Conjunctival Goblet Cell Mucin Secretion in Inflammation and Its Resolution

炎症中结膜杯状细胞粘蛋白的分泌及其解决

• 批准号: 9195092
• 负责人: Darlene A Dartt
• 金额: $ 52.82万
• 依托单位: SCHEPENS EYE RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9195092
• 关键词: 1-Phosphatidylinositol 3-Kinase; ADRBK1 gene; Acute; Adenoviruses; Adrenergic Receptor; Allergens; Allergic; Allergic Conjunctivitis; American; Anti-Inflammatory Agents; Anti-inflammatory; Antihistamines; Aspirin; Cell Proliferation; Cell secretion; Cells; Chinese Hamster Ovary Cell; Chronic; Clinical; Clinical Trials; Conjunctival Epithelium; Cornea; Cyclic AMP-Dependent Protein Kinases; Disease; Dominant-Negative Mutation; Epithelial; Eye; GPR32 gene; Generations; Goals; Goblet Cells; Grant; HRH2 gene; Histamine; Histamine H1 Receptors; Histamine H2 Receptors; Histamine Receptor; Human; Hypersensitivity; Individual; Inflammation; Inflammation Mediators; Inflammatory; Inflammatory Response; Laboratories; Leukotriene Receptor; Leukotrienes; Leukotrienes A; Lipids; Lung; MAPK3 gene; MUC5AC gene; Mast Cell Stabilizer; Measurement; Measures; Mediator of activation protein; Molecular; Mucins; Mucous body substance; Mus; Nose; Ovalbumin; Pathway interactions; Penetration; Peptides; Pharmacology; Phospholipase C; Phospholipase D; Production; Prostaglandin D2; Protein Isoforms; Protein Kinase; Protein Kinase C; Proto-Oncogene Proteins c-akt; Protocols documentation; Pruritus; Receptor Signaling; Recruitment Activity; Redness; Regulation; Research; Resolution; Role; STK6 gene; Series; Signal Pathway; Signal Transduction; Skin; Small Interfering RNA; Source; Staining method; Stains; Swelling; Symptoms; Work; conjunctiva; cytokine; effective therapy; emergency service responder; eye dryness; improved; in vivo; inhibitor/antagonist; lipid mediator; mast cell; mouse model; novel; ocular surface; prevent; public health relevance; receptor; receptor function; reduce symptoms; response; symptomatic improvement

DESCRIPTION (provided by applicant): Although 15-20% of Americans suffer from allergic conjunctivitis, current treatments are limited to anti-histamines and mast cell stabilizers that ar often ineffective. The long-term goal of the proposal is to develop a new treatment for allergic conjunctivitis that could also be used for allergies of the lung, skin, and nose along with other inflammatory diseases of the ocular surface, such as dry eye. The new treatment is an anti-inflammatory and pro-resolution lipid mediator, the D-series resolvins compared to E-series resolvins currently in a clinical trial. Conjunctival goblet cell over-secretion of mucous includin the mucin MUC5AC is one of the symptoms of allergic conjunctivitis that include itching, conjunctival redness, tearing, and chemosis. D-, but not E-, series resolvins have two modes of action. They block histamine and leukotriene over-stimulation of goblet cell secretion and themselves activate a small amount of secretion to protect the ocular surface while the over-secretion is being reset. E-series do not increase secretion. The current proposal will focus on determining how pro-resolution mediators act at a molecular level to counter-regulate proinflammatory mediator stimulation of goblet cell mucin secretion both in culture and in vivo in a mouse model of allergic conjunctivitis. Research will focus on the following aims: 1. Determine the cellular mechanisms the D- and E-series resolvins use to stimulate goblet cell mucin secretion and if goblet cells are the source of the resolvins and other lipid mediators; 2. Unravel the molecular mechanisms D- and E-series resolvins use to activate protein kinases to interact with histamine and leukotriene receptors to counter-regulate their action; and 3. Ascertain the efficacy of D- compared to E-series resolvins to terminate goblet cell secretion and improve symptoms in a murine model of allergic conjunctivitis. For aim 1 RvD1, aspirin triggered (AT)-RvD1, and RvE1 will be used to stimulate human conjunctival goblet cells in culture. The receptors and cellular signaling pathways activated by the resolvins including phospholipase C and D and PI-3K will be investigated using siRNA, pharmacological inhibitors, and adenovirus constructs. In addition, the production of pro-inflammatory and pro-resolution mediators will be determined by lipidomic measurements on goblet cells in culture. For aim 2, CHO cells transfected with a pro-inflammatory histamine or leukotriene receptor and a pro-resolution receptor and cultured human goblet cells will be used. Protein kinases predicted to phosphorylate the pro-inflammatory mediator receptors will be studied to determine if RvD1, AT-RvD1, and RvE1 activate the protein kinases and counter-regulate the histamine or leukotriene receptor to block its activity and terminate mucin secretion. siRNA, pharmacological inhibitors, and adenovirus constructs will be used for this aim. For aim 3, a mouse model of severe allergic conjunctivitis will be used with ovalbumin as the allergen. Resolvins compared to anti-histamines will be used to determine if they decrease MUC5AC secretion and goblet cell proliferation, ameliorate the clinical symptoms of allergic conjunctivitis, and terminate infiltraton of inflammatory cells.

