HCV Hepatocytes Kill HIV Hepatocytes through TRAIL Figure 2. HIV gp120 Does not Increase TRAIL. (A and B) Huh7 cells were incubated with increasing concentrations of HIV gp120 and analyzed for TRAIL expression by Flow cytometry (C and D) and Western blot. doi:10.1371/journal.pone.0027171.g002 R2 while HIVgp120 increases TRAIL-R2 and confers an acquired state of TRAIL sensitivity, while not impacting TRAIL levels. HIV increases TRAIL-R2 and HCV Increases TRAIL in vivo In our model, HIV gp120 treatment increases TRAIL-R2 expression on Huh7 cells, and HCV core protein increases TRAIL expression. In order to confirm that these changes also occur in vivo, liver samples from 10 uninfected individuals, 10 HIV infected, 10 HCV infected and 10 HIV/HCV co-infected individuals were stained by immunohistochemistry for TRAIL-R2, TRAIL and cleaved PARP as a measure of apoptosis (Fig. 4). Normal liver samples taken from individuals not infected with HIV or HCV have minimal TRAIL-R2, and minimal TRAIL expression. TRAIL-R2 levels are increased in liver samples from HIV- infected individuals and TRAIL staining is increased in HCV infected livers. Liver samples from HIV/HCV co-infected individuals have increased TRAIL and TRAIL-R2 staining (Fig. 4). Furthermore, HIV/HCV co-infected livers have greater hepatocyte apoptosis, as determined by staining of cleaved PARP, than HIV mono-infected, HCV mono-infected or uninfected livers, consistent with previous reports[17,31]. Co-incubation of HIV and HCV Treated Huh7 cells Results in a TRAIL Mediated Apoptosis Our observation of increased TRAIL-R2 expression and enhanced TRAIL sensitivity induced by HIV and increased TRAIL expression induced by HCV core, predicts that the presence of both viruses should result in augmented hepatocyte death. To test this hypothesis, Huh7 cells were stained with the lipophilic dye Dio, treated with HIV gp120 and then co-incubated at increasing effector-to-target ratios with HCV core protein transfected Huh7 cells. Following an overnight incubation, cells were analyzed by flow cytometry for active-caspase-3 expression to determine the levels of apoptosis in the Dio positive (HIV gp120 treated) and Dio negative (HCV core expressing) cells. There was no Dio leak between hepatocytes (data not shown). HIV core expressing (Dio positive) target cells had greater apoptosis than HCV treated hepatocytes and this effect increased with increasing E:T ratios (Fig. 5A). The effect was not present when target cells were treated with control protein rather than HIV gp120 (Fig. 5B). The HCV core expressing (Dio negative) effector cells had no greater apoptosis than untreated cells (data not shown). Pre- incubating the Huh7 cells with the TRAIL-R2-Fc fusion protein, blocked the increased apoptosis that resulted from co-incubating HIV and HCV treated hepatocytes (Fig. 5C and 5D). These data demonstrate that HIV gp120 increases TRAIL-R2 expression and TRAIL confers sensitivity in treated hepatocytes; HCV core protein increases TRAIL expression in hepatocytes and co- incubating the HIV gp120 and HCV core expressing liver cells results in an accelerated TRAIL mediated apoptosis, selectively in the HIV treated cells. The relevance of this in vivo is suggested by similar changes being observed in liver biopsies from patients with HIV infection, HCV infection or both. Discussion It is clear that HIV worsens the course of HCV infection by increasing hepatocyte apoptosis, liver fibrosis and inflammation and quickening the progression to end stage liver disease [6]. It remains unclear how HIV propagates HCV liver disease, but clinical evidence demonstrates that decreasing HIV viral replica- tion and improving CD4+ T cell counts, improves markers of liver disease [5,6]. PLoS ONE | www.plosone.org 3 November 2011 | Volume 6 | Issue 11 | e27171