Cyclic AMP, Cytokine Modulation and Alcoholic Liver Disease

描述(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，目前仍没有FDA批准的治疗方法。细胞因子代谢异常是ALD的主要特征。已有报道酒精性肝炎和/或肝硬变患者血清肿瘤坏死因子及肿瘤坏死因子诱导的促炎细胞因子/趋化因子，如IL-8和IL-18浓度升高，其水平与急性期反应、肝功能和临床转归相关。同样，关键的抗炎细胞因子IL-10水平也很低。动物模型研究支持细胞因子在ALD肝损伤中的病因学作用。在这项建议中要研究的主要问题是这种细胞因子代谢失调的机制(S)，我们的总体目标是开发新的治疗干预措施。肌萎缩侧索硬化症与氧化应激增加有关，同时伴随着细胞抗氧化剂的减少，如谷胱甘肽，更重要的是，由于合成异常，S-腺苷-L-蛋氨酸减少。临床研究表明，SAM对包括ALD在内的许多肝脏疾病都有有益的作用，大量的动物研究也清楚地证明了SAM对多种肝毒素的保护作用。我们小组以前的研究评估了SAM在积极调节内毒素(LPS)刺激的与ALD相关的TNF和IL-10产生中的作用。我们发现，SAM减少了内毒素刺激的肿瘤坏死因子的产生，增加了内毒素刺激的IL-10的产生，并增加了细胞内cAMP(CAMP)的水平，cAMP是已知的正向调节细胞因子产生的物质。因此，我们开始评估cAMP在ALD失调的细胞因子产生中的作用，这是本提案的研究重点。我们提供了令人信服的初步数据，即在酒精中长期培养的巨噬细胞系和长期灌胃酒精的小鼠的Kupffer细胞中cAMP减少。我们的工作假设是磷酸二酯酶4B(PDE4B)和cAMP代谢的改变导致了肿瘤坏死因子和白介素10代谢的异常，这在ALD的发生和发展中起着至关重要的作用。这项建议的具体目的是：1.记录酒精性肝炎(AH)和酒精性肝硬变患者外周血单核细胞PDE4B表达/活性增加和cAMP减少，并评估单核细胞与特异性磷酸二酯酶抑制剂体外孵育是否纠正cAMP和细胞因子代谢失调；2.确定PDE4(特别是PDE4B)抑制是否能阻止慢性酒精性肝病小鼠胃内酒精喂养模型和酒精性肝病小鼠模型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学机制；以及4.评估在正常志愿者和稳定期酒精性肝硬变患者中联合使用商业上可用的腺苷环化酶激动剂(米索前列醇-增加cAMP产生)和磷酸二酯酶抑制剂(戊氧富宁-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，目前仍没有FDA批准的治疗方法。细胞因子代谢异常是ALD的主要特征。已有报道酒精性肝炎和/或肝硬变患者血清肿瘤坏死因子(TNF)及肿瘤坏死因子诱导的促炎细胞因子/趋化因子(如IL-8和IL-18)水平升高，其水平与急性期反应、肝功能和临床转归相关。同样，关键的抗炎细胞因子IL-10水平也很低。动物模型研究支持细胞因子在ALD肝损伤中的病因学作用。在这项建议中要研究的主要问题是这种细胞因子代谢失调的机制(S)，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.