Abstract
Purpose :
Unchecked inflammatory reactions of the ocular surface can cause tissue damage that endangers one's vision. In corneal epithelial cells and other stratified epithelia, type II cytokeratin K6a is extensively expressed. We discovered that bacterial ligand-induced corneal inflammation was exacerbated in K6a-KO mice. Here, we examine a novel mechanism by which endogenous K6a in corneal epithelial cells controls inflammation in a way that is cell-intrinsic.
Methods :
Human telomerase-immortalized corneal epithelial cells (hTCEpi) were either untreated or treated with P. aeruginosa culture supernatant. We used siRNA knockdown (KD) followed by western blotting, immunofluorescence microscopy, transmission electron microscopy (TEM), and Immuno-TEM to check the expression and subcellular localization of LC3-II(marker for autophagosomes), p62 (substrate for degradative autophagy), interleukin-8 (IL-8) and other autophagy-related proteins. To detect acidity of autophagosomes, we transduced cells with a baculovirus system producing an autophagy tandem sensor (pH-sensitive tagRFP-eGFP-LC3B). The level of cytokines secreted was assessed using ELISA. K6a-HA expressing hTCEpi stable cell line was used to identify physical interactors of K6a by affinity purification followed by mass spectrometry (MS).
Results :
Under both basal and inflammatory conditions, K6a-KD hTCEpi cells secreted more proinflammatory cytokines (IL-8, IL-1a, CXCL1, CCL20) and had higher LC3-II expression compared to WT cells while p62 levels remained unaffected. Most LC3-II puncta in K6a-KD cells were limited to autophagosomes rather than acidic autolysosomes. Sec16a, a protein implicated in secretory autophagy, was identified as K6a's interaction partner. Since IL-8 and LC3-II were discovered to co-localize, IL-8 was exploited as a phenotypic readout for autophagy. The elevating effect of K6a-KD on IL-8 secretion was eliminated by simultaneously knocking down individual proteins involved in autophagosome formation (ATG5) and secretory autophagy (GRASP55, Rab8a, and sec16a).
Conclusions :
The finding suggests that K6a controls basal and bacterial ligand-induced secretory autophagy in corneal epithelial cells, which in turn regulates the release of proinflammatory mediators. These findings contribute to our understanding of the innate immune-regulatory role of endogenous K6a and the intrinsic mechanisms of inflammation control in corneal epithelial cells.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.