestive of autoimmune disease. A control group, the manufacturer’s instructions. consisting of 30 healthy individuals (19 males and 11 females; mean age 42 6 17.2 y) with no history of liver disease and who were sex- and age- Real-time PCR matched with the patient samples, was randomly selected from blood Total RNA was extracted with TRIzol reagent (Invitrogen). following donation centers in Wuhan, China. the manufacturer’s instructions. Real-time quantitative RT-PCR was Written informed consent was obtained from each patient. The study was performed using the Roche LC480 and SYBR RT-PCR kits (DBI Bio- conducted according to the principles of the Declaration of Helsinki and science, Ludwigshafen, Germany) in a reaction mixture of 20 mlSYBR approved by the Institutional Review Board of the College of Life Sciences, Green PCR master mix, 1 ml DNA diluted template, and RNase-free Wuhan University, in accordance with its guidelines for the protection of water to complete the 20 ml volume. Real-time PCR was carried out as human subjects. follows: step 1, preincubation, 95˚C, 5 min; step 2, amplification and quantification, 95˚C, 10 s, primer-dependent annealing temperature, Isolation of PBMCs 15 s, 72˚C 15 s, with a single fluorescence measurement, for 40 cycles; PBMCs were isolated by density centrifugation of fresh peripheral venous step 3, melting curve, 60–95˚C with a heating rate of 0.1˚C/s and blood samples diluted 1:1 in pyrogen-free PBS over Histopaque (Haoyang continuous fluorescence measurement; and step 4, cooling, 40˚C. Real- Biotech). Cells were washed twice in PBS and resuspended in culture time PCR primers were designed by Primer Premier 5.0 and their medium (RPMI 1640) supplemented with penicillin (100 U/ml) and sequences were as follows: IFN-a sense: 59-TTTCTCCTGCCTGA- streptomycin (100 mg/ml). AGGACAG-39,IFN-a antisense: 59-GCTCATGATTTCTGCTCTGA- CA-39;IFN-b sense: 59-TGGGAGGCTTGAATACTGCCTCAA-39,IFN- Generation of human immature DCs b antisense: 59-TCCTTGGCCTTCAGGTAATGCAGA-39; IFN-l1 sense: 59-CTTCCAAGCCCACCACAACT-39, IFN-l1 antisense: 59-GGCCTC- Monocytes were isolated from PBMCs by adhesion to plastic dishes for CAGGACCTTCAGC-39; PKR sense: 59-AAAGCGAACAAGGAGTAAG- .2 h at 37˚C, as described previously (41). To derive immature DCs, 39, PKR antisense: 59-GATGATGCCATCCCGTAG-39; OAS1 sense: 59- monocytes were cultured at 1 3 106 in RPMI 1640 medium containing TTCCGTCCATAGGAGCCAC-39, OAS1 antisense: 59-AAGCCCTACGAA- 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin, 50 ng/ml GM-CSF, GAATGTC-39; Mx1 sense: 59-ACCACAGAGGCTCTCAGCAT-39,Mx1 The Journal of Immunology 693 antisense: 59-CTCAGCTGGTCCTGGATCTC-39;IL-28R1sense:59-A- 4˚C. The lysates were sonicated on ice, and the debris was removed by CCTATTTTGTGGCCTATCAGAGCT-39, IL-28R1 antisense: 59-CGG- centrifugation at 12,000 3 g for 15 min at 4˚C. One fourth of the super- CTCCACTTCAAAAAGGTAAT-39; IL-10Rb sense: 59-CTGCTTCGC- natant was used as DNA input control. The remaining sample was diluted CTTGCTGTG-39, IL-10Rb antisense: 59-GATTCTGCTTGCCGCTCT- 10-fold with dilution buffer (0.01% SDS, 1% Triton X-100, 1 mM EDTA, 39;andGAPDHsense:59-GGAAGGTGAAGGTCGGAGTCAACGG-39, 10 mM Tris-HCl [pH 8], and 150 mM NaCl), followed by overnight in- GAPDH antisense: 59-CTCGCTCCTGGAAGATGGTGATGGG-39. cubation at 4˚C with Ab against NF-kB p65/p50, IRF3, or nonspecific IgG, Data were normalized to the GAPDH expression level in each sample. respectively. Immunoprecipitated complexes were collected using protein A/G-Sepharose. The pellets were washed four times with dialysis buffer HBV protein–expression assay (2 mM EDTA and 50 mM Tris-HCl [pH 8]). After washing, the precipitates were incubated in elution buffer (1% SDS and 0.1 M NaHCO3) at room HepG2 cells and Huh7 cells were transfected with plasmid pHBV1.3 and temperature. Supernatants were transferred to clean tubes, and RNase A treated with rhIL-27 protein. HepG2.2.15 cells also were treated with rhIL- was added to destroy bound RNA in the sample. Samples were incubated 27 protein. At 24 h posttreatment, HBeAg and HBsAg protein levels in cell at 65˚C for 5 h to reverse formaldehyde cross-links, and DNA was pre- culturemediaweredeterminedbyELISAusinganHBVHBeAgand cipitated with ethanol and extracted twice with phenol/chloroform. Finally, HBsAg diagnostic kit (KeHua Biotech, Shanghai, China), respectively. pellets were resuspended in Tris-EDTA buffer and subjected to PCR am- plification using the following primers: IFN-l1 proximal: 59-GCCAG- HBV DNA assay by real-time PCR TTGGCTGAAAGCTGCCCA-39 (sense) and 59-GGCAGGGCCAAGT- Capsid-associated DNA was extracted as described previously (42), with GAGCTGGGA-39 (antisense); IFN-l1 distal: 59-TTTAAGGGCAGGTG- modifications. Equivalent amounts of posttreatment cells were homoge- CAGGGTGTC-39 (sense) and 59-TTACCCAATGTGGTGGGCACCATC-39 nized in 1 ml lysis buffer (50 mM Tris [pH 7.5], 0.5% Nonidet P-40, 1 mM (antisense); and IRF7 59-CATAGTGAGACCCCTATCTCTACAA-39 (sense) EDTA, and 100 mM NaCl) and mixed gently at 4˚C for 1 h. Next, 10 ml and 59-ACTGGGTTTTACTATGTTGGTCACG-39 (antisense). 1 M MgCl2 and 10 ml DNase (10 mg/ml) were added and incubated for 2 h at 37˚C. Viral cores were precipitated by adding 35 ml 0.5 M EDTA and Immunofluorescence Downloaded from 225 ml 35% polyethylene glycol and incubating them at 4˚C for $30 min, HepG2 cells and Huh7 cells were treated with rhIL-27 protein (20 ng/ml) or after which the cores were concentrated by centrifugation, and the pellet PBS as a negative control. At 3 h posttreatment, cells were fixed with 4% was resuspended in resuspending buffer (10 mM Tris, 100 mM NaCl, paraformaldehyde for 15 min, washed three times with PBS, permeabilized 1 mM EDTA, 1% SDS, and 25 mg/ml proteinase K) and incubated over- with PBS containing 0.5% Triton X-100 for 5 min, washed three times with night. Viral DNA released from lysed cores was extracted with phenol and PBS, and finally blocked with PBS containing 4% BSA for 1 h at room chloroform, precipitated with isopropanol, and resuspended in Tris-EDTA temperature. The cells were then incubated with the primary Ab overnight at buffer. 4˚C, followed by incubation with Alexa Fluor 492–labeled secondary Abs http://www.jimmunol.org/ Capsid-associated DNA was quantified by quantitative PCR performed (Proteintech Group) for 1 h. Mounting was done with VECTASHIELD in a Light Cycler 480 real-time PCR instrument (Roche, Basel, Switzer- mounting medium with DAPI (Vector Laboratories, Burlingame, CA), and land) using the following set of TaqMan real-time PCR primers and probe: the cells were visualized by confocal laser microscopy (FluoView FV1000; primer P1: 59-AGAAACAACACATAGCGCCTCAT-39; primer P2: 59- Olympus, Tokyo, Japan). TGCCCCATGCTGTAGATCTTG-39; and probe: 59-TGTGGGTCACCA- TATTCTTGGG-39. Flow cytometry The PCR reaction was performed as follows: 1 cycle at 50˚C for 2 min, 1 cycle at 95˚C for 10 min, and 40 cycles at 95˚C for 15 s and 60˚C for HepG2 and Huh7 cells were Fc blocked by treatment with 1 mg human IgG/ 60 s. Plasmid pHBV1.3 was diluted over a range of 1027 to 100 and used 105 cells for 15 min at 4˚C prior to staining. A total of 5 3 105 to 1 3 106 as a standard, and all samples were analyzed in triplicate. cells in cold PBS buffer (supplemented with 2% BSA) was incubated with

