HCV in hepatic and stellate cell lines reveals cooperative profibrotic transcriptional activation between viruses and cell types

HIV/HCV co-infection accelerates progressive liver fibrosis, however the mechanisms remain poorly understood. HCV and HIV independently induce profibrogenic markers TGFβ1 (mediated by reactive oxygen species (ROS)) and NFκB in hepatocytes and hepatic stellate cells (HSC) in monoculture, however, they do not account for cellular cross-talk that naturally occurs. We created an in vitro co-culture model and investigated the contributions of HIV and HCV to hepatic fibrogenesis. GFP reporter cell lines driven by functional ROS (ARE), NFκB, and SMAD3 promoters were created in Huh7.5.1 and LX2 cells, using a transwell to generate cocultures. Reporter cells lines were exposed to HIV, HCV or HIV/HCV. Activation of the 3 pathways were measured, and compared according to infection status. Extracellular matrix products (Col1A1 and TIMP1) were also measured. Both HCV and HIV independently activate TGFβ1 signaling via ROS (ARE), NFκB, and SMAD3 in both cell lines in co-culture. Activation of Page 2 of 33 Hepatology Hepatology This article is protected by copyright. All rights reserved. these profibrotic pathways was additive following HIV/HCV co-exposure. This was confirmed when examining Col1A1 and TIMP1, where mRNA and protein levels were significantly higher in LX2 cells in co-culture following HIV/HCV co-exposure compared with either virus alone. In addition, expression of these profibrotic genes was significantly higher in the co-culture model compared to either cell type in monoculture, suggesting an interaction and feedback mechanism between Huh7.5.1 and LX2 cells. We conclude that HIV accentuates an HCV-driven profibrogenic program in hepatocyte and HSC lines through ROS, NFκB and TGFβ1 upregulation. Furthermore, co-culture of hepatocyte and HSC lines significantly increased expression of Col1A1 and TIMP1. Our novel co-culture reporter cell model represents an efficient and more authentic system for studying transcriptional fibrosis responses, and may provide important insights into hepatic fibrosis. Introduction Of the approximately one million people infected with human immunodeficiency virus (HIV) in the United States, thirty percent are co-infected with the hepatitis C virus (HCV) due to shared routes of transmission [1]. Chronic infection with HCV is well recognized to be associated with significant liver-related morbidity and mortality. In the highly active anti-retroviral treatment (HAART) era, liver disease is now the third leading cause of mortality among HIV-infected patients after acquired immunodeficiency syndrome (AIDS)-related causes and non-AIDSdefining cancers [1]. HIV co-infection with concomitant HCV infection has been shown to accelerate HIV/HCV-related fibrosis progression compared to HCV monoinfection [2-4]. In this context, HCV-related liver disease has rapidly contributed to a dramatic rise in the proportion of patients with advanced fibrosis or cirrhosis, an increase in the number of co-infected patients who have been referred for liver transplantation, a rise in the wait-list mortality among those awaiting liver transplantation, and a sharp rise in liver-related mortality [5]. Hepatic fibrosis is a wound-healing response to chronic liver injury that results in the accumulation of extracellular matrix (ECM) products, including collagen, fibronectin and proteoglycans. Hepatic stellate cells (HSC) are the primary source of ECM, and therefore are the main cell type within the liver that drive hepatic fibrogenesis[6]. Overexpression of the cytokine transforming growth factor beta-1 (TGFβ1) has been widely demonstrated following liver injury. Induction of TGFβ1 occurs in multiple cell types through several pathways, including Page 3 of 33 Hepatology Hepatology This article is protected by copyright. All rights reserved. reactive oxygen species (ROS), extracellular signal-regulated kinases (ERK), Jun aminoterminal kinases (JNK), and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) [7-9]. TGFβ1 also can activate HSC [10], which in turn further upregulates TGFβ1 production [11], and induces type I alpha 1 collagen (Col1A1) and tissue inhibitor of metalloproteinase 1 (TIMP1) expression, leading to deposition of ECM resulting in hepatic fibrogenesis [12]. Both HCV and HIV can establish infection within the liver through parenchymal and nonparenchymal cells, respectively. Previously, we have demonstrated that HCV and HIV each contribute to fibrogenesis via the induction of profibrogenic cytokines, such as TGFβ1, in monoculture models of hepatocyte or HSC lines infected with HCV or HIV [7]. Several other groups have also demonstrated that HCV induces mitochondrial oxidative stress and ROS [1315]. In a follow-on study, we found that HCV upregulates TGFβ1 via the induction of ROS, as well as through a p38 mitogen-activated protein kinases, JNK, and ERK1/2-dependent pathway [16]. We also identified that the induction of TGFβ1 