Side-Directed Release of Differential Extracellular Vesicle-associated microRNA Profiles from Bronchial Epithelial Cells of Healthy and Asthmatic Subjects.

Viktoria E M Schindler,Bilal Alashkar Alhamwe,Fahd Alhamdan,Christian Preußer,Lukas Hintz,Thorsten Stiewe,Elke Pogge Von Strandmann,Andrea Nist,Daniel P Potaczek,Clemens Thölken,Holger Garn

doi:10.3390/biomedicines10030622

Abstract

Extracellular vesicles (EVs) are released by virtually all cells and may serve as intercellular communication structures by transmitting molecules such as proteins, lipids, and nucleic acids between cells. MicroRNAs (miRNAs) are an abundant class of vesicular RNA playing a pivotal role in regulating intracellular processes. In this work, we aimed to characterize vesicular miRNA profiles released in a side-directed manner by bronchial epithelial cells from healthy and asthmatic subjects using an air−liquid interface cell culture model. EVs were isolated from a culture medium collected from either the basolateral or apical cell side of the epithelial cell cultures and characterized by nano-flow cytometry (NanoFCM) and bead-based flow cytometry. EV-associated RNA profiles were assessed by small RNA sequencing and subsequent bioinformatic analyses. Furthermore, miRNA-associated functions and targets were predicted and miRNA network analyses were performed. EVs were released at higher numbers to the apical cell side of the epithelial cells and were considerably smaller in the apical compared to the basolateral compartment. EVs from both compartments showed a differential tetraspanins surface marker expression. Furthermore, 236 miRNAs were differentially expressed depending on the EV secretion side, regardless of the disease phenotype. On the apical cell side, 32 miRNAs were significantly altered in asthmatic versus healthy conditions, while on the basolateral cell side, 23 differentially expressed miRNAs could be detected. Downstream KEGG pathway analysis predicted mTOR and MAPK signaling pathways as potential downstream targets of apically secreted miRNAs. In contrast, miRNAs specifically detected at the basolateral side were associated with processes of T and B cell receptor signaling. The study proves a compartmentalized packaging of EVs by bronchial epithelial cells supposedly associated with site-specific functions of cargo miRNAs, which are considerably affected by disease conditions such as asthma.

Highlights

Asthma is a common non-communicable inflammatory disease of the airways, affecting more than 339 million people worldwide, and is a major cause of morbidity around the globe [1,2]
The size of particles retrieved from the apical cell side wash corresponded to the typical size range of exosomes with a mean median size of 75 nm (62–95 nm)
No differences in concentration or size range were observed between particles from healthy and asthmatic subjects

Summary

Introduction

Asthma is a common non-communicable inflammatory disease of the airways, affecting more than 339 million people worldwide, and is a major cause of morbidity around the globe [1,2]. Disease pathogenesis in asthma involves the interaction of many different cell types within the respiratory tract, including CD4+ T-cells, granulocytes, dendritic cells, macrophages, myeloid-derived regulatory cells, natural killer cells, smooth muscle cells, and airway epithelial cells [3]. These various types of cells communicate via many different signaling mechanisms, such as soluble factors, including cytokines and chemokines. A new mechanism of intercellular communication by extracellular vesicles (EVs) was discovered [4] They have been found in all body fluids, including in blood [5], urine [6], and bronchoalveolar lavage fluid (BALF) [7]. Expressed exosomal proteins such as the tetraspanins CD9, CD63, and CD81 are commonly used as exosomal markers to distinguish exosomes from other EV subsets [11], conflicting data describe the expression on both exosomes and microvesicles [12]

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