STING agonists induce an innate antiviral immune response against hepatitis B virus Fang Guo1 ...

34 Pages·2014·1.08 MB·English

AAC Accepts, published online ahead of print on 15 December 2014
Antimicrob. Agents Chemother. doi:10.1128/AAC.04321-14
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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4 STING agonists induce an innate antiviral immune response against hepatitis B virus
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7 Fang Guo1#, Yanxing Han2,3#*, Xuesen Zhao1,4, Jianghua Wang5, Fei Liu1, Chunxiao Xu1, Lai D
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8 Wei5, Jian-Dong Jiang2,3, Timothy M. Block1,4, Ju-Tao Guo1*, Jinhong Chang1* w
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10 1Institute for Biotechnology and Virology Research, Department of Microbiology and o
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11 Immunology, Drexel University College of Medicine, Doylestown, Pennsylvania, USA. t
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12 2Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union a
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13 Medical College, Beijing, China. s
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14 3Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical g
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15 College, Beijing, China.
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16 4Baruch S. Blumberg Institute, Hepatitis B Foundation, Doylestown, Pennsylvania, USA il 5
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17 5Peking University People’s Hospital, Peking University Hepatology Institute, Beijing, China. 19

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19 Running Title: STING agonists induce antiviral response against HBV
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21 # These authors contributed equally to this work.
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23 * Corresponding authors, mailing address:
24 Jinhong Chang, Department of Microbiology and Immunology, Drexel University College of
25 Medicine, Doylestown, PA 18902. Fax: [phone]. E-mail: [email protected]
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26 Ju-Tao Guo, Department of Microbiology and Immunology, Drexel University College of o
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27 Medicine, Doylestown, PA 18902. Fax: [phone]. E-mail: [email protected] lo
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28 Yanxing Han, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking d

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29 Union Medical College, Beijing 100050, China. Fax: [phone]. E-mail: m

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30 [email protected] tp
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32 .
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33 ABSTRACT
34 Chronicity of hepatitis B virus (HBV) infection is due to the failure of a host to mount a
35 sufficient immune response to clear the virus. The aim of our study is to identify small
36 molecular agonists of pattern recognition receptor (PRR)-mediated innate immune
37 response to control HBV infection. To achieve this goal, a coupled mouse macrophage and D
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38 hepatocyte culture system mimicking the intrahepatic environment was established and n
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39 used to screen small molecular compounds that activate macrophages to produce cytokines, d
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40 which in turn suppress HBV replication in hepatocyte-derived stable cell line supporting f
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41 HBV replication in a tetracycline inducible manner. An agonist of the mouse stimulator of

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42 interferon genes (STING), 5,6-Dimethylxanthenone-4-acetic acid (DMXAA), was found to p
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43 induce a robust cytokine response in macrophages, which efficiently suppressed HBV a
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44 replication in mouse hepatocytes by reducing the amount of cytoplasmic viral sm
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45 nucleocapsids. Profiling of cytokines induced by DMXAA and agonists of representative r
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46 toll-like receptors (TLRs) in mouse macrophages revealed that unlike TLR agonists that n

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47 induced a predominant inflammatory cytokine/chemokine response, the STING agonist r
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48 induced a cytokine response dominated by type I interferons (IFN). Moreover, as ,
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49 demonstrated in a HBV hydrodynamic mouse model, intraperitoneal administration of 9

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50 DMXAA significantly induced the expression of IFN-stimulated genes and reduced HBV
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51 DNA replication intermediates in the livers of mice. This study thus proves the concept that s
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52 activation of STING pathway induces an antiviral cytokine response against HBV and the
53 development of small molecular human STING agonists as immunotherapeutic agents for
54 treatment of chronic hepatitis B is warranted.
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56 Hepatitis B virus (HBV) is a non-cytopathic hepadnavirus that chronically infects more than
57 350 million people worldwide. The outcomes and pathogenesis of HBV infection are largely
58 determined by the nature and magnitude of host antiviral immune response (1), which is
59 generally related to the age at the time of infection. While over 95% of adult-acquired infections
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60 are spontaneously cleared within 6 months by a vigorous and polyclonal HBV-specific T cell w
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61 response, more than 90% of exposed neonates and approximately 30% of children aged 1–5 lo
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62 years develop chronic infection (2, 3), which is associated with a weaker and often barely d

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63 detectable viral specific T cell response. m

