Hepatitis B Induces a Novel Inflammation Network Involving Three Inflammatory Factors, IL-29, IL-8, and Cyclooxygenase-2 This information is current as of September 27, 2021. Yi Yu, Rui Gong, Yongxin Mu, Yanni Chen, Chengliang Zhu, Zhichen Sun, Mingzhou Chen, Yingle Liu, Ying Zhu and Jianguo Wu J Immunol 2011; 187:4844-4860; Prepublished online 28 September 2011; Downloaded from doi: 10.4049/jimmunol.1100998 http://www.jimmunol.org/content/187/9/4844 http://www.jimmunol.org/ References This article cites 55 articles, 29 of which you can access for free at: http://www.jimmunol.org/content/187/9/4844.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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Hepatitis B Virus Induces a Novel Inflammation Network Involving Three Inflammatory Factors, IL-29, IL-8, and Cyclooxygenase-2

Yi Yu,*,†,‡ Rui Gong,*,† Yongxin Mu,*,† Yanni Chen,*,†,‡ Chengliang Zhu,*,† Zhichen Sun,*,† Mingzhou Chen,*,†,‡ Yingle Liu,*,†,‡ Ying Zhu,*,†,‡ and Jianguo Wu*,†,‡

Chronic inflammation induced by (HBV) is a major causative factor associated with the development of cirrhosis and . In this study, we investigated the roles of three inflammatory factors, IL-8, IL-29 (or IFN-l1), and cyclooxygenase-2 (COX-2), in HBV infection. We showed that the expression of IL-29, IL-8, and COX-2 was enhanced in HBV-infected patients or in HBV-expressing cells. In HBV-transfected human lymphocytes and hepatocytes, IL-29 activates the

production of IL-8, which in turn enhances the expression of COX-2. In addition, COX-2 decreases the production of IL-8, which Downloaded from in turn attenuates the expression of IL-29. Thus, we proposed that HBV infection induces a novel inflammation network involving three inflammatory factors that regulate each other in the order IL-29/IL-8/COX-2, which involves positive regulation and negative feedback. In addition, we also demonstrated that COX-2 expression activated by IL-8 was mediated through CREB and C/EBP, which maintains the inflammatory environment associated with HBV infection. Finally, we showed that the ERK and the JNK signaling pathways were cooperatively involved in the regulation of COX-2. We also demonstrated that IL-29 inhibits

HBV replication and that IL-8 attenuates the expression of IL-10R2 and the anti-HBV activity of IL-29, which favors the http://www.jimmunol.org/ establishment of persistent viral infection. These new findings provide insights for our understanding of the mechanism by which inflammatory factors regulate each other in response to HBV infection. The Journal of Immunology, 2011, 187: 4844–4860.

epatitis B virus (HBV) is a hepatotropic, non-cytopathic levels, effects on cellular pathways, and genetic imbalances. The virus that can cause hepatitis, cirrhosis, and hepatocel- role of any given cytokine may be specific or overlapping to H lular carcinoma (HCC). Currently, ∼350 million people generate downstream effects that result from a maze of complex worldwide are chronically infected with HBV (1–3). HBV is interactions (4). considered to be one of the major causative factors associated IL-8 is a 71-aa belonging to the CXC chemokine with the development of HCC, particularly as a result of its induc- family. It is produced by many cell types, including , by guest on September 27, 2021 tion of chronic inflammation. are critical components epithelial cells, fibroblasts, and hepatocytes. It has been dem- of the immune system and play a significant role against HCC onstrated that IL-8 elicits many effects, including neutrophil, development, as evidenced by the changes in their expression T lymphocyte, and basophil chemotaxis, degranulation, oxidative bursts, and lysosomal-enzyme release (5). Furthermore, IL-8 has been shown to be an important mediator of the inflammatory *State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072, People’s Republic of China; †Chinese-French Liver Disease responses to many and bacteria. For example, IL-8 is in- Research Institute, Zhongnan Hospital, Wuhan University, Wuhan, Hubei 430072, duced in response to expression of the NS5A protein of the People’s Republic of China; and ‡Wuhan Institutes of Biotechnology, Wuhan, Hubei 430075, People’s Republic of China (HCV) (6–8) and replication of HCV (9). The X protein of HBV (HBx) transactivates IL-8 expression Received for publication April 6, 2011. Accepted for publication August 25, 2011. through NF-kB and C/EBP-like cis-elements (10). Moreover, IL-8 This work was supported by research grants from the National Mega Project on Major Infectious Diseases Prevention (2012ZX10002006 and 2012ZX10004207), level in patients with chronic hepatitis B was significantly higher the National Mega Project on Major Drug Development (2009ZX09301-014 and than that in healthy controls, and large fluctuations in IL-8 con- 2011ZX09401-302), the Major State Basic Research Development Program (973 centrations in sera of patients in association with the hepatic flares Program; 2007CB512803 and 2012CB518900), the National Natural Science Foun- dation of China (30730001 and 81171525), the Key Project of the Chinese Ministry were observed in a previous study (11). IL-8 increased with ex- of Education (204114208), the Department of Science Technology of Hubei Province acerbation of liver damage, reached the peak when the liver (2005ABC003), the Science and Technology Programs of Wuhan (200760323102), the Fundamental Research Funds for the Central Universities (1102001), and the damage was most severe, and decreased when patient conditions Specialized Research Fund for the Doctoral Program of Higher Education improved (12). Indeed, IL-8 is commonly described as a leukocyte (20090141110033). chemotactic molecule that is responsible for maintaining the in- Address correspondence and reprint requests to Prof. Jianguo Wu, State Key Labo- flammatory environment associated with HBV infection and that ratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072, People’s Republic of China. E-mail address: [email protected] may play a role in the development of (13). Cyclooxygenase (COX) is the rate-limiting enzyme in the bio- Abbreviations used in this article: ATF4, activating transcription factor 4; COX, cyclooxygenase; COX-1, cyclooxygenase-1; COX-2, cyclooxygenase-2; CRE, cAMP synthetic pathway of PGs and thromboxanes from arachidonic response element; DC, dendritic cell; HBV, hepatitis B virus; HBx, X protein of acid. PGs play important roles in many biological processes. HBV; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; IRF3, IFN regulatory factor 3; IRF7, IFN regulatory factor 7; ISRE, IFN stimulation response element; LIP, Changes in the production of prostanoids, potent lipid in- liver-enriched inhibitory protein; mCREB, mutant of CREB; 2959OAS, 29-59-oligoa- flammatory mediators, are associated with a variety of illnesses, denylate synthetase; PKR, dsRNA-activated protein kinase; RNAi, RNA interference; including acute and chronic inflammation, cardiovascular disease, shRNA, short hairpin RNA; siRNA, small interfering RNA. and colon cancer (14). Two COX isoforms have been discovered: Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100998 The Journal of Immunology 4845

