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

描述(申请人提供)：在美国，长期酗酒是导致肝硬变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、纤维化和肝硬变。目前的概念是，慢性酒精摄入会增加肠道通透性，导致门静脉内毒素(内毒素，LPS)水平升高。门静脉内毒素增加通过Toll样受体(TLR)4刺激枯否细胞，TLR 4是一种内毒素受体，可促进肝脏炎症，导致酒精性肝损伤。在酒精性肝硬变患者中，不仅内毒素水平升高，血和腹水中细菌DNA水平也升高。细菌DNA由TLR9识别，TLR9广泛表达于免疫细胞，包括库普弗细胞，肝脏中的驻留巨噬细胞。另一方面，我们最近发现TLR4在肝纤维化中直接激活肝星状细胞(HSCs)。我们假设，过量的酒精摄入破坏了肠道上皮屏障，导致肠道微生物来源的内毒素和细菌DNA移位到肝脏。脂多糖和细菌DNA分别激活表达在Kupffer细胞和HSCs中的TLR4和TLR9，进而产生炎症和纤维化介质，导致酒精性脂肪变性、脂肪性肝炎和纤维化。TLR4和TLR9的协同作用，以及肝细胞对细胞死亡的敏感性增加，可能进一步加重ASH和纤维化的程度。酒精处理后，肝脏中的Kupffer细胞产生炎性细胞因子，这可能与包括脑损伤在内的全身器官损伤有关。基于这些假设，本方案的目的是：目的1：确定TLR4在ASH中Kupffer细胞和HSCs激活中的作用；TLR4-骨髓嵌合体将被产生并经胃内乙醇喂养处理。将对脑损伤和肠道通透性进行评估。目的#2：确定TLR9在ASH中的作用；将在ASH模型中评估肝脏中TLR9的负责细胞类型。目的#3：确定TLR4和TLR9在Kupffer细胞和HSC中的协同作用。目的#4：探讨ASH患者肝细胞对细胞死亡的敏感性是否增加。我们将使用连续灌胃乙醇的模型来测试这些特定的目标。这项拟议的研究将为深入了解多个TLR信号在酒精诱导的发病机制中肠道-肝脏-大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世界范围内巨大的卫生保健负担。本研究的目的是探讨TLR4和TLR9是否协同促进酒精性脂肪性肝炎和继发性脑损伤。本研究结果为酒精性脂肪性肝炎和脑损伤等脏器病变的治疗提供了潜在的靶向。

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.