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64 Sustained suppression of viral replication with long-term nucleos(t)ide analogue therapy or tt
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65 through a finite-duration of pegylated alpha interferon (IFN-α) therapy has been associated with a
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66 improvement of liver diseases, prevention of liver decompensation and reduction of a
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67 hepatocellular carcinoma morbidity and mortality (4, 5). However, a “functional cure”, .o
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68 characterized by normalization of liver function, HBV surface antigen (HBsAg) seroconversion o
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69 and durable immune control of HBV replication, is rarely achieved with the current therapies (6,
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70 7). Hence, restoration of host innate as well as HBV-specific adaptive immune responses is il 5
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71 essential for a functional cure of chronic HBV infection (8). 0
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72 Although hepatocytes are the primary host cells of HBV, hepatic non-parenchymal cells b
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73 (NPCs) have been shown to play a critical role in priming an effective HBV-specific antiviral u
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74 immunity (9, 10). For instance, it was demonstrated recently that activation of hepatic
75 macrophages induces the expression of a distinct profile of cytokines/chemokines that regulate
76 the priming of successful immune response against HBV in the livers of mice (10). It is,
77 therefore, conceivable that pharmacological activation of Kupffer cells and/or intrahepatic
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78 dendritic cells with agonists of TLRs or other pattern recognition receptors (PRRs) may facilitate
79 the intrahepatic priming of an anti-HBV immune response. In support of this hypothesis, it has
80 been demonstrated recently in woodchucks and chimpanzees that administration of TLR7
81 agonists not only induced an innate cytokine response that suppresses HBV replication, but also
82 shaped the adaptive immune response to achieve a durable control of HBV infection (11). In
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83 addition, studies in HBV transgenic mice also demonstrated that activation of myeloid dendritic w
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84 cells with an agonistic anti-CD40 antibody not only induced an intrahepatic cytokine response to lo
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85 control HBV replication (12, 13), but also restored HBV-specific, PD-1-mediated CD8⁺ T cell d

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86 exhaustion (14). m

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87 Stimulator of interferon genes (STING) is the adaptor protein of multiple cytoplasmic DNA tt
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88 receptors and a PRR recognizing bacterial second messengers cyclic di-adenosine a
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89 monophosphate (c-di-AMP) and cyclic di-guanosine monophosphate (c-di-GMP) (15). It was a
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90 discovered recently that cytoplasmic DNA activates cyclic guanosine monophosphate-adenosine .o
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91 monophosphate synthase (cGAS) to produce cGAMP, which subsequently binds to STING and o
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92 induces IFNs and other cytokines (16) (17). The fact that STING can be activated by cyclic di-
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93 nucleotides implies that like TLR7/8, STING might be activated by other small molecules and il 5
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94 thus a potential target for pharmacological activation of the innate immune response as well as 0
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95 priming of an adaptive immune response. Indeed, some previously identified flavonoid IFN- b
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96 inducers, including 10-(carboxymethyl)-9(10H)acridone (CMA) and 5,6-dimethylxanthenone-4- u
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97 acetic acid (DMXAA), were recently identified as murine STING agonists (18, 19). DMXAA
98 (Vadimezan or ASA404) was initially developed as a vascular disrupting agent with antitumor
99 activity partially through activation of NK and tumor-associated macrophages in various mouse
100 models, and had been evaluated in phase II clinical trials for treating cancers (18). In studies
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101 reported herein, we demonstrated that DMXAA activated a STING-dependent signaling pathway
102 to induce a type I-IFN-dominant cytokine response in mouse macrophages, which efficiently
103 suppressed HBV replication in cultured murine hepatocytes and in the livers of mice by reducing
104 the amount of cytoplasmic viral nucleocapsids. Our work has thus validated STING as a valuable
105 target for the immunotherapy of chronic hepatitis B, and the development of small molecular
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106 human STING agonists as immunotherapeutics against chronic HBV infections is warranted. w
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108 MATERIALS AND METHODS d

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109 Cell culture. Murine macrophage cell line RAW264.7 (ATCC TIB-71) and GP2-293 cells m

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110 (Clontech) were maintained in Dublecco’s modified Eagle medium (DMEM) supplemented with tp
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111 10% fetal bovine serum (FBS). AML12HBV10, an immortalized murine hepatocyte (AML12)- a
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112 derived cell line supporting high level of HBV replication in a tetracycline (tet)-inducible s
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113 manner, was maintained as previously described (20, 21). or
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114 n