COX-1 is constitutively expressed in almost all human tissues (15, donation center. The collection of blood samples for research was ap- 16). COX-2 is the inducible form of the enzyme, and its expres- proved by the Institutional Review Board of the College of Life Sciences, sion is significantly activated by inflammatory stimuli, resulting in Wuhan University, in accordance with guidelines for the protection of human subjects. Written informed consent was obtained from each par- increasing synthesis of prostanoids in inflamed tissues. Accumu- ticipant. lated evidence has shown that viral proteins can stimulate COX-2 expression; for example, latent membrane protein 1 of the EBV Isolation of PBMCs (17), core and NS5A proteins of HCV (18), and HBx (19). COX-2 PBMCs were obtained by density centrifugation of blood samples diluted is overexpressed in liver cirrhosis, contributing to PG overpro- 1:1 in pyrogen-free saline over Histopaque (Haoyang Biotech). Cells were duction, which may be a major component of the inflammation washed twice in saline and suspended in culture medium (RPMI 1640) m and hyperdynamic circulation associated with HCC development supplemented with penicillin (100 U/ml) and streptomycin (100 g/ml). in cirrhosis (20). Moreover, COX-2 and its downstream PG re- Generation of human immature dendritic cells ceptor EP1-mediated signaling pathway accelerate LPS-induced liver injury (21). Both IL-8 and COX-2 are stimulated by HBV Monocytes were isolated from PBMCs by adhesion to plastic dishes for .2 h at 37˚C as previously described (29). Immature dendritic cells (DCs) proteins and associated with inflammatory processes. We need to were generated from monocytes by culturing in RPMI 1640 medium elucidate whether there is a connection between these two proteins containing 10% FBS, 1000 U/ml GM-CSF, 500 U/ml IL-4 (R&D Systems, in the host inflammatory response to HBV infection. Minneapolis, MN), and antibiotics for 7 d. Medium was changed 2, 4, and IL-29 belongs to the IFN-l gene family and is composed of 6 d after culturing. l l three distinct genes: IFN- 1 (IL-29), IFN- 2 (IL-28A), and IFN- Reagents l3 (IL-28B) (22, 23). The IFN-l receptor complex consists of the Downloaded from unique ligand-binding chain IFN-lR, also known as IL-28R, and Kinase inhibitors NS398, U0126, SB203580, SP600125, and GF109203 the accessory receptor chain IL-10R2. Although almost any cell were purchased from Sigma Chemical Company (St. Louis, MO). All l protein kinase inhibitors were dissolved in DMSO and used at a final type is able to express IFN- after viral infection, dendritic cells concentration of 10 mM for NS398, U0126, and SB203580, 30 mM for appear to be major producers of IFN-l (24). IFN-lRs are ex- SP600125, and 50 mM for GF109203. Escherichia coli LPS (No. L-2880; pressed at variable levels in most cell types. Furthermore, IFN-l Sigma) dissolved in PBS was added at a final concentration of 1 mg/ml. Recombinant human IL-29 was purchased from eBioscience (San Diego, was shown to inhibit the replication of a number of viruses, in- http://www.jimmunol.org/ CA), and recombinant human IL-8 was purchased from R&D Systems. cluding vesicular stomatitis virus, encephalomyocarditis virus, Ab against COX-2 was purchased from Cayman Chemical (Ann Arbor, HCV, and HBV (25). Previous investigations have demonstrated MI). Abs specific for b-actin, ERK, phospho-ERK, phospho-JNK, IFN elevated serum levels of IL-8 in patients with HCV infection and regulatory factor 3 (IRF3), IFN regulatory factor 7 (IRF7), IL-10R2, partial inhibition of IL-8 on the antiviral actions of IFN-a in vitro dsRNA-activated protein kinase (PKR), 29-59-oligoadenylate synthetase 9 9 (26). However, the effect of IL-8 on the antiviral activity of IFN-l (2 5 OAS), CREB, and C/EBP were purchased from Santa Cruz Bio- technology (Santa Cruz, CA). Ab specific for JNK was purchased from is still unclear. Technology (Beverly, MA). Ab against IL-29 was obtained Our previous studies have shown that influenza A virus infection from R&D Systems. activates IL-32, inducible NO synthase, and COX-2 expression by a complex mechanism, in which the three proinflammatory fac- Cell culture by guest on September 27, 2021 tors regulate each other. This prompted us to investigate the in- The human hepatoma cell lines HepG2 and Huh7 were grown in DMEM flammatory cytokine network induced by viral infection and aroused supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, and m our curiosity regarding the situation of HBV infection (27, 28). 100 g/ml streptomycin sulfate at 37˚C with 5% carbon dioxide. The HepG2.2.15 cell line was derived from HepG2 cells and stably expresses Because IL-8 is a key mediator in liver inflammation associated HBV (ayw) and was maintained in DMEM containing 400 mg/ml G418. with HBV infection, the aim of this study was to investigate the role of IL-8 in the inflammatory response to HBV infection and identify Plasmid construction the related molecular mechanism. In this study, we demonstrated HBV-1.2, a plasmid carrying a greater-than-unit-length (129%) HBV ge- that expression levels of IL-29, IL-8, and COX-2 were elevated in nome (payw1.2; subtype ayw) and its control vector were described pre- HBV patients and HBV-transfected hepatoma cells. In addition, we viously (30, 31) and were obtained from Dr. Robert Schneider (New York University Medical Center). The IFN stimulation response element (ISRE) revealed a temporal correlation between HBV protein levels and luciferase reporter plasmid was a gift from Dr. Hongbing Shu (Wuhan IL-29, IL-8, and COX-2 levels. Because IL-29, IL-8, and COX-2 University). The promoter regions of human IL-29 and IL-8 were ampli- are all involved in regulation of inflammation, the question arises fied by genomic PCR: IL-29 sense, 59-GCACGCGTCAATCCAAGATGA- whether they are independent or not in the host inflammatory TGTG-39; IL-29 antisense, 59-TAGGTACCGACTAGATAGATGAGAG- 9 9 9 response to viral infection. Our results show that HBV infection 3 . IL-8 sense, 5 -ATCTCGAGGCTCTGCTGTCTCTGAA-3 ; IL-8 anti- sense, 59-TAGGTACCAACCCAGGCATTATTTT-39. activates the expression of IL-29, IL-8, and COX-2 by an un- The PCR fragment was inserted into the MluI-KpnI and KpnI-XhoI sites of recognized mechanism, in which these inflammatory factors reg- the luciferase vector [pGL3-IL-29(21806/+38)-Luc and pGL3-IL-8(21534/ ulate each other in the order IL-29/IL-8/COX-2 with positive 227)-Luc, respectively]. The luciferase reporter vector (pGL3) containing regulation and negative feedback. Furthermore, we investigated a COX-2 promoter region (2891/+9) and its site-specific mutants were reported previously (32). pcDNA-CREB-1–dominant negative mutant of the molecular mechanism that underlines the effects of IL-8 on CREB (mCREB) expresses CREB-1 dominant-negative mutant (S133A), as the regulation of IL-29 and COX-2, respectively. described previously (33, 34). Construction of the plasmid expressing the mutant of C/EBPb (liver-enriched inhibitory protein; LIP) was described Materials and Methods previously (35). Mutants of ERK1 and ERK2 were gifts from Dr. Melanie H. Clinical samples Cobb (University of Texas Southwestern Medical Center, Dallas, TX), and mutants of JNK were gifts from Dr. Michael Karin (University of California Peripheral blood samples were obtained from 20 patients (13 males and 7 at San Diego, San Diego, CA). The plasmid expressing the IL-29 gene, pEF- females with mean age 40.3 6 12.7 y) with chronic hepatitis B admitted to SPFL-IL-29, and its vector plasmid pEF-SPFL were obtained from Dr. RenMin Hospital (Wuhan University). All patients were confirmed HBV Sergei V. Kotenko (University of Medicine and Dentistry of New Jersey). positive (but negative for HCV, HDV, and HIV), not suffering from any An IL-8 construct was created by RT-PCR amplification of the open reading concomitant illness, and did not show any serological markers suggestive frame from human HepG2 hepatoma cells. To create the IL-8–encoding of . To match for sex and age, 20 healthy individuals vector, the IL-8 gene was amplified using the primers IL-8 sense 59- (16 males and 4 females with mean age 42.8 6 11.5 y) with no history of TTGGATCCATGACTTCCAAGCTGGCCGTGGCTC-39 and IL-8 anti- liver disease were randomly selected as controls from the local blood sense 59-GGCTCGAGTTATGAATTCTCAGCCCTCTTCAAA-39, in which 4846 HBV INDUCES A NOVEL INFLAMMATORY NETWORK

BamHI and XhoI sites were introduced, respectively. The PCR product was AGCTGGAA-39; IRF7 antisense, 59-GATGTCGTCATAGAGGCTGTT- cloned into BamHI and XhoI sites of pCMV-tag2B to generate the plasmid GG-39. PKR sense, 59-AAAGCGAACAAGGAGTAAG-39; PKR antisense, pCMV-tag2B-IL-8, in which IL-8 was tagged with FLAG. The resulting 59-GATGATGCCATCCCGTAG-39.2959OAS sense, 59-ACCCAACCAA- construct was confirmed by DNA sequencing. CREB, activating tran- TAATGTGAG-39;2959OAS antisense, 59-AGGAATGTACGGATGATGT- scription factor 4 (ATF4), C/EBPa, and C/EBPb short hairpin RNA 39. GAPDH sense, 59-AAGGCTGTGGGCAAGG-39; GAPDH antisense, (shRNA) plasmids were constructed by ligating the corresponding pairs of 59-TGGAGGAGTGGGTGTCG-39. oligonucleotide (Invitrogen) to pSilencerTM 2.1-U6 neo (Ambion, Austin, Data were normalized by the level of GAPDH expression in each sample TX). The sequences of 64-nt primers were as follows: CREB sense, 59- as described earlier. GATCCGGAGTCAGTGGATAGTGTATTCAAGAGATACACTATCCAC- 9 9 TGACTCCTTTTTTGGAAA-3 ; CREB antisense, 5 -AGCTTTTCCAA- Assay for HBV protein expression AAAAGGAGTCAGTGGATAGTGTATCTCTTGAATACACTATCCACT- GACTCCG-39. ATF4 sense, 59-GATCCGCACTTCAAACCTCATGGG- Forty-eight hours postinfection, levels of HBsAg and HBeAg proteins in TTCAAGAGACCCATGAGGTTTGAAGTGCTTTTTTGGAAA-39; ATF4 cell culture media were determined by ELISA using an HBV HBsAg Ag and antisense, 59-AGCTTTTCCAAAAAAGCACTTCAAACCTCATGGGTC- HBeAg Ag diagnostic kit (Shanghai KeHua Biotech), respectively. TCTTGAACCCATGAGGTTTGAAGTGCG-39. C/EBPa sense, 59-GAT- CCGAAGTCGGTGGACAAGAACTTCAAGAGAGTTCTTGTCCAC- CGACTTCTTTTTTGGAAA-39; C/EBPa antisense, 59-AGCTTTTCC- Analysis of HBV DNA by real-time PCR AAAAAAGAAGTCGGTGGACAAGAACTCTCTTGAAGTTCTTGTC- Capsid-associated DNA was extracted as described previously, with CACCGACTTCG-39. C/EBPb sense, 59-GATCCGGCCAACTTCTAC- modifications. Equivalent amounts of HepG2 cells were homogenized in 1 TACGAGTTCAAGAGACTCGTAGTAGAAGTTGGCCTTTTTTGGAA- ml lysis buffer (50 mM Tris, pH 7.5, 0.5% Nonidet P-40, 1 mM EDTA, and A-39; C/EBPb antisense, 59-AGCTTTTCCAAAAAAGGCCAACTTCT- 100 mM NaCl) and mixed gently at 4˚C for 1 h. Next, 10 ml 1 M MgCl2 and ACTACGAGTCTCTTGAACTCGTAGTAGAAGTTGGCCG-39. 10 ml DNase (10 mg/ml) were added and incubated for 2 h at 37˚C. Viral IL-8 small interfering RNA (siRNA) and negative control were syn- cores were then precipitated by adding 35 ml (0.5 M) EDTA and 225 ml Downloaded from thesized by RiBo Biotech (GuangZhou RiBo Biotech) based on the se- 35% polyethylene glycol and incubating them at 4˚C for at least 30 min, quence described previously (36). IL-29 shRNA, its negative control after which the cores were concentrated by centrifugation and the pellet (shRNA-control) and positive control (shRNA-GAPDH) were from Gen- was resuspended in 10 mM Tris, 100 mM NaCl, 1 mM EDTA, 1% SDS, ePharma (Shanghai GenePharma) and prepared by ligation of the corre- and 20 ml proteinase K (25 mg/ml) and incubated overnight. Viral DNA sponding pairs of oligonucleotides to PGPU6/GFP/Neo. The shRNA-IL-29 released from lysed cores was extracted with phenol and chloroform, target sequence was 59-GCCACATTGGCAGGTTCAAAT-39, the shRNA- precipitated with isopropanol, and resuspended in Tris-EDTA. control target sequence was 59-GTTCTCCGAACGTGTCACGT-39, and

