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: 9980375
• 负责人: Jay Thiagarajah
• 金额: $ 16.07万
• 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/9980375
• 关键词: Affect; American; Apical; Area; Bacteria; Biology; Biotinylation; Body Surface; Carrier Proteins; Cell Line; Cell membrane; Cell surface; Cells; Citrobacter rodentium; Colon; Colon Carcinoma; Complex; Data; Environment; Enzymes; Epithelial; Epithelial Cells; Epithelium; Exhibits; Exposure to; 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; Injury; Innate Immune Response; Intestinal Diseases; Intestinal Mucosa; Intestines; Ion Channel; Knowledge; Lactobacillus; Lamina Propria; Lead; Location; Measures; Mediating; Methods; Microbe; Modeling; Morbidity - disease rate; Mucous Membrane; Mus; NADPH Oxidase; NADPH Oxidase 1; Natural Immunity; Oxidases; Pathogenesis; Pathway interactions; Patients; Process; Production; Proteins; Reactive Oxygen Species; Receptor Activation; Regulation; Research; Role; Shapes; Signal Transduction; Signaling Molecule; Skin; Source; Surface; Testing; Toll-like receptors; Training; Urinary tract; aquaporin 3; career development; commensal bacteria; commensal microbes; cytokine; early onset; environmental transport; epithelial wound; experimental study; extracellular; fluorescence imaging; gut microbiota; host-microbe interactions; hydrogen transport; immune function; improved; in vivo; in vivo evaluation; inflammatory disease of the intestine; innate immune function; intestinal epithelium; loss of function mutation; member; microbial; new therapeutic target; novel; paracrine; pathogen; pathogenic bacteria; pathogenic microbe; prevent; protein expression; repaired; response; response to injury; water channel; wound healing

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.

项目摘要 结肠上皮形成了结肠腔复杂微生物环境之间的第一道细胞屏障 还有尸体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 控制炎症过氧化氢（H2 O2）是细胞中重要的信号分子， 参与上皮修复和宿主防御。研究表明，早发性炎症患者 肠道疾病和结肠癌对H2 O2的调节受损。细胞外H2 O2是由细胞产生的。 膜氧化酶（例如NADPH氧化酶1（NOX 1））、分泌型氧化酶和细菌 在肠道内。H2 O2在肠道环境中向肠上皮细胞发出信号的机制 肠是知之甚少，并代表了一个重要的知识差距。水通道蛋白3（AQP 3）， 已知质膜通道的水通道蛋白家族传导H2 O2，并且在细胞膜中高度表达。 结肠上皮我以前的研究表明，H2 O2通过AQP 3的转运对上皮细胞的增殖和分化非常重要。 损伤后的修复以及对病原微生物的炎症反应。然而， AQP 3调节这些先天性免疫功能的机制尚不清楚。本提案的总体目标是 因此，了解AQP 3调节的H2 O2信号传导如何影响上皮细胞对损伤的反应， 微生物-肠道中对炎症和传染性肠道疾病很重要的关键过程。 目的1研究H2 O2通过AQP 3转运如何影响结肠中的信号转导。 上皮及其在创伤修复中的作用。具体来说，我将研究：如何来源和位置的H2 O2 信号影响AQP 3依赖性细胞信号传导; AQP 3是否与NOX 1共调节;以及AQP 3是否 通过产生H2 O2的微生物促进伤口修复。目的2研究AQP 3介导的 H2 O2转运影响上皮对病原菌和肠道细菌的炎症反应。具体地说， 我将研究病原体（啮齿类）是否诱导H2 O2产生NOX 1，并诱导 通过AQP 3的炎症信号传导，以及是否与Toll样受体激活或H2 O2产生相关。 大肠杆菌可以改变AQP 3介导的炎症信号。为了实现这些目标，野生型 (AQP3+/+/N 0X 1 +/+）、AQP 3-/-和N 0X 1-/-小鼠和原代结肠类肠。荧光 成像将用于测量H2 O2，内窥镜损伤模型将用于体内评估伤口 修复.将使用蛋白质表达和生物素化研究分析细胞信号传导的激活。的 从该建议中产生的信息可以为治疗肠道疾病提供新的治疗靶标。 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， 肠子这些结果也可能更广泛地适用于H2 O2在其他微生物定植的细胞中是如何调节的。 身体的表面，如泌尿道，皮肤和气道，因此可能会产生影响， 胃肠道疾病