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115 Reagents. DMXAA, CMA and 2,7-Bis[2-(diethylamino)ethoxy]-9-fluorenone (tilorone) were il
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116 purchased from Sigma-Aldrich. TLR1/2 agonist Pam3CSK4, TLR3 agonist poly I:C, TLR4 2
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117 agonist lipopolysaccharide (LPS) and TLR7 agonist gardiquimod were from Invivogen.
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118 Recombinant murine IFN-α, IL-1, IL-6 and TNF-α were from PBL InterferonSource. Antibody g
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119 against carboxyl terminal 14 amino acid of HBV core protein was described previously (21). t
120 Antibodies against β-actin and mouse IFNAR-1 were obtained from Sigma-Aldrich and Santa
121 Cruz Biotechnology, respectively. Antibodies against mouse STING, TBK1, S172-phosphorylated
122 TBK1, IkBα, p38, phosphorylated-p38, JNK, phosphorylated-JNK, ERK, and phosphorylated-
123 ERK, were purchased from Cell Signaling Technology. Plasmids pTmcs-HBV1.3 and pCMV-
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124 SB were kind gifts of Dr. Francis V. Chisari (The Scripps Research Institute, La Jolla, California,
125 USA).
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127 Analyses of HBV DNA, RNA and nucleocapsids. HBV core DNA extraction from
128 AML12HBV10 cells as well as analyses by Southern blot hybridization and real-time PCR D
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129 assays were as described previously (21). Total cellular RNA was extracted using TRIzol reagent n
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130 (Invitrogen). HBV RNA was analyzed by Northern blot hybridization with an α-32P-UTP-labeled d
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131 full-length minus-stranded RNA probe. HBV capsid and capsid-associated viral DNA were f
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132 analyzed by a native agarose gel electrophoresis followed by transfer to a nitrocellulose
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133 membrane. HBV capsids were detected by probing the membrane with an antibody against HBV :
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134 core protein followed by visualizing with the LI-COR Odyssey system. Capsid-associated DNA c
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135 was detected by hybridization with radioactively labeled HBV riboprobe (21). m
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136 g
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137 Real-Time RT-PCR assays. For cytokine gene expression analysis, total RNA was extracted A
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138 using TRIzol reagent. cDNAs were synthesized using SuperScript III (Invitrogen). Quantitative il
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139 real-time PCR analysis was performed using a LightCycler 480 II (Roche). Primer sequence 0
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140 information is available upon request. b
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141 e
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142 Generation of STING knockdown cell line. A plasmid expressing shRNA specifically
143 targeting murine STING was constructed by inserting the following cDNA sequence into pRS
144 vector (Origene): tcaatcagctacataacaactcgagttgttatgtagctgattga. A control plasmid expressing
145 scrambled shRNA from pRS vector was purchased from Origene. Package of VSV G protein
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146 pseudotyped retroviruses in GP2-293 cells using pCMV/VSV-G and pRS vector-derived plasmid
147 expressing a scrambled shRNA or STING specific shRNA was essentially as reported previously
148 (22). RAW264.7 cells were transduced with pseudotyped retroviruses expressing each of the
149 shRNA, as previously described (22).
150 D
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151 Antiviral efficacy of DMXAA in HBV DNA hydrodynamic mouse model. Ten-week-old lo
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152 female NOD/SCID mice were purchased from Vital River Laboratory Animal Technology Co. e
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153 Ltd. All experiments were conducted with Institutional Animal Care and Use Committee ro
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154 approval. To establish HBV hydrodynamic model, 13.5 μg of plasmid pTmcsHBV1.3 expressing h
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155 1.3mer HBV genome and 4.5 μg of pCMV-SB expressing the Sleeping Beauty transposase were :/
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156 injected through tail vein, according to procedures previously described (23, 24). To test the c.
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157 efficacy of DMXAA in vivo, 7 days post injection, mice were treated with either DMXAA (in
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158 PBS with 7.5% sodium Bicarbonate) at 25mg/kg or vehicle, via intraperitoneal injection. Mice g
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159 were sacrificed 24 h after treatment. Intrahepatic HBV core DNA was extracted and quantified
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160 by using a real-time PCR assay. Intrahepatic total RNA was extracted and the interferon il
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161 stimulated genes (ISGs) OAS1b and viperin mRNAs were determined by real-time RT-PCR 2
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162 assay. Weight of individual mouse was monitored before and 24 h after the treatment.
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164 t
165 RESULTS
166 Establishment of a cell culture system for evaluation of PRR agonist-induced antiviral
167 response against HBV Unlike RIG-I-like receptors that are ubiquitously expressed in many
168 types of somatic cells, expression of other PRRs, such as TLRs and cGAS, is usually restricted to
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169 macrophages, dendritic cells and a few other cell types. For instance, due to the lack or low level
170 expression of TLRs in hepatocytes, direct treatment of hepatocytes with TLR agonists induces a
171 negligible cytokine response. However, some hepatic NPCs, particularly Kupffer cells and
172 dentritic cells, express high levels of TLRs (25, 26). Hence, our strategy toward a “functional
173 cure” of chronic HBV infection is to activate intra-hepatic macrophages and/or dendritic cells
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174 with the agonist of toll-like receptors (TLR) or other PRRs, that should not only induce an w
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175 antiviral cytokine response to suppress HBV replication in hepatocytes (26-28), but also lo
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176 promotes the priming and activation of HBV-specific adaptive immune response to ultimately d