Resuspended capsid-associated HBV DNAs were quantified by real-time http://www.jimmunol.org/ the shRNA-GAPDH target sequence was 59-GTATGACAACAGCCTC- PCR as described by the manufacturer (PG Biotech, Shenzhen, China). AAG-39. Primers used in real-time PCR were as follows: P1, 59-ATCCTG- CTGCTATGCCTCATCTT-39; and P2, 59-ACAGTGGGGAAAGCCC- Luciferase assay TACGAA-39. The probe was 59-TGGCTAGTTTACTAGTGCCATTTTG- 9 HepG2 cells were cotransfected with reporter plasmids and their corre- 3 . PCR was carried out and analyzed using a Roche LC480. sponding expression plasmids. Cells were lysed with luciferase cell culture lysis reagent (Promega, Madison, WI). Cell lysates and luciferase assay substrate (Promega) were mixed, and the light intensity was detected by a luminometer (Turner T20/20). Assays were performed in triplicate and expressed as means relative to the vector control (100%). by guest on September 27, 2021 ELISA HepG2 cells were transfected with HBV-1.2. Culture supernatants were harvested at 6, 12, 24, 36, 48, and 72 h posttransfection. For the quanti- fication of IL-29 and IL-8 released in culture supernatants, a Human IL-29 Platinum ELISA Kit (eBioscience) and a Human IL-8 Instant Elisa Kit (eBioscience) were used according to the manufacturer’s instructions. PGE2 level was assayed with the Biotrak PG E2 Enzyme Immunoassay system (R&D Systems) according to the manufacturer’s protocol. Semiquantitative RT-PCR analysis Total RNA was isolated from HepG2 cells using the TRIzol reagent (Invitrogen, Carlsbad, CA); it was treated with DNase I and reverse- transcribed with MLV reverse transcriptase (Promega) and random pri- mers (Takara). PCR was performed in 25-ml reactions with the following detection primer pairs: IL-8 sense, 59-ATGACTTCCAAGCTGGCCGT- GCT-39; IL-8 antisense, 59-TCTCAGCCCTCTTCAAAAACTTCTC-39. IL-10R2 sense, 59-GGCTGAATTTGCAGATGAGCA-39; IL-10R2 anti- sense, 59-GAAGACCGAGGCCATGAGG-39. Detection primers for COX-2 and b-actin were described previously (37). b-Actin was amplified by PCR for normalization in all. Real-time PCR Total RNAwas extracted with the TRIzol reagent (Invitrogen) following the manufacturer’s instructions. Real-time quantitative RT-PCR analysis was performed using the Roche LC480 and SYBR RT-PCR kits (DBI Bio- science) in a reaction mixture of 20 ml containing 0.5 mM of each PCR primer, 10 ml of SYBR Green PCR master mix, 1 ml of DNA diluted FIGURE 1. Analysis and comparison of the mRNA levels of IL-29, m template, and RNase-free water to complete the 20 l volume. Real-time IL-8, and COX-2 expressed in HBV patients and healthy individuals. primers were as follows: IL-29 sense, 59-TTCCAAGCCCACCACAAC-39; Peripheral blood samples were obtained from 20 patients with chronic IL-29 antisense, 59-TCCCTCACCTGGAGAAGC-39.IL-8sense,59- CATACTCCAAACCTTTCCACCCC-39; IL-8 antisense, 59-TCAGCC- hepatitis B and 20 healthy individuals. The PBMCs were isolated from the CTCTTCAAAAACTTCTCCA-39. COX-2 sense, 59-TGCATTCTTTGC- peripheral blood samples and lysed. IL-29 (A), IL-8 (B), and COX-2 (C) CCAGCACT-39; COX-2 antisense, 59-AAAGGCGCAGTTTACGCTGT- mRNA was detected, respectively, from total RNA extracted from the 39. IRF3 sense, 59-ACCAGCCGTGGACCAAGAG-39; IRF3 antisense, 59- PBMC lysates by real-time PCR. Light gray, healthy individuals; dark TACCAAGGCCCTGAGGCAC-39. IRF7 sense, 59-TGGTCCTGGTGA- gray, HBV patients. The Journal of Immunology 4847

Nuclear extraction Results Cells were incubated in serum-free media for 24h, washed twice with cold IL-29, IL-8, and COX-2 expression was upregulated in patients PBS, and scraped into 1 ml cold PBS. Cells were harvested by centrifu- with HBV infection gation (15 s) and incubated in two packed cell volumes of buffer A (10 mM To determine the effects of HBV infection on the expression of HEPES, pH 8, 0.5% Nonidet P-40, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and 200 mM sucrose) for 5 min at 4˚C with flipping of the tube. The inflammatory factors, we measured and compared IL-29, IL-8, and crude nuclei were collected by centrifugation (30 s); pellets were rinsed COX-2 mRNA levels in PBMCs of 20 patients with chronic with buffer A, resuspended in one packed cell volume of buffer B (20 mM hepatitis B and 20 healthy individuals by real-time PCR. Results HEPES, pH 7.9, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, and 1.0 showed that IL-29 mRNA levels were significantly higher in mM DTT), and incubated on a shaking platform for 30 min at 4˚C. Nuclei were centrifuged (5 min), and supernatants were diluted 1:1 with buffer C patients with chronic hepatitis B than those in healthy individuals (20 mM HEPES, pH 7.9, 100 mM KCl, 0.2 mM EDTA, 20% glycerol, and (means 6 SEM: 0.05085 6 0.02616 versus 0.21433 6 0.20918, 1 mM DTT). Cocktail protease inhibitor tablets were added to each type of p , 0.01) (Fig. 1A). IL-8 mRNA levels were also much higher in buffer. Nuclear extracts were snap-frozen in liquid nitrogen and stored at patients than in healthy individuals (means 6 SEM: 0.9405 6 270˚C until use. 0.43984 versus 2.06597 6 0.96463, p , 0.001) (Fig. 1B). COX-2 Western blot analysis mRNA levels were higher in patients than in healthy individuals 6 6 6 , Whole-cell lysates were prepared by lysing cells with PBS, pH 7.4, (means SEM: 2.045 0.6266 versus 4.414 2.87483, p containing 0.01% Triton X-100, 0.01% EDTA, and 10% mixture pro- 0.001) (Fig. 1C). These results suggested that HBV infection may tease inhibitor (Roche). Protein concentration was determined by the result in the upregulation of IL-29, IL-8, and COX-2 expression. Bradford assay (Bio-Rad). Cultured cell lysates (100 mg) were elec- trophoresed in 12% SDS-PAGE gel and transferred to a nitrocellulose HBV activates the expression of IL-29, IL-8, and COX-2 and Downloaded from membrane (Amersham). Nonspecific sites were blocked with 5% nonfat the production of PGE2 dried milk before being incubated with an Ab used in this study. Protein bands were detected using SuperSignal Chemiluminescent (Pierce, To verify the above results from clinical analysis, we further de- Rockford, IL). termined and compared IL-29, IL-8, COX-2, and PGE2 levels in human hepatoma HepG2.2.15 cells that carry an integrated HBV Statistical analysis genome and human hepatoma HepG2 cells without the HBV ge- All experiments were reproducible and were carried out in duplicate or nome. Results from real-time PCR analyses showed that relative http://www.jimmunol.org/ quadruplicate. Each set of experiments was repeated at least three times mRNA levels of IL-29, IL-8, and COX-2 were much higher in with similar results, and representative experiments are shown. The results are presented as means. Student t test for paired samples was used to HepG2.2.15 cells than those in HepG2 cells (Fig. 2A). The pro- determine statistical significance. Differences were considered statistically duction of IL-29, IL-8, and PGE2 was also upregulated in culture significant at a p value # 0.05. supernatants of HepG2.2.15 cells compared with that in culture by guest on September 27, 2021

FIGURE 2. Determination of the effect of HBV on the expression of IL-29, IL-8, and COX-2 and the production of PGE2 in cultured cells. A, HepG2 or HepG2.2.15 cells were serum-starved for 24 h and then lysed. IL-29, IL-8, and COX-2 mRNA in the cell lysates were detected by real-time PCR. B, HepG2 or HepG2.2.15 cells were serum-starved for 24 h. IL-29,

IL-8, and PGE2 proteins in the culture supernatants were detected by ELISA. C, HepG2 or HepG2.2.15 cells were serum-starved for 24 h and then lysed. COX- 2 and b-actin proteins in the cell lysates were detected by Western blot. Data shown are mean 6 SE; n =3. *p , 0.05. D, HepG2 cells (left panel) or Huh7 cells (right panel) were cotransfected with reporters, pGL3- IL-29-Luc, pGL3-IL-8-Luc, or pGL3-COX-2-Luc, and plasmid pHBV-1.2 or its control. Activities of IL-29, IL-8, and COX-2 promoters in the transfected cells were determined by luciferase activity assays. Data shown are mean 6 SE; n =3.*p , 0.05. E, HepG2 cells (left panel) and Huh7 cells (right panel) were transfected with pHBV-1.2 and harvested at indicated time points. IL-29, IL-8, and PGE2 proteins in the culture supernatants were determined by ELISA. F, HepG2 cells (left panel) and Huh7 cells (right panel) were transfected with pHBV-1.2 for different times as indicated. LPS 100 ng/ml was added as positive con- trol. Proteins were prepared from transfected cells and used for Western blot using Ab to COX-2 or b-actin, respectively. 4848 HBV INDUCES A NOVEL INFLAMMATORY NETWORK

