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

**Viktoria E. M. Schindler 1,†, Fahd Alhamdan 1,†, Christian Preußer 2,3, Lukas Hintz 1, Bilal Alashkar Alhamwe 2,4, Andrea Nist 5, Thorsten Stiewe 5, Elke Pogge von Strandmann 2,3, Daniel P. Potaczek 1, Clemens Thölken 6,† and Holger Garn 1,\*,†**


**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, miRNAassociated 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.

**Citation:** Schindler, V.E.M.; Alhamdan, F.; Preußer, C.; Hintz, L.; Alashkar Alhamwe, B.; Nist, A.; Stiewe, T.; Pogge von Strandmann, E.; Potaczek, D.P.; Thölken, C.; et al. Side-Directed Release of Differential Extracellular Vesicle-associated microRNA Profiles from Bronchial Epithelial Cells of Healthy and Asthmatic Subjects. *Biomedicines* **2022**, *10*, 622. https://doi.org/10.3390/ biomedicines10030622

Academic Editors: Berislav Bošnjak and Stanislawa Bazan-Socha

Received: 16 February 2022 Accepted: 27 February 2022 Published: 7 March 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Keywords:** bronchial epithelial cells; extracellular vesicles; miRNAs; airway epithelium; asthma; cellular compartmentalization

### **1. 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. In the past decade, 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]. All EVs are composed of a lipid bilayer containing transmembrane proteins and can be classified into exosomes, microvesicles, and apoptotic bodies depending on size, structural components, and generation process [8]. While exosomes are approximately 30–150 nm in diameter and are derived from the exocytosis of multivesicular bodies, microvesicles are shed at the cell surface and are 50–1000 nm in size [9,10]. EVs express membrane proteins, which frequently have been used as surface markers to identify specific EV subsets such as exosomes, or to trace the cell of origin. Universally expressed exosomal proteins such as the tetraspanins CD9, CD63, and CD81 are therefore commonly used as exosomal markers to distinguish exosomes from other EV subsets [11], although conflicting data describe the expression on both exosomes and microvesicles [12].

EVs function as intercellular communicators transporting diverse lipids, proteins, and nucleic acids, such as DNA and certain types of RNA including messenger RNA (mRNA) and small RNAs, such as microRNAs (miRNAs), small interfering RNAs (siRNAs), transfer RNAs (tRNAs), and PIWI-associated RNAs (piRNAs) [13]. Small RNAs are less than 200 nucleotides long and are not translated into proteins, but rather regulate biological processes by interfering with mRNA translation. MiRNAs are defined as short non-coding single-stranded RNAs with a length of approximately 22 nucleotides. They target mRNA, inducing mRNA degradation or inhibiting protein translation, and thereby regulate gene expression [14]. Vesicular miRNAs are protected from degradation by RNA-degrading enzymes (RNAses) in body fluids due to the protective shell provided by the vesicles lipid bilayer, and therefore can be shuttled between cells [15].

The airway epithelium is known to account for a variety of abnormal responses in asthma, such as epithelial mucus metaplasia [16,17]. Increasing evidence further suggests an active role of lung epithelial cells in the initiation and perpetuation of local immune mechanisms not only by the secretion of cytokines, but also as a major producer of EVs [18–21]. Many studies have described a potential proinflammatory role of EVs in allergy and asthma, but with few studies specifically addressing the role of EVs derived from airway epithelial cells. Vesicles secreted by lung epithelial cells have been shown to prime immune cells toward proinflammatory features [22]. Furthermore, a differential expression of extracellular miRNAs in asthmatic patients compared to non-asthmatics with a downstream regulatory impact on inflammation has been described [23,24]. However, to the best of our knowledge, so far, no study has explicitly distinguished between vesicular miRNA profiles on the apical and basolateral cell side of airway epithelial cells.

The purpose of this study was to investigate vesicle characteristics and vesicular miRNA profiles associated with EVs derived from the airway epithelial cells of healthy and asthmatic subjects depending on the cell side of secretion. Therefore, an air−liquidinterface cell culture model of airway epithelial cells was used for sample collection. We then performed small RNA sequencing and conducted extensive bioinformatic analyses

to identify vesicular miRNA signatures. Furthermore, miRNA associated roles, functions, and targets were predicted by associated target genes, and miRNA network analysis was conducted to reveal closely related functional clusters within the identified set.