1 mg/ml specific Ab for 1 h at 4˚C. Cells were washed with cold PBS by guest on September 23, 2021 Nuclear extraction buffer (supplemented with 2% BSA) twice, and then 2.5 3 104 cells/ sample were analyzed by flow cytometry using a FACSCalibur (Beckman HepG2 cells and Huh7 cells were incubated in serum-free media for 24 h Coulter). and then treated with rhIL-27 protein at 20 ng/ml or PBS as a negative control for 3 h. Cells were washed with cold PBS and scraped into 1 ml cold Statistical analysis PBS, followed by centrifugation at 2000 3 g for 5 min in a microcentrifuge and incubation in two packed-cell volumes of buffer A (10 mM HEPES All experiments were reproducible and were carried out in triplicate. Each [pH 8], 0.5% Nonidet P-40, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, set of experiments was repeated at least three times with similar results, and and 200 mM sucrose) for 10 min on ice, with flipping of the tube. Nuclei representative experiments are shown. The results are presented as means. were collected by centrifugation (12,000 3 g for 30 s). Pellets were rinsed ANOVA test for paired samples was used to determine statistical signifi- with buffer A, resuspended in buffer B (20 mM HEPES [pH 7.9], 1.5 mM cance. Differences were considered statistically significant at p , 0.05. MgCl2, 420 mM NaCl, 0.2 mM EDTA, and 1.0 mM DTT), and incubated on a rocking platform at 4˚C for 30 min. Nuclei were clarified by centri- fugation (12,000 3 g for 10 min), and the supernatants were diluted 1:1 Results with buffer C (20 mM HEPES [pH 7.9], 100 mM KCl, 0.2 mM EDTA, IL-27 represses HBV protein expression and capsid-associated 20% glycerol, and 1 mM DTT). A mixture of protease inhibitors (Roche) DNA replication was added to each type of buffer. Nuclear extracts were stored at 270˚C until use. We reported previously that IL-27 protein was elevated in patients with HBV infection and that HBV enhances IL-27 expression in Western blotting several cell types, including THP1, HepG2, Huh7, and HEK293T cells Whole-cell lysates were prepared by lysing cells with PBS (pH 7.4) (36). A correlation between HBV infection and IL-27 expression was containing 0.01% Triton X-100, 0.01% EDTA, and 10% protease inhibitor also shown (43). In this study, we explored the impact of IL-27 on mixture (Roche). Protein concentration was determined by the Bradford HBV replication. HepG2.2.15 cells, which carry HBV genomic DNA assay (Bio-Rad, Richmond, CA). The samples from cell lysates were (ayw subtype) on their , were treated with different separated on 12% SDS polyacrylamide gels and transferred electrically to nitrocellulose membranes (Amersham, Piscataway, NJ). Nonspecific sites concentrations of rhIL-27, as indicated. In addition, HepG2 cells were blocked by 5% skim milk before being incubated with an Ab in this and Huh7 cells were transfected with pHBV-1.3, which carries HBV study. Protein bands were detected using SuperSignal Chemiluminescent genomic DNA (ayw subtype) on its chromosome, and then substrate (Pierce, Rockford, IL). were treated with rhIL-27 at different concentrations, as indicated. Chromatin immunoprecipitation assay ELISA results showed that HBV HBeAg protein (Fig. 1A) and HBsAg protein (Fig. 1B) were downregulated by rhIL-27 in a dose- HepG2 cells were treated for 3 h with rhIL-27 protein at 20 ng/ml or PBS as dependent fashion in HepG2.2.15, pHBV-1.3-transfected HepG2, a negative control. Formaldehyde was added to the culture medium to a final concentration of 1%. The cells were washed twice with PBS, scraped, and and pHBV-1.3–transfected Huh7 cells. Similarly, real-time PCR lysed in lysis buffer (1% SDS, 10 mM Tris-HCl [pH 8], 10% protease results revealed that HBV capsid–associated DNA (Fig. 1C) was inhibitor mixture, and 50 mg/ml both aprotinin and leupeptin) for 10 min at downregulated by rhIL-27 in a concentration-dependent manner in 694 IL-27 INHIBITS VIRUS REPLICATION BY ACTIVATING IFN-l1 Downloaded from http://www.jimmunol.org/ FIGURE 1. Evaluation of the effects of IL-27 on HBV protein expression and viral capsid–associated DNA replication. HepG2.2.15 cells were treated with rhIL-27 at different concentrations, as indicated. HepG2 cells and Huh7 cells were transfected with plasmid pHBV-1.3 and treated with rhIL-27 protein at 6 h posttransfection. At 24 h posttreatment, cell culture supernatants were collected, and HBV capsid–associated DNA was extracted. The levels of secreted HBeAg (A) and HBsAg (B) were determined by ELISA, and the expression of capsid-associated DNA (C) was assessed by real-time PCR. *p , 0.05. all three cell types. We also showed that the cell viability was not transfected with the reporter pISRE-Luc, in which expression of affected under these experimental conditions (Supplemental Fig. 1A), the luciferase gene is under the control of ISRE, and then treated indicating that the reduction in HBV protein expression and capsid- with rhIL-27 or PBS. Results from the luciferase assay showed by guest on September 23, 2021 associated DNA replication were not due to alterations in cell via- that ISRE activity was stimulated by rhIL-27, but not by PBS, in bility caused by rhIL-27. These results suggested that IL-27 has both HepG2 and Huh7 cells (Fig. 2B), indicating that IL-27 plays inhibitory effects on HBV protein expression, as well as on HBV a positive role in the regulation of ISRE activity. capsid–associated DNA replication. IFN-a plays an essential role in both the innate immune re- sponse and the adaptive immunity against viral infections by IL-27 activates ISG expression by activating STAT1/2 stimulating a variety of antiviral proteins and cytokines, leading to Upon detection of viral components, host cells are known to the protection of the host from further viral infection. We inves- activate antiviral responses via TLRs and the IFN cascade. In the tigated the effects of IL-27 on the expression of antiviral proteins IFN antiviral response, IFN-a initially binds to IFNAR1 and PKR, Mx1, and OAS1. Cells were treated with PBS, rhIL-27, or IFNAR2, thereby activating the receptor complex, resulting in rhIFN-a for different times. RT-PCR results indicated that PKR induction of the phosphorylation of STAT1/2 and the expres- mRNA was upregulated by IL-27 and IFN-a, reaching a peak at sion of ISGs, which, in turn, causeanantiviralstateintarget 24 and 12 h posttreatment, respectively (Fig. 2C, left panel); Mx1 cells (21). mRNA was enhanced by IL-27 and IFN-a, reaching a peak at 24 To explore the regulatory mechanism involved in the down- and 12 h posttreatment, respectively (Fig. 2C, middle panel), whereas regulation of HBV replication mediated by IL-27, we first evaluated OAS1 mRNA was activated by IL-27 and IFN-a, reaching a peak the effect of IL-27 on the phosphorylation status of STAT1/2 at 24 h posttreatment (Fig. 2C, right panel). Moreover, Western proteins. HepG2 cells were treated with rhIL-27 for different blot results revealed that PKR, Mx1, and OAS1 proteins were ac- times, as indicated, or were treated with IFN-a protein as a positive tivated by IL-27 or IFN-a but not by PBS (Fig. 2D, Supplemental control. Western blot results showed that p-STAT1 and p-STAT2 Fig. 2B). These data indicate that both IL-27 and IFN-a can activate proteins were upregulated by rhIL-27 and rhIFN-a, respectively, ISG expression, but IFN-a induces a stronger and earlier response whereas STAT1, STAT2, and b-actin proteins remained relatively than does IL-27. unchanged under the same conditions (Fig. 2A, Supplemental l Fig. 2A). These results suggested that, similar to type I IFN (IFN-a), IL-27 expression and IFN- 1 production are highly correlated IL-27 has a stimulatory effect on the activation of STAT1/2. in patients infected with HBV, and IL-27 upregulates IFN genes Viral infection triggers the production of type I IFN (IFN-a/b), in vitro which leads to the activation of several hundred ISGs through We showed previously that HBVupregulates IL-27 expression both ISRE on the promoters of ISGs. It was reported that IL-27 can in vivo and in vitro (36) and demonstrated that HBV induces exert IFN-like function in liver cells and contribute to the antiviral a novel inflammation network involving three inflammatory fac- response in these cells (44). To investigate the role of IL-27 in tors: IFN-l1, IL-8, and COX-2 (37). However, the correlation of regulating the activity of ISRE, HepG2 and Huh7 cells were IL-27 and IFN-l1 has not been studied. In this study, we evaluated The Journal of Immunology 695

