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

Abstract

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-κB/ RelA-dependent cytokine and chemokine expression. Moreover, proteomic screen reveals that K6a interacts with ELKS, a critical NEMO-binding scaffold that links between canonical IKKα/β and the principal cytoplasmic inhibitor of RelA, i.e. IκBα., to promote its phosphorylation and degradation. Surprisingly, K6a does not antagonize any of these canonical NF-κB signaling events. Instead, we found that ELKS in addition to canonical IKKs interacts with the atypical IKK member IKKϵ. Furthermore, knockdown of K6a in hTCEpi cells promotes ELKS-dependent phosphoactivation of IKKϵ, 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-κB 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-κB in corneal epithelial cells. Using both IKK classes other than only canonical IKKs for TLR/NF-κB 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.

Epithelial cytokeratin 6a restricts secretory autophagy of proinflammatory cytokines by interacting with Sec16A

“…Our investigation started with multiplex assays and conventional ELISA measuring cytokine secretion from hTCEpi cells untreated or treated with cell-free bacterial culture supernatants containing a mixture of inflammatory Toll-like receptor ligands. As multiple cytokines released from K6a-KD cells are upregulated as compared to WT cells, these findings are consistent with our recent report, which has shown that K6a knockdown causes increased NF-kB activation and cytokine expression in hTCEpi cells and mouse corneal epithelium challenged with purified LPS and LTA, and that mechanistically, ELKS-interacting K6a is a newfound negative regulator of non-canonical NF-kB signaling for preventing excessive expression of proinflammatory cytokines and chemokines at the transcriptional level 27 . In this study, K6a interactome analysis has identified Sec16A and showed various autophagy and secretory pathways enriched in hTCEpi cells; so silencing K6a has led to elevated LC3-II level (lysate protein and cytoplasmic puncta), whether or not the cells are stimulated with inflammatory ligands, suggesting a possible relationship between K6a, autophagy and cytokine secretion.…”

“…Similarly, upon stimulation by 20% PAO1 culture supernatant, K6a-KD cells also secreted higher levels of IL-1a, IL-6, IL-8, GROa/CXCL-1 as compared to wild type cells (Figure 1A). Next, we validated some of these findings by traditional ELISA (Figure 1B), which was in line with our recent findings 27 . These results indicated that K6a in corneal epithelial cells negatively regulates secreted levels of proinflammatory mediators.…”

“…In a mouse model of P. aeruginosa and S. aureus keratitis, we demonstrated that treatment with KAMPs not only kills bacteria in the infected corneas but also controls infiltration of neutrophils and macrophages leading to significant reduction of vision-threatening corneal opacification and tissue damage 26 . Recently, we also reported that C57BL/6 mice with K6a gene knockout exhibit higher severity of corneal inflammation with and without infection, and that cytosolic K6a in corneal epithelial cells interacts with ELKS to suppress Toll-like receptor-mediated IKKedependent (non-canonical) NF-kB activation, thereby controls inflammatory cytokine expression 27 .…”