Synergistic actions by multiple Toll-like receptors in alcoholic liver disease

描述（由申请人提供）：慢性酒精滥用是美国成年患者肝硬化和肝功能衰竭的主要原因。酒精性肝病患者从脂肪变性发展为脂肪性肝炎、纤维化和肝硬化。目前的概念是，慢性酒精摄入增加肠道通透性，导致门静脉内毒素（脂多糖，LPS）水平升高。通过门静脉增加的内毒素通过Toll样受体（TLR）4刺激枯否细胞，TLR 4是LPS的受体，其促进肝脏炎症，导致酒精性肝损伤。酒精性肝硬化患者血液和腹水中不仅LPS，而且细菌DNA水平也升高。细菌DNA被TLR 9识别，TLR 9在免疫细胞中广泛表达，包括枯否细胞，肝脏中的常驻巨噬细胞。另一方面，我们最近发现TLR 4直接激活肝纤维化中的肝星状细胞（HSC）。我们推测过量的酒精摄入破坏了肠上皮屏障，导致肠道微生物来源的LPS和细菌DNA易位到肝脏中。LPS和细菌DNA分别激活库普弗细胞和HSC中表达的TLR 4和TLR 9，这反过来又产生炎症和纤维化介质，导致酒精性脂肪变性、脂肪性肝炎（ASH）和纤维化。TLR 4和TLR 9之间的协同相互作用以及肝细胞对细胞死亡的敏感性增加可能进一步加剧ASH和纤维化的程度。在乙醇处理后，肝脏中的枯否细胞产生炎性细胞因子，其可能与包括脑损伤在内的全身器官损伤相关。基于这些假设，本提案的目的是：目的#1：确定TLR 4在ASH中对枯否细胞和HSC活化的作用;将产生TLR 4-骨髓嵌合体并用胃内乙醇喂养处理。将评估脑损伤和肠通透性。目的#2：确定TLR 9在ASH中的作用;将在ASH模型中评估肝脏中负责TLR 9的细胞类型。目的#3：确定TLR 4和TLR 9在枯否细胞和HSC中的协同作用。目的#4：确定在ASH中肝细胞对细胞死亡的敏感性是否增加。我们将使用连续胃内乙醇喂养模型来测试这些特定目的。这项研究将为深入了解多种TLR信号在酒精诱导的发病机制中肠-肝-脑相互作用中的作用提供分子机制。 公共卫生相关性：酒精性脂肪性肝炎是全球范围内巨大的卫生保健负担。本研究的目的是研究TLR 4和TLR 9是否协同促进酒精性脂肪性肝炎和随后的脑损伤。本研究结果将为酒精性脂肪性肝炎和脑损伤等器官病变的治疗提供潜在的靶点。

DESCRIPTION (provided by applicant): Chronic alcohol abuse is the major cause of cirrhosis and liver failure in adult patients in the United States. Alcoholic liver disease in patients progresses from steatosis to steatohepatitis, fibrosis, and cirrhosis. The current concept is that chronic alcoholic intake increases intestinal permeability that leads to an elevation of endotoxin (lipopolysaccharide, LPS) levels in the portal vein. Increased endotoxin via the portal vein stimulates Kupffer cells through Toll-like receptor (TLR) 4, a receptor for LPS, which promotes hepatic inflammation resulting in alcoholic liver injury. Not only LPS, but also bacterial DNA levels in blood and ascites are elevated in patients with alcoholic-induced liver cirrhosis. Bacterial DNA is recognized by TLR9 that is widely expressed in immune cells including Kupffer cells, resident macrophages in the liver. On the other hand, we have recently shown that TLR4 directly activates hepatic stellate cells (HSCs) in hepatic fibrosis. We hypothesize that excessive alcohol intake disrupts intestinal epithelial barrier leads to translocation of intestinal microflora- derived LPS and bacterial DNA into the liver. LPS and bacterial DNA activate TLR4 and TLR9, respectively, expressed in Kupffer cells and HSCs, which in turn produce inflammatory and fibrogenic mediators, resulting in alcoholic steatosis, steatohepatitis (ASH) and fibrosis. Synergistic interaction between TLR4 and TLR9, and increased sensitivity of hepatocytes to cell death might further exacerbate the degrees of ASH and fibrosis. Upon ethanol treatment, Kupffer cells in the liver produce inflammatory cytokines, which could be associated with systemic organ injury including brain injury. Based on these hypotheses, the aims of this proposal are: Aim #1: To determine the role of TLR4 on the activation of Kupffer cells and HSCs in ASH; TLR4-bone marrow chimera will be generated and treated with intragastric ethanol feeding. Brain injury and intestinal permeability will be assessed. Aim #2: To determine the role of TLR9 in ASH; The responsible cell types for TLR9 in the liver will be assessed in ASH models. Aim #3: To determine the synergistic actions by TLR4 and TLR9 in Kupffer cells and HSCs. Aim #4: To determine whether the sensitivity of hepatocytes to cell death is increased in ASH. We will test these specific aims using the continuous intragastric ethanol feeding model. The proposed study will provide insight into the molecular mechanism underlying the role of multiple TLR signaling in Gut- Liver-Brain interaction in alcohol-induced pathogenesis. PUBLIC HEALTH RELEVANCE: Alcoholic steatohepatitis represents a massive health care burden worldwide. The goal of this study is to investigate whether TLR4 and TLR9 synergistically promote alcoholic steatohepatitis and subsequent brain injury. The results from this study will provide the potential targeting for the therapy of alcohol-induced organ pathogenesis including steatohepatitis and brain injury.