Pradip B. Devhare

Knockdown of Autophagy Inhibits Infectious Hepatitis C Virus Release by the Exosomal Pathway.

ABSTRACT Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma in humans. We showed previously that HCV induces autophagy for viral persistence by preventing the innate immune response. Knockdown of autophagy reduces extracellular HCV release, although the precise mechanism remains unknown. In this study, we observed that knockdown of autophagy genes enhances intracellular HCV RNA and accumulates infectious virus particles in cells. Since HCV release is linked with the exosomal pathway, we examined whether autophagy proteins associate with exosomes in HCV-infected cells. We observed an association between HCV and the exosomal marker CD63 in autophagy knockdown cells. Subsequently, we observed that levels of extracellular infectious HCV were significantly lower in exosomes released from autophagy knockdown cells. To understand the mechanism for reduced extracellular infectious HCV in the exosome, we observed that an interferon (IFN)-stimulated BST-2 gene is upregulated in autophagy knockdown cells and associated with the exosome marker CD63, which may inhibit HCV assembly or release. Taken together, our results suggest a novel mechanism involving autophagy and exosome-mediated HCV release from infected hepatocytes. IMPORTANCE Autophagy plays an important role in HCV pathogenesis. Autophagy suppresses the innate immune response and promotes survival of virus-infected hepatocytes. The present study examined the role of autophagy in secretion of infectious HCV from hepatocytes. Autophagy promoted HCV trafficking from late endosomes to lysosomes, thus providing a link with the exosome. Inhibition of HCV-induced autophagy could be used as a strategy to block exosome-mediated virus transmission.

Exosome-Mediated Intercellular Communication between Hepatitis C Virus-Infected Hepatocytes and Hepatic Stellate Cells

ABSTRACT Fibrogenic pathways in the liver are principally regulated by activation of hepatic stellate cells (HSC). Fibrosis is associated with chronic hepatitis C virus (HCV) infection, although the mechanism is poorly understood. HSC comprise the major population of nonparenchymal cells in the liver. Since HCV does not replicate in HSC, we hypothesized that exosomes secreted from HCV-infected hepatocytes activate HSC. Primary or immortalized human hepatic stellate (LX2) cells were exposed to exosomes derived from HCV-infected hepatocytes (HCV-exo), and the expression of fibrosis-related genes was examined. Our results demonstrated that HCV-exo internalized to HSC and increased the expression of profibrotic markers. Further analysis suggested that HCV-exo carry miR-19a and target SOCS3 in HSC, which in turn activates the STAT3-mediated transforming growth factor β (TGF-β) signaling pathway and enhances fibrosis marker genes. The higher expression of miR-19a in exosomes was also observed from HCV-infected hepatocytes and in sera of chronic HCV patients with fibrosis compared to healthy volunteers and non-HCV-related liver disease patients with fibrosis. Together, our results demonstrated that miR-19a carried through the exosomes from HCV-infected hepatocytes activates HSC by modulating the SOCS-STAT3 axis. Our results implicated a novel mechanism of exosome-mediated intercellular communication in the activation of HSC for liver fibrosis in HCV infection. IMPORTANCE HCV-associated liver fibrosis is a critical step for end-stage liver disease progression. However, the molecular mechanisms for hepatic stellate-cell activation by HCV-infected hepatocytes are underexplored. Here, we provide a role for miR-19a carried through the exosomes in intercellular communication between HCV-infected hepatocytes and HSC in fibrogenic activation. Furthermore, we demonstrate the role of exosomal miR-19a in activation of the STAT3–TGF-β pathway in HSC. This study contributes to the understanding of intercellular communication in the pathogenesis of liver disease during HCV infection.