supernatants of HepG2 cells (Fig. 2B). Western blot analyses also The levels of PGE2 were accumulated as transfection time in- showed that COX-2 protein level was increased in HepG2.2.15 creased in both cells (Fig. 2E). In addition, the expression of cells (Fig. 2C). COX-2 protein was examined in cell lysates by Western blot To determine whether HBV directly activates the expression of analyses, which showed that COX-2 protein levels increased as the IL-29, IL-8, and COX-2, we performed an additional experiment. transfection time increased and reached in both HepG2 (Fig. 2F, HepG2 cells and Huh7 cells were cotransfected with plasmid left panel) and Huh7 (Fig. 2F, right panel) cells. These results pHBV-1.2 (which carries 1.2-fold length of the HBV genome and suggested that HBV activates IL-29, IL-8, and COX-2 expression has the ability to produce mature HBV virions) or its parental and PGE2 production. plasmid pRL-TK without the HBV genome, along with the reporter plasmids pGL3-IL-29-Luc, pGL3-IL-8-Luc, or pGL3-COX-2-Luc, IL-29 stimulates IL-8 expression in human immature DCs and respectively. Results from luciferase activity analyses indicated hepatoma cells that IL-29, IL-8, and COX-2 promoter activities were stimulated in IL-29 is one of several novel IL-10–related cytokines that have the presence of HBV in both hepatoma cells, HepG2 (Fig. 2D, left recently been discovered. Proinflammatory functions for IL-10– panel) and Huh7 (Fig. 2D, right panel). related cytokines, such as IL-22, have been investigated (38). The effects of HBV infection on the expression of IL-29, IL-8, Previous studies have shown that IL-29, when binding to its receptor and COX-2 proteins or the production of PGE2 were further complex, activates ERK-1/2, stress-activated protein kinase/JNK assessed. HepG2 and Huh7 cells were transfected with pHBV-1.2. MAPKs, and Akt, resulting in the activation of IL-8 protein The culture supernatants and the cell pellets of transfected cells expression in intestinal epithelial cell line HT-299 (39). were harvested at 12, 24, 36, 48, and 72 h posttransfection. The To identify the role of IL-29 in the expression of the proin- Downloaded from production of IL-29, IL-8, and PGE2 was measured in culture flammatory factor IL-8, human blood -derived DCs were supernatants. Results showed that the level of IL-29 protein in- stimulated with recombinant human IL-29 protein at different creased as transfection time increased and reached the peak at 12 h concentrations for 24 h. IL-8 mRNA and protein was detected by posttransfection in HepG2 cells (Fig. 2E, left panel) and at 24 h real-time PCR and ELISA, respectively. Results showed that IL-8 posttransfection in Huh7 cells (Fig. 2E, right panel). The level of mRNA (Fig. 3A) and protein (Fig. 3B) levels were upregulated by

IL-8 protein increased as transfection time increased and reached IL-29 protein in a dose-dependent manner. Similar results were http://www.jimmunol.org/ a peak at 36 h posttransfection in HepG2 cells (Fig. 2E, left panel) also obtained in two hepatoma cell lines, HepG2 and Huh7. Cells and at 48 h posttransfection in Huh7 cells (Fig. 2E, right panel). were treated with recombinant human IL-29 protein at different

FIGURE 3. Determination of the role of IL- by guest on September 27, 2021 29 in the regulation of IL-8 expression in blood monocyte-derived DCs and hepatoma cells. A and B, DCs were generated from monocytes, which were isolated from PBMCs, and then in- cubated with recombinant human IL-29 protein at different concentrations as indicated for 24 h. Cell lysates and culture supernatants were pre- pared and collected. IL-8 mRNA (A) and protein (B) were detected by real-time PCR and ELISA, respectively. C and D, HepG2 cells (left panels) or Huh7 cells (right panels) were incubated with recombinant human IL-29 protein at different concentrations as indicated for 24 h. Cell lysates and culture supernatants were prepared and collected. IL-8 mRNA (C) and protein (D) were detected by real-time PCR and ELISA, re- spectively. E, HepG2 cells (left panel) or Huh7 cells (right panel) were cotransfected with re- porter pGL3-IL-8-Luc and pEF-SPFL-IL-29 or pEF-SPFL. IL-8 promoter activity was de- termined by luciferase activity assay at 48 h posttransfection. Data shown are mean 6 SE; n =3.*p , 0.05. F, HepG2 or Huh7 cells were transfected with pEF-SPFL-IL-29 at different concentrations as indicated. IL-8 protein (left and middle panels) and IL-29 protein (right panel) in culture supernatants were detected by ELISA at 48 h posttransfection, respectively. Data shown are mean 6 SE; n =3.*p , 0.05. The Journal of Immunology 4849 concentrations for 24 h. Results from real-time PCR analyses The effects of shRNA-IL-29 on the activity of IL-8 gene pro- showed that relative IL-8 mRNA levels were increased as the moter and the expression of IL-8 protein in HBV-transfected concentrations of IL-29 increased in both HepG2 cells (Fig. 3C, HepG2 and Huh7 cells were further investigated. Cells were left panel) and Huh7 cells (Fig. 3C, right panel). In an add-on, cotransfected with pHBV-1.2, pGL3-IL-8-Luc, and shRNA-IL-29 ELISA results indicated that IL-8 protein levels were increased as or shRNA-control. Luciferase activity assays showed that IL-8 the concentrations of IL-29 increased in both HepG2 cells (Fig. promoter activity was decreased in HepG2 cells (Fig. 4B, left 3D, left panel) and Huh7 cells (Fig. 3D, right panel). panel) and Huh7 cells (Fig. 4B, right panel) in the presence of To confirm further the effect of IL-29 on IL-8 expression, we shRNA-IL-29. ELISA results indicated IL-8 protein level was also tested the role of IL-29 in the regulation of IL-8 gene promoter reduced in the presence of shRNA-IL-29 in both HepG2 and Huh7 activity and protein expression in human hepatoma cells. HepG2 cells (Fig. 4C). These results suggested that activation of IL-8 and Huh7 cells were cotransfected with the reporter plasmid pGL3- regulated by HBV may require IL-29. IL-8-Luc and pEF-SPFL-IL-29 or its control pEF-SPFL. Results IL-8 inhibits IL-29 expression during HBV infection showed that IL-8 promoter was activated in the presence of IL-29 in HepG2 cells (Fig. 3E, left panel) and Huh7 cells (Fig. 3E, right We also investigated the role of IL-8 in the regulation of IL-29 panel). The effect of IL-29 on the expression of IL-8 protein was expression mediated by HBV. HepG2 and Huh7 cells were then investigated in cells transfected with pEF-SPFL-IL-29 at treated by recombinant human IL-8 protein at different concen- different concentrations. ELISA results revealed that IL-8 protein trations. Results from RT-PCR showed that IL-29 mRNA levels levels were increased as IL-29 concentrations increased in the were reduced as the concentrations of IL-8 increased in both supernatants of transfected HepG2 and Huh7 cells (Fig. 3F). HepG2 cells (Fig. 5A, left panel) and in Huh7 cells (Fig. 5A, right Downloaded from panel). Similarly, IL-29 protein levels were also decreased when HBV activates IL-8 expression through IL-29 the concentrations of IL-8 protein increased in both cell types The effect of IL-29 on the expression of IL-8 in hepatoma cells was (Fig. 5B). These results suggest that IL-8 represses the expression further evaluated using an additional approach, RNA interfer- of IL-29 mRNA and protein. ence (RNAi). Cells were transfected with shRNA-control, shRNA- To determine the effect of IL-8 on the activity of IL-29 pro-

GAPDH, and shRNA-IL-29, respectively. ELISA results showed moter in the presence of HBV, HepG2 and Huh7 cells were http://www.jimmunol.org/ that IL-29 protein released in the supernatants of transfected cells cotransfected with pCMV-tag2B-IL-8 at different concentrations, was reduced by treatment with shRNA-IL-29 but not affected pHBV-1.2, and pGL3-IL-29-Luc. Results from the luciferase ac- by shRNA-control or shRNA-GAPDH (Fig. 4A, top panel), in- tivity assay demonstrated that the activity of IL-29 promoter was dicating shRNA-IL-29 was specific and effective. In addition, we decreased in both HepG2 cells (Fig. 5C, left panel) and Huh7 cells showed that GAPDH protein level was decreased in the presence (Fig. 5C, middle panel) as the concentration of IL-8 increased of shRNA-GAPDH (Fig. 4A, bottom panel). (Fig. 5C, right panel). Similarly, the effect of IL-8 on the expression by guest on September 27, 2021