FIGURE 2. Determination of the roles of IL-27 in the regulation of STAT1/2 activation and IFN-inducible antiviral protein expression. (A)HepG2 cells were incubated with rhIL-27 (20ng/ml)for10–60minandIFN-a (as a positive control; 100 U/ml) for 20 min. Cells were lysed, and the levels of p-STAT1, nonphosphorylated STAT1, p-STAT2, nonphosphorylated STAT2, and b-actin proteins were assessed by Western blot. (B) HepG2 cells (left panel) and Huh7 cells (right panel) were transfected with the reporter pISRE-Luc. At 12 h posttransfection, rhIL-27 protein (20 ng/ml) was added to cell culture supernatant for 24 h. Downloaded from ISRE activity was measured by lucif- erase activity assays. Data are mean + SE (n = 3). (C) HepG2 cells were treated with PBS, rhIL-27 protein (20 ng/ml), or IFN-a (300 UI/ml) for different times, as indicated. (D)HepG2 cells were incubated with rhIL-27 pro- http://www.jimmunol.org/ tein (20 ng/ml) for 24 h. The mRNA (C) and protein (D) levels of IFN-inducible antiviral genes, such as Mx1, OAS1, and PKR were assessed by real-time PCR and Western blot, respectively. Data are mean + SE (n = 3). *p , 0.05. by guest on September 23, 2021 the expression status of IL-27 and IFN-l1 in patients with CHB IFN-a and IFN-l1 are involved in the repression of HBV infection and determined the correlation between IL-27 expression replication mediated by IL-27 and IFN-l1 production during HBV infection. IFN-a mediates the antiviral response by binding to a common PBMCs were isolated from patients with HBV infection (n = heterodimeric receptor consisting of two subunits (IFNAR1 and 30) and healthy individuals (n = 30). Total mRNA were extracted IFNAR2) (45), whereas IFN-l1 regulates its biological function from isolated PBMCs and assessed by real-time PCR. Results through binding to a distinct receptor complex consisting of two showed that the mRNA levels of IFN-l1 and the two subunits of subunits (IL28R1 or IFN-lR1 and IL10R2) (24). However, IFN-l1 IL-27 (p28 and EBI3) were significantly higher in patients with can activate signal-transduction pathways similar to those ac- 6 6 CHB than in healthy individuals (mean SEM, IFN-l1: 8.379 tivated by IFN-a (23, 24), and it displays antiviral activity similar 6 , 6 0.875 versus 1.325 0.0877, p 0.05; p28: 5.648 0.4068 versus to IFN-a (28). Because our results revealed that IL-27 upregulates 6 , 6 6 2.297 0.1936, p 0.05; EBI3: 5.891 0.2983 versus 2.656 the expression of IFN-a, IFN-b, and IFN-l1, we further evaluated 0.1238, p , 0.05) (Fig. 3A). Further analysis revealed that ele- the effects of IFN-a/b and IFN-l1 on IL-27 function. vated levels of IFN-l1 were tightly correlated with increased The roles of IFN-a/b and IFN-l1 in IL-27 activity were in- levels of p28 and EBI3 in the PBMCs of patients with CHB (Fig. vestigated initially using an RNA-interfering approach. The effi- 3B). These results revealed that IFN-l1, p28, and EBI3 are up- ciencies of small interfering RNA (siRNA) molecules were regulated in PBMCs of HBV-infected patients and that IFN-l1 evaluated in HepG2 cells transfected with small interfering (si)– expression and IL-27 production are highly correlated in vivo. negative control (NC), si-IFNAR1, si-IFNAR2, short hairpin However, we do not know whether an unknown factor (HBV in- (sh)–NC, and sh–IFN-l1. Real-time PCR results indicated that fection or type I IFNs) causes the observed simultaneous induction IFNAR1 mRNA and IFNAR2 mRNA were significantly down- of IL-27 and IFN-l1. regulated in the presence of si-IFNAR1 and si-IFNAR2, respec- In addition, we evaluated the effect of IL-27 on IFN expression tively (Supplemental Fig. 3A). Flow cytometry results showed that and antiviral protein production. PBMCs were treated with rhIL- IFNAR1 and IFNAR2 proteins were not affected by the treatment 27 for different times, as indicated. Real-time PCR results showed with si-IFNAR1 and si-IFNAR2 (Supplemental Fig. 3B). Simi- that IL-27 activates the expression of IFNs (IFN-a . IFN-b . larly, real-time PCR and ELISA results revealed that both IFN-l1 IFN-l1) in a time-dependent manner at 24 h posttreatment in mRNA (Supplemental Fig. 3C, left panel) and IFN-l1 protein PBMCs (Fig. 3C). In addition, IL-27 also stimulates the ex- (Supplemental Fig. 3C, right panel) were significantly downreg- pression of ISGs (OAS1 . Mx1 . PKR) in a time-dependent ulated in the presence of sh–IFN-l1. These results indicated that manner at 24 h posttreatment in PBMCs (Fig. 3D). si-IFNAR1, si-IFNAR2, and sh–IFN-l1 are specific and efficient. 696 IL-27 INHIBITS VIRUS REPLICATION BY ACTIVATING IFN-l1

