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

Abstract

Background: 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. 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. Methods: Bronchial 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. Results: Reduced 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. Conclusion: Our results suggest that decreased level of miR-146a in HBECs from patients with asthma may contribute to the development of neutrophilic phenotype of asthma.