Hepatitis C virus–induced CCL5 secretion from macrophages activates hepatic stellate cells

Hepatitis C virus (HCV)–mediated chronic liver disease is a serious health problem around the world and often causes fibrosis/cirrhosis and hepatocellular carcinoma. The mechanism of liver disease progression during HCV infection is still unclear, although inflammation is believed to be an important player in disease pathogenesis. We previously reported that macrophages including Kupffer cells exposed to HCV induce proinflammatory cytokines. These secreted cytokines may activate hepatic stellate cells (HSCs) toward fibrosis. In this study, we examined crosstalk between macrophages and HSCs following HCV infection. Primary human HSCs and immortalized HSCs (LX2 cells) were incubated with conditioned medium derived from HCV‐exposed human macrophages. Expression of inflammasome and fibrosis‐related genes in these cells was examined, with increased expression of inflammatory (NLR family pyrin domain containing 3, interleukins 1β and 6, and cysteine‐cysteine chemokine ligand 5 [CCL5]) and profibrogenic (transforming growth factor β1, collagen type 4 alpha 1, matrix metalloproteinase 2, and alpha‐smooth muscle actin) markers. Further investigation suggested that CCL5, secreted from HCV‐exposed macrophages, activates inflammasome and fibrosis markers in HSCs and that neutralizing antibody to CCL5 inhibited activation. Conclusion: Together, our results demonstrate that human macrophages exposed to HCV induce CCL5 secretion, which plays a significant role in hepatic inflammation and fibrosis. (Hepatology 2017;66:746–757).

Zika virus infection dysregulates human neural stem cell growth and inhibits differentiation into neuroprogenitor cells.

The current outbreak of Zika virus-associated diseases in South America and its threat to spread to other parts of the world has emerged as a global health emergency. A strong link between Zika virus and microcephaly exists, and the potential mechanisms associated with microcephaly are under intense investigation. In this study, we evaluated the effect of Zika virus infection of Asian and African lineages (PRVABC59 and MR766) in human neural stem cells (hNSCs). These two Zika virus strains displayed distinct infection pattern and growth rates in hNSCs. Zika virus MR766 strain increased serine 139 phosphorylation of histone H2AX (γH2AX), a known early cellular response proteins to DNA damage. On the other hand, PRVABC59 strain upregulated serine 15 phosphorylation of p53, p21 and PUMA expression. MR766-infected cells displayed poly (ADP-ribose) polymerase (PARP) and caspase-3 cleavage. Interestingly, infection of hNSCs by both strains of Zika virus for 24 h, followed by incubation in astrocyte differentiation medium, induced rounding and cell death. However, astrocytes generated from hNSCs by incubation in differentiation medium when infected with Zika virus displayed minimal cytopathic effect at an early time point. Infected hNSCs incubated in astrocyte differentiating medium displayed PARP cleavage within 24–36 h. Together, these results showed that two distinct strains of Zika virus potentiate hNSC growth inhibition by different mechanisms, but both viruses strongly induce death in early differentiating neuroprogenitor cells even at a very low multiplicity of infection. Our observations demonstrate further mechanistic insights for impaired neuronal homeostasis during active Zika virus infection.

Hepatitis C virus–induced tumor‐initiating cancer stem–like cells activate stromal fibroblasts in a xenograft tumor model

Hepatitis C virus (HCV) often causes persistent infection and is an increasingly important factor in the etiology of fibrosis/cirrhosis and hepatocellular carcinoma, although the mechanisms for the disease processes remain unclear. We have shown previously that HCV infection generates an epithelial–mesenchymal transition state and tumor‐initiating cancer stem–like cells in human hepatocytes. In this study, we investigated whether HCV‐induced tumor‐initiating cancer stem–like cells when implanted into mice activate stromal fibroblasts. A number of fibroblast activation markers, including matrix metalloproteinase 2, were significantly increased at the mRNA or protein level in the xenograft tumors, suggesting the presence of tumor‐associated fibroblasts. Fibroblast activation markers of murine origin were specifically increased in tumor, suggesting that fibroblasts migrate to form stroma. Next, we demonstrated that conditioned medium from HCV‐infected human hepatocytes activates fibrosis‐related markers in hepatic stellate cells. We further observed that these HCV‐infected hepatocytes express transforming growth factor beta, which activates stromal fibroblast markers. Subsequent analysis suggested that anti–transforming growth factor beta neutralizing antibody, when incubated with conditioned medium from HCV‐infected hepatocytes, inhibits fibrosis marker activation in primary human hepatic stellate cells. Conclusion: HCV‐infected hepatocytes induce local fibroblast activation by secretion of transforming growth factor beta, and a preneoplastic or tumor state of the hepatocytes influences the network for the tumor‐associated fibroblast environment. (Hepatology 2017;66:1766–1778)

Differential Expression of MicroRNAs in Hepatitis C Virus-Mediated Liver Disease Between African Americans and Caucasians: Implications for Racial Health Disparities.