FIGURE 3. Investigation of the correlation between IL-27 expression and IL-29 production in patients with HBV infection. Peripheral blood samples were obtained from 25 healthy individuals and 20 patients with CHB infection. PBMCs were isolated from peripheral blood samples and lysed. (A) IL-29, p28, and EBI3 mRNA were detected by real-time PCR from total RNA ex- tracted from PBMC lysates using the respective pri- mers. *p , 0.05, **p , 0.01. (B) Significant positive correlations were found between p28 mRNA and IL-29 mRNA levels in HBV-infected patients (R2 = +0.773, p , 0.05), as well as between EBI3 mRNA and IL-29 levels (R2 = +0.5962, p , 0.05). The solid line repre- sents the regression. (C and D) PBMCs from healthy Downloaded from individuals were isolated and treated with PBS or rhIL- 27 protein (20 ng/ml) for different times, as indicated. The mRNA levels of IFN-a/b/l1(C) and ISGs PKR, OAS1, and Mx1 (D) were determined by real-time PCR. http://www.jimmunol.org/

The role of IFN-l1 in the regulation of HBV replication was IL-27 specifically activates IFN-l1 expression in immature determined by knockdown and overexpression of IFN-l1. HepG2 DCs and hepatocytes cells were cotransfected with pHBV1.3 and sh–IFN-l1 or treated We revealed that IL-27 induces an antiviral response similar to by guest on September 23, 2021 with rhIFN-l1. Results showed that HBV HBeAg protein, HBsAg IFN-a, showed that IFN-l1 expression is well correlated with IL- protein, and capsid-associated DNA were upregulated by sh–IFN- 27 expression in HBV-infected patients, and demonstrated that l1 (Fig. 4A) but downregulated by rhIFN-l1 (Fig. 4B), suggesting IFN-l1 is involved in regulating the anti-HBV function of IL-27. that IFN-l1 represses HBV replication. These facts suggested that there is a close correlation between IL- Next, the effect of IFN-a/b on the expression of IFN-l1 induced 27 and IFN-l1 and prompted us to investigate the role of IL-27 on by IL-27 was determined. Cells were treated with rhIL-27, si- the expression and function of IFN-l1. IFNAR, or the neutralizing Ab specific to IFNAR2 (anti-IFNAR2). Human immature DCs, HepG2 cells, and Huh7 hepatocytes were Results indicated that IFN-l1 mRNA (Fig. 4C) and protein (Fig. 4D) treated with rhIL-27 for different times or at different concen- were upregulated by rhIL-27, but such activation was repressed by trations, as indicated. Real-time PCR results showed that IFN-l1 si-IFNAR or anti-IFNAR2. In addition, IFN-l1 mRNA (Fig. 4C) and mRNA was upregulated by IL-27 in a time-dependent fashion protein (Fig. 4D) were downregulated by si-IFNAR or anti-IFNAR2 in the absence of rhIL-27 (Fig. 4C, 4D). These results suggested that (Fig. 5A) and in a dose-dependent manner (Fig. 5C) in DCs, type I IFN is involved in the activation of IFN-l1 induced by IL-27. HepG2 cells, and Huh7 cells. ELISA results indicated IFN-l1 Next, we evaluated the effects of IFN-l1 and IFNAR on the protein was enhanced by IL-27 in a time-dependent manner (Fig. regulation of IL-27 antiviral activity. HepG2 cells were cotrans- 5B) and in a dose-dependent fashion (Fig. 5D) in all three cell fected with pHBV1.3 and sh–IFN-l1 or si-IFNAR and then treated types. Similarly, results also indicated that IFN-a mRNA and with rhIL-27. ELISA results showed that HBV HBeAg protein, IFN-b mRNA were upregulated by IL-27 in a time-dependent HBsAg protein, and capsid-associated DNA were downregulated fashion (Supplemental Fig. 4A) and in a dose-dependent manner by IL-27, and such repression was suppressed by sh–IFN-l1 (Fig. (Supplemental Fig. 4B). These results demonstrated that IL-27 4E) and si-IFNAR (Fig. 4F). These results indicated that IFN-l1 activates IFN-l1 expression in human immature DCs and hepa- and IFN-a are involved in the downregulation of HBV replication toma cells. mediated by IL-27. IL-27 mediates its biological function through binding to a specific Moreover, HepG2 cells were transfected with pHBV1.3, treated receptor (IL-27R) consisting of TCCR (WSX-1 or IL-27Ra)and with anti-IFNAR2 or with neutralizing Ab specific to IFN-l1 a common receptor subunit of the IL-6 family cytokine (gp130) (3, (anti–IFN-l1), and incubated with rhIL-27. Results revealed that 46–48). Like other class I cytokine receptor chains, IL-27Ra het- HBV HBeAg protein (Fig. 4G, left panel), HBsAg protein (Fig. 4G, erodimerizes with gp130 to form a functional IL-27R (2). middle panel), and capsid-associated DNA (Fig. 4G, right panel) The role of IL-27 in the activation of IFN-l1 was evaluated were downregulated by IL-27, but this repression was suppressed further by an RNA-interference approach, using siRNA specific to by anti-IFNAR2 or by anti–IFN-l1. These results confirmed that IL-27Ra (si–WSX-1). To determine the efficiency of siRNAs, IFN-a and IFN-l1 are involved in the repression of HBV repli- HepG2 and Huh7 cells were transfected with si–WSX-1 or its cation mediated by IL-27. control si-NC. Real-time PCR results (Supplemental Fig. 3D) and The Journal of Immunology 697 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 4. Blocking IFN-a/b and IFN-l1 signals suppresses the host anti-HBV effect mediated by IL-27. (A) HepG2 cells were cotransfected with pHBV1.3 and sh–IFN-l1 (or sh-NC). At 24 h posttreatment, the levels of HBeAg protein and HBsAg protein of HBV in culture supernatants were measured by ELISA, and