Cyclic AMP, Cytokine Modulation and Alcoholic Liver Disease

描述（由申请人提供）：酒精性肝病（ALD）仍然是美国肝脏相关死亡的主要原因，目前还没有FDA批准的治疗方法细胞因子代谢异常是ALD的主要特征。据报道，酒精性肝炎和/或肝硬化患者血清中肿瘤坏死因子（TNF）和TNF诱导的促炎细胞因子/趋化因子（如IL-8和IL-18）浓度升高，且水平与急性期反应、肝功能和临床结局的标志物相关。类似地，关键的抗炎细胞因子IL-10的水平降低。动物模型研究支持细胞因子在酒精性肝损伤中的病因作用。在这个提议中要研究的主要问题是这种失调的细胞因子代谢的机制，我们的总体目标是开发新的治疗干预措施。ALD与氧化应激的增加相关，伴随着细胞抗氧化剂的减少，如谷胱甘肽（GSH），重要的是，S-腺苷-L-甲硫氨酸（SAM），由于低于正常的合成。临床研究表明，SAM在许多肝脏疾病（包括ALD）中具有有益作用，许多动物研究已清楚地证明SAM对各种肝毒素的保护作用。我们小组先前的研究评估了SAM在正性调节内毒素（LPS）刺激的与ALD相关的TNF和IL-10产生中的作用。我们发现，SAM减少LPS刺激的TNF产生和增加LPS刺激的IL-10产生，SAM增加细胞内cAMP（cAMP）的水平，这是已知的积极调节细胞因子的产生。因此，我们开始评估cAMP在ALD中失调的细胞因子产生中的作用，这是当前提案的研究重点。我们目前令人信服的初步数据表明，cAMP减少在长期培养的巨噬细胞系在酒精中，并在库普弗细胞从长期喂食乙醇灌胃小鼠。我们的工作假设是磷酸二酯酶4 B（PDE 4 B）和cAMP代谢的改变导致TNF和IL-10代谢异常，这在ALD的发展和持续中起着关键作用。本提案的具体目标是：1.记录来自酒精性肝炎（AH）和酒精性肝硬化患者的外周血单核细胞中增加的PDE 4 B表达/活性和减少的cAMP，并评估单核细胞与特异性磷酸二酯酶抑制剂的体外孵育是否校正失调的cAMP和细胞因子代谢; 2.确定在慢性ALD的鼠胃内乙醇喂养模型和AH的鼠模型中，PDE 4（并且具体地，PDE 4 B）抑制是否阻断酒精诱导的肝损伤的发展; 3.评估酒精性肝病中cAMP代谢改变的潜在分子和表观遗传机制;和4.评价在正常志愿者和稳定期酒精性肝硬化患者中联合使用市售腺苷酸环化酶激动剂（米索前列醇-增加cAMP生成）和磷酸二酯酶抑制剂（Pentoxyfilline -减少cAMP分解）的效果。 公共卫生相关性：酒精性肝病（ALD）仍然是美国肝病死亡的主要原因，并且仍然没有FDA批准的治疗方法。细胞因子代谢异常是ALD的主要特征。据报道，酒精性肝炎和/或肝硬化患者血清中肿瘤坏死因子（TNF）和TNF诱导的促炎细胞因子/趋化因子（如IL-8和IL-18）浓度水平升高，且水平与急性期反应、肝功能和临床结局的标志物相关。类似地，关键的抗炎细胞因子IL-10的水平降低。动物模型研究支持细胞因子在酒精性肝损伤中的病因作用。在这个提议中要研究的主要问题是这种失调的细胞因子代谢的机制，我们的总体目标是开发新的治疗干预措施。

DESCRIPTION (provided by applicant): Alcoholic Liver Disease (ALD) remains a leading cause of liver-related deaths in the U.S., and there is still no FDA-approved therapy. Abnormal cytokine metabolism is a major feature of ALD. Elevated serum concentrations of tumor necrosis factor (TNF) and TNF-inducible proinflammatory cytokines/chemokines, such as IL-8 and IL-18 have been reported in patients with alcoholic hepatitis and/or cirrhosis, and levels correlated with markers of the acute phase response, liver function, and clinical outcome. Similarly, there are depressed levels of the critical anti-inflammatory cytokine IL-10. Studies in animal models support an etiologic role for cytokines in the liver injury of ALD. The major problem to be investigated in this proposal is the mechanism(s) for this dysregulated cytokine metabolism, and our overall goal is the development of novel therapeutic interventions. ALD is associated with an increase in oxidative stress with a concomitant decrease in cellular antioxidants, such as glutathione (GSH) and importantly, S-adenosyl-L-methionine (SAM), due to subnormal synthesis. Clinical studies have suggested that SAM has beneficial effects in many hepatic disorders including ALD, and numerous animal studies have clearly demonstrated the protective effects of SAM against a variety of hepatotoxins. Previous studies from our group evaluated the role of SAM in positively modulating endotoxin (LPS)-stimulated TNF and IL-10 production related to ALD. We showed that SAM decreased LPS stimulated TNF production and increased LPS stimulated IL-10 production, and that SAM increased cellular levels of cyclic AMP (cAMP), which is known to positively modulate cytokine production. We therefore began an evaluation of the role of cAMP in dysregulated cytokine production in ALD, which is the research focus of this current proposal. We present compelling preliminary data that cAMP is decreased in a macrophage cell line chronically cultured in alcohol, and in Kupffer cells from mice chronically fed ethanol intragastrically. Our working hypothesis is that altered phosphodiesterase 4B (PDE4B) and cAMP metabolism cause abnormal TNF and IL-10 metabolism, which play a critical role in the development and perpetuation of ALD. Specific Objectives for this proposal are to: 1. Document increased PDE4B expression/activity and decreased cAMP in peripheral blood monocytes from patients with alcoholic hepatitis (AH) and alcoholic cirrhosis, and evaluate whether in vitro incubation of monocytes with specific phosphodiesterase inhibitors corrects dysregulated cAMP and cytokine metabolism; 2. Determine whether PDE4 (and specifically, PDE4B) inhibition blocks the development of alcohol-induced liver injury in a murine intra-gastric ethanol-feeding model of chronic ALD and in a murine model of AH; 3. Evaluate potential molecular and epigenetic mechanisms whereby cAMP metabolism is altered in alcoholic liver disease; and 4. Evaluate the effects of combined consumption of a commercially- available adenyl cyclase agonist (Misoprostol - increases cAMP production) and a phosphodiesterase inhibitor (Pentoxyfilline - decreases cAMP breakdown) in normal volunteers and in patients with stable alcoholic cirrhosis. PUBLIC HEALTH RELEVANCE: Alcoholic Liver Disease (ALD) remains a leading cause of death from liver disease in the U.S. and there is still no FDA-approved therapy. Abnormal cytokine metabolism is a major feature of ALD. Elevated serum concentration levels of tumor necrosis factor (TNF) and TNF-inducible proinflammatory cytokines/chemokines, such as IL-8 and IL-18 have been reported in patients with alcoholic hepatitis and/or cirrhosis, and levels correlated with markers of the acute phase response, liver function, and clinical outcome. Similarly, there are depressed levels of the critical anti-inflammatory cytokine IL-10. Studies in animal models support an etiologic role for cytokines in the liver injury of ALD. The major problem to be investigated in this proposal is the mechanism(s) for this dysregulated cytokine metabolism, and our overall goal is the development of novel therapeutic interventions.