权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Epithelial cytokeratins control corneal inflammation through intrinsic mechanisms

上皮细胞角蛋白通过内在机制控制角膜炎症

基本信息

批准号：
10200071
负责人：
K. P. Connie Tam
金额：
$ 49.13万
依托单位：
CLEVELAND CLINIC LERNER COM-CWRU
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-01 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10200071
关键词：
Acute Adrenal Cortex Hormones Animal Model Anti-Inflammatory Agents Autophagocytosis Autophagosome Bacterial Model Binding Biological Assay Blindness Cell Culture Techniques Cells Cervix Uteri Chronic Clinical Clustered Regularly Interspaced Short Palindromic Repeats Co-Immunoprecipitations Confocal Microscopy Cornea Corneal Injury Cytokeratin Cytokine Gene Data Development Disease Progression Down-Regulation Epithelial Epithelial Cells Eyedrops Filament Flow Cytometry Gene Expression Goals Homeostasis Human I Kappa B-Alpha IL8 gene Immune Immunobiology Immunoprecipitation Impairment In Vitro Infection Infiltration Inflammation Inflammation Mediators Inflammatory Inflammatory Response Injury Innate Immune Response Intermediate Filament Proteins Intervention Keratin Keratitis Knock-out Knockout Mice Knowledge Lead Ligands Lysosomes Mass Spectrum Analysis Mediating Mediator of activation protein Medical MicroRNAs Molecular Molecular Chaperones Mus Neutrophil Infiltration Oral cavity Outcome Pathologic Pathway interactions Patient Care Pharmaceutical Preparations Pharmacologic Substance Phosphorylation Phosphotransferases Physiologic Intraocular Pressure Post-Transcriptional Regulation Production Proteins Pseudomonas aeruginosa Pseudomonas aeruginosa infection Regulation Reporter Reporting Role Scientist Severity of illness Signal Pathway Signal Transduction Signaling Molecule Site Skin Societies Sterility Stomach T-Lymphocyte TRIM Gene Testing Therapeutic Therapeutic Effect Tissues Topical Corticosteroids Topical application Tumor-infiltrating immune cells United States Vision Visit WNT Signaling Pathway Work base beta catenin cell type chemokine clinical practice corneal epithelial wound healing corneal epithelium cytokine depolymerization epithelial wound experimental study immunoregulation improved in vivo inflammatory milieu innovation knock-down mouse model nanoparticle neutrophil novel pre-clinical public health relevance recruit response response to injury side effect socioeconomics standard of care targeted treatment tool wound wound healing

项目摘要

PROJECT SUMMARY/ABSTRACT Epithelial Cytokeratins Control Corneal Inflammation Through Intrinsic Mechanisms Keratitis, both infectious and sterile, can cause serious tissue destruction to the cornea and jeopardize vision. In clinical practice, the current standard of care for corneal inflammation is topical corticosteroids. Although powerful, corticosteroids are associated with a number of serious side effects. Therefore, the search for new and better therapeutic options to control inflammation is necessary to improve patient care and clinical outcomes. Keratin 6a (K6a), a major intermediate filament protein, is ubiquitous and wound-induced in many epithelial cell types. We have demonstrated that inflammatory bacterial components induce phosphorylation of K6a that promotes its filament depolymerization and hence increases its cytosolic level. Preliminary data showed that corneal K6a knockdown in vitro and in vivo augments inflammatory responses to injury and bacterial components, as evidenced by the increased secretion of proinflammatory cytokines and chemokines. K6a deficiency also leads to increased secretion of DKK-1, the inhibitory ligand of Wnt/β-catenin signaling pathway. In animal models, K6a deficiency exacerbates LPS-induced sterile corneal inflammation and Pseudomonas aeruginosa keratitis, as well as impairs corneal epithelial wound healing. By immunoprecipitation-mass spectrometry, we have identified interaction partners of cytosolic K6a in corneal epithelial cells, including major regulators of NF-κB signaling and selective degradative autophagy. This proposal aims to characterize the immunoregulatory function of this abundant pool of cytosolic K6a through investigating its physical and functional partnerships with key pathway regulators; as the knowledge will facilitate development of anti-inflammatory drugs that are more tissue-specific with less systemic side-effects. The central hypothesis is that K6a partners with major pathway regulators to control inflammatory responses such that inflamed and/or barrier-disrupted corneas can return to homeostasis. Our specific aims will test the hypotheses that (1) cytosolic K6a antagonizes NF-κB pathway by directly sequestering ELKS (IKK activator) and hnRNPA1 (IκBα destabilizer); (2) cytosolic K6a activates Wnt/β-catenin pathway by facilitating miR-152- mediated downregulation of DKK-1; (3) cytosolic K6a promotes TRIM- and/or HSPA8 chaperone-mediated degradative autophagy to control inflammatory mediator production; and (4) topical delivery of K6a-loaded nanoparticles controls the inflammatory milieu of the cornea during P. aeruginosa infection and epithelial wound healing. Both cell culture and corneal epithelial specific K6a knockout mice will be used in the studies. Results are expected to define the anti-inflammatory role of cytokeratins in epithelial immunobiology and may lead to specific therapeutic strategies to restore homeostasis of the cornea and other sites during acute and chronic inflammation.

