Retinoic acid improves baseline barrier function and attenuates TNF-α-induced barrier leak in human bronchial epithelial cell culture model, 16HBE 14o.

Patrick J Callaghan,Elizabeth Rybakovsky,Bryan Ferrick,Sunil Thomas,James M Mullin,Michael Koval

doi:10.1371/journal.pone.0242536

Patrick J Callaghan, Elizabeth Rybakovsky + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0242536

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Abstract

Retinoic acid (RA) has been shown to improve epithelial and endothelial barrier function and development and even suppress damage inflicted by inflammation on these barriers through regulating immune cell activity. This paper thus sought to determine whether RA could improve baseline barrier function and attenuate TNF-α-induced barrier leak in the human bronchial epithelial cell culture model, 16HBE14o- (16HBE). We show for the first time that RA increases baseline barrier function of these cell layers indicated by an 89% increase in transepithelial electrical resistance (TER) and 22% decrease in 14C-mannitol flux. A simultaneous, RA-induced 70% increase in claudin-4 attests to RA affecting the tight junctional (TJ) complex itself. RA was also effective in alleviating TNF-α-induced 16HBE barrier leak, attenuating 60% of the TNF-α-induced leak to 14C-mannitol and 80% of the leak to 14C-inulin. Interleukin-6-induced barrier leak was also reduced by RA. Treatment of 16HBE cell layers with TNF-α resulted in dramatic decrease in immunostaining for occludin and claudin-4, as well as a downward “band-shift” in occludin Western immunoblots. The presence of RA partially reversed TNF-α’s effects on these select TJ proteins. Lastly, RA completely abrogated the TNF-α-induced increase in ERK-1,2 phosphorylation without significantly decreasing the TNF-driven increase in total ERK-1,2. This study suggests RA could be effective as a prophylactic agent in minimizing airway barrier leak and as a therapeutic in preventing leak triggered by inflammatory cascades. Given the growing literature suggesting a “cytokine storm” may be related to COVID-19 morbidity, RA may be a useful adjuvant for use with anti-viral therapies.

Highlights

Epithelial tissues are physical barriers between the outside environment and our systemic circulation and interstitial tissue
At a concentration of 50μM, Retinoic acid (RA) increased transepithelial electrical resistance (TER) by 89% (Fig 1A) and reduced mannitol flux by 22% (Fig 1B), indicating a true increase in barrier function
Retinoic acid produced the following effects on 16HBE cell layers: 1) increase of basal barrier strength evidenced by an 89% increase in TER and 22% decrease in mannitol flux (Fig 1); 2) moderate but significant suppression of endogenous TNF-α production (Fig 2); 3) attenuation of TNF-α-induced barrier leak to mannitol, lactulose and inulin(Figs 3 and 4); 4) attenuation of the IL-6-induced increase in barrier leak to mannitol; 5) preservation of occludin and claudin-4 immunostaining in the presence of TNF-α (Fig 5); 6) reversal of the downward “band-shift” of occludin triggered by TNF-α (Fig 6); and 7) inhibition of the increase in ERK-1,2 phosphorylation induced by TNF-α (Fig 7)

Summary

Introduction

Epithelial tissues are physical barriers between the outside environment and our systemic circulation and interstitial tissue. Examples are the epidermis of the skin and the linings of organs defining luminal spaces in our body such as our gastrointestinal tract, renal tubes, and alveoli in the lungs [1]. Cells constituting these protective sheets are unique in that they polarize Retinoic acid and inflammation-induced epithelial barrier leak

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