the levels of HBV capsid–associated DNA in transfected cells were assessed by real-time PCR. (B) HepG2 cells were cotransfected with pHBV1.3 and then treated with rhIFN-l1 protein (20 ng/ml) for 24 h. The levels of HBeAg protein and HBsAg protein of HBV in culture supernatants were measured by ELISA, and the levels of HBV capsid–associated DNA in transfected cells were assessed by real-time PCR. (C and D) HepG2 cells were treated or not with neutralizing Ab to IFNAR2 (10 mg/ml) for 30 min or were transfected with si-IFNAR (or si-NC) for 24 h before rhIL-27 (50 ng/ml) treatment. IFN-l1 expression was determined by real-time PCR (C) and ELISA (D) 24 h post–IL-27 treatment. (E and F) HepG2 cells were cotransfected with pHBV1.3 and si-IFNAR1/2 (or si-NC) or sh–IFN-l1 (or sh-NC) and then treated with rhIL-27 protein (50 ng/ml) at 12 h post- transfection. At 24 h posttreatment, the levels of HBeAg protein and HBsAg protein of HBV in culture supernatants were measured by ELISA, and the levels of HBV capsid–associated DNA in transfected cells were assessed by real-time PCR. (G) HepG2 cells were transfected with pHBV1.3 and treated with neutralizing Ab to IFNAR2 (10 mg/ml) or neutralizing Ab or IFN-l1, which was added 30 min before rhIL-27 (50 ng/ml) treatment. The HBeAg and HBsAg levels were determined by ELISA, and the levels of capsid-associated DNA in transfected cells were assessed by real-time PCR. Data are mean + SE (n = 3). *p , 0.05, **p , 0.01. 698 IL-27 INHIBITS VIRUS REPLICATION BY ACTIVATING IFN-l1

FIGURE 5. Determination of the role of IL-27 in regulation of IFN-l1 expression in immature DCs and hepatocytes. DCs were generated from monocytes, which were iso- lated from PBMCs of healthy donors. (A and B)DCs(left panels), HepG2 cells (middle panels), and Huh7 cells (right panels) were treated with rhIL-27 protein or PBS at 20 ng/ml for different times, as indicated. Cell culture supernatant was collected, and total mRNA was extracted. IFN-l1 protein levels were assessed by ELISA (A), and IFN-l1 mRNA levels were determined by Downloaded from real-time PCR (B). Data shown are mean 6 SE (n =3).(C and D)DCs(left panels), HepG2 cells (middle panels), and Huh7 cells (right panels) were treated with rhIL-27 protein or LPS at 20 ng/ml. At 24 h post- treatment, cell culture supernatant was col-

lected, and total mRNA was extracted. http://www.jimmunol.org/ IFN-l1 protein levels were assessed by ELISA (C), and IFN-l1 mRNA levels were determined by real-time PCR (D). (E and F) HepG2 cells and Huh7 cells were transfected with si–WSX-1 or si-NC. rhIL-27 protein (50 ng/ml) was added at 12 h posttransfec- tion, and IFN-l1 expression was assessed by real-time PCR (E) and ELISA (F)24h posttreatment. Data are mean + SE (n = 3). *p , 0.05. by guest on September 23, 2021

flow cytometry results (Supplemental Fig. 3E) showed that IL- HepG2 and Huh7 cells were treated with PBS, rhIL-27, or 27Ra RNA and protein were downregulated by si–WSX-1, indi- IFN-a. Cytoplasm extracts and nuclear extracts were prepared, and cating that it is effective. HepG2 and Huh7 cells were transfected proteins were detected by Western blot using specific Abs. Results with si–WSX-1 or si-NC and treated with rhIL-27 or PBS. Results revealed that, in the presence of IL-27, the p65, p50, and IRF7 from real-time PCR and ELISA showed that IFN-l1 mRNA (Fig. proteins were decreased in the cytoplasm and increased in the 5E) and protein (Fig. 5F) were significantly upregulated by IL-27, nucleus of HepG2 and Huh7 cells, whereas the IRF3 and GAPDH but downregulated by si–WSX-1, in both HepG2 and Huh7 cells. proteins were relatively unchanged in the cytoplasm, and the IRF3 These results suggested that IL-27 is specifically involved in the and lamin proteins were relatively unchanged in the nucleus of regulation of IFN-l1 expression. both cell types (Fig. 6A, Supplemental Fig. 2C). In addition, immunofluorescence assays clearly showed that, in the absence of l k IL-27 activates IFN- 1 by stimulating NF- B and IRF7 IL-27, the p65 and p50 proteins were distributed mainly in the l nuclear translocation and IFN- 1 promoter recognition cytosol of cells; however, in the presence of IL-27, the majority of It was reported that NF-kB and IRF3/7 are required for LPS- the p65 and p50 proteins were translocated to the nucleus of cells induced activation of IFN-l1 expression (26). The phosphoryla- (Fig. 6B). These results indicated that IL-27 enhances NF-kB p65 tion of IRF3/7 on specific serine residues results in a homo- or and NF-kB p50 translocation from the cytosol to the nucleus. heterodimer formation (45), which then translocates to the nucleus Moreover, the effect of NF-kBonIFN-l1 expression regulated and activates the transcription of IFN-l1. To understand the by IL-27 was verified further by assessing the ability of p65 and mechanism by which IL-27 activates IFN-l1 expression, we p50 binding to the IFN-l1 promoter. Results from chromatin assessed the effect of IL-27 on the activities of NF-kB and IRF3/7 immunoprecipitation assays showed that IL-27 enhances the binding by evaluating its role in the nuclear translocation of p65 and p50 of p65 and p50 to the distal (Fig. 6C, left panel) and the prox- (the two subunits of NF-kB) and IRF3/7. imal (Fig. 6C, middle panel)NF-kB binding sites on the IFN-l1 The Journal of Immunology 699

