Purchase this article with an account.
K P Connie Tam, Karthikeyan Bose, David Man, Jonathan K Chan; Keratin 6A regulation of corneal epithelial-derived inflammatory mediators during homeostasis and antigen challenge. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5768.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
We reported that cytoskeletal keratin 6A (K6A) mediates host defense via its antimicrobial c-terminal fragments. Increasing evidence also shows that keratin proteins can interact with endogenous factors and signaling molecules to mediate various cellular processes. Here, we explored whether K6A has an immunoregulatory role in corneal immunity and inflammation.
Human corneal epithelial (hTCEpi) cells were treated with control or K6A siRNA, followed by 18 h incubation with Pseudomonas aeruginosa culture supernatant (antigens) or vehicle control. Secretion of inflammatory mediators into the cell culture media was assessed by the Proteome Profiler Human Cytokine Array (R&D Systems), followed by densitometric analysis by ImageJ. To knock down K6A in vivo, C57BL/6 mice were subconjunctivally injected with siRNA once a day for 2 days. Mice were inoculated with 104 CFU amikacin-resistant P. aeruginosa clinical isolate after corneal scarification and 4 h epithelial healing. Cornea pathology was scored daily (0-16 points). At the time of sacrifice (48 h post infection), enucleated eyes were homogenized in PBS to quantify viable bacterial load.
Knocking down K6A in hTCEpi cells upregulated the basal secretion of interleukin (IL)-8, IL-1α, CCL20, CXCL1 and the Wnt signaling antagonist Dickkopf-1 (DKK-1), and downregulated chitinase 3-like 1 (CHI3L1), insulin-like growth factor binding protein 3 (IGFBP3) and soluble ST2 protein (sST2). In response to antigen challenge, differential secretion profiles of these inflammatory mediators between K6A knockdown and control cells were maintained, except IGFBP3. In vivo, mice treated with K6A siRNA had significantly more severe keratitis and higher bacterial load. At 48 h post infection, median [upper, lower quartile] scores and bacterial burden were 12 [13.25, 10.5] points and 8.35 [10.75, 6.18]x104 CFU/eye for K6A knockdown mice, and 4.5 [6, 4] points and 2.41 [3.78, 1.4]x104 CFU/eye for control mice (P=0.0022 and 0.0087 in each case, Mann-Whitney).
K6A regulates production of both pro- and anti-inflammatory mediators by corneal epithelial cells to maintain immune homeostasis during health and control host responses during disease. Our data highlights the novel role of keratins in immunoregulation. The molecular mechanisms and pathways by which K6A prevents overactivation of the immune system remain to be determined.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
This PDF is available to Subscribers Only