Increased Sensitivity to Binge Alcohol-Induced Gut Leakiness and Inflammatory Liver Disease in HIV Transgenic Rats

Abstract

The mechanisms of alcohol-mediated advanced liver injury in HIV-infected individuals are poorly understood. Thus, this study was aimed to investigate the effect of binge alcohol on the inflammatory liver disease in HIV transgenic rats as a model for simulating human conditions. Female wild-type (WT) or HIV transgenic rats were treated with three consecutive doses of binge ethanol (EtOH) (3.5 g/kg/dose oral gavages at 12-h intervals) or dextrose (Control). Blood and liver tissues were collected at 1 or 6-h following the last dose of ethanol or dextrose for the measurements of serum endotoxin and liver pathology, respectively. Compared to the WT, the HIV rats showed increased sensitivity to alcohol-mediated gut leakiness, hepatic steatosis and inflammation, as evidenced with the significantly elevated levels of serum endotoxin, hepatic triglycerides, histological fat accumulation and F4/80 staining. Real-time PCR analysis revealed that hepatic levels of toll-like receptor-4 (TLR4), leptin and the downstream target monocyte chemoattractant protein-1 (MCP-1) were significantly up-regulated in the HIV-EtOH rats, compared to all other groups. Subsequent experiments with primary cultured cells showed that both hepatocytes and hepatic Kupffer cells were the sources of the elevated MCP-1 in HIV-EtOH rats. Further, TLR4 and MCP-1 were found to be upregulated by leptin. Collectively, these results show that HIV rats, similar to HIV-infected people being treated with the highly active anti-retroviral therapy (HAART), are more susceptible to binge alcohol-induced gut leakiness and inflammatory liver disease than the corresponding WT, possibly due to additive or synergistic interaction between binge alcohol exposure and HIV infection. Based on these results, HIV transgenic rats can be used as a surrogate model to study the molecular mechanisms of many disease states caused by heavy alcohol intake in HIV-infected people on HAART.

Fig 1. Binge alcohol-mediated hepatic fat accumulation and inflammation in HIV-Tg rats compared to WT rats.

(A-F) Representative photomicrographs of H&E stained liver sections from the indicated rat livers are presented: (A) vehicle-control WT, (B) vehicle-control HIV-Tg rats, (C, E) ethanol-exposed WT rats, and (D, F) ethanol-exposed HIV-Tg rats. Insets represent enlarged portions of liver sections to show accumulated fat. Arrows (in E and F) represent accumulated neutrophils. (G) Hepatic triglyceride levels in WT or HIV-Tg rats exposed to dextrose control or ethanol, as indicated. *, # Significantly different from the corresponding dextrose controls and ethanol-exposed WT counterparts, respectively.

Fig 2. Binge ethanol-mediated increment of inflammatory foci and neutrophils in HIV-Tg rats compared to the corresponding WT rats.

(A-C) Representative liver sections stained with chloroacetate esterase indicating inflammatory foci are presented: (A) WT-EtOH, (B) HIV-EtOH, and (C) quantification of inflammatory foci. (D-F) Increase neutrophils stained with F4/80 in liver sections of each group are presented, as indicated: (D) WT-EtOH, (E) HIV-EtOH, and (F) Quantification of F4/80 positive cells per hpf.

Fig 3. Binge ethanol-mediated elevation of serum endotoxin and TLR4 mRNA levels in HIV-Tg rats compared to WT.

Increased gut leakage in ethanol-exposed HIV-Tg rats. (A) Serum endotoxin levels and (B) hepatic levels of TLR4 mRNA in samples collected at 1 and 6 h, respectively, after the last ethanol dose in the indicated groups are presented.

Fig 4. Binge ethanol-mediated elevation of hepatic MCP-1 levels in HIV-Tg rats compared to WT.

Changes in MCP-1 levels in HIV-Tg or WT rats following exposure to dextrose control or EtOH are presented. The levels of (A) serum MCP-1, (B) hepatic MCP-1 mRNA, (C) hepatic CCR2 mRNA, (D) MCP-1 mRNA in primary hepatocytes, and (E) MCP-1 mRNA in Kupffer cells in the indicated groups are presented.

Fig 5. Binge ethanol-mediated elevation of hepatic leptin in HIV-Tg rats compared to WT.

Changes in the leptin and its related factors in HIV-Tg or WT rats following exposure to dextrose control or EtOH are presented. The mRNA levels of (A) hepatic leptin, (B) leptin receptor, (C) TRL4 in hepatocytes treated with leptin and EtOH, alone or in combination, (D) MCP-1 in hepatocytes treated with EtOH, leptin, leptin+EtOH or neutralizing anti-leptin (nLeptin) antibody are shown.

Fig 6. Schematic diagram describing a potential mechanism for binge alcohol-mediated gut leakiness, inflammation and steatosis in HIV-Tg rats.

Binge alcohol can increase gut leakiness, serum endotoxin levels, the expression of TLR4 receptor, contributing to activation of Kupffer cells, producing greater amounts of MCP-1 and CCR2 for neutrophil infiltration and subsequently inflammatory fatty liver injury in HIV-Tg rats compared to the corresponding WT.