that the ERK1/2-signaling pathway is involved in the activation of IFN-l1 expression regulated by IL-27. Second, the role of ERK1/2 signaling in the IL-27–activated expression of IFN-l1 was evaluated further by introducing two dominant kinase-inactive mutants, mERK1 and mERK2, which block ERK activity by competing with endogenous kinases (49). HepG2 and Huh7 cells were transfected with the plasmids ex- pressing mERK1 or mERK2 and then treated with rhIL-27. Results revealed that IFN-l1 mRNA (Fig. 7B) and IFN-l1 pro- tein (Fig. 7C) were stimulated by IL-27, but such activation was blocked by mERK1 and mERK2 in both cell types. These results further supported that ERK1 and ERK2 are involved in the acti- vation of IFN-l1 expression regulated by IL-27. Third, the role of IL-27 in the phosphorylation of ERK1/2 was determined. HepG2 and Huh7 cells were treated with rhIL-27 for different times, as indicated. Results showed that p-ERK protein was upregulated by IL-27 in both HepG2 cells and Huh7 cells, whereas ERK and b-actin proteins were relatively unchanged

under the same conditions (Fig. 7D, Supplemental Fig. 2D), Downloaded from suggesting that IL-27 stimulates the phosphorylation of ERK protein. Finally, the effects of IL-27 and ERK1/2 on the nuclear trans- location of NF-kB p65 and p50 were investigated. HepG2 and Huh7 cells were transfected with plasmids expressing mERK1 or

mERK2 and then treated with rhIL-27. Results revealed that nu- http://www.jimmunol.org/ clear translocation of p65 and p50 was enhanced by IL-27; however, such activation was reduced, in part, by mERK1 and mERK2 in both cell types (Fig. 7E, Supplemental Fig. 2E), sug- gesting that the role of NF-kB in IFN-l1 expression is regulated by ERK1/2. Taken together, these results demonstrated that the FIGURE 6. Evaluation of the effects of NF-kB and IRF3/7 on regulat- ing the expression of IFN-l1 mediated by IL-27. (A and B) HepG2 cells ERK1/2-signaling pathway and NF-kB protein are involved in the and Huh7 cells were incubated with PBS, rhIL-27 protein (20 ng/ml), or activation of IFN-l1 regulated by IL-27. rhIFN-a (300 UI/ml) for 3 h. Nuclear and cytoplasm protein extracts were l