by ROS is NFκB dependent [16]. In contrast to HCV, which establishes infection within hepatocytes, HIV cannot infect hepatocytes. However recent evidence supports that HIV is able to infect HSC through a chemokine receptor-independent mechanism [17]. In addition, HIV-1 is also capable of interacting with and can signal through cysteine-X-cysteine receptor 4 (CXCR4) and chemokine motif receptor 5 (CCR5), the co-receptors required for HIV entry, which are expressed on both HSCs and hepatocytes [18-20]. Furthermore, infection with HIV has been shown to induce oxidative stress and upregulate TGFβ1 expression [17]. Therefore, HIV-1 may act both directly and indirectly to contribute to hepatic fibrosis in HIV/HCV co-infected patients. Although HIV itself and its envelope glycoprotein gp120 can each increase TGFβ1 levels, they also augment HCV-induced TGFβ1 expression in infectious JFH1 HCV tissue culture models of HIV/HCV coinfection [17]. In addition, TGFβ1 produced by hepatocytes and other resident liver cell populations further upregulate HCV replication in hepatocytes, thereby augmenting responses to HCV infection [18]. Previously, we provided evidence that both HIV and HCV independently regulate hepatic fibrosisby regulating the production and deposition of components of ECM, including Col1A1 and TIMP1 via induction of ROS [7]. This regulation occurs in an NFκB-dependent fashion in both hepatocytes and HSCs. In addition, we have shown that HIV/HCV co-exposure enhances Page 4 of 33 Hepatology Hepatology This article is protected by copyright. All rights reserved. the production of these profibrogenic cytokines, leading to greater induction of Col1A1 and TIMP1 expression in both hepatocytes and HSCs. These data demonstrate that HIV and HCV both contribute to hepatic fibrogenesis in several cell types within the liver, and that HIV and HCV act cooperatively to induce hepatic fibrogenesis in the context of HIV/HCV co-exposure compared to exposure with either virus. Cell-cell interactions are central to the function and responses of many organ systems, and the liver is a prime example of where cross talk between cell types is critical. This is further complicated by the lack of an adequate small animal model to study HIV/HCV co-infection; chimpanzees are the only robust animal model that supports HIV and HCV infection, which is costly and inaccessible to many. Therefore, there is a great need for cell culture systems that more realistically recapitulate the in vivo liver milieu during HIV/HCV co-infection, which could also apply more broadly to other liver diseases. The transwell co-culture system provides the unique ability to assess cell-to-cell interactions within specific cell types of interest in a real-time, high throughput manner. Understanding how cell-to-cell interactions modulate or amplify tissue responses to viral infection may provide further insight into the complex cellular processes that accelerate liver disease and identify novel targets that may be amenable for therapeutic applications. To further explore the mechanisms by which HIV and HCV collaborate to stimulate liver fibrogenesis using conditions more closely approximating the in vivo liver environment, we developed reporter cell lines for hepatocytes and HSCs and placed them in co-culture. Material and Methods Cell culture Experiments we performed using Huh7.5.1 cells (kindly provided by Dr Francis Chisari, Scripps Institute, La Jolla, CA, USA) – a sub-line of Huh7 human hepatoma cells that are highly permissive for HCV replication [21], LX2 cells – immortalized human hepatic stellate cells [22], and 293T Human Embryonic Kidney cells. Primary hepatic stellate cells were isolated from the non-parenchymal cell fraction from HCV/HIV seronegative donor livers (Triangle Research Labs, Durham, NC, USA). HSCs were isolated as previously described [23]. Cells were cultured in Dulbecco's modified Eagle's medium (Mediatech, Manassas, VA, USA) supplemented with 10% fetal bovine serum (Mediatech, Manassas, VA, USA), 100 U/mL of penicillin and 100 μg/mL of streptomycin (Lonza/BioWhittaker, Walkersville, MD, USA) and were maintained at 37°C in humidified air containing 5% carbon dioxide. Page 5 of 33 Hepatology Hepatology This article is protected by copyright. All rights reserved. Viral stocks The infectious JFH1 genotype 2a clone [24], a generous gift from Dr Takaji Wakita, was used to prepare cell-culture derived HCV particles as previously described [24]. The HIV NL4-3 virus clone, a molecularly cloned highly cytopathic CXCR4-tropic virus, was obtained from the Ragon Institute (MGH Harvard, Boston, MA, USA). HIV stock virus was generated as previously described [25]. Construction of reporter plasmids Reporter plasmids for the transcription factor response elements of the three major pathways of interest were constructed as previously described [26,27]. The first reporter plasmid is antioxidant response elements (ARE, representing ROS response), second is NFκB, and third is SMAD3 representing TGFβ1 response.