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      CCR5 receptor antagonism inhibits hepatitis C virus (HCV) replication in vitro

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          Abstract

          Background and aim

          The hepatitis C virus (HCV) is a single-strand RNA virus that infects millions of people worldwide. Recent advances in therapy have led to viral cure using two- and three- drug combinations of direct acting inhibitors of viral replication. CCR5 is a chemokine receptor that is expressed on hepatocytes and represents a key co-receptor for HIV. We evaluated the effect of CCR5 blockade or knockdown on HCV replication in Huh7.5 JFH1 cells.

          Methods

          Cells were exposed to varying concentrations of maraviroc (CCR5 inhibitor), cenicriviroc (CCR2/CCR5 inhibitor), sofosbuvir (nucleotide polymerase inhibitor), or raltegravir (HIV integrase inhibitor).

          Results

          HCV RNA was detected utilizing two qualitative strand-specific RT-PCR assays. HCV core antigen and NS3 protein was quantified in the supernatant and cell lysate, respectively. siRNA was utilized to knockdown CCR5 gene expression in hepatocytes. Alternatively, anti-CCR5 antibodies were employed to block the receptor. Supernatant levels of HCV RNA (expressed as fold change) were not reduced in the presence of raltegravir but were reduced 8.55-fold and 12.42-fold with cenicriviroc and maraviroc, respectively. Sofosbuvir resulted in a 16.20-fold change in HCV RNA levels. HCV core and NS3 protein production was also reduced in a dose-dependent manner. Two distinct anti-CCR5 antibodies also resulted in a significant reduction in HCV protein expression, as did siRNA knockdown of CCR5 gene expression.

          Conclusions

          These data provide evidence that CCR5 modulation could have a significant effect on HCV replication in an in vitro system. Further evaluation of the role of CCR5 inhibition in clinical settings may be warranted.

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          Most cited references33

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          The Role of Chemokines in Acute Liver Injury

          Chemokines are small molecular weight proteins primarily known to drive migration of immune cell populations. In both acute and chronic liver injury, hepatic chemokine expression is induced resulting in inflammatory cell infiltration, angiogenesis, and cell activation and survival. During acute injury, massive parenchymal cell death due to apoptosis and/or necrosis leads to chemokine production by hepatocytes, cholangiocytes, Kupffer cells, hepatic stellate cells, and sinusoidal endothelial cells. The specific chemokine profile expressed during injury is dependent on both the type and course of injury. Hepatotoxicity by acetaminophen for example leads to cellular necrosis and activation of Toll-like receptors while the inciting insult in ischemia reperfusion injury produces reactive oxygen species and subsequent production of pro-inflammatory chemokines. Chemokine expression by these cells generates a chemoattractant gradient promoting infiltration by monocytes/macrophages, NK cells, NKT cells, neutrophils, B cells, and T cells whose activity are highly regulated by the specific chemokine profiles within the liver. Additionally, resident hepatic cells express chemokine receptors both in the normal and injured liver. While the role of these receptors in normal liver has not been well described, during injury, receptor up-regulation, and chemokine engagement leads to cellular survival, proliferation, apoptosis, fibrogenesis, and expression of additional chemokines and growth factors. Hepatic-derived chemokines can therefore function in both paracrine and autocrine fashions further expanding their role in liver disease. More recently it has been appreciated that chemokines can have diverging effects depending on their temporal expression pattern and the type of injury. A better understanding of chemokine/chemokine receptor axes will therefore pave the way for development of novel targeted therapies for the treatment of liver disease.
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            Activation of chemokine and inflammatory cytokine response in hepatitis C virus-infected hepatocytes depends on Toll-like receptor 3 sensing of hepatitis C virus double-stranded RNA intermediates.

            Chemokines and inflammatory cytokines are key regulators of immunity and inflammation during viral infections. Hepatitis C virus (HCV) is a hepatotropic RNA virus frequently associated with chronic liver inflammation and hepatocellular carcinoma. Intrahepatic levels of chemokines and cytokines are elevated in chronic HCV infections, but the underlying mechanisms remain unclear. We found that Toll-like receptor-3 (TLR3) senses HCV infection in cultured hepatoma cells, leading to nuclear factor kappa B (NF-κB) activation and the production of numerous chemokines and inflammatory cytokines, such as regulated on activation normal T cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1α, MIP-1β, IP-10, and interleukin-6. The chemokine/cytokine induction occurred late in HCV infection and was abrogated when HCV was ultraviolet-inactivated before infection, indicating a dependence on the cellular recognition of HCV replication products. Gel-shift and chromatin immunoprecipitation assays revealed that NF-κB plays a pivotal role in HCV-induced chemokine/cytokine transcription. Mutations specifically disrupting the double-stranded RNA (dsRNA)-binding activity of TLR3 ablated the chemokine/cytokine response to HCV infection, indicating that HCV dsRNA was the pathogen-associated molecular pattern triggering TLR3 signaling. In vitro synthesized HCV dsRNAs, with a minimal length of ∼80-100 base pairs, activated TLR3-dependent chemokine expression, regardless of the genome position from which they derived. In contrast, HCV single-stranded RNAs, including those derived from the structured 3'nontranslated region highly potent for RIG-I activation, failed to do so. Moreover, robust production of chemokines and inflammatory cytokines was also observed in primary human hepatocytes after stimulation with extracellular poly-I:C, a TLR3 ligand. Our data suggest that TLR3-mediated chemokine and inflammatory cytokine responses may play an important role in host immune responses to HCV and the pathogenesis of HCV-associated liver diseases. Copyright © 2011 American Association for the Study of Liver Diseases.
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              Human hepatic stellate cells express CCR5 and RANTES to induce proliferation and migration.

