Tam, Connie1; Bhushan, Anand1; Chan, Jonathan1; Sun, Yan1
1Cleveland Clinic Cole Eye Institute, Cleveland, OH
Purpose: Epithelial cells form a crucial barrier against harmful microbes and inflammatory stimuli. Restraining inflammatory responses at the corneal barrier is necessary for avoiding sight-threatening tissue damage. Yet, epithelial cell-intrinsic mechanisms that dampen inflammation are largely unexplored. Keratin 6a (K6a) is a common type II cytokeratin highly expressed in corneal and other stratified epithelial cells. In a mouse model of sterile corneal inflammation, we observed disease exacerbation in K6a knockout mice. Here, we investigate a novel cell-intrinsic mechanism by which cytoplasmic K6a curbs corneal inflammation via regulation of secretory autophagy of cytokines.
Methods: Human telomerase-immortalized corneal epithelial cells (hTCEpi) were either untreated or treated with inflammatory P. aeruginosa culture supernatant. Specific gene expression was knocked down by siRNA. Cytokines secreted to culture media were measured by ELISA. Protein expression was assessed by Western blotting of cell lysates. Subcellular localization of LC3-II, interleukin-8 (IL-8) and other autophagy-related proteins was detected by immunofluorescence confocal microscopy and transmission electron microscopy (TEM). To detect acidity of autophagosomes, cells were transduced with a baculovirus system producing an autophagy tandem sensor (RFP-eGFP-LC3B). Stable hTCEpi cell line expressing HA tagged-K6a was used to identify physical interactors of K6a by affinity purification followed by mass spectrometry.
Results: Under both basal and inflammatory conditions, K6a-knockdown (K6a-KD) hTCEpi cells secreted higher levels of cytokines and chemokines (IL-1a, IL-6, IL-8, CXCL1, CCL20) as compared to wildtype cells. K6a-KD cells also had increased expression of LC3-II, a marker for autophagosomes, while the level of sequestosome-1 (SQSTM1/p62), a substrate for degradative autophagy, remained unchanged. In K6a-KD cells, the majority of LC3-II puncta were associated with non-acidified autophagosomes rather than acidified autolysosomes. Upon stimulation, IL-8 was found to co-localize with LC3-II. Mass spectrometric analysis of K6a immunoprecipitates identified Sec16a, a protein involved in secretory autophagy, as an interacting partner of K6a. Further experiments showed that knocking down key proteins involved in autophagosome formation (ATG5) and secretory autophagy process (GRASP55, Rab8a, Sec16a) abolished the augmentative effect of K6a-KD on cytokine and chemokine secretion.
Conclusions: These findings reveal a novel repressive role of K6a in secretory autophagy-mediated proinflammatory mediator secretion and provide new insights into cell-intrinsic mechanisms of inflammation control at epithelial barriers.
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