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Efficient Attenuation of NK Cell−Mediated Liver Injury through Genetically Manipulating Multiple Immunogenes by Using a Liver-Directed Vector This information is current as of September 30, 2021. Jianlin Geng, Xuefu Wang, Haiming Wei, Rui Sun and Zhigang Tian J Immunol published online 3 April 2013 http://www.jimmunol.org/content/early/2013/04/03/jimmun ol.1203129 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2013/04/03/jimmunol.120312 Material 9.DC1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 30, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published April 3, 2013, doi:10.4049/jimmunol.1203129 The Journal of Immunology Efficient Attenuation of NK Cell–Mediated Liver Injury through Genetically Manipulating Multiple Immunogenes by Using a Liver-Directed Vector Jianlin Geng, Xuefu Wang, Haiming Wei, Rui Sun, and Zhigang Tian Adenovirus or adenoviral vectors were reported to induce serious liver inflammation in an NK cell–dependent manner, which limits its clinical applicability for liver gene therapy. We tried to develop an efficient liver-directed therapeutic approach to control hepatic NK cell function via simultaneously manipulating multiple immune genes. Based on our previous study, we found that CCL5 knockdown synergistically enhanced the attenuating effect of silencing CX3CL1 (fractalkine [FKN]) in adenovirus-induced acute liver injury. In addition, the combined treatment of human IL-10 expression with FKN knockdown would further strengthen the protective effect of silencing FKN. We used a hepatocyte-specific promoter to construct a hepatocyte-specific Downloaded from multiple function vector, which could simultaneously overexpress human IL-10 and knock down CCL5 and FKN expression. This vector could attenuate adenovirus-induced acute hepatitis highly efficiently by reducing liver NK cell recruitment and serum IFN-g and TNF-a. The multiple function vectors could be delivered by nonviral (hydrodynamic injection) and viral (adenovirus) approaches, and maintained long-term function (more than 1 month in mice). Our results suggest a possible strategy to ameliorate the acute liver injury induced by adenovirus by modulating multiple immune genes. The novel multifunction vector has an extensive and practical use for polygenic and complex liver diseases such as malignancies and hepatitis, which correlate with http://www.jimmunol.org/ multiple gene disorders. The Journal of Immunology, 2013, 190: 000–000. he liver is the largest solid organ in the body, and its ef- been developed and demonstrated to be an attractive gene therapy ficient protein synthetic capacity makes the liver an at- vector with highly reduced toxicity and long-term transduction ef- T tractive target organ for gene therapy (1, 2). Although ex- ficiency (5, 6). Second, chemically engineered vectors such as poly- citing results were obtained in animal models and in some clinical ethylene glycol modified adenoviruses exhibit receptor-specific trials, many obstacles remain in clinical practice. Among these cell transduction and reduced vector toxicity (7). Third, because obstacles, liver-specificity and immune injury are the most chal- adenovirus-induced strong innate responses are mainly mediated by lenging (3). Due to its liver tropism feature, ability to infect qui- innate immune cells such as NK cells, cell activities and functions by guest on September 30, 2021 escent hepatocytes, and high capacity for transgenes, the adenoviral can be regulated to ameliorate the liver injury. Blocking of NKG2D vector is now the most widely used vector in clinical trials (2). receptor inhibited NK cell activation upon adenovirus infection, Systemic infection of high-dose adenoviruses will initiate innate leading to a delay in adenovirus clearance and NK cell–mediated and adaptive immune responses against adenovirus that causes se- cytolysis (8). Knockdown of chemokines such as fractalkine (FKN) vere allergic reaction and inactivation of adenoviral vectors, which to inhibit NK cell recruitment and activity could protect the acute is the major drawback of adenovirus-mediated gene therapy (4). liver injury induced by adenoviral vector (9). Inflammatory cyto- A variety of strategies have been used to reduce the immunoge- kines such as IFN and TNF had important roles in adenovirus- nicity of adenovirus