To our knowledge, this is the first study to report on the “effect of training” on UK Thoroughbred national hunt racehorses, determined by both transcriptomic and proteomic analysis of TW samples harvested before and during a period of training, thus permitting an assessment on the basal gene expression of principal innate immune cells of the equine airway. TW samples are routinely collected in contrast to the less readily available and more technically invasive bronchoalveolar lavage (BAL)-derived samples, which have previously been favoured for transcriptomic and proteomic analyses (42–44). The transcriptome was derived from high throughput sequencing, and results were analysed with a paired design to account for individual variation. Animals were of similar age, same sex and breed and lived in a similar environment. All animals recruited in this study were from the same yard and subject to the same general management structure. RNA-seq data were analysed using DEseq2 software, a widely used tool in high-throughput RNA sequencing studies using raw counts (26). Compared with a single-end strategy, the paired-end strategy used in this study has been recommended as it minimises the likelihood of false positives (45); moreover, the validity of the data, in terms of differential expression, was supported by the RT-qPCR results. However, irrespective of the methodology used, considering the genetic variability among individuals and the limited sample size, the potential for false-positive results remains, necessitating caution when interpreting individual gene expression data in RNAseq-based studies. Similar caution should be exercised when interpreting the differential expression of individual proteins in the proteomic dataset. Thus, the consideration of linked, rather than individual, genes and proteins conveys more confidence when interpreting these kind of data (45).
Our experimental protocol allowed us to investigate the effect of training on airway innate immune responses at both the gene and protein level. Although this revealed significantly limited overlap between the RNAseq and proteomic datasets, this was not unexpected as such a lack of concordance between gene and protein expression has been well documented (46, 47) and can be attributed to a variety of factors such as post-transcriptional machinery, variable half-lives, molecular degradation, or even sampling bias (37). Additionally, certain factors specific to the current study design may also have contributed to this discordance in gene and protein expression. Firstly, unlike the transcriptomic analysis, proteomic analysis was not restricted to the cellular component of the TW sample, thus permitting the detection of secreted proteins. This approach was considered more likely to provide a more holistic assessment of the airway immune status as well as offering a greater potential to identify biomarkers with potential clinical and/or training applications. Secondly, gene expression data was obtained from each individual animal; whereas, protein analysis was conducted on pooled samples at each time point. This approach was justified on financial grounds due to the high cost associated with the application of these technologies. It should also be noted that, due to insufficient protein yield, samples from Horses 3, 7 and 12 were not included in the proteomic analysis for both time points. Thirdly, the lack of overlap in gene/protein expression in the current comparison can also be explained by the high ratio (approx. 27) between the expressed genes (21,357) and proteins (802) detected using the applied methodologies. Lastly, with such transcriptomic studies, the conclusions that can be derived from the data generated are highly dependent on the quality of the annotations of the genome used, which for the horse remains challenging (48). Overall, consideration of these study-related factors and the recognised discordance between gene and protein expression does significantly question the validity of inferring changes in protein expression from transcriptomic data alone. Rather, this study highlights the value of applying a combined transcriptomic and proteomic approach when studying cellular mechanisms in both health and disease. Despite the lack of agreement at the individual gene and protein level, significant similarities were observed at a pathway and cascade level.
We demonstrated a clear modification of both mRNA and protein expression in TW derived samples during the training period. Based on the multi-omics methodologies applied, we identified 2,138 differentially expressed equine genes and 260 proteins during this period. Our findings are consistent with those derived from comparable studies in humans and horses (44, 49, 50), and support a clear association between intense training and immune system deregulations, haemopoietic and metabolic abnormalities and cellular stress at the level of the airway. It is conceivable that such training-associated changes may play a role in increasing susceptibility to opportunistic infection and airway inflammation in racehorses.
Local immune responses in tracheal airway immune cells during training
Using a multi -omics approach (RNAseq and proteomics) we observed an alteration in a number of gene and protein pathways in association with training (time point T1); many of these pathways were associated with important aspects of airway immunity, such as antigen presentation, complement activation, immune cell chemotaxis, cellular stress, response to bacterial/viral infection and regulation of IFN signalling (Figs. 5 and 7, Additional file 3, 4). Analysis also revealed the increased expression of several myeloid cell chemoattractants (e.g., CCL2, CSF3R, S100A9 and other related S100 genes) at time point T1. Various relevant gene ontology terms were observed, such as neutrophil and monocyte chemotaxis, highlighting a level of immune cell activation in the airways. Additionally, acute phase response, cellular stress and oxidative phosphorylation also characterise the response to intense training.