IL-27 enhances the expression of IFN- 1 receptors IL-28R1 by guest on September 23, 2021 A prepared. ( )NF-kB p65, NF-kB p50, IRF3, IRF7, lamin A/C, and and IL-10Rb GAPDH proteins were detected by Western blotting. (B) The nuclear translocations of NF-kB p65/p50 were obtained using immunofluorescence IFN-l1 and IFN-a/b share the same JAK/STAT-signaling pathway assays. Original magnification 3100. (C) HepG2 cells were incubated with in the induction and expression of a common set of ISGs. However, rhIL-27 protein or PBS at 20 ng/ml for 6 h. The binding activities of IFN-l1 binds to a distinct receptor complex consisting of the unique NF-kB p65 and p50 to the distal and proximal NF-kB binding sites and ligand-binding subunit IL-28R1 (also known as IFN-lR1) and the IRF7 to the ISRE sites of the promoter of IFN-l1 gene were evaluated accessory receptor subunit IL-10Rb (also known as IFN-lR2). by chromatin immunoprecipitation assay. To investigate the roles of IL-27 in the expression of IL-28R1 and IL-10Rb, HepG2 and Huh7 cells were incubated with PBS or rhIL-27. Real-time PCR and Western blot results showed that promoter, as well as stimulates the binding of IRF7 to the ISRE IL-28R1 and IL-10Rb mRNAs (Fig. 8A) and IL-28R1 and IL- sites on the IFN-l1 promoter (Fig. 6C, right panel). These results 10Rb proteins (Fig. 8B, Supplemental Fig. 2F) were upregulated demonstrated that IL-27 activates IFN-l1 expression by enhanc- by IL-27 in both HepG2 cells and Huh7 cells. To evaluate the ing the binding of transcription factor NF-kB and IRF7 to the biological relevance of IL-27–activated IL-28R1 and IL-10Rb promoter of IFN-l1 gene. expression, we determined whether IL-28R1 and IL-10Rb pro- teins were expressed on the cell surface of IL-27–stimulated cells. ERK1/2 and NF-kB are involved in the activation of IFN-l1 Thus, FACS analyses of cell surface expression of IL-28R1 and regulated by IL-27 IL-10Rb proteins were carried out. Flow cytometry results Next, we investigated the molecular mechanism and signaling revealed that IL-28R1 and IL-10Rb proteins were stimulated on pathway involved in the activation of IFN-l1 regulated by IL-27. the cell surface by IL-27 (Fig. 8C). Thus, these results demon- First, the effects of different inhibitors of signaling components strated that IL-27 activates the expression of the IFN-l1 receptors on IL-27–activated IFN-l1 expression were evaluated. Cells were IL-28R1 and IL-10Rb. Taken together, we propose a hypothetical treated with DMSO (negative control), U0126 (ERK1/2 inhibitor), model (Fig. 9) that HBV infection could activate the expression of LY294002 (PI3K inhibitor), H89 (PKA inhibitor), SP600125 IL-27 and IFN-l1, and they inhibit the HBV replication through (JNK inhibitor), or SB203589 (p38 inhibitor). RT-PCR results type I IFN. revealed that IFN-l1 mRNA was upregulated by IL-27, and this activation was not affected by DMSO or LY294002, was slightly Discussion reduced by H89, SP600125, and SB203589, and was significantly Cytokines play a critical role in the control of the innate and adaptive inhibited by U0126 (Fig. 7A). We also noticed that these inhibitors immune responses. Members of the IL-12 cytokine family (IL-12, had no significant effect on cell viability under the experimental IL-23, IL-27, and IL-35) are heterodimeric, which allows unique conditions used (Supplemental Fig. 1B). These results revealed functional interactions not shared by other cytokine families. Despite 700 IL-27 INHIBITS VIRUS REPLICATION BY ACTIVATING IFN-l1 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 7. Determination of signaling pathways involved in the activation of IFN-l1 expression regulated by IL-27. (A) HepG2 cells were pretreated with DMSO (control) or one of the specific kinase inhibitors (ERK inhibitor U0126 [10 mM], PI3K inhibitor LY294002 [25 mM], PKA inhibitor H-89 [10 mM], JNK inhibitor SP600125 [30 mM], or p38 inhibitor SB203580 [10 mM]) for 2 h, and rhIL-27 protein (50 ng/ml) was added to the cell culture medium for 24 h. Cells were harvested, and total mRNA was extracted. IFN-l1 mRNA levels were assessed by real-time PCR. Data are mean 6 SE (n = 3). (B and C) HepG2 cells (left panels) and Huh7 cells (right panels) were transfected with two dominant-negative mutants of ERK1 (mERK1) and ERK2 (mERK2). At 12 h posttransfection, rhIL-27 protein or PBS was added to the cell culture medium at 50 ng/ml for 24 h. IFN-l1 mRNA levels were determined by real- time PCR (B), and protein levels were determined by ELISA (C). Data are mean 6 SE (n = 3). (D) HepG2 cells (upper panel) and Huh7 cells (lower panel) were incubated with rhIL-27 or PBS at 50 ng/ml for different times, as indicated. Proteins were prepared, and the levels of p-ERK1/2, ERK, and b-actin in cell lysates were detected by Western blot using the appropriate Abs. The blot is a representative of three experiments with similar results. (E) HepG2 cells (upper panels) and Huh7 cells (lower panels) were transfected with two dominant-negative mutants of ERK1 (mERK1) and ERK2 (mERK2). At 24 h posttransfection, rhIL-27 protein (50 ng/ml) was added to the cell culture media for 3 h. Nuclear extracts and cytoplasm protein were prepared. NF-kB 65, NF-kB p50, lamin A/C, and GAPDH proteins were detected by Western blot using the corresponding Abs. The blot is a representative of three experiments with similar results. *p , 0.05. many shared structural features and molecular partners, members of Serum IL-27 triggers an earlier immune response to prevent hepatic the IL-12 family mediate surprisingly diverse functions. IL-27 is injury in different clinical-pathologic stages of HBV-infected a heterodimeric anti-inflammatory cytokine composed of EBI3 and patients (50). The receptor complex for IL-27 is composed of the p28, which regulates Th cell function during autoimmune and cytokine receptor IL-27Ra (WSX-1 or TCCR) and gp130 (2). pathogen-induced immune responses (1, 3, 4). IL-27 exhibits vari- We recently reported that IL-27 is elevated in the serum of ous therapeutic capabilities, such as regulating antiviral ability (12, patients infected with IAV and demonstrated that IL-27 is highly 13), enhancing antitumor immune responses (14), directing anti- induced through COX-2– and the PKA-signaling pathways during proliferative effects on tumors (15–17), and reducing the inflam- IAV infection in human lung epithelial cells (A549) and human mation associated with experimental autoimmune arthritis (18, 19). PBMCs infected with IAV and revealed that activated IL-27 The Journal of Immunology 701 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021 FIGURE 8. Determination of the role of IL-27 in the expression of the IFN-l1 receptors IL-28R1 and IL-10Rb.(A and B) HepG2 cells (left panels) and Huh7 cells (right panels) were incubated with rhIL-27 protein or PBS at 50 ng/ml for 24 h. (A) IL-28R1 and IL-10Rb mRNA levels were assessed by real- time PCR. IL-28R1, IL-10Rb, and b-actin protein levels were determined by Western blot (B) and flow cytometry (C). Experiments were performed three times with similar results. Data are mean 6 SE (n = 3). *p , 0.05. inhibits IAV replication by activating STAT1/2 phosphorylation have a similar effect on the activation of STAT1/2. It is known that and PKR phosphorylation (20). We also showed that IL-27 protein IFN-a activates several hundred ISGs through ISRE. Our results levels were elevated in patients with HBV infection and that HBV showed that ISRE activity was stimulated by IL-27, indicating that enhances the expression of IL-27 in several cell types, including IL-27 also plays a positive role in the regulation of ISRE activity. THP-1, HepG2, Huh7, and HEK293T cells (36). A correlation IFN-a is essential for the induction of adaptive immunity against between HBV infection and IL-27 expression was also reported viral infections by stimulating a variety of antiviral proteins and (43). These results suggested that IL-27 plays an important role in cytokines, leading to protection of the host from further viral viral infections. However, the function of IL-27 in HBV replica- infections. In this study, we revealed that the expression of IFN- tion and infection has not been reported. regulated antiviral proteins Mx1, OAS1, and PKR is enhanced by This study explores the effect of IL-27 on HBV replication. Our IL-27 and stimulated by IFN-a. Thus, our data demonstrated that results showed that IL-27 represses the expression of HBV proteins IL-27 displays an IFN-a–like ability to induce the expression of and the replication of HBV capsid–associated DNA in HepG2.2.15, IFN-regulated antiviral proteins. HepG2, and Huh7 cells in a dose-dependent fashion, suggesting that Our results indicated that both IFN-l1 mRNA and IFN-l1 protein IL-27 plays an inhibitory role in HBV replication. IL-27 was re- were significantly upregulated by IL-27, but this activation was ported to exhibit antiviral activity by inhibiting replication of HIV repressed by anti-IFNAR2 Ab, indicating that IFN-a is involved in and HCV (12, 13) and to produce IFN-inducible antiviral proteins the activation of IFN-l1 mediated by IL-27. These results were by eliciting a type I IFN-like response (51, 52), in which IFN-a consistent with previous findings that showed that neutralization of initially binds to the type I IFN receptor, IFNAR1/2, and induces type I IFN Ab blocks the IL-27–induced antiviral state (52). the phosphorylation of STAT1/2 and the expression of ISGs, Chronic viral hepatitis is the leading cause of liver disease and which, in turn, initiate an antiviral state in target cells. may play a role in the pathogenesis of lesions characteristic of After determining that IL-27 inhibits HBV replication, we in- cirrhosis and hepatocellular carcinoma. HBV and HCV are the two vestigated the mechanism involved in such regulation. We revealed major causes of chronic viral hepatitis. Therapeutic IFN-a is used that phosphorylation of STAT1 and STAT2 was stimulated by IL-27 widely to treat HBV and HCV infections with considerable clinical and IFN-a proteins, respectively, suggesting that IL-27 and IFN-a success (38, 53). However, IFN-a is not effective for all infected 702 IL-27 INHIBITS VIRUS REPLICATION BY ACTIVATING IFN-l1