and inhibit the liver injury induced by adeno- induced acute liver injury; therefore, overexpression of inhibitory virus. First, gutless adenovirus lacking all viral coding regions has cytokines IL-10 has also been used to modulate the injury (10, 11). Finally, to achieve liver specificity in gene therapy, liver-specific promoters cannot only achieve prolonged transgene expression but Department of Immunology, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China; and Hefei National Laboratory also reduce side effects caused by transgene expression in non–liver for Physical Sciences at Microscale, Hefei, Anhui 230027, China cells (12, 13). Various liver-specific promoter elements and chi- Received for publication November 12, 2012. Accepted for publication March 4, meric promoters have been evaluated for high liver specificity and 2013. activity (14). This work was supported by the Ministry of Science and Technology of China (973 Because liver diseases, such as malignancies and hepatitis, Basic Science Project 2010CB911901), the Natural Science Foundation of China correlate with several immune gene disorders, a vector containing (91029303, 30911120480, and 31021061), and National Science and Technology Major Projects (2012ZX10002014, 2012ZX10002006). combinational gene intervention (e.g., simultaneous overexpression Address correspondence and reprint requests to Dr. Zhigang Tian and Dr. Rui Sun, and knockdown of multiple genes) is urgently needed to modulate School of Life Sciences, University of Science and Technology of China, 443 the inflammatory liver microenvironment during disease (15). Huangshan Road, Hefei 230027, China. E-mail addresses: [email protected] (Z.T.) Adenovirus-mediated coexpression of TRAIL and a short hairpin and [email protected] (R.S.). RNA (shRNA) containing a sequence to silence COX-2 exhibited The online version of this article contains supplemental material. great synergistic antitumor activity in experimental hepatocellular Abbreviations used in this article: ALT, alanine transaminase; ALP, alkaline phos- phatase; AST, aspartate aminotransferase; BILI, bilirubin; CRE, creatinine; DC, carcinoma (16). Thus, development of vectors that can handle mul- dendritic cell; FCM, flow cytometry; FKN, fractalkine; GGT, g-glutamyltranspepti- tiple genes is practically needed. dase; huIL-10, human IL-10; miRNA, microRNA; RNAi, RNA interference; shRNA, In the current study, we found that synergistically interfering short hairpin RNA; VP, viral particle. CCL5 and FKN or the combined overexpression of human IL-10 Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 (huIL-10) with FKN knockdown could enhance the protective ef- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203129 2 ATTENUATING LIVER INJURY THROUGH MANIPULATING MULTIPLE GENES fect of silencing FKN alone in adenovirus-induced acute liver injury. Cryostat sections (6 mm thick) were prepared using a Leica CM1950 Next, we constructed a novel multifunction plasmid vector using an cryostat. EGFP and DsRed2 fluorescence protein expression was analyzed efficient hepatocyte-specific promoter, which could simultaneously with a fluorescence microscope (Carl Zeiss AXIOSCOP2, Oberkochen, Germany). overexpress and knock down multiple immune genes in a liver- directed manner. Our data show that simultaneous overexpression Mouse hepatic cell isolation of huIL-10 and knockdown of endogenous CCL5 and FKN highly Liver leukocytes were isolated similar to what has been described previ- attenuated adenovirus-mediated acute liver injury by inhibiting NK ously (18). Livers were passed through a 200-gauge stainless steel mesh. cell recruitment and activation and inflammatory cytokines pro- Cells were resuspended in 40% Percoll (Life Technologies BRL) and 3 duction. This vector could be delivered by viral and nonviral ap- centrifuged at 1260 g for 15 min at room temperature. Pelleted leuko- cytes were washed in PBS and used to evaluate cellularity and for flow proaches and exert function in vivo for a long time. cytometry (FCM) staining. Materials and Methods NK cell depletion Mice and i.v. injection Mice were injected i.v. with 50 mg anti-asialo-GM1 Ab (Wako, Tokyo, Japan) or 50 mg rabbit IgG control (Calbiochem, La Jolla, CA) per mouse C57BL/6 mice were purchased from the Experimental Animal Center 24 h before adenovirus infection. The elimination