Recent Advances in understanding Alcoholic Liver Disease Tímea Óvári, Romane Marc, Côme Julienne JPEMS students Supervisor: Zsolt Bagosi, M.D., Ph.D. October 8, 2015

Summary Introduction to Alcoholic Liver Disease Role of intestinal permeability and endotoxemia in alcoholic liver disease Role of the hepatic stellate cells in the alcoholic liver cirrhosis Molecular mechanisms of alcoholic fatty liver Effects of ethanol on liver regeneration Innate immune response in Alcoholic Liver Disease Dysregulated cytokine metabolism Possible therapeutic molecules

Alcoholic Liver Disease • Hepatitis : inflammation in the liver  leads to cell injury. Symptoms : jaundice, fever, pain. Recovery in 10-50% of cases if alcohol abstinence • Steatosis : Lipid accumulation. Reversible process. • Cirrhosis : Chronic inflammation induce fibrosis process : production of collagen. Surviving hepatocytes try to regenerate the hepatic parenchyma  regenerative nodule formation. Complications : portal hypertension, liver failure and ascite formation. Irreversibility.

Ethanol induce increasing endotoxemia Role of intestinal permeability and endotoxemia in alcoholic liver disease Ethanol induce increasing endotoxemia 3 hypothesis : The localization of the disruption seems to be correlate with the type of use : Acute  duodenal localization Chronic  intestinal localization +++ Tight junction disruption MLCK : myosine light chain kinase TJ : tight junction AJ : adherence junction (R. K. Rao, A. Seth, P. Sheth , 2004)

Central role of LPS – endotoxemia in ALD  activate the Kupffer cells and other cells  Kupffer cell begin to produce pro-inflammatory cytokines.

2. Stellate cell : key role in the fibrogenesis Role of the hepatic stellate cells in the alcoholic liver cirrhosis 1. Physiological role of the stellate cells • Quiescent cells : storage of retinol • Located in the Disse space 2. Stellate cell : key role in the fibrogenesis Activation of hepatic stellate cell involve all components of the liver after alcohol exposure : • Liver sinusoidal endothelial cells • Kuppfer cells • Hepatocytes • Autocrin loop  auto-activation

• Liver sinusoidal endothelial cells  defenestration  lack of retinol • Kuppfer cells pro-inflammatory cytokines - TGFß : fibrogenic factor - PDGF : mitogenic factor • Injured hepatocytes  produce ROS • Autocrin loop : activated stellate cell produce their own TNF alpha and TGFß  accelerate the differentiation. (Guo and Friedmana, 2010)

Molecular mechanisms of alcoholic fatty liver

Two main pathways Ethanol decreases oxidation of the lipids by distorting PPARα, and increases lipogenesis by modifying SRBP1. PPARα : Peroxysome proliferator activating receptor α SREBP1 : Sterol regulatory element binding protein 1

Inhibition of PPARα by ethanol PPARα is a nuclear receptor which stimulates transcription of genes involved in free fatty acid transport and oxidation. To be activated it has to dimerize with RXR High endotoxin level in portal blood induced by ethanol decreases RXRα levels (www.wikipedia.org) RXRα : Retinoïd X Receptor α

Alteration of PPARα, SREBP1 and AMPK leads to steatosis AMPK : same effect as PPARα and inhibits SREBP1 Ethanol reduces AMPK activity ² ² ²

Effects of ethanol on liver regeneration

Different responses of a cell to an alcoholic stress Death of cells by necrosis  signal for regeneration Too damaged cells start apoptosis Surviving progenitors start repair mechanisms and then enlargement and division Surviving hepatocytes go into replicative senescence Response of a cell to a stress depend of the concentration of ROS Alcohol injuried liver can only count on progenitor cells to regenerate  alcoholics are more sensible to liver injuries

Liver stem cells Little is known about liver stem cells Come from the liver ? Fusion of myeloid progenitor and resident liver cells ? Injured progenitor cells express Notch and Jagged factors showing a reactivation of foetal pathways

Innate immune response in alcoholic liver diseases ETHANOL Innate immune response in alcoholic liver diseases ALD LPS CD14/TLR4 KUPFFER 1 h ↓ 24 h ↑ IRAK T cell transplantation Inflammatory mediators (TNF-α, IL-2, IL-8) Superoxide T CELL Kupffer cells are the main effectors of innate immune response in ALD. They are activated by gut-derived endotoxin. ETHANOL CD14/TLR4 TGF-β STELLATE collagen production (FIBROSIS)

Dysregulated cytokine metabolism LPS SINUSOIDAL CELLS Adhesion molecules KUPFFER NEUTROPHIL ROS Transmigration Oxidative stress IL-8 IL-18 Inflammatory mediators (TNF-α, IL-2, IL-8) Superoxide T CELL HEPATOCYTE Cell death STELLATE

Possible therapeutic molecules Resistance: Deficiency in CD14/TLR4 pathway Deficiency in TNF-α receptor 1 Deficiency in p47phox (NADPH oxidase) LPS Gadolinium chloride Antioxidants KUPFFER Anti-TNF-α Ab Adenoviral overexpression of SOD Inflammatory mediators (TNF-α, IL-2, IL-8) Superoxide Non-absorbable antibiotics Probiotics T CELL Kupffer cells are the main effectors of innate immune response in ALD. They are activated by gut-derived endotoxin. ETHANOL TGF-β STELLATE collagen production (FIBROSIS)

Thank you for your attention! Tímea Óvári, Romane Marc, Côme Julienne Acknowledgement: Supervisor: Zsolt Bagosi M.D. Ph.D. Department of Pathophysiology, University of Szeged Professor Dr. habil Gyula Szabó, M.D., Ph.D., D.Sc. Professor Dr. habil Márta Széll M.D., Ph.D.