Hepatitis B Virus 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 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 27, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 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 hepatitis B virus (HBV) is a major causative factor associated with the development of cirrhosis and hepatocellular carcinoma. 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 genes 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 cytokine 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 chemokine 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 monocytes, by guest on September 27, 2021 tion of chronic inflammation. Cytokines 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 viruses 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 hepatitis C virus (HCV) (6–8) and replication of HCV (9). The X protein of HBV (HBx) transactivates IL-8 gene 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 cancer (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.