Background:  Patients co-infected with HIV/hepatitis C virus (HCV) develop more rapid fibrosis than patients mono-infected with HCV. Fibrosis progression correlates with HIV viremia suggesting a direct role of HIV in liver fibrogenesis. CCR5 and CXCR4, the 2 major co-receptors required for HIV entry into cells, are expressed on hepatic stellate cells (HSC), the cell central to the fibrotic process. The aims of this study were to examine whether HIV enters HSC and actively replicates, and to characterize the effect of HIV and gp120 (HIV envelope protein) on HSC biology.

Methods:  First the capacity of HIV IIIB (CXCR4-tropic or X4) and HIV Bal (CCR5-tropic or R5) to infect HSC was assessed by enzyme-linked immunosorbent assay (ELISA) for supernatant p24. LX2 cells, a human HSC line, were infected and washed to remove unbound virus. Significant concentrations of p24 (>2 ng/mL) were detected on all days examined (until 7 days). Since X4 viruses predominate later in the course of HIV disease coincident with chronic liver disease in patients with HCV/HIV, we examined whether HIV IIIB infects primary human HSC (passage #3) and replicates using p24 assay and qRT-PCR for unspliced and multiply-spliced HIV-1 RNA. The detection of p24(>8 ng/mL) associated with intracellular unspliced and multi-spliced HIV suggests active replication (confirmed by sequencing MS HIV-1). The ability of HIV to infect HSC was confirmed by challenging the cells with a recombinant HIV expressing GFP in place of the early gene nef. The finding of GFP-positive cells indicates HIV entry and early gene expression. Because HIV infection may be either CD4-dependent or -independent, CD4 expression by HSCs was documented by immunofluorescence.

Results:  CD4-blocking experiments revealed that HIV IIIB entry into HSC was CD4-independent. HIV infection promoted HSC activation, since qRT-PCR demonstrated a 1.6-fold (p <0.0001) and 1.5-fold (p <0.0001) increase in collagen I and α-SMA mRNA levels, respectively. Furthermore, incubation with either monomeric gp120(X4) or AT-2 treated X4 (oligomeric gp120) for 1 to 24 hours resulted in a 2.1-fold (p <0.007) and 1.4-fold (p <0.004) increase in collagen I mRNA levels, respectively.

Conclusions:  HIV enters and actively replicates within HSC independent of CD4. Both viral entry as well as exposure of cells to viral envelope glycoproteins can promote activation and collagen induction in HSC. These results suggest that direct infection or Env-mediated activation of HSC may contribute to rapid development of fibrosis in patients co-infected with HIV/HCV.