              Activated hepatic stellate cells (HSCs) are the main producers of extracellular matrix in the fibrotic liver and are involved in the regulation of hepatic inflammation. The aim of this study was to characterize the role of regulated on activation, normal T-cell expressed, and presumably secreted (RANTES) in activated HSCs. RANTES mRNA and protein secretion were strongly induced after stimulating HSCs with TNF-alpha, IL-1beta, or CD40L. RANTES production was NF-kappaB dependent, because inhibitor-kappaB (IkappaB) superrepressor and dominant-negative IkappaB kinase-2 almost completely blocked RANTES expression. NF-kappaB activation was sufficient to drive RANTES expression as demonstrated by the strong induction of RANTES in HSCs expressing NF-kappaB-inducing kinase. The JNK/activator protein-1 pathway also contributed to RANTES expression as demonstrated by the blocking effects of the JNK inhibitor SP600125. HSCs responded to stimulation with recombinant human (rh)RANTES with an increase in intracellular calcium concentration and a rapid increase in free radical formation. Furthermore, rhRANTES induced ERK phosphorylation, ERK-dependent [3H]thymidine incorporation, and HSC proliferation. Additionally, rhRANTES induced focal adhesion kinase phosphorylation and a substantial increase in HSC migration. HSCs functionally expressed chemokine receptor-5 (CCR5), as shown by flow-cytometric analysis and RT-PCR, and the inhibitory effects of a blocking CCR5 antibody on rhRANTES-induced ERK activation, proliferation, and migration. Diphenylene iodonium and N-acetylcysteine inhibited rhRANTES-induced ERK activation and HSC proliferation, indicating that NADPH oxidase-dependent production of reactive oxygen species was required. In conclusion, RANTES and CCR5 represent potential mediators of 1) HSC migration and proliferation and 2) a cross-talk between HSCs and leukocytes during fibrogenesis.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ResourcesRole: SupervisionRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: InvestigationRole: MethodologyRole: Validation
                Role: Data curationRole: InvestigationRole: MethodologyRole: Writing – original draftRole: Writing – review & editing
                Role: Formal analysisRole: MethodologyRole: Software
                Role: Formal analysisRole: MethodologyRole: Software
                Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: MethodologyRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: Project administrationRole: ResourcesRole: SupervisionRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                29 October 2019
                2019
                : 14
                : 10
                : e0224523
                Affiliations
                [1 ] Division of Digestive Diseases, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH, United States of America
                [2 ] Digestive Health Center, Cincinnati Children’s Hospital, Cincinnati, OH, United States of America
                [3 ] Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, United States of America
                [4 ] Neurology Division, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States of America
                [5 ] University of Maryland, Baltimore, MD, United States of America
                Saint Louis University, UNITED STATES
                Author notes

                Competing Interests: Dr Sherman has received research grants or contracts awarded to his institution from AbbVie, Gilead Sciences, Inc, Merck, Intercept Pharmaceuticals, Inc, and Inovio. He has served as an advisory board member or a consultant to Abbott Laboratories and uniQure. He has also served on data and safety monitoring boards for Watermark (Allergan) and MedPace. This does not alter our adherence to PLOS ONE policies on sharing data and materials. All other authors have nothing to disclose.

                Author information
                http://orcid.org/0000-0003-1907-0570
                http://orcid.org/0000-0002-0560-0019
                Article
                PONE-D-19-19731
                10.1371/journal.pone.0224523
                6818973
                31661521
                cbc841bf-7b39-4323-9ca1-6ebbe9a34f79
                © 2019 Blackard et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 12 July 2019
                : 15 October 2019
                Page count
                Figures: 5, Tables: 0, Pages: 11
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100000060, National Institute of Allergy and Infectious Diseases;
                Award ID: R01AI065256
                Award Recipient :
                This work was supported in part by grant NIAID R01AI065256 to KES. ( https://www.niaid.nih.gov/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Cenicriviroc was provided under an MTA between the University of Cincinnati and Tobira (now Allergan).
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