Alcoholic liver disease (ALD) is the second highest cause of liver transplants in the United States, yet the mechanisms that underlie ethanol-induced liver damage remain poorly understood. Clinical studies of ALD recognize intestinal dysfunction as a critical player in the progression of disease. Chronic ethanol consumption induces transient losses in the intestinal barrier, which correlate with significant increases in circulating microbial byproducts such as lipopolysaccharide (LPS). Increases in LPS are associated with activation of toll like receptor 4 (TLR4), which stimulates the release of inflammatory cytokines from intestinal and liver resident macrophages, and results in the dysfunction of the liver parenchyma. Cobalt protoporphyrin (CoPP), a global inducer of heme oxygenase-1 (HO-1), inhibits the production of inflammatory cytokines in Kupffer cells and significantly reduces markers of liver injury. However, CoPP also induces HO-1 in the hepatocytes and the intestinal epithelium. Preliminary data in caco-2 cells suggest that epithelial HO-1 expression protects tight junctions. We hypothesize that the induction of HO-1 within the intestinal epithelium protects the liver from inflammation and damage by preserving the function of the intestinal tight junctions during ethanol feeding. To investigate the role of HO-1 in maintaining the intestinal barrier, the localization of tight junctions will be determined in the jejunum, ileum, proximal, and distal colo. The modified lactulose-mannitol test will be used to assess permeability of the fore, mid, and hindgut. Barrier measurements will be compared with circulating endotoxin to determine the impact of global HO-1 induction on endotoxin translocation. To determine the impact of epithelial HO-1 on liver protection, conditional HO-1 knockout mice will be generated for the intestinal epithelium, and the HO-1 will be induced with CoPP during the chronic ethanol diet. Hepatocyte, and monocyte conditional HO-1 knockouts will be generated to determine the impact of HO-1 in the liver parenchyma. The permeability of caco-2 monolayers in response to ethanol will be determined under conditions with or without CoPP and compared with the localization and relative expression of tight junction proteins. The impact of HO-1 on tight junctions will be determined by treating with inhibitors of HO-1 expression or enzymatic activity. HO-1 catalyzes the degradation of heme into carbon monoxide, biliverdin, and molecular iron, which is associated with upregulation of ferritin heavy chain. The impact of these products on the localization of tight junctions and barrier permeability will be determined using pharmacologic inhibitors, overexpression plasmids, shRNA and scrambled controls. During these experiments, the transepithelial electrical resistance, immunohistochemistry and FITC-dextran permeability assays will be used to assess barrier function.
Alcohol abuse is a leading cause of morbidity and mortality worldwide. Recent data indicate that alcoholic liver disease (ALD) affects over 10 million Americans. The long-term goals of this research project are to identify the mechanisms of HO-1 that are responsible for protection from inflammation and liver damage. Previous work has identified the intestine as a critical contributor to ALD progression. Increases in intestinal permeability and changes in the bacterial microbiota following chronic ethanol result in elevated levels of bacterial products such as lipopolysaccharide (LPS) in both alcoholic patients and animal models of ALD. The recognition of LPS by Toll-like receptor 4 (TLR4) on liver resident Kupffer cells, promotes the release of inflammatory cytokines that impact the survival of hepatocytes. Preliminary data indicate that the protection of intestinal tight junctions contribute to the protection by HO-1. Therefore, the overall objective of this proposal is to investigate the impact of HO-1 in preserving the function of the intestinal barrier during chronic ethanol consumption. Understanding the protective mechanisms within the intestinal liver axis will provide the foundation for the future development therapeutic interventions to slow or reverse alcoholic liver disease.
| Saikia, Paramananda; Bellos, Damien; McMullen, Megan R et al. (2017) MicroRNA 181b-3p and its target importin ?5 regulate toll-like receptor 4 signaling in Kupffer cells and liver injury in mice in response to ethanol. Hepatology 66:602-615 |
