HIV induces TRAIL sensitivity in hepatocytes

Abstract

Background: HIV infected patients have an increased susceptibility to liver disease due to Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), alcoholic, and non-alcoholic steatohepatitis. Clinically, this results in limited options for antiretroviral therapy and accelerated rates of liver disease, causing liver disease to be the second leading cause of death for HIV infected patients. The mechanisms causing this propensity for liver dysfunction during HIV remains unknown.

Methodology/principal findings: We demonstrate that HIV and/or the HIV glycoprotein gp120 ligation of CXCR4 on hepatocytes selectively up-regulates TRAIL R2 expression and confers an acquired sensitivity to TRAIL mediated apoptosis which is mediated by JNK II, but not p38 nor G-proteins.

Conclusions/significance: These findings suggest that HIV infection renders hepatocytes more susceptible to liver injury during disease states associated with enhanced TRAIL production such as HBV, HCV, or steatohepatitis.

Figure 1. HIV IIIb makes Huh7 cells sensitive to TRAIL mediated apoptosis.

Huh7 cells were pre-incubated or not with AMD3100 for one hour followed by treatment with purified HIV IIIb or mock virus for six hours. Selective points were then treated with skTRAIL for twelve hours. Cells were then fixed, permeabilized, and stained with fluorescently labeled anti-active caspase-3 antibodies and analyzed by flow cytometry. Camptothesin (10 µM) was used as positive control for caspase-3 activation. (A) A single representative dot plot for each group is given. In each plot, the percentage of cells positive for active caspase-3 is shown in the upper right quadrant. (B) Values are expressed as the mean±SD of three independent experiments.

Figure 2. HIV gp120 up-regulates TRAIL R2 expression in Huh7 cells.

(A) Huh7 cells were treated with HIV gp120 or control protein for six hours, stained with fluorescently conjugated antibodies specific for TRAIL receptors and analyzed by flow cytometry. BSA control protein is shown as a dotted line, and HIV gp120 is shown as a solid line. A single representative histogram from three independent experiments is given for each group. (B) Huh7 cells were treated with HIV gp120 for one, two, or three hours, lysed, and resolved on 15% SDS-PAGE, electrotransferred to PVDF membrane, and probed for TRAIL R2. Densitometer readings are presented as the relative intensity of TRAIL R2 normalized to actin. The data represents the mean±SD of three independent experiments. (C) Huh7 cells were pre-incubated or not with AMD3100 for one hour followed by treatment with HIV gp120 or BSA and analyzed for TRAIL R2. Data of mean channel fluorescence represents the mean±SD of three independent experiments.

Figure 3. HIV gp120 induces TRAIL sensitivity in Huh7 cells in a JNK dependent manner.

(A) Huh7 cells were pre-incubated or not with AMD3100 for one hour and then treated with HIV gp120 or BSA for six hours followed by treatment of skTRAIL. Cell viability was determined by reduction of MTS by live cells. (B) Huh7 cells were pre-incubated with JNK II inhibitor, p38 inhibitor or the G-protein inhibitor, Pertussis toxin, for one hour followed by treatment with HIV gp120 or control protein for six hours. Cell viability was determined by reduction of MTS in live cells. Values are expressed as the mean±SD of three independent experiments from duplicate wells.

Figure 4. siRNA inhibition of JNK II inhibits gp120 induced TRAIL R2 upregulation.

Huh7 cells were treated with either BSA or gp120 in the presence or absence siRNA constructs for JNK I, JNK II, and p38; and analyzed for TRAIL R2 content (A). The results presented below are pooled TRAIL R2 densitometry normalized to Actin. Parallel experiments demonstrate that siRNA for JNK I, JNK II, and p38 did not alter TRAIL R2 expression (B), and these siRNA constructs were specific for the proteins against which they were directed (C).

Figure 5. Role of TRAIL in HIV/HCV co-infection.

Schematic model of synergy between HIV gp120/CXCR4-induced TRAIL sensitivity and other proapoptotic stimuli (e.g., HBV, HCV, Bile salts), causing hepatocyte death and consequent cirrhosis.