项目总结/摘要上皮细胞角蛋白通过内在机制控制角膜炎症感染性和无菌性角膜炎可导致角膜严重组织破坏并危及视力。在临床实践中，目前角膜炎症的护理标准是局部皮质类固醇。虽然强效皮质类固醇与许多严重的副作用有关。因此，寻找新的更好的治疗选择，以控制炎症是必要的，以改善病人的护理和临床结果。角蛋白6a（Keratin 6a，K6 a）是一种主要的中间丝蛋白，广泛存在于许多组织中，上皮细胞类型。我们已经证明，炎症性细菌成分诱导磷酸化， K6 a促进其丝解聚，因此增加其胞质水平。初步数据表明角膜K6 a在体外和体内的敲低增强了对损伤的炎症反应，细菌组分，如通过促炎细胞因子和趋化因子的分泌增加所证明的。 K6 a缺陷还导致Wnt/β-catenin信号传导的抑制性配体DKK-1分泌增加通路在动物模型中，K6 a缺乏加剧了LPS诱导的无菌性角膜炎症，铜绿假单胞菌角膜炎，以及损害角膜上皮伤口愈合。通过免疫沉淀-质谱，我们已经确定了在角膜中的细胞溶质K6 a的相互作用伙伴，上皮细胞，包括NF-κB信号传导和选择性降解性自噬的主要调节剂。这一项提案旨在通过以下方式来表征这种丰富的胞质K6 a库的免疫调节功能：调查其与关键途径监管机构的物理和功能伙伴关系;因为知识将促进开发更具有组织特异性且全身副作用更少的抗炎药物。中心假设是K6 a与主要途径调节剂合作来控制炎症反应使得发炎和/或屏障破坏的角膜可以恢复稳态。我们的具体目标将考验假设：（1）胞浆K6 a通过直接隔离ELKS（IKK激活剂）拮抗NF-κB通路和hnRNPA 1（IκBα去稳定剂）;（2）胞浆K6 a通过促进miR-152- 1的表达激活Wnt/β-catenin通路。（3）胞质K6 a促进TRIM和/或HSPA 8分子伴侣介导的DKK-1下调; 降解性自噬以控制炎性介质产生;和（4）局部递送负载K6 a的纳米颗粒控制铜绿假单胞菌感染和上皮细胞感染期间角膜的炎症环境伤口愈合细胞培养和角膜上皮特异性K6 a敲除小鼠将用于研究。这些结果有望明确细胞角蛋白在上皮免疫生物学中的抗炎作用，导致特定的治疗策略，以恢复角膜和其他部位在急性和慢性炎症。