FIGURE 4. Analysis of the role of IL-29 in the regulation of IL-8 expression mediated by HBV. A, Determination of the effectiveness and specificity of shRNA-IL-29 on IL-29 . HepG2 cells were transfected with shRNA-control, shRNA- GAPDH, and shRNA-IL-29, respectively. IL-29 pro- tein in culture supernatants was detected by ELISA (top panel). GAPDH in cell lysates was detected by Western blot (bottom panel). B, HepG2 cells (left panel) or Huh7 cells (right panel) were cotransfected with pGL3-IL-8-Luc, shRNA-IL-29, or shRNA-con- trol, and pHBV-1.2. Luciferase activity was measured at 48 h posttransfection. Data shown are mean 6 SE; n =3.*p , 0.05. C, HepG2 cells (left panel) or Huh7 cells (right panel) were transfected with shRNA-IL-29 at different concentrations as indicated, and pHBV-1.2. IL-8 protein released in the culture supernatants was measured by ELISA at 48 h posttransfection. Data shown are mean 6 SE; n =3.*p , 0.05. 4850 HBV INDUCES A NOVEL INFLAMMATORY NETWORK Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 5. Determination of the effect of IL-8 on the regulation of IL-29 expression mediated by HBV. A, HepG2 cells (left panel) and Huh7 cells (right panel) were incubated with recombinant human IL-8 protein at different concentrations as indicated for 24 h. IL-29 mRNA in cell lysates was detected by real-time PCR. B, HepG2 cells (left panel) and Huh7 cells (right panel) were incubated with recombinant human IL-8 protein at different concentrations as indicated for 24 h. IL-29 protein in cell supernatants was detected by ELISA. C, HepG2 and Huh7 cells were cotransfected with pCMV-tag2B-IL-8 at different concentrations as indicated, pGL3-IL-29-Luc, and pHBV-1.2. Luciferase activity was measured at 48 h postinfection in HepG2 cells (left panel) and Huh7 cells (middle panel). IL-8 protein was determined by ELISA (right panel). D, HepG2 cells and Huh7 cells were cotransfected with pCMV-tag2B- IL-8 at different concentrations and pHBV-1.2. Forty-eight hours posttransfection, IL-29 proteins were detected by Western blot for HepG2 cells (left panel) or by ELISA for Huh7 cells (right panel). E, HepG2 cells were transfected with shRNA-control, shRNA-GAPDH, siRNA-control, or siRNA-IL-8. IL-8 protein in culture supernatants was detected by ELISA (top panel). GAPDH in cell lysates was detected by Western blot (bottom panel). F, HepG2 cells (left panel) and Huh7 cells (right panel) were cotransfected with pHBV-1.2 and siRNA-IL-8 or siRNA-control. The level of IL-29 mRNA was examined by real- time PCR. G, HepG2 and Huh7 cells were cotransfected with pHBV-1.2 and siRNA-IL-8 or siRNA-control. The level of IL-29 protein was detected by Western blot for HepG2 cells (left panel) or by ELISA for Huh7 cells (right panel). Data shown are mean 6 SE; n =3.*p , 0.05. of IL-29 protein regulated by HBV was also determined. HepG2 siRNA-control, and siRNA-IL-8, respectively. ELISA results and Huh7 cells were cotransfected with pCMV-tag2B-IL-8 at showed that IL-8 protein level was decreased in the presence of different concentrations and pHBV-1.2. Results from Western blot siRNA-IL-8 (Fig. 5E, top panel) and that GAPDH protein level analysis showed that IL-29 protein was decreased as the concen- was decreased in the presence of siRNA-GAPDH (Fig. 5E, bottom trations of IL-8 increased in HepG2 cells (Fig. 5D, left panel). panel), indicating the two siRNA molecules were effective and ELISA results indicated that IL-29 level was reduced as the con- specific. centrations of IL-8 increased in Huh7 cells (Fig. 5D, right panel). The effects of siRNA-IL-8 on the expression of IL-29 mRNA and The regulation of IL-29 gene expression mediated by IL-8 protein induced by HBV infection were then determined. HepG2 was further confirmed by RNAi approach using siRNA specific and Huh7 cells were cotransfected with pHBV-1.2 and siRNA-IL-8 to IL-8 (siRNA-IL-8) and its control siRNA (siRNA-control). To or siRNA-control. Results of real-time PCR showed that relative determine the effectiveness and specificity of siRNA-IL-8, HepG2 IL-29 mRNA level was significantly increased in the presence of cells were transfected with shRNA-control, shRNA-GAPDH, siRNA-IL-8 in both HepG2 cells (Fig. 5F, left panel) and Huh7 The Journal of Immunology 4851 cells (Fig. 5F, right panel). Western blot results showed that IL-29 In addition, HepG2 cells and Huh7 cells were also treated with protein was stimulated by treatment with siRNA-IL-8 in HepG2 recombinant human IL-8 protein for 24 h. Similar results were cells (Fig. 5G, left panel), and ELISA results indicated that IL-29 obtained from the treated hepatoma cells. In treated HepG2 cells, protein was increased in Huh7 cells treated with siRNA-IL-8 (Fig. the levels of COX-2 mRNA (Fig. 6D, left panel), COX-2 protein 5G, right panel). These results indicate that IL-8 is able to inhibit (Fig. 6E, left panel), and PGE2 (Fig. 6F, left panel) were stimu- the expression of IL-29 and demonstrate that IL-8 has an in- lated by IL-8 in a dose-dependent manner. In treated Huh7 cells, hibitory effect on the expression of IL-29 protein. the levels of COX-2 mRNA (Fig. 6D, right panel), COX-2 protein (Fig. 6E, right panel), and PGE (Fig. 6F, right panel) were en- IL-8 stimulates COX-2 expression in human PBMCs and 2 hanced by IL-8 in a concentration-dependent fashion. hepatoma cells To confirm the effect of IL-8 on COX-2 expression, we further The above results demonstrated that HBV stimulates the expression investigated the role of pCMV-tag2B-IL-8 in the activation of the of IL-8 and COX-2 proteins. The next question we asked is whether COX-2 gene promoter activity, mRNA expression, and protein the two proteins interact with each other or act as independent production in HepG2 and Huh7 cells. Cells were cotransfected with effectors in the progression of chronic inflammation induced by pGL3-COX-2-Luc, and pCMV-tag2B-IL-8 or pCMV-tag2B. Results HBV infection. Previous study showed that IL-8 enhances amy- of luciferase activity assay, semiquantitative RT-PCR, and Western loid-b (Ab1–42)-induced expression of COX-2 in cultured human blot showed that the levels of COX-2 promoter activity (Fig. 6G), microglia (40). In this study, we investigated the role of IL-8 in mRNA expression (Fig. 6H), and protein production (Fig. 6I)were COX-2 expression in human PBMCs and hepatoma cells. enhanced in cells transfected with pCMV-tag2B-IL-8. These results

Human PBMCs were treated with recombinant human IL-8 demonstrate that IL-8 stimulates COX-2 expression. Downloaded from protein for 24 h, and COX-2 mRNA and protein levels were de- termined. Results from real-time PCR indicated that COX-2 mRNA IL-8 enhances COX-2 expression regulated by HBV level was increased as the concentrations of IL-8 increased (Fig. To investigate the role of IL-8 in the regulation of COX-2 during 6A). Western blot analysis showed that COX-2 protein expression HBV infection, the RNAi approach was applied. To test the effect was stimulated by IL-8 in a dose-dependent manner (Fig. 6B). The of siRNA-IL-8 on the regulation of COX-2 expression mediated

production of PGE2 was also enhanced by treatment with IL-8 in by HBV infection, HepG2 and Huh7 cells were cotransfected http://www.jimmunol.org/ a concentration-dependent fashion (Fig. 6C). with pGL3-COX-2-Luc, pHBV-1.2, and siRNA-IL-8 or siRNA- by guest on September 27, 2021

FIGURE 6. Determination of the effect of IL-8 on the regulation of COX-2 expression mediated by HBV. A–C, PBMCs were incubated with recombinant human IL-8 protein at different concentrations as indicated or treated with 100 ng/ml LPS as positive control for 24 h. Cell lysates and culture supernatants were prepared and collected. The levels of COX-2 mRNA (A), COX-2 protein (B), and PGE2 (C) were detected by real-time PCR, Western blot, and ELISA, respectively. Data shown are mean 6 SE; n =3.*p , 0.05. D–F, HepG2 cells (left panels) or Huh7 cells (right panels) were incubated with recombinant human IL-8 protein at different concentrations as indicated for 24 h. Cell lysates and culture supernatants were prepared and collected. COX-2 mRNA (D),

COX-2 protein (E), and PGE2 (F) were detected by real-time PCR, Western blot, and ELISA, respectively. Data shown are mean 6 SE; n =3.*p , 0.05. G–I, HepG2 cells (left panels) and Huh7 cells (right panels) were cotransfected with pGL3-COX-2-Luc and pCMV-tag2B-IL-8 or pCMV-tag2B for 48 h. COX-2 promoter activity (G), COX-2 mRNA (H), and COX-2 protein (I) were determined by luciferase activity analysis, semiquantitative RT-PCR, and Western blot analysis, respectively. Data shown are mean 6 SE; n =3.*p , 0.05. 4852 HBV INDUCES A NOVEL INFLAMMATORY NETWORK

FIGURE 7. Analysis of the role of IL-8 in the reg- ulation of COX-2 expression mediated by HBV. A, Analysis of the effect of siRNA-IL-8 on the regula- tion of COX-2 expression mediated by HBV. HepG2 cells (left panel) and Huh7 cells (right panel) were cotransfected with siRNA-IL-8 or siRNA-control, pGL3-COX-2-Luc, and HBV-1.2 for 48 h. COX-2 promoter activity in transfected cells was determined by luciferase activity assays. Data shown are mean 6 SE: n =3.*p , 0.05. B and C, HepG2 cells (left panels) and Huh7 cells (right panels) were cotrans- fected with siRNA-IL-8 or siRNA-control and HBV- 1.2. Forty-eight hours posttransfection, total RNA ex- Downloaded from tracts (B) and protein extracts (C) were prepared, and the levels of mRNA and proteins of COX-2 and b-actin were determined by semiquantitative RT-PCR and Western blot, respectively. http://www.jimmunol.org/

control. Luciferase activity analysis results indicated that results suggest that IL-8 facilitates the expression of COX-2 COX-2 promoter activity was stimulated by HBV and inhibited activated by HBV infection. by siRNA-IL-8 in both HepG2 cells (Fig. 7A, left panel)and by guest on September 27, 2021 Huh7 cells (Fig. 7A, right panel). In addition, the levels of COX-2 inhibits IL-8 expression induced by HBV COX-2 mRNA (Fig. 7B) and protein (Fig. 7C) were activated by In a previous study, we have reported that inducible NO synthase HBV and also reduced by siRNA-IL-8 in both cell types. These negatively regulates IL-32 expression mediated by influenza A

FIGURE 8. Determination of the effect of COX-2 on the regulation of IL-8 expression mediated by HBV. A and B, PBMCs were treated with the inhibitor of COX- 2, NS398, at different concentrations as indicated or treated with 100 ng/ml LPS as a positive control. Cell lysates and culture supernatants were prepared and collected 48 h posttreatment. The levels of IL-8 mRNA (A) and IL-8 protein (B) were detected by real-time PCR and ELISA, respectively. C and D, HepG2 cells (left panels) and Huh7 cells (right panels)were transfected with pHBV-1.2 for 12 h and then treated with NS398 at different concentrations as indicated. Thirty-six hours posttreatment, the mRNA levels (C) and protein levels (D) of IL-8 and b-actin were ex- amined by semiquantitative RT-PCR and ELISA, re- spectively. Data shown are mean 6 SE; n =3.*p , 0.05. The Journal of Immunology 4853

FIGURE 9. Analysis of the roles of IL-8 in the regulation of ISRE and IRF3/7. A, The role of IL-8 in the activation of ISRE. HepG2 cells were cotrans- fected with pCMV-tag2B-IL-8 at different concen- trations and the reporter pISRE-Luc. ISRE promoter activity was measured by luciferase activity assays at 48 h posttransfection. Data shown are mean 6 SE; n =3.*p , 0.05. B–D, The effects of IL-8 and siRNA- IL-8 on the expression of IRF3. HepG2 cells were cotransfected with pCMV-tag2B-IL-8 or pCMV-tag2B and siRNA-IL-8 or siRNA-control. Forty-eight hours posttransfection, IRF3 mRNA (B) and IRF3 protein (C, D) were examined by real-time PCR and Western 6 blot, respectively. Data shown are mean SE; n =3. Downloaded from *p , 0.05. E–G, The effects of IL-8 and siRNA-IL-8 on the expression of IRF7. HepG2 cells were co- transfected with pCMV-tag2B-IL-8 or pCMV-tag2B and siRNA-IL-8 or siRNA-control. Forty-eight hours posttransfection, IRF7 mRNA (E) and IRF7 protein (F, G) were examined by real-time PCR and Western blot, respectively. Data shown are mean 6 SE; n =3. http://www.jimmunol.org/ *p , 0.05. by guest on September 27, 2021 virus infection (28). In this study, we speculated that there is also different concentrations for 24 h. Results from real-time PCR and a feedback regulation between IL-8 and COX-2 during HBV in- ELISA showed that the levels of IL-8 mRNA (Fig. 8A) and protein fection. The effect of COX-2 on IL-8 expression induced by HBV (Fig. 8B) were increased in cells treated with NS398 in a concen- was then investigated in this study. Human PBMCs were transfected tration-dependent manner. These results suggested that COX-2 has with pHBV1.2 and treated with NS398 (an inhibitor of COX-2) at a negative effect on IL-8 expression induced by HBV.

