Exposure to human immunodeficiency virus/hepatitis C virus in hepatic and stellate cell lines reveals cooperative profibrotic transcriptional activation between viruses and cell types

Abstract

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 co-cultures. 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 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.

Human immunodeficiency virus and liver disease: A comprehensive update

“…In addition, by developing a coculture system, the group recently was able to demonstrate that both HCV and HIV independently activate transforming growth factor-b1 signaling through reactive oxygen species in both cell lines, and activation of these profibrotic pathways was additive following exposure to both viruses. (7) Expression of these profibrotic genes was significantly higher in the coculture model compared to either cell type in monoculture, suggesting an interaction and feedback mechanism between Huh7.5.1 and LX2 cells. Thus, it appears that HIV exacerbates a profibrogenic program in hepatocyte and hepatic stellate cell lines, and this model is at least relevant to HCV-related liver fibrosis progression (see Fig.…”

“…In a series of elegant in vitro studies, Chung and coworkers showed that the HIV envelope stimulates greater transforming growth factor‐β1 production by Huh7.5.1 cells and together with HCV enhances collagen and tissue inhibitor of metalloproteinase‐1 production by stellate cells through the production of reactive oxygen species. In addition, by developing a coculture system, the group recently was able to demonstrate that both HCV and HIV independently activate transforming growth factor‐β1 signaling through reactive oxygen species in both cell lines, and activation of these profibrotic pathways was additive following exposure to both viruses . Expression of these profibrotic genes was significantly higher in the coculture model compared to either cell type in monoculture, suggesting an interaction and feedback mechanism between Huh7.5.1 and LX2 cells.…”

Critical role of CREBH‐mediated induction of transforming growth factor β2 by hepatitis C virus infection in fibrogenic responses in hepatic stellate cells

“…In HCV‐mediated fibrogenesis, up‐regulation of fibrogenic factors was reported in a co‐culture of HSCs with HCV Core‐expressing cells, and TGF‐β1 secreted from the E2‐expressing cells activated the HSCs . HCV/human immunodeficiency virus co‐exposure in hepatocytes and HSC revealed cooperative transcriptional activation of profibrotic pathways . However, the impact of knockdown or knockout of the key molecules that were identified in their study on fibrogenic responses were not determined in the co‐culture setting.…”

Hepatic stellate cells as key target in liver fibrosis

“…HIV can directly infect HSCs, and viral envelop protein, gp120, promotes collagen I expression via CXCR4 [104, 105]. Exposure to HIV induces TGFβ, ROS, and NF-κB in a hepatocyte/LX-2 co-culture reporter cell model [106]. …”