Keratin 6a attenuates Toll-like receptor-triggered proinflammatory response in corneal epithelial cells by suppressing ELKS/IKKϵ-dependent activation of NF-κB

The corneal epithelium at the ocular surface is constantly exposed to the environment and represents the first line of defense against infection, mechanical injury or chemical irritation. Through TLR-mediated recognition of pathogen- and damage-associated molecular patterns, it engages in direct antimicrobial responses and alerts the immune system on intruder and tissue damage by secreting pro-inflammatory and chemotactic cytokines that promote immune cell infiltration. How the corneal epithelium downregulates TLR signaling is unclear, yet it highly expresses keratin 6a (K6a), a cytoskeletal protein that has emerged to play essential regulatory roles in corneal innate immune response. Here we report that mice harboring genetic deletion of K6a are more susceptible to developing bacterial keratitis with unresolved corneal opacification and higher bacterial load. Such disease phenotype is caused by the increased pro-inflammatory cytokine and chemokine secretions from the K6a-null corneal epithelium, which further promotes the infiltration of immune cells and their associated pro-inflammatory response. Using human corneal epithelial cells immortalized by telomerase reverse transcriptase (hTCEpi cells), we demonstrated that knocking down K6a enhances NF-{kappa}B/ RelA-dependent cytokine and chemokine expression. Moreover, proteomic screen reveals that K6a interacts with ELKS, a critical NEMO-binding scaffold that links between canonical IKK/{beta} and the principal cytoplasmic inhibitor of RelA, i.e. I{kappa}B., to promote its phosphorylation and degradation. Surprisingly, K6a does not antagonize any of these canonical NF-{kappa}B signaling events. Instead, we found that ELKS in addition to canonical IKKs interacts with the atypical IKK member IKK{varepsilon}. Furthermore, knockdown of K6a in hTCEpi cells promotes ELKS-dependent phosphoactivation of IKK{varepsilon}, which in turn phosphorylates and activates RelA. Our study thus demonstrated an unexpected role of cytosolic K6a as a novel negative regulator of TLR/NF-{kappa}B signaling in preventing excess proinflammatory cytokine and chemokine expressions. It further highlighted the functional importance of ELKS as a common signaling scaffold for both canonical and atypical IKK-dependent activation of NF-{kappa}B in corneal epithelial cells. Using both IKK classes other than only canonical IKKs for TLR/NF-{kappa}B induction as in other cell types including myeloid immune cells suggest that the cornea epithelium is more flexible in modulating its inflammatory response, which could greatly minimize corneal damage while preserving its essential functions for barrier protection and light refraction.