Reduced expression of miR-146a in human bronchial epithelial cells alters neutrophil migration

Anet Kivihall,Marek Sanak,Alan Altraja,Bogdan Jakiela,Jerzy Soja,Grazyna Bochenek,Ana Rebane,Alar Aab,Krzysztof Sładek

doi:10.1186/s13601-019-0301-8

Abstract

BackgroundThe role of miRNAs in the pathogenesis and determining the phenotypes of asthma is not fully elucidated. miR-146a has been previously shown to suppress inflammatory responses in different cells. In this study, we investigated the functions of miR-146a in human bronchial epithelial cells (HBECs) in association with neutrophilic, eosinophilic, and paucigranulocytic phenotypes of asthma.MethodsBronchial brushing specimens and brochial mucosal biopsy samples were collected from adult patients with asthma and from age- and gender-matched non-asthmatic individuals. The expression of miR-146a in bronchial brushing specimens, bronchial biopsy tissue sections or cultured primary bronchial epithelial cells was analyzed by RT-qPCR or by in situ hybridization. The expression of direct and indirect miR-146a target genes was determined by RT-qPCR or ELISA. The migration of neutrophils was studied by neutrophil chemotaxis assay and flow cytometry. For statistical analysis, unpaired two-way Student’s t test, one-way ANOVA or linear regression analysis were used.ResultsReduced expression of miR-146a was found in bronchial brushing specimens from asthma patients as compared to non-asthmatics and irrespective of the phenotype of asthma. In the same samples, the neutrophil attracting chemokines IL-8 and CXCL1 showed increased expression in patients with neutrophilic asthma and increased IL-33 expression was found in patients with eosinophilic asthma. Linear regression analysis revealed a significant negative association between the expression of miR-146a in bronchial brushings and neutrophil cell counts in bronchoalveolar lavage fluid of patients with asthma. In bronchial biopsy specimens, the level of miR-146a was highest in the epithelium as determined with in situ hybridization. In primary conventional HBEC culture, the expression of miR-146a was induced in response to the stimulation with IL-17A, TNF-α, and IL-4. The mRNA expression and secretion of IL-8 and CXCL1 was inhibited in both stimulated and unstimulated HBECs transfected with miR-146a mimics. Supernatants from HBECs transfected with miR-146a had reduced capability of supporting neutrophil migration in neutrophil chemotaxis assay.ConclusionOur results suggest that decreased level of miR-146a in HBECs from patients with asthma may contribute to the development of neutrophilic phenotype of asthma.

Highlights

The role of miRNAs in the pathogenesis and determining the phenotypes of asthma is not fully elucidated. miR-146a has been previously shown to suppress inflammatory responses in different cells
When patients were subgrouped based on inflammatory phenotypes, we observed miR-146a downregulation in airway epithelial cells from patients with eosinophilic, neutrophilic and paucigranulocytic asthma phenotype as compared to non-asthmatic controls (Fig. 1b)
The strongest miR-146a staining was confined to the bronchial (Fig. 1d) and bronchiolar epithelium (Additional file 1: Figure S1C), suggesting that in the lungs, miR-146a is mainly expressed in the bronchial epithelium

Summary

Introduction

The role of miRNAs in the pathogenesis and determining the phenotypes of asthma is not fully elucidated. miR-146a has been previously shown to suppress inflammatory responses in different cells. We investigated the functions of miR-146a in human bronchial epithelial cells (HBECs) in association with neutrophilic, eosinophilic, and paucigranulocytic phenotypes of asthma. As asthma is a highly heterogeneous disease, attempts have been made to define asthma phenotypes and/or endotypes on the basis of involved immune cells or molecular processes, respectively [4,5,6]. As one option, this allows categorization of asthmatics into eosinophilic, neutrophilic, mixed granulocytic and paucigranulocytic asthma phenotypes [4, 5]. Independent from phenotype, airways of all asthma patients are influenced by chronic inflammation, which leads to the changes in genes affecting cell proliferation, such as HBEGF and FGF2 and eventually, airway remodeling may take place [1,2,3]

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Conclusion