MicroRNA signatures of equine asthma phenotypes in serum and bronchoalveolar lavage fluid

Equine asthma (EA) is a highly prevalent, chronic, inflammatory disease of the lower airways ranging from mild-to-moderate to severe clinical presentations. Diagnosis currently relies on bronchoalveolar lavage fluid (BALF) cytology, an invasive method associated with interobserver variability, which highlights the need for more reproducible approaches. MicroRNAs (miRNAs) are small noncoding RNAs involved in gene regulation. They are stable and readily detectable in body fluids and have shown promising results as biomarkers in human asthma. The aim of this pilot study was to characterize miRNA expression profiles in BALF and serum from horses with distinct EA phenotypes to evaluate their biomarker potential and explore their putative involvement in disease pathogenesis. A total of 43 horses were included and classified as either EA ( n = 32) or controls ( n = 11), based on clinical examination and BALF cytology. The EA horses were further divided into three phenotypes based on BALF inflammatory cell composition: neutrophilic asthma ( n = 10), mastocytic asthma ( n = 15), and mixed asthma ( n = 7). RNA was isolated from both serum and BALF samples and analyzed by quantitative real-time PCR (qPCR) targeting 103 miRNAs linked to asthma and pulmonary inflammation in humans. Differential miRNA expression was analyzed across EA phenotypes. The most significantly differentially expressed miRNAs were used for hypothesis-generation by in silico target prediction and pathway enrichment exploratory analyses. Horses with mixed EA had significantly lower levels of eca-miR-125a-3p and eca-miR-125b-5p in BALF compared to controls. Additionally, eca-miR-146a-5p expression was significantly increased in BALF from horses with neutrophilic EA compared to mastocytic EA. For serum, none of the miRNAs showed significant differential expression within the EA phenotypes. Target and pathway enrichment analyses for eca-miR-146a-5p identified immune-relevant pathways supporting its possible involvement in inflammatory processes associated with asthma. This study identified three promising candidates, eca-miR-125a-3p, eca-miR-125b-5p, and eca-miR-146a-5p, as potential biomarkers in BALF associated with different EA phenotypes. These miRNAs are interesting candidates for further validation in an independent cohort before they can be used in diagnostics.