Aquaporin 3 dependent responses of colon epithelial cells to microbes and injury

结肠上皮细胞对微生物和损伤的水通道蛋白3依赖性反应

• 批准号: 9292930
• 负责人: Jay Thiagarajah
• 金额: $ 16.65万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9292930
• 关键词: Affect; American; Apical; Area; Bacteria; Biology; Biotinylation; Body Surface; Carrier Proteins; Cell Line; Cell membrane; Cell surface; Cells; Colon; Colon Carcinoma; Complex; Data; Environment; Enzymes; Epithelial; Epithelial Cells; Epithelium; Exhibits; Family; Gastrointestinal Diseases; Generations; Goals; Host Defense; Hydrogen Peroxide; Immune response; Immune signaling; Immunity; Impairment; In Vitro; Infection; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Inflammatory Infiltrate; Inflammatory Response; Inflammatory disease of the intestine; Injury; Innate Immune Response; Intestinal Diseases; Intestines; Ion Channel; Knowledge; Lactobacillus; Lamina Propria; Lead; Location; Measures; Mediating; Methods; Microbe; Modeling; Morbidity - disease rate; Mus; NADPH Oxidase; NADPH Oxidase 1; Natural Immunity; Oxidases; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Peroxides; Process; Production; Proteins; Reactive Oxygen Species; Receptor Activation; Regulation; Research; Role; Shapes; Signal Transduction; Signaling Molecule; Skin; Source; Surface; Symbiosis; Testing; Toll-like receptors; Training; Urinary tract; Wound Healing; aquaporin 3; career development; commensal microbes; cytokine; early onset; environmental transport; experimental study; extracellular; fluorescence imaging; gut microbiota; host-microbe interactions; hydrogen transport; immune function; improved; in vivo; innate immune function; loss of function mutation; member; microbial; new therapeutic target; novel; paracrine; pathogen; pathogenic bacteria; prevent; protein expression; protein transport; repaired; response; response to injury; water channel

PROJECT SUMMARY The colonic epithelium forms the first cellular barrier between the complex microbial milieu of the colonic lumen and the body. How the epithelium senses and responds to this environment is critical to protecting the body and controlling inflammation. Hydrogen peroxide (H2O2) is an important signaling molecule in cells and is involved in epithelial repair and host defense. Studies have shown that patients with early-onset inflammatory bowel disease and colon cancer have impaired regulation of H2O2. Extracellular H2O2 is produced by cell- membrane oxidases (e.g. NADPH oxidase 1 (NOX1)), secreted oxidase enzymes, and commensal bacteria within the gut. The mechanism by which H2O2 in the intestinal environment signals to the epithelium of the intestine is poorly understood and represents an important gap in knowledge. Aquaporin 3 (AQP3), one of the aquaporin family of plasma membrane channels, is known to conduct H2O2 and is highly expressed in the colonic epithelium. My previous studies show that H2O2 transport through AQP3 is important for epithelial repair after injury and for the inflammatory response to a pathogenic microbe. However, the mechanisms by which AQP3 modulates these innate immune functions is not well understood. The overall aim of this proposal is therefore to understand how AQP3-regulated H2O2 signaling might shape epithelial responses to injury and microbes – critical processes in the gut that are important for inflammatory and infectious intestinal disease. Aim 1 of my proposal investigates how H2O2 transport through AQP3 affects signal transduction in the colonic epithelium and its role in wound repair. Specifically, I will examine: how the source and location of the H2O2 signal impacts AQP3-dependent cell signaling; if AQP3 is co-regulated with NOX1; and whether AQP3 amplifies wound repair by H2O2-producing commensal microbes. Aim 2 investigates how AQP3-mediated H2O2 transport impacts epithelial inflammatory responses to pathogenic and commensal bacteria. Specifically, I will be looking at whether a pathogen (c.rodentium) induces NOX1 generation of H2O2 and induces inflammatory signaling via AQP3, and whether associated Toll-like receptor activation or a H2O2-producing commensal bacterium can alter AQP3-mediated inflammatory signaling. To carry out these aims, wild-type (AQP3+/+/ NOX1+/+), AQP3-/- and NOX1-/- mice and primary colonic enteroids will be used. Fluorescence imaging will be used to measure H2O2 and an endoscopic injury model will be used in vivo to assess wound repair. Activation of cellular signaling will be analyzed using protein expression and biotinylation studies. The information arising from this proposal may provide new therapeutic targets for treatment of intestinal inflammatory disorders and lead to a better understanding of the role of aquaporins at the mucosal surface of the intestine. The results may also apply more broadly to how H2O2 is regulated at other microbe-colonized surfaces of the body, such as the urinary tract, skin, and airways, and therefore may have an impact beyond gastrointestinal disease.

项目总结 结肠上皮在结肠腔的复杂微生物环境之间形成第一层细胞屏障。 还有身体。上皮细胞如何感知和响应这种环境对保护身体至关重要 和控制炎症。过氧化氢(H_2O_2)是细胞内一种重要的信号分子。 参与上皮修复和宿主防御。研究表明，早发性炎症性疾病患者 肠道疾病和结肠癌损害了过氧化氢的调节。细胞外的过氧化氢是由细胞- 膜氧化酶(例如NADPH氧化酶1(NOX1))、分泌的氧化酶和共生菌 在肠子里。肠道环境中的过氧化氢向肠道上皮细胞发出信号的机制 人们对肠道知之甚少，它代表着知识上的一个重要缺口。水通道蛋白3(AQP3)是 水通道蛋白家族是一类质膜通道，可传导H_2O_2，在 结肠上皮。我以前的研究表明，H_2O_2通过AQP3转运对上皮细胞很重要 用于损伤后的修复和对致病微生物的炎症反应。然而，这些机制通过 目前还不清楚是哪种AQP3调节了这些先天免疫功能。这项建议的总体目标是 因此，要了解AQP3调节的过氧化氢信号如何影响上皮细胞对损伤和 微生物-肠道中的关键过程，对炎症性和传染性肠道疾病非常重要。 我建议的第一个目标是研究过氧化氢通过AQP3的运输如何影响结肠中的信号转导 上皮及其在伤口修复中的作用。具体地说，我将研究：过氧化氢的来源和位置 信号影响AQP3依赖的细胞信号；AQP3是否与NOX1共同调节；以及AQP3是否 通过产生过氧化氢的共生微生物放大伤口修复。AIM 2研究AQP3是如何介导的 过氧化氢转运影响上皮细胞对致病菌和共生菌的炎症反应。具体来说， 我将研究病原体(轮状芽孢杆菌)是否会诱导NOX1产生过氧化氢，并诱导 通过AQP3的炎症信号，以及相关的Toll样受体激活或过氧化氢产生 共生菌可以改变AQP3介导的炎症信号。为了实现这些目标，野人 将使用(AQP3+/+/NOX1+/+)、AQP3-/-和NOX1-/-小鼠和初级结肠肠样。荧光 成像将用于测量过氧化氢，内窥镜损伤模型将用于活体评估伤口 修理。细胞信号的激活将通过蛋白质表达和生物素化研究来分析。这个 这一提议提供的信息可能为肠道疾病的治疗提供新的治疗靶点。 炎症紊乱，并导致更好地了解水通道蛋白在胃粘膜表面的作用 肠子。这一结果也可能更广泛地适用于其他微生物定植的过氧化氢的调节方式 身体表面，如尿路、皮肤和呼吸道，因此可能会产生超越 胃肠道疾病。