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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：
10002241
负责人：
K. P. Connie Tam
金额：
$ 45.08万
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/10002241
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 Epithelium 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.

项目摘要/摘要上皮细胞角蛋白通过内源性机制控制角膜炎症感染性和无菌性角膜炎可对角膜造成严重的组织破坏，危害视力。在临床实践中，目前治疗角膜炎症的标准是外用皮质类固醇。虽然强效的皮质类固醇与一些严重的副作用有关。因此，寻找新的而控制炎症的更好的治疗方案对于改善患者护理和临床是必要的结果。角蛋白6a(keratin6a，K6a)是一种主要的中间丝蛋白，广泛存在于多种组织中。上皮细胞类型。我们已经证明，炎性细菌成分诱导磷酸化。 K6a促进其细丝解聚，从而增加其胞浆水平。初步数据研究表明，在体外和体内，角膜K6a基因敲除增强了对损伤和炎症的反应细菌成分，如促炎细胞因子和趋化因子分泌增加。 K6a缺乏还会导致Wnt/β-连环蛋白信号的抑制配体DKK-1分泌增加路径。在动物模型中，K6a缺乏加剧了内毒素诱导的不孕角膜炎症和铜绿假单胞菌引起角膜炎，并损害角膜上皮伤口的愈合。通过免疫沉淀-质谱法鉴定角膜细胞内K6A的相互作用伙伴上皮细胞，包括核因子-κB信号的主要调节者和选择性降解自噬。这该提案旨在通过以下途径表征这一丰富的胞浆K6a池的免疫调节功能调查其与关键途径调节者的物理和功能伙伴关系；正如所知的那样促进抗炎药物的开发，这些药物更具组织特异性，全身副作用较少。中心假说是K6a与主要途径调节因子合作控制炎症反应。使得发炎和/或屏障破坏的角膜可以恢复内稳态。我们的具体目标将考验假设(1)胞内K6a通过直接隔离ELK(IKK激活剂)而拮抗NF-κB途径和hnRNPA1(IκBα失稳因子)；(2)胞内K6a通过促进miR-152激活Wnt/β-连环蛋白途径。 DKK-1介导的下调；(3)胞内K6a促进TRIM和/或HSPA8伴侣介导降解性自噬以控制炎症介质的产生；以及(4)局部递送负载K6a 纳米颗粒控制铜绿假单胞菌感染时角膜和上皮的炎症环境伤口愈合。细胞培养和角膜上皮特异性K6a基因敲除小鼠都将用于研究。预计结果将确定细胞角蛋白在上皮免疫生物学中的抗炎作用，并可能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。