FIGURE 10. Determination of the effect of IL-8 on IL-10R2 expression. A and B, The effect of pCMV- tag2B-IL-8 on IL-10R2 expression. HepG2 cells were transfected with pCMV-tag2B-IL-8 at different concen- trations. Forty-eight hours posttransfection, the levels of IL-10R2 mRNA were examined by real-time PCR (A) and semiquantitative RT-PCR (B), respectively. C, The effect of pCMV-tag2B-IL-8 on IL-10R2 expres- sion. HepG2 cells were transfected with pCMV-tag2B- IL-8 at different concentrations. Forty-eight hours posttransfection, the levels of IL-10R2 and b-actin proteins were examined by Western blot using Ab to IL-10R2 or b-actin, respectively. D–F, The effect of siRNA-IL-8 on IL-10R2 expression. HepG2 cells were transfected with siRNA-IL-8 or siRNA-control. Forty- eight hours posttransfection, the levels of IL-10R2 mRNA and protein were examined by real-time PCR (D), semiquantitative RT-PCR (E), and Western blot (F), respectively. Data shown are mean 6 SE; n =3. *p , 0.05. 4854 HBV INDUCES A NOVEL INFLAMMATORY NETWORK

To confirm the effect of COX-2 on IL-8 expression, HepG2 cells IL-8 inhibits the activity of ISRE on IL-29 promoter and the and Huh7 cells were also transfected with pHBV1.2 and treated expression of IRF3/7 genes with NS398 at different concentrations for 24 h. Both semi- Previous studies showed that IL-29 (IFN-l1) gene expression quantitative RT-PCR (Fig. 8C) and ELISA (Fig. 8D) results was mediated by spatially separated promoter elements that in- showed that the levels of IL-8 mRNA and protein were enhanced dependently interact with IFN regulatory factor and NF-kB. Both by NS398 in a dose-dependent fashion in HepG2 cells (Fig. 8C, NF-kB and IRF3/7 were required for transcriptional regulation 8D, left panels) and Huh7 cells (Fig. 8C,8D, right panels). Thus, of the IL-29 gene (41). NF-kB and IRF3/7 also bind to the NF- our results demonstrate that COX-2 acts as a negative regulator of kB element and ISRE of the IL-29 promoter in IFN-a–primed IL-8 expression during HBV infection. cells (42). Additionally, we demonstrated that IL-8 inhibits IL-29 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 11. Determination of the effects of CREB and C/EBP on the expression of COX-2 regulated by IL-8. A, Diagrams of COX-2 core promoters, which includes the wild-type COX-2 promoter (WT, 2891/+9), a COX-2 promoter with a C/EBP mutated (Mut1, 2124/+132), a COX-2 promoter with a CRE mutated (Mut2, 253/+59), and COX-2 promoter with a double mutant of C/EBP and CRE (Mut3, 2124/+132 and 253/+59). B, The effect of IL-8 on the activation of COX-2 promoter and its mutants. HepG2 cells were cotransfected with pCMV-tag2B-IL-8 or its control and plasmids containing the luciferase reporter gene under the control of different COX-2 promoters, WT, Mut1, Mut2, and Mut3. Forty-eight hours posttransfection, COX-2 promoter activity was determined by luciferase activity assay. Data shown are mean 6 SE; n =3.*p , 0.05. C, The effects of mCREB and LIP on the activation of COX-2 promoter and its mutants. Cells were cotransfected with pCMV-tag2B-IL-8, pGL3-COX-2-Luc, and different amounts of mCREB and LIP, respec- tively. Forty-eight hours posttransfection, COX-2 promoter activity was determined by luciferase activity assay. Data shown are mean 6 SE; n =3.*p , 0.05. D and E, The effects of siRNA on the expression of COX-2. HepG2 cells were cotransfected with pCMV-tag2B-IL-8 along with siRNA specific to CREB, ATF4, C/EBPa, and C/EBPb, respectively. The levels of COX-2 mRNA (D) and protein (E) were examined by semiquantitative RT-PCR and Western blot, respectively. Results represent means of three independent experiments. F, The role of IL-8 in the expression of CREB. Cells were transfected with pCMV-tag2B-IL-8 for different times as indicated. Protein extracts were prepared, and the levels of CREB protein were determined by Western blot. G, The role of IL-8 in the translocation of CREB. HepG2 cells were transfected with pCMV-tag2B-IL-8 for different times as indicated. Protein extracts were prepared from cytosol and nucleus of transfected cells. CREB protein in nucleus (top panel) and cytosol (bottom panel) was detected by Western blot using Ab to CREB. The blot is a representative of three experiments with similar results. H, The role of IL-8 in the translocation of C/EBP. HepG2 cells were transfected with pCMV-tag2B-IL-8 for different times as indicated. Protein extracts were prepared from whole cell and nucleus of transfected cells. C/EBP protein in nucleus (top panel) and whole cell (bottom panel) was detected by Western blot using Ab to C/EBP. The blot is a representative of three experiments with similar results. The Journal of Immunology 4855 expression during HBV infection. Thus, we speculated that IL-8 IL-28R1 and IL-10R2 bind to the same receptor pair to activate may also regulate IL-29 expression through IRF3/7. downstream signaling pathways. To demonstrated this speculation, HepG2 cells were cotrans- To investigate the regulatory effect of IL-8 on the expression of fected with pCMV-tag2B-IL-8 at different concentrations and with IL-10R2 gene, HepG2 cells were transfected with pCMV-tag2B- a reporter, pISRE-Luc. Results of the luciferase assay showed that IL-8. Results of real-time PCR (Fig. 10A) and semiquantitative ISRE activity was decreased in the presence of IL-8 in a dose- RT-PCR (Fig. 10B) and Western blot (Fig. 10C) showed that the dependent manner (Fig. 9A), suggesting IL-8 plays a negative levels of IL-10R2 mRNA and protein were decreased as the role in the regulation of ISRE activity. concentrations of IL-8 increased. In addition, HepG2 cells were To investigate the effects of IL-8 on the expression of IRF3 and treated with siRNA-control or siRNA-IL-8. Results of real-time IRF7, two approaches were used: overexpression of IL-8 and PCR (Fig. 10D), semiquantitative RT-PCR (Fig. 10E), and West- knockdown of IL-8. HepG2 cells were cotransfected with pCMV- ern blot (Fig. 10F) showed that the levels of IL-10R2 mRNA tag2B-IL-8 or pCMV-tag2B and siRNA-IL-8 or siRNA-control. and protein were increased in the presence of siRNA-IL-8. These The effects of IL-8 on the expression of IRF3 and IRF7 mRNA results demonstrate that IL-8 has an inhibitory effect on IL-10R2 expression in HepG2 cells. were determined by real-time PCR, which showed that the levels of IRF3 mRNA (Fig. 9B) and IRF7 mRNA (Fig. 9E) were reduced in CREB and C/EBP recognition sites are required for the cells transfected with pCMV-tag2B-IL-8 and increased in cells activation of COX-2 regulated by IL-8 treated with siRNA-IL-8. These results suggested that IL-8 in- Regulation of COX-2 gene expression relies on many consensus hibits the transcription of IRF3 and IRF7 genes. cis-elements, including the C/EBP and CREB binding sites in the Downloaded from The effects of IL-8 on the expression of IRF3 and IRF7 proteins promoter (43, 44). Our above results suggested that IL-8 activates were determined by Western blot using Abs to IRF3, IRF7, or the expression of COX-2. We next investigated the roles of cis- b-actin (as a control), respectively. The results showed that protein regulatory elements in the activation of COX-2 expression regu- levels of IRF3 (Fig. 9C) and IRF7 (Fig. 9F) were reduced in cells lated by IL-8 using a mutation analysis approach. A C/EBP transfected with pCMV-tag2B-IL-8. However, protein levels of binding site mutant (Mut1), a cAMP response element (CRE)

IRF3 (Fig. 9D) and IRF7 (Fig. 9G) were increased in cells treated binding site mutant (Mut2), and a C/EBP binding site plus CRE http://www.jimmunol.org/ with siRNA-IL-8. Taken together, these results demonstrate that binding site double mutant (Mut3) were generated by site-specific IL-8 inhibits the expression of IRF3 and IRF7. mutagenesis. Reporter plasmids were then constructed, in which the luciferase gene was under the control of wild-type, Mut1, Mut2, IL-8 inhibits the expression of IL-29 receptor, IL-10R2 and Mut3 promoter of COX-2 gene, respectively (Fig. 11A). IL-10R2 is one of the receptor chains of IFN-ls, which is also HepG2 cells were cotransfected with pCMV-tag2B-IL-8 and each called “type III IFN.” It is a member of a newly identified IFN of the reporters. Results from luciferase activity analysis indicated family composed of three members: IL-29, IL-28A, and IL-28B. that Mut1, Mut2, and Mut3 resulted in the reduction of COX-2 by guest on September 27, 2021

