Although sEA’s heritability has been shown in several horse breeds, and a familial aggregation has long been ascertain, external factors, such as environment, increase the likelihood of expressing the disease [
3,
21,
22].
The chromosome region ECA13 has been associated with sEA in one family of Swiss Warmbloods, while region ECA15 has been implicated in a different family of the same breed. The inheritance mode differed between both families, being autosomal recessive in the first family and autosomal dominant in the second [
25]. Additionally, in the first family of horses the Interleukin 4 receptor (IL-4R) gene and its neighboring regions in ECA13 appeared to contribute to disease in some individuals [
26,
27,
28]. In humans, polymorphic differences in the Interleukin 4 receptor α chain (IL4Rα) gene play an important role in the development of asthma, since they induce the isotopic switch to immunoglobulin E (IgE) and the differentiation of T-helper type 2 (Th2) lymphocytes [
29,
30].
Racine and colleagues described an interaction between IL-4R and products of the
SOCS5 gene, which may influence the molecular cascades involving nuclear factor (NF)-κB [
31]. The gene coding for SOCS5 is located in the ECA15 chromosome region, and it is predominantly expressed by Th1 cells while further inhibiting Th2 differentiation. The inhibitory effect of SOCS5 on IL-4 signaling contributes to the non-Th2 cytokine profile observed in human non-allergic asthma [
32], and may explain further similarities between both species.
In a genome wide association study (GWAS), the gene responsible for the TXNDC11 protein, also located in the ECA13 region, has been linked to sEA [
33]. In humans, TXNDC11 controls the production of hydrogen peroxide in the respiratory epithelium [
34], as well as the expression of MUC5AC mucin, which has been shown to play a significant role on airway hyperreactivity in mice [
35]. In sEA-affected horses MUC5AC is upregulated, thus contributing to the mucus plugging observed in the disease [
36].
The analysis of genomic copy number variants did not reveal any relevant variant regions which could be associated with the sEA, although a copy number loss was reported on chromosome 5 involving the gene NME7 [
37]. The expression of this gene is necessary for ciliary function in the lungs and may be involved in sEA, since in knockout mice it induces primary ciliary dyskinesia [
38]. Also, using RNA sequencing technique, a single point substitution was detected in the PACRG and RTTN genes in asthmatic horses, predictively altering their proteins, which are related to ciliary function [
39].
In a gene set enrichment analysis of the bronchial epithelium after hay dust exposure, asthmatic horses presented upregulated genes of the E2F transcription factor family, which contribute to cell cycle regulation. Thus, asthmatic horses may suffer from impaired bronchial epithelial regeneration associated to subepithelial remodeling [
40].
These recent studies have shown that the respiratory epithelium contributes to the immunological response observed in severely asthmatic horses.
Furthermore, an analysis of expression quantitative trait loci (eQTLs) allied with GWAS did not find a significant association between observed genetic variants and sEA, except for a disease-genetic variant in CLEC16A gene, which regulates gene expression of dexamethasone-induced protein (DEXI) [
41]. This is of special importance in comparative pathology, as DEXI has also been reported in human asthma [
42], although in horses it appears to not be a reliable indicator of sEA [
41].
The identification and differential expression analysis of microRNAs (miRNAs) present in the serum of sEA-affected horses, showed a downregulation of miR-128 and miR-744. These findings suggest that a Th2/Th17 immunological response may characterize sEA [
10,
43].
Additionally, a recent work on Polish Konik horses aimed to detect the effects of inbreeding on sEA, however no effects were observed at the individual level [
44].
Although most of these findings relate to certain families of Swiss Warmblood horses, they illustrate the complex genetic heterogeneity of sEA, which most likely results from the interaction of different genes. However, the use of such specific horse families and the likelihood of high variety of genetic background mechanisms contributing to the disease limits the application of these findings to the general equine population.
3. Immunological Phenotypes and Endotypes
Phenotype is the term used to describe the observable clinical characteristic of a disease, whereas endotype, a subclass of phenotype, refers to its molecular and genetic mechanism or treatment response [
45].
As stated in the 2016 consensus, equine asthma (EA) is currently defined by two major phenotypes, which differ according to disease onset, clinical presentation and its severity—mild/moderate EA (mEA) and the already described sEA, which is the focus of this review [
1]. However, phenotypes are insufficient when deciding upon the appropriate therapeutic management or determining the prognosis, which mainly depend on the immunological mechanisms of the disease.
Human asthma is usually considered to be a type 1 hypersensitivity, due to increased levels of IgE associated with a Th2 response, resulting in the recruitment of eosinophils into the airways [
46]. However, an endotype which does not appear to be associated to a Th2 response has also been identified. As such, human asthma is divided into two major endotypes according to cytokine profile: Th2 and non-Th2 type asthma [
47]. The Th2 type asthma is considered an allergic phenotype with the aforementioned eosinophil involvement and because its cytokine profile has been thoroughly described, several biomarkers are available for characterizing the disease and will be addressed bellow.
However, sEA is typically characterized by a neutrophilic response [
1,
48], and appears to not have the typical presentation of a type 1 hypersensitivity [
49]. Although a Th2 cytokine profile has been described in sEA-affected horses, these animals do not display an early phase response [
50,
51]. However, a late phase response leading to neutrophilic bronchiolitis, associated with an increase in CD4+ T cells in the bronchoalveolar lavage fluid (BALF), has been described [
50,
52,
53]. These features have led to the hypothesis that a type 3 hypersensitivity response, resulting in antibody-antigen complexes and activation of complement cascade, were involved in the disease’s immunology [
54]. However, because sEA does not possess most of the features described in type 3 hypersensitivities, it is unlikely that this type of response accounts for the main immunological features of the disease [
55].
Still the precise cytokine profile of sEA remains unclear, with a multitude of reports pointing to either a Th1, a Th2, a Th17 or a mixed mediated response.
Table 1 illustrates the cytokines reported in sEA-affected horses.
Table 1. Cytokines reported in sEA-affected horses according to T helper subtype [
10,
43,
49,
52,
56,
57,
58,
59,
60,
61,
62].
Th2 |
Th17 |
Th1/Th2 |
Th1/Th17 |
Th2/Th17 |
Undefined |
↑ IL-4 1(r) |
↑ CXCL13 2(r) |
↑ IL-4 1(r) |
↑ IL-1β1(r) |
↓ miR-197 2(r) |
↓ IFN-γ 1(r) |
↑ IL-5 1(r) |
|
↑ IFN-γ 1(r) |
↑ IL-8 1(r); 3(r); 3(p) |
↑ miR-744 2(r) |
↓ IL-4 1(r) |
↓ IFN-γ 1(r) |
|
|
↑ IFN-γ 1(r) |
↓ miR-26a 4(r) |
↓ IL-5 1(r) |
|
|
|
↑ TNF-α 1(r) |
↑ miR-31 4(r) |
↓ IL-13 1(r) |
|
|
|
↑ IL-17 1(r) |
↓ TNF-α 4(r) |
|
|
|
|
|
↑ IL-4R 4(r) |
|