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177 control the virus infection (25, 26, 29). m

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178 Accordingly, we have developed a cell-based assay mimicking the intrahepatic environment tp
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179 to screen small molecular PRR agonists for treatment of chronic hepatitis B. Specifically, as a
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180 depicted in Fig. 1A, mouse macrophages (RAW 264.7) were treated with testing compounds and s
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181 the conditioned media of treated macrophages were then applied to immortalized mouse or
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182 hepatocytes harboring HBV (AML12HBV10) to test the compound-induced antiviral cytokine o
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183 response in macrophages. The assay system was first validated with known TLR agonists. As p
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184 shown in Fig. 1B, while direct treatment of AML12HBV10 cells with agonists of TLR1/2, ,

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185 TLR3, TLR4 or TLR7 did not apparently inhibit HBV DNA replication, treatment of 1
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186 AML12HBV10 cells with the conditioned media harvested from TLR agonist-treated y

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187 macrophages (indirect treatment) effectively inhibited HBV DNA replication. The results thus e
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188 confirmed and extended previous findings that although failed to directly activate an antiviral
189 response in hepatocytes, TLR agonists induced macrophages to produce soluble factors that
190 suppress HBV replication in hepatocytes (25, 29).
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192 DMXAA-induced antiviral response in macrophages suppressed HBV replication in
193 hepatocytes. Using the assay system described above, we initially screened 75 flavonoids in
194 MicroSource compound collection (MicroSource Discovery Systems, Inc.) and identified a
195 flavonoid, 7, 2’-dihydroxyflavone, that demonstrated an indirect, but not direct, antiviral activity
196 against HBV (data not shown). Encouraged by this finding, literature search for additional
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197 flavonoids with interferon-inducing activity revealed three compounds, including tilorine, CMA w
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198 and DMXAA (Fig. 2A), that were identified as potent IFN inducers in mice several decades ago lo
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199 (30-32). While tilorine neither directly nor indirectly inhibited HBV replication, both CMA and d

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200 DMXAA demonstrated a strong indirect anti-HBV activity in our assay system (Fig. 2B). m

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201 Interestingly, it was recently reported by several groups that both CMA and DMXAA are mouse tt
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202 STING agonists (18, 19, 33). In support of this notion, we show in Fig. 2C that DMXAA, but not a
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203 LPS, efficiently induces phosphorylation of STING in RAW264.7 cells. Consistent with the fact a
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204 that AML12HBV10 cells express undetectable level of STING (Fig. 2C), direct treatment of the .o
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205 hepatocytes with DMXAA did not inhibit HBV DNA replication. However, the conditioned o
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206 media harvested from DMXAA-treated RAW264.7 cells inhibited HBV DNA replication in the
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207 mouse hepatocytes in a dose-dependent manner, with an estimated EC50 of 10 µM (Fig. 2D). The il 5
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208 cytotoxicity of DMXAA in both mouse macrophage (RWA246.7) and hepatocyte 1
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209 (AML12HBV10) cell lines has been tested under the conditions described for indirect and direct y

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210 treatments by microscopic inspection and MTT assay. No cytotoxic effect was observed at e
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211 concentration up to 500 µM of DMXAA.
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213 DMXAA-induced antiviral response reduced the amount of cytoplasmic HBV capsids. In
214 order to map the HBV replication step(s) being inhibited by DMXAA-induced antiviral response
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