HCV Hepatocytes Kill HIV Hepatocytes through TRAIL Figure 5. Co-incubation of HIV/HCV treated hepatocytes kills HIV treated hepatocytes via TRAIL. (A) Huh7 cells expressing HCV core were co-incubated with HIV gp120 treated hepatocytes at increasing effector-to-target ratios, and cell death of HIV treated (Dio positive) hepatocytes were determined by flow cytometry for active caspase 3 expression. (B) HIV gp120 or BSA control treated cells were incubated with HCV core or mock transfected cells and death analyzed in the gp120 or BSA treated cells. (C and D) HIV gp120 or BSA treated cells were incubated with HCV core or mock transfected cells, in the presence or absence of a TRAIL-R2-Fc fusion protein, or control-Fc fusion protein, and cell death analyzed in the gp120 or BSA treated cells. doi:10.1371/journal.pone.0027171.g005 cells [34]. HIV gp120 also induces death of non-immune cells, including neurons and hepatocytes [10,24,35,36]. Therefore, HIV gp120 causes at least two distinct effects upon the liver: 1) low level of background death [24] which may account for the increased liver disease in HIV mono-infected patients compared to uninfected patients, and 2) increasing TRAIL-R2 expression, and co-incident acquired sensitivity to TRAIL mediated apoptosis [14], which likely contributes to the accelerated course of co- morbid liver disease during HCV co-infection. Within our proposed model, one would expect increased hepatocyte apoptosis during any disease states in which TRAIL and TRAIL-R2 are increased. In addition to HIV gp120, a number of other stimuli can increase TRAIL-R2 expression or TRAIL sensitivity in hepatocytes. The Hepatitis B virus protein MHBs(t) sensitizes human hepatocytes to TRAIL [37] and is mediated via Bax signaling [38]. Free fatty acid induced steatosis causes TRAIL sensitivity in human hepatocytes [39] which is exacerbated by treatment with alcohol [22]. In addition, bile acids increase TRAIL-R2 and induce TRAIL sensitivity in human hepatocytes. This process is mediated via a c-Jun N-terminal kinase-dependent pathway involving Sp1 [12]. Medications can also sensitize cells to TRAIL mediated apoptosis and are being investigated as a means to treat otherwise TRAIL resistant cancers. Doxorubicin treatment, for instance, sensitizes glioblas- toma cells [40] and liver cancer cells to TRAIL via the BH3-only proteins Bim and Bid [41]. At supra-therapeutic levels, even HIV protease inhibitors, such as Nelfinavir, can sensitize cells to TRAIL [42]. This may in part explain why there is worse liver disease during Hepatitis C Virus infection and any of these co-existing conditions. Several reports indicate that hepatocytes obtained from patients with HCV have increased TRAIL expression compared to hepatocytes from uninfected controls [21,22]. Our data expands upon these observations with the novel findings that this effect localizes to the HCV core protein. Moreover, we now report that increased apoptosis occurs in hepatocytes when HCV core expressing hepatocytes produce TRAIL which induces fratricidal killing of HIV gp120 primed hepatocytes that manifest both increased TRAIL-R2 as well as enhanced TRAIL sensitivity. This model is further supported by immunohistochemistry in tissues from HIV or HCV mono-infection, or HIV/HCV co-infection where similar changes occur in vivo as what we observed in vitro. While we demonstrate that HCV core protein alone is sufficient to increase TRAIL expression, it is possible that other HCV structural or non-structural proteins may also alter TRAIL expression. Furthermore, our model does not exclude the possibility that HCV infection triggers an indirect effect that in turn regulates TRAIL expression. HCV infection results in a number of alterations in the immune system most notably, HCV causes a Type 1 interferon response. Type 1 interferon induces TRAIL expression on NK cells which is associated with control of HCV infection in patients [43]. Furthermore, in a cell culture model, the HCV isolate JFH-1 resulted in an interferon response which caused apoptosis of infected cells [44]. Interestingly, amino PLoS ONE | www.plosone.org 5 November 2011 | Volume 6 | Issue 11 | e27171