House dust mite and Th2 cytokine-mediated epithelial barrier dysfunction attenuation by KL001 in 16-HBE cells

ABSTRACT

House dust mite (HDM) is a common aeroallergen that can disrupt the airway epithelial barrier leading to dysregulated immune response, resulting in allergic lung diseases such as asthma. Cryptochrome (CRY), a circadian clock gene, plays an important role in the regulation of metabolism, and immune response. It remains unclear whether stabilizing CRY using KL001 can attenuate HDM/Th2 cytokine-induced epithelial barrier dysfunction in 16-HBE cells. We evaluate the effect of KL001 (20 µM) pre-treatment (4 hrs) in HDM/Th2 cytokine (IL-4 or IL-13)-mediated change in epithelial barrier function. HDM and Th2 cytokine-induced changes in transepithelial electrical resistance (TEER) were determined by an xCELLigence real-time cell analyzer and delocalization of adherens junction complex (AJC: E-cadherin and β-catenin) and tight junction proteins (TJP: Occludin and Zonula occludens-1) by immunostaining and confocal microscopy. Finally, quantitative real-time PCR (qRT-PCR) and Western blotting were used to measure altered gene expression and protein abundance of the epithelial barrier function and core clock genes, respectively. HDM and Th2 cytokine treatment significantly decreased TEER associated with altered gene expression and protein abundance of the selected epithelial barrier function and circadian clock genes. However, pre-treatment with KL001 attenuated HDM and Th2 cytokine-induced epithelial barrier dysfunction as early as 12–24 hrs. KL001 pre-treatment showed attenuation of HDM and Th2 cytokine-induced alteration in the localization and gene expression of AJP and TJP (Cdh1, Ocln, and Zo1) and core clock genes (Clock, Arntl/Bmal1, Cry1/2, Per1/2, Nr1d1/Rev-erbα, and Nfil3). We demonstrate, for the first time, the protective role of KL001 in HDM and Th2 cytokine-mediated epithelial barrier dysfunction.

GRAPHICAL ABSTRACT

KEYWORDS:

• allergic asthma
• circadian clock
• epithelial barrier dysfunction
• house dust mite
• KL001
• transepithelial electrical resistance

Acknowledgments

The authors like to acknowledge Mr. Jason Shield and Dr. Nikhil Mittal for providing us with the xCELLigence RTCA MP Instrument and accessories (Agilent Technologies, Inc.) to conduct this study. We authors like to specially thank Dr. Matthias A. Salathe for providing access to utilize their confocal microscopy facility to conduct imaging as part of this study. The graphical abstract figure presented in this manuscript is prepared using Servier Medical Art (https://smart.servier.com).

Disclosure statement

No potential conflict of interest was reported by the authors.

Author contribution

SKD, AS, IKS: conception and design of the in vitro study and performed experiments.

SKD, IKS: Data analysis, interpretation, and manuscript drafting.

SKD, AS: acquisition of data, and partly conducted the experiments within the study.

All authors have checked the revised manuscript and approved this for publication.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21688370.2023.2203841.

Additional information

Funding

This work was partly supported by the National Institute of Health NIH R01 HL142543 (I.K.S) and the University of Kansas Medical Center, School of Medicine, Internal Medicine Start-Up Funds (I.K.S.).