Neutrophil-Epithelial Interaction Mediated by Adenosine

描述(由申请人提供)：炎症性腹泻疾病是美国和世界范围内的一个主要公共卫生问题和经济负担。腺苷是一种内源性信号分子，在炎症活动时，其在结肠组织和腔液中的水平升高。在肠道中，腺苷2B受体(A2BAR)是结肠上皮细胞表达的主要腺苷受体，并介导腺苷的作用。我们的研究得到了NIH K08和R01的资助，重点研究了结肠上皮A2BAR的信号机制和生物学功能。这一竞争性的更新应用建立在一系列体外和体内观察的基础上，这些观察表明结肠上皮A2BAR具有促腹泻和促炎作用。总之，我们的研究表明，A2BAR的激活通过协调上皮细胞和中性粒细胞之间的相互作用信号来协调急性炎症反应：i)A2BAR处于静止状态并在激动剂刺激下被募集到膜上；ii)在炎症性肠病(IBD)模型中，A2BAR的mRNA和蛋白水平在急性炎症发作时上调；iii)A2BAR介导顶部极化的IL-6和纤维连接蛋白的分泌，进而分别介导中性粒细胞的激活和细菌的黏附/侵袭；iv)A2BAR促进IL-8的分泌并介导中性粒细胞在炎症刺激下的募集；v)A2BAR拮抗或基因缺失抑制中性粒细胞的渗透；6)基因消融小鼠的A2BAR可防止细菌和化学诱导的腹泻和炎症；以及7)使用高度特异的抑制剂抑制A2BAR可缓解小鼠的急性和慢性炎症。尽管我们已经在了解上皮A2BAR在协调粘膜炎症反应中的作用方面取得了重大进展，但A2BAR的生理作用、其在炎症过程中的调节以及免疫细胞A2BAR在炎症中的作用尚不清楚。本研究的目的是阐明A2BAR的生理功能，调节结肠上皮细胞A2BAR的表达，并确定免疫细胞A2BAR在炎症性腹泻疾病中的作用。我们推测A2BAR在调节正常结肠的液体分泌中起中心作用。肠道炎症时上皮细胞和免疫细胞中A2BAR的表达和功能上调在炎症反应中起重要作用，因此A2BAR拮抗是治疗炎症性腹泻疾病的有效方法。这些拟议的研究不仅将解决有关A2BAR在调节结肠液分泌和炎症反应中的生理意义的问题，而且还将为在一个为肠道炎症疾病提供有限治疗方式的领域开发A2BAR靶向疗法提供“原则证据”。公共卫生相关性：炎症性腹泻病是美国和世界范围内严重的发病率和死亡率的原因。这些疾病的发病机制尚不清楚。腺苷是一种自然产生的代谢物，在活动性肠道炎症期间产生，其水平在炎症期间增加。在肠道，腺苷2b受体(A2B受体)介导腺苷的作用。这一建议将阐明腺苷2b受体在腹泻和炎症中的功能、调节和生物学作用。重要的是，从该项目获得的信息将有助于制定旨在预防和/或治疗肠道炎症和腹泻疾病的新战略。

DESCRIPTION (provided by applicant): Inflammatory diarrheal illnesses are a major public health concern and economic burden in the United States and worldwide. Adenosine is an endogenous signaling molecule whose level is increased in the colonic tissue and luminal fluid during active inflammation. In the intestine, the adenosine 2B receptor (A2BAR) is the predominant adenosine receptor expressed in colonic epithelial cells and mediates the effects of adenosine. Our research, supported by NIH K08 and R01 funding, has focused on characterizing the signaling mechanisms and biological function of colonic epithelial A2BAR. This competing renewal application is built on a series of in vitro as well as in vivo observations that demonstrate a pro-diarrheogenic and pro-inflammatory role for colonic epithelial A2BAR. Together, our studies demonstrate that the activation of A2BAR orchestrates acute inflammatory response by coordinating interactive signaling between epithelial cells and neutrophils: i) A2BAR is intracellular at rest and is recruited to the membrane upon agonist stimulation; ii) A2BAR mRNA and protein levels are upregulated during acute flares in human and animal models of inflammatory bowel disease (IBD); iii) A2BAR mediates apically polarized IL-6 and fibronectin secretion, which in turn mediate neutrophil activation and bacterial adhesion/invasion respectively; iv) A2BAR augments IL-8 secretion and mediates neutrophil recruitment in response to inflammatory stimuli; v) A2BAR antagonism or gene deletion inhibits neutrophil infiltration; vi) genetic ablation of A2BAR in mice protects against bacterial and chemically-induced diarrhea and inflammation; and vii) inhibition of A2BAR using highly specific inhibitors ameliorates acute and chronic inflammation in mice. Although we have made significant advances in understanding the role of epithelial A2BAR in orchestrating mucosal inflammatory response, the physiological role of A2BAR, its regulation during inflammation and the contribution of immune cell A2BAR in inflammation is unknown. The goal of the studies proposed herein is to elucidate the physiological function of the A2BAR, regulation of the colonic epithelial A2BAR expression, and determine the role of immune cell A2BAR in inflammatory diarrheal illnesses. We hypothesize that the A2BAR plays a central role in mediating fluid secretion in the normal colon. Upregulation of A2BAR expression and function in epithelial and immune cells during intestinal inflammation plays an important role in inflammatory response; therefore A2BAR antagonism is an effective treatment for inflammatory diarrheal illnesses. These proposed studies will not only resolve the questions concerning the physiologic significance of A2BAR in the regulation of colonic fluid secretion and inflammatory response, but will also provide "proof-of-principle" for the development of A2BAR-targeted therapies in an arena that offers limited treatment modalities for intestinal inflammatory disorders. PUBLIC HEALTH RELEVANCE: Inflammatory diarrheal diseases are a cause of significant morbidity and mortality in U.S. and worldwide. The pathogenesis of these illnesses is unknown. Adenosine, is a naturally occurring metabolite that is generated during active intestinal inflammation, and its levels increase during inflammation. In the intestine, adenosine 2b receptor (A2B receptor) mediates the effects of adenosine. This proposal will elucidate the function, regulation and biological actions of adenosine 2b receptor in diarrhea and inflammation. Importantly, information gained from this project will be instrumental for the generation of new strategies aimed at preventing and/or treating intestinal inflammatory and diarrheal illnesses.