BMC Seminar - Non-antibiotic effects of macrolides on airway epithelial differentiation

BMC Seminar Thursday 27th of Novemeber at 12:00 - 13:00 in Læknagarður, room 201

Presenter: Árni Ásbjarnarson, PhD student, supervised by Prof. Þórarinn Guðjónsson, Faculty of Medicine, University of Iceland

Title: Non-antibiotic effects of macrolides on airway epithelial differentiation and metabolism

Abstract: The respiratory epithelium maintains the barrier against inhaled harmful agents. Barrier failure occurs in several respiratory diseases resulting in acute or chronic inflammation leading to further destructive effects and exacerbations. Macrolides are used to treat a spectrum of infections but are also known for off-label use. Some macrolides, particularly azithromycin (AZM), reduce exacerbations in chronic obstructive pulmonary disease (COPD), whereby its efficacy is thought to be due to its effects on inflammation and oxidative stress. In vitro data indicate that AZM reduces epithelial barrier failure, evidenced by increased transepithelial electrical resistance (TEER). We have compared the effects of different macrolides on differentiation and barrier integrity in VA10 cells, a bronchial epithelial cell line for 14 and 21 days. Erythromycin, clarithromycin, roxithromycin, AZM, solithromycin, and tobramycin (an aminoglycoside) were analyzed using RNA sequencing, barrier integrity assays, and immunostaining to evaluate effects on the epithelium. Furthermore, we have analysed the effects of macrolides on oxidative phosphorylation, mitochondrial phenotype, and oxidative stress in bronchial epithelial cells using real-time cell metabolism analysis and fluorescent probes. Lastly, we examined if and how AZM may inhibit EMT in vitro, both in a 2D and 3D context. Phenotypic changes were evaluated by RT-PCR, Western blot, immunostaining and RNA seq. EMT was induced in VA10 cells via exposure of 10 ng/ml TGF-β1.

All macrolides affected the gene expression of pathways involved in epithelial-to-mesenchymal transition, metabolism, and immunomodulation. Treatment with AZM, clarithromycin, and erythromycin raised TEER and induced phospholipid retention. AZM treatment was distinct in terms of enhancement of the epithelial barrier, retention of phospholipids, vesicle build-up, and its effect on gene sets related to keratinocyte differentiation and establishment of skin barrier. All macrolides lowered respiration, AZM and SOLI caused phenotypic changes to mitochondria. However, at 72 hours, AZM was the only macrolide to increase reactive oxygen species (ROS). In longer treatment 14 and 21 days AZM produces antioxdants indicating a process of mitohormesis. We also demonstrate that AZM is an efficient inhibitor of EMT in lung epithelial cells in vitro and we propose CAV-1 interactions with AZM as a potential mechanism, as inhibition of CAV-1 is known to decrease SNAI1 expression. Cholesterol depletion/trafficking should also be considered as it may affect a variety of membrane bound signaling molecules.

Bio: Árni obtained both his bachelor’s degree in Biochemistry and Molecular biology and a Master’s degree in Biology, at the University of Iceland. He did his Master‘s project at Guðmundur Hrafn Guðmundsson´s lab working on LL-37 immunomodulation and Bordetella infection. After his Master‘s degree, he worked as a laboratory research associate at the Magnúsdóttir lab and later at the SCRU lab. He is now pursuing Ph.D. studies at Þórarinn Guðjónssons lab where he is studying the mechanism of action for disease modifying effects of macrolides.