FIGURE 12. Analysis of the effects of ERK and JNK signaling pathways in the expression of COX-2 regulated by IL-8. A, The effects of inhibitors of signaling components on the regulation of COX-2 promoter activity mediated by IL-8. Cells were cotransfected with pCMV-tag2B-IL-8 and pGL3-COX-2- Luc for 12 h and then treated with DMSO (control) or inhibitors of signaling components U0126, SB203580, SP600125, and GF109203 for 36 h. The cells were lysed, and COX-2 promoter activity was measured by luciferase activity assays. Data shown are mean 6 SE; n =3.*p , 0.05. B, The effects of the mutants of ERK1/2 and JNK on the activity of COX-2 promoter. HepG2 cells were cotransfected with pCMV-tag2B-IL-8, pGL3-COX-2-Luc, and each of the three dominant-negative mutants of ERK1 (mERK1), ERK2 (mERK2), and JNK (mJNK). Forty-eight hours posttransfection, COX-2 promoter activity was determined by luciferase activity assays. Data shown are mean 6 SE; n =3.*p , 0.05. C, The effect of IL-8 on the phosphorylation of ERK. HepG2 cells were transfected with pCMV-tag2B-IL-8 or pCMV-tag2B for 48 h. Proteins were then prepared from transfected cells, and the levels of phospho- ERK1/2 and nonphospho-ERK in cell lysates were detected by Western blot using Ab to phospho-ERK (P-ERK) or ERK, respectively. The blot is a representative of three experiments with similar results. D, The effect of IL-8 on the phosphorylation of JNK. HepG2 cells were transfected with pCMV- tag2B-IL-8 or pCMV-tag2B. At 48 h posttransfection, proteins were prepared from transfected cells, and the levels of phospho-JNK and nonphospho-JNK in cell lysates were detected by Western blot using Ab to phospho-JNK (P-JNK) or JNK, respectively. The blot is a representative of three experiments with similar results. 4856 HBV INDUCES A NOVEL INFLAMMATORY NETWORK promoter activity to the basal level (Fig. 11B). These results in- siRNA-C/EBPb (Fig. 11E, lane 6), but not by siRNA-ATF4 (Fig. dicated that C/EBP and CRE recognition sites were required for 11E, lane 4). These results confirm that CREB and C/EBP play activating COX-2 expression regulated by IL-8. important roles in the activation of COX-2 regulated by IL-8. The roles of CREB and C/EBP in the activation of COX-2 We next investigated the effect of IL-8 on the expression of mediated by IL-8 were also examined by introducing two CREB protein. HepG2 cells were transfected with pCMV-tag2B- dominant-negative mutants, mCREB and LIP, of CREB and IL-8 for different times. CREB or b-actin proteins were then de- C/EBP. HepG2 cells were cotransfected with pCMV-tag2B-IL-8, termined by Western blot, which showed that CREB protein ex- pGL3-COX-2-Luc, and each of the two mutants at different pression was activated in the presence of IL-8 in a time-dependent concentrations. Luciferase activity analysis results showed that manner, whereas the expression of b-actin protein remained COX-2 promoter activity was stimulated by IL-8 (Fig. 11C, lane 2 unchanged (Fig. 11F). It has been demonstrated that localization to versus lane 1), but such activation was repressed by mCREB (Fig. the nucleus is essential for CREB and C/EBP functions. Thus, we 11C, lanes 3, 4, and 5) and LIP (Fig. 11C, lanes 6, 7, and 8)in further examined whether IL-8 was involved in the translocation a dose-dependent manner. These results again suggested that of CREB and C/EBP from cytosol to nucleus. Results showed that C/EBP and CRE recognition sites were required for regulating in the presence of IL-8, the level of CREB protein in the nucleus COX-2 mediated by IL-8. was gradually increased while the level of CREB protein in the The roles of CREB and C/EBP in the activation of COX-2 cytosol remained relatively unchanged as transfection time in- mediated by IL-8 were further determined by the RNAi ap- creased (Fig. 11G). Furthermore, we observed a similar effect of proach. HepG2 cells were cotransfected with pCMV-tag2B-IL-8 IL-8 on the translocation of C/EBP from cytosol to nucleus, and siRNA specific to CREB, ATF4, C/EBPa, and C/EBPb, re- whereas the levels of C/EBP protein in the whole-cell lysates Downloaded from spectively. Semiquantitative RT-PCR results showed that the level remained relatively unchanged (Fig. 11H). Taken together, these of COX-2 mRNA was increased in the presence of IL-8 (Fig. 11D, results demonstrated that during HBV infection, IL-8 stimulates lane 2 versus lane 1), and such activation was attenuated by the translocation of CREB and C/EBP from cytosol to nucleus, siRNA-CREB (Fig. 11D, lane 3), siRNA-C/EBPa (Fig. 11D, lane resulting in the activation of COX-2 expression. 5), and siRNA-C/EBPb (Fig. 11D, lane 6), but not by siRNA- IL-8 stimulates COX-2 expression through activating ERK and ATF4 (Fig. 11D, lane 4). Western blot also showed that the http://www.jimmunol.org/ level of COX-2 protein was enhanced by IL-8 (Fig. 11E, lane 2 JNK signaling pathways versus lane 1). Such regulation was affected by siRNA-CREB Members of the MAPK family have been shown to regulate the (Fig. 11E, lane 3), siRNA-C/EBPa (Fig. 11E, lane 5), and activities of several transcription factors that are important in the by guest on September 27, 2021