African Americans (AAs) have higher hepatocellular carcinoma (HCC) mortality rates than Caucasian Americans (CAs). Chronic hepatitis C virus (HCV) infection leads to cirrhosis and HCC. HCV infection is highly prevalent in the AA population compared to other racial groups. AAs are also less likely to naturally clear HCV, potentially contributing to higher prevalence of HCV. However, the explanation for this disparity is currently unknown. Circulating microRNAs (miRNAs) in the blood are emerging as biomarkers for pathological conditions. Expression analysis of miRNAs in major racial groups would be important for optimizing personalized treatment strategies. Here we assessed the differential expression of circulatory miRNAs from HCV-infected AA and CA patients. We identified increased expression of miR-146a, miR-150, and miR-155 in HCV-infected AA patient sera compared to that of CA. Further analysis demonstrated that these miRNAs were significantly elevated in AA patients diagnosed with HCV-mediated HCC. Higher expression of miR-150 was also noted in cirrhosis and HCC in AA patients, which may serve as a predictor of liver disease progression in this population. The differential expression of miRNAs suggests that these miRNAs and their target genes could be useful to gain further mechanistic insight of racial disparity associated with HCV-mediated pathogenesis.

Correction for Devhare et al., “Exosome-Mediated Intercellular Communication between Hepatitis C Virus-Infected Hepatocytes and Hepatic Stellate Cells”

This correction does not change the interpretation of data. Citation Devhare PB, Sasaki R, Shrivastava S, Di Bisceglie AM, Ray R, Ray RB. 2017. Correction for Devhare et al., “Exosome-mediated intercellular communication between hepatitis C virusinfected hepatocytes and hepatic stellate cells.” J Virol 91:e00349-17. https://doi.org/10.1128/ JVI.00349-17. Copyright

Abstract 5724: Circulatory microRNA expression profiling between HCV-infected African & Caucasian Americans: implications for racial health disparities

The purpose of the study: Hepatocellular carcinoma (HCC) is global problem and incidence of HCC is increasing in last several years. Hepatitis C virus (HCV) infection is one of the major cause towards development of HCC. HCV infection is highly prevalent in African American population compared to other ethnic groups and these people are less likely to naturally clear HCV. Higher incidence of HCC and mortality was noted in HCV infected African Americans as compared to Caucasians. However, the explanation for this disparity and the molecular mechanisms behind this are currently unknown. Circulating microRNAs in the blood are emerging as biomarkers for pathological conditions. Differential expression of miRNAs among ethnic groups would be important for optimizing personalized treatment strategies. Experimental procedures: In this study, we assessed the differential expression of circulatory miRNAs from HCV infected African Americans and Caucasians by cancer specific miRNA array profiling. Expression of significantly altered miRNAs was validated by qRT-PCR. Results: We identified increased expression of miR-146a, miR-150 and miR-155 in HCV infected African Americans patient sera as compared to sera of Caucasians. Further analysis demonstrated that these miRNAs were significantly elevated in African Americans diagnosed with HCV-mediated HCC. Higher expression of miR-150 was also noted in cirrhosis and HCC in African Americans, which may serve as predictor of liver disease progression in this population. Conclusion: The differential expression of miRNAs suggests that these miRNAs and their target genes could be useful to gain further mechanistic insight of racial disparity associated with HCV pathogenesis. Citation Format: Pradip Devhare, Robert Steele, Adrian M. Di Bisceglie, David E. Kaplan, Ratna B. Ray. Circulatory microRNA expression profiling between HCV-infected African & Caucasian Americans: implications for racial health disparities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5724. doi:10.1158/1538-7445.AM2017-5724