the organization of IFN-l1 regulatory sequences appears to be different from that of IFN-b (26). In this study, we evaluated the effect of IL-27 on the activities of NF-kB and IRF3/7 by assessing the role of IL-27 in the nuclear translocation of NF-kB and IRF3/7. Our results revealed that NF-kB p65, NF-kB p50, and IRF7 proteins are accumulated in the nucleus and decreased in the cytosol in the presence of IL-27, whereas the levels of IRF3 and lamin A proteins are relatively unchanged in the cytosol and the nucleus. We also showed that NF-kBp65andNF-kB p50 were distributed mainly in the cytosol in the absence of IL-27, but they translocated to the nucleus in its presence. In addition, IL-27 enhances the binding of NF-kBtotheIFN-l1promoter. These results demonstrated that IL-27 activates IFN-l1 expression through enhancing nuclear translocation of NF-kB and the binding of this transcription factor to the IFN-l1 promoter and, thus, stim- ulates IFN-l1 expression. To explore the signaling pathways involved in IL-27–mediated IFN-l1 expression, two approaches were used. First, we evaluated

the effects of IL-27–regulated signaling components on IFN-l1 Downloaded from l FIGURE 9. A novel mechanism by which a cytokine limits virus rep- expression. The results showed that IFN- 1expressionisen- lication through regulating a type III IFN in a manner similar to type I IFN. hanced by IL-27; such activation was not affected by DMSO or During HBV infection, the virus activates cytokine (IL-27) expression, LY294002; was slightly reduced by H89, SP600125, and SB203589; which, in turn, stimulates type III IFN (IFN-l1) production through reg- and was significantly repressed by U0126. These results suggested ulating ERK1/2 signaling and enhancing transcription factor (NF-kB) that ERK1/2 is required for the activation of IFN-l1 expression

nuclear translocation and its binding to IFN-l1 promoter. In contrast, IL- regulated by IL-27. Moreover, expression of IFN-l1stimulatedby http://www.jimmunol.org/ 27–activated IFN-l1 upregulates the production of IFN-l1 receptors (IL- IL-27 is blocked by two dominant ERK mutants: mERK1 and 28R1 and IL-10Rb), resulting in activation of the STAT1/2 pathway, mERK2. Second, we determined the effect of IL-27 on ERK1/2 which, in turn, induces the expression of antiviral proteins (PKR, OAS1, phosphorylation and revealed that, in the presence of IL-27, p-ERK and Mx1) by a manner similar to type I IFN and, finally, inhibits HBV is increased, but ERK protein is relatively unchanged. In addition, protein expression and viral DNA replication. nuclear translocation of NF-kB is induced by IL-27, but such regulation is partially blockedbymERK1andmERK2.Thus, individuals (39, 54), and it may trigger some serious side effects, we demonstrated that the ERK-signaling pathway is required for including fatigue, anorexia, fever, depression, and myelosuppres- the activation of IFN-l1 regulated by IL-27. by guest on September 23, 2021 sion (55, 56). Consequently, improved alternative therapies are re- IFN-l1andIFN-a/b share the same JAK/STAT-signaling path- quired for clinical application. Because IL-27 displays IFN-a–like way, which leads to the expression of a common set of genes. activities, IL-27 may be used as a potential antiviral agent. However, IFN-l1 binds to a distinct receptor complex consisting We showed previously that HBVenhances IL-27 expression both of IL-28R1 (IFN-lR1) and IL-10Rb (IFN-lR2). In this study, we in vivo and in vitro (36) and demonstrated that HBV induces a demonstrated that IL-27 activates the expression of IL-28R1 and novel inflammation network, including IFN-l1 (37). However, the IL-10Rb in hepatocytes and suggested that IL-27 plays a role in correlations between IL-27 and IFN-l1 expression and function magnifying the antiviral responsiveness of IFN-l1. have not been studied. In this study, we investigated the expression In conclusion, we revealed that HBV infection activates cytokine status of both IL-27 and IFN-l1 in patients with HBV infection (IL-27) and type III IFN (IFN-l1) expression and demonstrated and evaluated the correlation between IL-27 and IFN-l1 expres- that viral-activated IL-27 and IFN-l1 are coordinated to inhibit sion during HBV infection. Our results showed that p28, EBI3, HBV replication through type I IFN (IFN-a/b). On one hand, and IFN-l1 mRNA levels were significantly higher in PBMCs HBV infection activates IL-27 expression, which, in turn, stim- isolated from patients with CHB compared with healthy individ- ulates IFN-l1 production through regulating ERK1/2 signaling uals. We also revealed that elevated IFN-l1 levels were tightly and enhancing NF-kB nuclear translocation and binding to the correlated with the elevated p28 and EBI3 levels in the PBMCs of IFN-l1 promoter. On the other hand, IL-27–activated IFN-l1 patients with CHB. These results suggested that expression of activates its receptors (IL-28R1 and IL-10Rb), resulting in acti- IL-27 and IFN-l1 was elevated in HBV-infected patients and vation of the STAT1/2 pathway, which, in turn, induces the ex- that IL-27 expression was highly correlated with IFN-l1 expression. pression of antiviral proteins (such as PKR, OAS1, and Mx1) by IFN-l1 is able to inhibit HBV and HCV replication (28, 30). a manner similar to IFN-a/b and, finally, inhibits HBV protein DCs are the major producers of IL-27 (1) and IFN-l1 (25). Be- expression and viral DNA replication. Thus, we identified a pre- cause both IL-27 and IFN-l1 play roles in antiviral responses, we viously unknown mechanism by which a cytokine stimulates an- investigated the correlation of IL-27 and IFN-l1 function. We tiviral responses to limit virus replication through regulating type showed that overexpression of IL-27 stimulates IFN-l1 expression III IFN and type I IFN (Fig. 9). in human immature DCs and hepatoma cells, whereas knockdown of IFN-l1 reduces the IL-27–mediated anti-HBV effect. These Acknowledgments results suggested that the antiviral ability of IL-27 was mediated We thank Dr. Melanie H. Cobb for providing the ERK1 and ERK2 mutants by IFN-l1 to some extent. and Dr. Hongbing Shu for providing pISRE-Luc. The mechanism underlying IL-27–mediated regulation of IFN-l1 expression was evaluated further. It was reported that NF-kBand Disclosures IRF3/7 are required for LPS-induced activation of IFN-l1 and that The authors have no financial conflicts of interest. The Journal of Immunology 703

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