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Jonathan Chan, Yan Sun, Karthikeyan Bose, William Carrera, K P Connie Tam; A novel role for cytoskeletal keratin 6a in regulating acute corneal inflammation. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4639. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Keratin 6a (K6a) is a major intermediate filament protein composing the cytoskeleton in corneal epithelial cells. These cells are critical to the orchestration of immune cell infiltration during corneal inflammation through secretion of proinflammatory and chemotactic cytokines. We previously reported that inflammatory bacterial components induce K6a phosphorylation, which in turn promotes its filament depolymerization and increases its subunit level in the cytosol, and that endogenous K6a regulates both basal and bacterial ligand-induced cytokine production from human corneal epithelial cells. Here, we explored the immunoregulatory role of K6a in mouse models of LPS-induced corneal inflammation and corneal epithelial wound healing.
Age- and sex-matched C57BL/6 mice were subconjunctivally injected with non-targeting control or K6a siRNA once a day for 2 days. Corneas of anesthetized control and K6a knockdown (KD) mice were scratched and inoculated with PBS or purified Pseudomonas aeruginosa LPS (20 ug). Mice were euthanized at 3 h and 24 h post-inoculation for corneal cytokine analysis (by ELISA) and infiltrating neutrophil detection (by immunofluorescence microscopy), respectively. In other experiments, the central corneal epithelium was removed by a blunt mini-blade to create a 2-mm diameter abrasion, and re-epithelialization was monitored by fluorescein staining of the wounds at 0, 24, 48, 72 h and 7 days post-abrasion.
Proinflammatory IL-1α, CXCL1 and CCL20, as well as neutrophil infiltration, were significantly higher in the scratched and LPS-inoculated corneas of K6a KD versus control mice. Mouse corneas that were scratched only also had increased levels of IL-1α and CXCL1 in K6a KD mice compared with control mice. At 48 h post-abrasion, re-epithelialization of corneas was nearly complete in control but not K6a KD mice, of which the wounds still failed to close after 7 days.
During LPS- or injury-induced corneal inflammation, K6a regulates the levels of proinflammatory and chemotactic cytokines in the cornea, in conjunction with neutrophil recruitment or epithelial wound healing. These findings added a new dimension to our knowledge of the functions of cytoskeletal keratin proteins, particularly in controlling inflammatory responses in epithelia. Future studies are needed to determine the molecular mechanism(s) for the immunoregulatory activity of K6a.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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