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/?-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-?B 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-?B pathway by directly sequestering ELKS (IKK activator) and hnRNPA1 (I?B? destabilizer); (2) cytosolic K6a activates Wnt/?-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.
Corneal keratitis is a major cause of blindness and presents significant challenges for treatment. This project aims to define a new and unexpected role for cytosolic keratin 6a and its intrinsic mechanisms in maintaining immune and tissue homeostasis in the cornea. Results of the proposed studies could lead to new anti- inflammatory therapeutics for controlling acute and chronic inflammation of the cornea and other sites.
