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.

项目概要 结肠上皮形成结肠腔复杂微生物环境之间的第一个细胞屏障 上皮细胞如何感知和响应这种环境对于保护身体至关重要。 过氧化氢 (H2O2) 是细胞中重要的信号分子。 研究表明，参与上皮修复和宿主防御的早发性炎症患者。 肠道疾病和结肠癌对 H2O2 的调节受损 细胞外 H2O2 是由细胞产生的。 膜氧化酶（例如 NADPH 氧化酶 1 (NOX1)）、分泌型氧化酶和共生细菌 肠道内 H2O2 向肠道上皮细胞发出信号的机制。 人们对肠道知之甚少，并且代表了水通道蛋白 3 (AQP3) 的一个重要知识空白。 质膜通道的水通道蛋白家族，已知可传导 H2O2，并在 我之前的研究表明，H2O2 通过 AQP3 的转运对于上皮细胞很重要。 然而，损伤后的修复和对病原微生物的炎症反应。 AQP3 调节这些先天免疫功能的机制尚不清楚。该提案的总体目标尚不清楚。 因此，我们的目标是了解 AQP3 调节的 H2O2 信号如何影响上皮对损伤的反应和 微生物——肠道中的关键过程，对炎症和感染性肠道疾病很重要。 我的提案的目标 1 研究 H2O2 通过 AQP3 的运输如何影响结肠中的信号转导 具体来说，我将研究：H2O2 的来源和位置。 信号影响 AQP3 依赖性细胞信号传导；AQP3 是否与 NOX1 共同调节； 通过产生 H2O2 的共生微生物来增强伤口修复作用，目标 2 研究 AQP3 是如何介导的。 H2O2 转运影响上皮细胞对致病菌和共生菌的炎症反应。 我将研究病原体（c.rodentium）是否会诱导 NOX1 产生 H2O2 并诱导 通过 AQP3 的炎症信号传导，以及是否相关 Toll 样受体激活或产生 H2O2 共生细菌可以改变 AQP3 介导的炎症信号传导，以实现这些目标。 将使用 (AQP3+/+/ NOX1+/+)、AQP3-/- 和 NOX1-/- 小鼠和原代结肠肠荧光。 成像将用于测量 H2O2，内窥镜损伤模型将用于体内评估伤口 将使用蛋白质表达和生物素化研究来分析细胞信号传导的激活。 该提案产生的信息可能为肠道疾病的治疗提供新的治疗靶点 炎症性疾病并导致更好地了解水通道蛋白在粘膜表面的作用 这些结果也可能更广泛地应用于其他微生物定殖时 H2O2 的调节。 身体表面，如尿道、皮肤和气道，因此可能产生的影响超出 胃肠道疾病。