描述（由申请人提供）：尽管 15-20% 的美国人患有过敏性结膜炎，但目前的治疗仅限于抗组胺药和肥大细胞稳定剂，而这些药物通常无效。该提案的长期目标是开发一种治疗过敏性结膜炎的新疗法，该疗法也可用于肺部、皮肤和鼻子过敏以及干眼等其他眼表炎症性疾病。新的治疗方法是一种抗炎和促消退脂质介质，D 系列消退素与目前正在进行临床试验的 E 系列消退素进行比较。结膜杯状细胞过度分泌粘液（包括粘蛋白 MUC5AC）是过敏性结膜炎的症状之一，包括瘙痒、结膜发红、流泪和结膜水肿。 D-（而非 E-）系列解析素有两种作用模式。它们阻止组胺和白三烯对杯状细胞分泌的过度刺激，并在重置过度分泌的同时激活少量分泌物以保护眼表面。 E系列不增加分泌。目前的提案将重点确定促消退介质如何在分子水平上发挥作用，以对抗调节促炎介质在过敏性结膜炎小鼠模型的培养物和体内刺激杯状细胞粘蛋白分泌的作用。研究将集中于以下目标： 1. 确定 D 系列和 E 系列分解素用于刺激杯状细胞粘蛋白分泌的细胞机制，以及杯状细胞是否是分解素和其他脂质介质的来源； 2. 解开谜团 D 系列和 E 系列解析素用于激活蛋白激酶与组胺和白三烯受体相互作用以反调节其作用的分子机制； 3. 确定与 E 系列消退素相比，D-在过敏性结膜炎小鼠模型中终止杯状细胞分泌并改善症状的功效。对于目标 1 RvD1，阿司匹林触发 (AT)-RvD1 和 RvE1 将用于刺激培养物中的人结膜杯状细胞。将使用 siRNA、药理学抑制剂和腺病毒构建体来研究由包括磷脂酶 C 和 D 以及 PI-3K 在内的解析素激活的受体和细胞信号传导途径。此外，促炎和促消退介质的产生将通过对培养的杯状细胞进行脂质组学测量来确定。对于目标 2，将使用用促炎组胺或白三烯受体和促消退受体转染的 CHO 细胞以及培养的人杯状细胞。将研究预计磷酸化促炎介质受体的蛋白激酶，以确定 RvD1、AT-RvD1 和 RvE1 是否激活蛋白激酶并反调节组胺或白三烯受体以阻断其活性并终止粘蛋白分泌。 siRNA、药理学抑制剂和腺病毒构建体将用于此目的。对于目标 3，将使用严重过敏性结膜炎的小鼠模型，并以卵清蛋白作为过敏原。与抗组胺药相比，Resolvins 将用于确定它们是否能减少 MUC5AC 分泌和杯状细胞增殖、改善过敏性结膜炎的临床症状并终止炎症细胞的浸润。