FIGURE 13. Determination of the roles of IL-29 and IL-8 in the expression of PKR and 2959OAS and the replication of HBV. A and B, The effects of IL-29 and IL-8 on PKR expression. HepG2 cells were cotransfected with pEF-SPFL-IL-29, pEF-SPFL-IL-29 plus pCMV-tag2B-IL-8, or its control. Forty-eight hours posttransfection, the levels of PKR mRNA (A) and protein (B) were determined by semiquantitative RT-PCR and Western blot, respectively. Data shown are mean 6 SE; n =3.*p , 0.05. C and D, The effects of IL-29 and IL-8 on 2959OAS (OAS) expression. HepG2 cells were cotransfected with pEF-SPFL-IL-29, pEF-SPFL-IL-29 plus pCMV-tag2B-IL-8, or its con- trol. Forty-eight hours posttransfection, the levels of 2959OAS mRNA (C) and protein (D) were determined by semiquantitative RT-PCR and Western blot, re- spectively. Data shown are mean 6 SE; n =3.*p , 0.05. E–G, The effects of IL-29 and IL-8 on HBV replication. HepG2 cells were cotransfected with pHBV1.2, pEF-SPFL-IL-29, and pCMV-tag2B-IL-8 or its control. Forty-eight hours posttransfection, the levels of HBeAg (E) and HBsAg (F) of HBV in culture supernatants were measured by ELISA, and the levels of HBV capsid-associated DNA (G) in transfected cells were assessed by real-time PCR. Data shown are mean 6 SE; n =3.*p , 0.05. The Journal of Immunology 4857 proinflammatory response (45). Regulation of COX-2 expression increased in the presence of IL-29 but reduced in the presence of depends on different kinase activators in various cell types (46, both IL-29 and IL-8. 47). Based on our findings that IL-8 activates COX-2 expression To investigate the effects of IL-29 and IL-8 on HBV protein and such regulation required C/EBP and CREB, we further in- expression and the viral DNA replication, HepG2 cells were vestigated the roles of the members of the MAPK family in the cotransfected with pEF-SPFL-IL-29 or pEF-SPFL and pCMV- regulation of COX-2 expression mediated by IL-8. HepG2 cells tag2B-IL-8 or pCMV-tag2B. Levels of HBV Ags, HBeAg and were cotransfected with pCMV-tag2B-IL-8 and pGL3-COX-2- HBsAg, and HBV capsid-associated DNA were determined in Luc and then treated with DMSO (as a control), U0126 (ERK- transfected cells. Results showed that in cells transfected with pEF- specific inhibitor), SB203580 (p38 MAPK-specific inhibitor), SPFL-IL-29, the levels of HBeAg (Fig. 13E) and HBsAg (Fig. SP600125 (JNK-specific inhibitor), and GF109203 (protein kinase 13F) were reduced, and the level of HBV DNA (Fig. 13G) was C-specific inhibitor), respectively. Results of luciferase activity significantly inhibited. In addition, results indicated that in cells analysis showed that in IL-8–transfected cells, COX-2 promoter transfected with both pEF-SPFL-IL-29 and pCMV-tag2B-IL-8, activity was increased in control cells (DMSO), decreased by the the levels of HBeAg (Fig. 13E) and HBsAg (Fig. 13F) were rel- treatment of U0126, SB203580, and SP600125, but not affected atively unchanged, whereas the level of HBV DNA (Fig. 13G) was by GF109203 (Fig. 12A). These results suggested that ERK and slightly reduced. Taken together, these results demonstrate that IL- JNK signaling pathways might be involved in the activation of 29 inhibits HBV replication, and IL-8 plays an negative role in the COX-2 expression regulated by IL-8. anti-HBV activity of IL-29. On the basis of this study, we propose The effects of ERK and JNK signaling pathways on the acti- a hypothetical model (Fig. 14) according to which hepatitis B vation of COX-2 induced by IL-8 were further investigated using virus infection triggers production of IL-29, IL-8, and COX-2 Downloaded from two different approaches. First, three dominant kinase-inactive resulting in a host inflammatory response. mutants, mERK1, mERK2, and mJNK, were introduced, whose expression blocks kinase activity by competing with endogenous Discussion kinases (48, 49). HepG2 cells were cotransfected with pCMV- Generally, the inflammatory response is beneficial to the host. tag2B-IL-8, pGL3-COX-2-Luc, and each of the three kinase Inflammatory cytokines are normally released in response to mutants, respectively. Results showed that COX-2 promoter ac- http://www.jimmunol.org/ tivity was enhanced in the presence of pCMV-tag2B-IL-8 (Fig. 12B, lane 2 versus lane 1), but such activation was reduced in the presence of mERK1 (Fig. 12B, lane 3), mERK2 (Fig. 12B, lane 4), and mJNK (Fig. 12B, lane 5). Second, the phosphorylation status of ERK1/2 and JNK was examined in HepG2 cells by Western blot analyses using Abs specific to phospho-ERK, ERK, phospho- JNK, or JNK, respectively. Results showed that both the levels of phosphorylated ERK (Fig. 12C) and phosphorylated JNK (Fig. 12D) were increased in the presence of pCMV-tag2B-IL-8, by guest on September 27, 2021 whereas ERK and JNK proteins remained relatively unchanged with or without IL-8. Taken together, these results suggested that IL-8 activates ERK and JNK signaling pathways, resulting in the stimulation of COX-2 expression. IL-29 inhibits HBV replication, and IL-8 plays a negative role in anti-HBV activity of IL-29 It has been shown that IL-29 initially binds to IL-28R1 and causes a conformational change that subsequently allows IL-10R2 to bind to IL-29. The receptor complex is then activated and induces the expression of many IFN-stimulated genes that in turn induces an antiviral state in target cells. Protein kinase regulated by dsRNA (PKR) and 2959OAS is well known to be induced by IL-29 stimulation. Upon binding to dsRNA, 2959OAS catalyzes the formation of 29-59-linked oligoadenylate and activates RNase L, which breaks down viral and cellular RNA (50). PKR is also ac- FIGURE 14. A proposed model for the regulation of an inflammatory tivated by dsRNA, which leads to the phosphorylation of its cytokine network mediated by HBV infection. HBV infection induces substrate, eukaryotic translation initiation factor 2a, inhibits the a regulatory loop of inflammatory cytokine network (I), in which three guanosine nucleotide exchange factor, eukaryotic translation ini- inflammatory factors regulate each other in the order IL-29/IL-8/COX-2, tiation factor 2b, and halts viral replication (51). which involves positive regulation and negative feedback. IL-29 activated Our results demonstrating that IL-8 inhibits the expression of by HBV stimulates the expression of IL-8 (II) that in turn represses IL-29 IL-29 and IL-10R2 led us to consider that such inhibition could expression (III). Moreover, IL-8 activated by HBV infection through IL-29 result in impairing the formation of the antiviral state. To assess stimulates the ERK and JNK signaling pathways (IV) and enhances the this speculation, HepG2 cells were cotransfected with pEF-SPFL, translocation of CREB and C/EBP transcriptional factors from cytosol to nucleus, resulting in the activation of COX-2 expression (V) that triggers pEF-SPFL-IL-29, or pCMV-tag2B-IL-29 plus pCMV-tag2B-IL-8. PGE2 production and finally inflammatory responses (VI). COX-2 also Results of real-time PCR showed that the levels of PKR mRNA inhibits the production of IL-8 (VII). In addition, IL-29 activates the ex- 9 9 (Fig. 13A) and 2 5 OAS mRNA (Fig. 13C) were enhanced in the pression of PKR and 2959OAS (OAS) (VIII) that in turn inhibits HBV presence of IL-29 but were reduced in the presence of both IL-29 replication (IX). Also, after activation by IL-29, IL-8 negatively feeds back and IL-8. Results of Western blot showed that the levels of both to inhibit the expression of IL-29 (X), resulting in the repression of PKR PKR protein (Fig. 13B) and 2959OAS protein (Fig. 13D) were also and 2959OAS expression and thus enhancing HBV replication. 4858 HBV INDUCES A NOVEL INFLAMMATORY NETWORK diverse kinds of cellular stimuli, including viral infection, and impairing induction of antiviral genes (PKR and 2959OAS) and function to stimulate host responses aimed at controlling cellular inhibiting HBV replication. These results indicate that HBV stress and minimizing cellular damage. However, when the damage inhibited the antiviral effect of type III IFN by altering IL-8 levels is not repaired, excessive immune cell infiltration and persistent to favor the establishment of persistent HBV infection. cytokine production can be provoked. In fact, the host immune IL-8, as a classic proinflammatory cytokine, also plays an im- responses to hepatitis viruses are fairly weak and are unable portant role in the inflammatory process. In the current study, we completely to downregulate and clear the infection, resulting in identified a novel IL-8 downstream proinflammatory factor, COX- chronic stimulation of the Ag-specific in per- 2, in the presence of HBV infection. Although adult hepatocytes do sistently infected patients. The continuous expression of cytokines not express COX-2 under normal conditions, the level of COX-2 in and recruitment of activated lymphomononuclear cells to the liver hepatocytes increases during chronic inflammatory liver diseases affects many cellular pathways and ultimately results in fibrosis, (20, 55). COX-2 is also overexpressed in liver cirrhosis and may cirrhosis, and/or HCC. Many studies have suggested that hepatitis contribute to HCC development. After being induced by HBV viral proteins play direct roles in interfering with cytokine pro- infection, IL-8 may trigger proinflammatory factor expression, duction. For example, HBx has been shown to interact directly including COX-2, and subsequent host inflammatory responses with mediators including IL-8, ICAM-1, and the MHC factor and to maintain the inflammatory status for the development of liver regulate their function in the inflammatory process. IL-8 has been damage. CREB and C/EBP have been reported to mediate COX-2 shown to be associated with HBV-infected patients, and higher activation induced by diverse stimulating agents. We demonstrated expression levels were observed in liver cirrhosis and HCC than in this study that CREB and C/EBP are also essential for IL-8– in healthy individuals (52, 53). Moreover, IL-8 levels in patients induced COX-2 expression because mutations in binding sites, Downloaded from with chronic hepatitis B were significantly higher than in healthy negative mutants, and specific siRNA of CREB and C/EBP controls, and large fluctuations in IL-8 concentrations in patients’ eliminated IL-8 function in the activation of COX-2. Based on sera associated with hepatic flares were observed. For most the findings that IL-8 activates COX-2 expression and CREB and patients, the peak serum level of IL-8 preceded the onset of the C/EBP are involved in such regulation, we further investigated the flare of liver inflammation (the alanine transaminase peak), either roles of different MAPKs on the activation of COX-2 regulated by

simultaneous with or immediately after a sharp increase in viral IL-8. Our results demonstrated that ERK and JNK were involved http://www.jimmunol.org/ load (11). Thus, IL-8 is thought to be an important inflammatory in the activation of COX-2 regulated by IL-8 because inhibition of mediator, responsible for maintaining the inflammatory environ- each of those kinases abolished the expression of COX-2 activated ment associated with HBV infection that may play a role in the by IL-8. pathogenesis of liver damage. We focused the current study on the This novel notion that HBV activates IL-29, IL-8, and COX-2 role of IL-8 in the inflammatory cytokine network induced by expression and that these three inflammatory cytokines regulate HBV infection. each other in the order IL-29/IL-8/COX-2, which involves positive Using an RNAi approach, we recently demonstrated that IL-29 regulation and negative feedback, expands our understanding of was upregulated and COX-2 was downregulated, respectively, after relevant, highly pathophysiological processes caused by HBV. IL- knocking down IL-8 in HBV-infected HepG2 or Huh7 cells. 8 may be a key mediator in the cytokine network induced by HBV, by guest on September 27, 2021 Furthermore, we demonstrated that HBV infection resulted in which not only impairs the antiviral activity of IL-29 and favors activation of IL-29, IL-8, and COX-2 by a heretofore unrecognized the establishment of persistent viral infection but also induces the mechanism. Based on these results, we proposed a model (Fig. 14) high expression of proinflammatory factor COX-2 to maintain the of the activation of IL-29, IL-8, and COX-2 by HBV infection, inflammatory environment associated with HBV infection (Fig. resulting in a host inflammatory response. The model indicates 14). This study may allow the rational development of immuno- that HBV infection stimulates an inflammatory cytokines network therapeutic strategies that enhance viral control while limiting or including at least three inflammatory factors. HBV stimulates the blocking liver inflammation. expression of IL-29 (I), IL-29 then upregulates the production of IL-8 (II), which in turn attenuates IL-29 production (III). Also, IL- Acknowledgments 8 activates ERK and JNK signaling pathways (IV) and enhances We are grateful to Dr. Robert Schneider of the New York University Medical the binding of CREP and C/EBP to COX-2 promoter (V) resulting Center for providing pHBV1.2, Dr. Melanie H. Cobb of the University of in the activation of COX-2 gene, which in turn stimulates PEG2 Texas Southwestern Medical Center for providing the mutants of ERK1 and production and induces inflammation (VI). COX-2 also inhibits ERK2 genes, Dr. Michael Karin of the University of California at San Diego the production of IL-8 (VII). In addition, IL-29 enhances the for providing the mutant of the JNK gene, Dr. Sergei V. Kotenko of the production of PKR and 2959OAS (VIII), which inhibit HBV rep- University of Medicine and Dentistry of New Jersey for providing the plas- lication (IX). Moreover, IL-8 has a negative effect on the anti- mid expressing the IL-29 gene (pEF-SPFL-IL-29) and its vector (pEF- SPFL), and Dr. Hongbing Shu of Wuhan University of China for providing HBV activity of IL-29 (X). pISRE-Luc. Although IL-8 inhibits the antiviral actions of type I IFN in vitro (8, 54), little is currently known regarding the effect of IL-8 on the antiviral response of type III IFN. Our study provides new Disclosures knowledge regarding the antiviral response of type III IFN. IL-29 The authors have no financial conflicts of interest. is able to inhibit the replication of a number of viruses, including vesicular stomatitis virus, encephalomyocarditis virus, HCV, and HBV. We further extended the antiviral activity of IL-29 on HBV References in HepG2 cells and demonstrated that IL-8 attenuated the antiviral 1. Rehermann, B., and M. Nascimbeni. 2005. Immunology of hepatitis B virus and hepatitis C virus infection. Nat. Rev. Immunol. 5: 215–229. activity of IL-29. We also provided evidence that IL-29 expression 2. Chisari, F. V., and C. Ferrari. 1995. Hepatitis B virus immunopathogenesis. was decreased by IL-8 through the inhibition of the expression of Annu. Rev. Immunol. 13: 29–60. IRF3 and IRF7. IL-10R2, one of the receptor chains of IL-29, was 3. Ganem, D., and A. M. Prince. 2004. Hepatitis B virus infection—natural history and clinical consequences. N. Engl. J. Med. 350: 1118–1129. also downregulated by IL-8 in HepG2 cells. Additionally, IL-8 4. Budhu, A., and X. W. Wang. 2006. The role of cytokines in hepatocellular was found to attenuate the antiviral responsiveness of IL-29 by carcinoma. J. Leukoc. Biol. 80: 1197–1213. The Journal of Immunology 4859

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