1. Introduction
Despite intensive efforts, the prevalence of allergies and asthma is still increasing worldwide [
1]. Although the therapy for asthma has improved over the years, asthmatic condition can still lead to a sudden death [
2,
3]. Asthma is a complex disease and hundreds of candidate genes have been proposed for this condition [
4,
5]. Apart from genetic predisposition, environmental factors seem to play a crucial role in asthma development, including the exposition to indoor and outdoor allergens, such as mites or pollens and irritants like lipopolysaccharides (LPS) [
6,
7]. Novel techniques and methods, such as next generation sequencing (NGS) have opened a new era in asthma research. Although many genome-wide association studies have been conducted, little replication has been observed [
8]. One possible explanation of this phenomenon could be the fact that multiple known phenotypes of human asthma are present and classification depends on clinical phenotypes (severity, treatment-resistance etc.), trigger (allergic/non-allergic asthma, aspirin-induced asthma etc.) and inflammatory phenotype (eosinophilic, neutrophilic, or paucigranulocytic asthma) [
9]. Due to the absence of standardized phenotype definitions in the past, interpretation as well as integration of findings proved to be challenging. The most consistently identified candidate genes (interleukin 13 (
IL13), interleukin 4 (
IL4)
, interleukin-4 receptor, alpha (
IL4RA), cluster of differentiation 14 (
CD14)
, Beta-2 adrenergic receptor (
ADRB2), membrane-spanning 4-domains subfamily A member 2 (
MS4A2/FCER1B), tumor necrosis factor (
TNF) superfamily, disintegrin and metalloproteinase domain-containing protein 33 (
ADAM33), and ORLDM Sphingolipid biosynthesis regulator 3 (
ORMDL3) do not show association in all of the populations studied or may only show small effects explaining only a very low percentage of the total phenotypic variance [
5,
10,
11].
Severe equine asthma (also called recurrent airway obstruction or heaves) occurs naturally and shares many features with human neutrophilic asthma [
12] and also shows parallels to human late-onset and severe asthma [
13]. Therefore, asthmatic horses are considered a good animal model for human asthma [
13,
14]. Asthma in horses has a large economic impact on horse breeding and equestrian sports. Until now, little has been done to prevent the development of asthma in horses. Treatment strategies are focused mostly on a decreased exposure of asthmatic horses to hay, which has been shown to be the major risk factor for asthma development in horses [
15,
16]. Even though a strong genetic predisposition to severe equine asthma has been reported [
17,
18,
19], excluding affected animals from breeding is difficult. Clinical signs of asthma often appear later than age eight, which is much higher than the average age at which horses are chosen for breeding. Hence, the search for non-invasive biomarkers is of great interest. Potential biomarkers discovered in the equine model could also be further investigated for their implication in human asthma and might even serve as novel therapeutic targets for both equine and human asthma.
Recently, microRNAs in serum (miRNAs) have received great attention as potential biomarkers for many diseases, e.g., neoplastic, cardiac, immune-related, pulmonary and other diseases [
20]. MicroRNAs are small RNA molecules that impact biologic responses through the regulation of mRNA transcription and/or translation. A single miRNA may regulate dozens of target genes and thus disrupt an entire genetic pathway leading to pathological features [
21].
MicroRNAs are very stable molecules compared to other RNA species and can be transported between cells, tissues and even organisms (mother and fetus) [
22]. Extracellular miRNAs can be deregulated in serum and other body fluids during the pathogenesis of many disorders. MicroRNAs from serum are thus of particular interest as promising non-invasive disease biomarkers [
23]. Our present understanding of their role in the regulation of allergic diseases is still very limited. However, differential miRNA expression has been shown in a wide range of tissues, cell types, biofluids and vesicles such as bronchoalveolar lavage fluid exosomes, airway T cells and serum from asthmatic patients [
24,
25]. Since distinct miRNA networks regulate CD4
+ T cell differentiation, miRNA differential expression studies have the potential to unravel aberrant molecular mechanisms underlying disorders of the immune system [
26]. Specifically, miR-155 plays a major role in both allergy and anti-parasitic immunity [
27].
Over 1000 miRNAs have been identified in the horse with distinct subsets of miRNAs differentially expressed in a tissue-specific manner [
28,
29]. Due to their conservation, a majority of equine mature miRNAs have been perfectly matched to human disease-associated miRNAs [
30], indicating the potential of investigating miRNA profiles in equine allergic and other conditions [
31].
We investigated serum miRNAs and compared the expression profiles of 37 asthmatic Warmblood horses in comparison with 35 unaffected control horses using miRNA-seq. As erythrocyte-derived miRNA may bias the expression profile of serum miRNA [
32], we took into account the level of hemolysis in our samples. Furthermore, we retrieved the potential targets of candidate miRNA biomarkers and investigated their expression in peripheral blood mononuclear cells (PBMCs) [
33] in correlation with the serum miRNA expression in the same individuals.