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

Bhushan, A.; Chan, J. K.; Sun, Y.; Ghiamati, F.; Crabb, J. S.; Jang, G.-F.; Willard, B.; Crabb, J. W.; Tam, C.

doi:10.1101/2024.01.04.574264

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bioRxiv · 10.1101/2024.01.04.574264

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

Bhushan, A.·Chan, J. K.·Sun, Y.·Ghiamati, F.·Crabb, J. S.·Jang, G.-F.·Willard, B.·Crabb, J. W.·Tam, C.

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Abstract

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, K6a knockout mice exhibit disease exacerbation. Here, we investigated cell-intrinsic mechanisms by which cytoplasmic K6a curbs corneal inflammation. We stimulated wild-type (WT) and K6a siRNA-knockdown (K6a-KD) human corneal epithelial (hTCEpi) cells with inflammatory P. aeruginosa culture supernatant. Our results showed that, under both basal and inflammatory conditions, K6a-KD cells secreted higher levels of cytokines and chemokines (IL-1, IL-6, IL-8, CXCL1, CCL20) as compared to WT cells. K6a-KD cells also had increased level of LC3-II, a marker for autophagosomes, while autophagic degradation of SQSTM1/p62 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 by confocal microscopy. Mechanistically, 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 the secretory autophagy process (Sec16A, GRASP55, Rab8) abolished the augmentative effect of K6a-KD on cytokine and chemokine secretion. These findings reveal a novel repressive role of K6a in secretory autophagy-mediated proinflammatory cytokine secretion and provide new insights into cell-intrinsic mechanisms of inflammation control at epithelial barriers.

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Bhushan, A., Chan, J. K., Sun, Y., Ghiamati, F., Crabb, J. S., Jang, G.-F., Willard, B., Crabb, J. W., Tam, C.. 2024-01-05. Epithelial cytokeratin 6a restricts secretory autophagy of proinflammatory cytokines by interacting with Sec16A. https://doi.org/10.1101/2024.01.04.574264

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