Overall, both the transcriptomic and proteomic data were consistent with a level of inflammation in the airways during training, a phenomenon very well documented in racehorses. Based on the TW differential cytology results, 3 out of 16 animals developed airway neutrophilic inflammation (as defined by neutrophil ratio > 20) at time point T1, consistent with a low level of airway inflammation during training, despite the absence of clinical signs or evidence of poor performance. In light of the well-recognised association between intense exercise and an acute phase response in both humans and horses (51), it is quite conceivable that repetitive periods of arduous exercise over a prolonged period of time may also result in low level airway inflammation. Indeed, recent equine studies have shown that an intense training programme will induce an “inflammation-like state” based on the measurement of acute phase protein SAA, which is greater than that induced by a lighter physical activity program (52). Serum amyloid A was also significantly induced during the training period (T1) in our study. Despite the relatively low number of horses with a significant elevation in TW neutrophil ratio at T1, we did detect a training-associated upregulation of myeloperoxidase (MPO) protein expression, potentially reflective of a low level of airway neutrophil presence/activity below the threshold of detection by TW cytological examination. Indeed, MPO has previously been proposed as a more sensitive marker of airway neutrophil presence compared to cytology (53). Despite its beneficial properties in host defence, overproduction of MPO may exert detrimental effects during inflammation (54), including tissue damage and vascular dysfunction through the generation of potent ROS and activation of MMPs (54, 55). Consistent with these downstream effects, we also detected the upregulation of several MMPs (including MMP9) and ROS in association with training (56–58).
In addition to inflammation, training was associated with evidence of tissue remodelling. Chitinase 3 like 1, ARG2 and MMP9, all upregulated during training, have previously been linked to airway remodelling and/or declining lung function (59). MMP9 has been shown to contribute to airway remodelling in severe equine asthma (49). Training was also associated with an upregulation of ORMDL3 and HLA, both of which are highly associated with human and murine asthma (59), with ORMDL3 playing an important role in airway remodelling (increased airway smooth muscle, sub-epithelial fibrosis and mucus) (60). Interleukin 4 induced 1, another gene which was highly expressed during training, has been shown to inhibit T cell proliferation, regulate the programming of macrophages towards a polarised M2 phenotype and downregulate LPS-induced TNF expression in murine macrophages (61). Indeed, the data suggests that training may induce an overall immunomodulatory milieu at the level of the airway, characterised by increased expression of IL4I1 (61) and ROS (62). This may explain the significant downregulation of TNF during the training period and the identification of gene ontology terms related with apoptosis and cell death, as ROS have been shown to regulate both necrotic and apoptotic T activated cell death via caspase activation (62).
Training-associated IFN response on equine airways
A large number of IFN regulated and stimulated genes were differentially expressed during the training period. The IFN response is considered a key driver of inflammation in the lung by stimulating the recruitment and activation of immune cells, often in response to viral infection. Despite these beneficial effects, the negative regulation of the IFN response is necessary to avoid excessive and continuous activation of IFN-induced pathways with associated collateral tissue damage (39). Moreover, there is recently increasing evidence that type I IFNs are not only induced by viral infections, but also by bacterial and fungal infections. Indeed, in the case of bacterial lung infections, the type I IFN mediated signalling may be detrimental (63).
Training resulted in a greater differential expression of genes related to the type 1 IFN response, compared to type II and III IFN responses. Many of the recently described and highly conserved mammalian type I IFN ISGs were differentially expressed during training; these were related with antiviral activity, antigen presentation, PAMP detection and apoptosis. Interestingly, we identified several of the ISGs upregulated during training to be associated with ubiquitination; these included the ring finger proteins RNF19A, RNF25, RNF181, RNF183 and highlighted protein reformation as part of the IFN response. This was also reflected in the pathway analysis, as protein synthesis was one of the top Biofunctions detected in our datasets. Recent findings derived from different mammalian species (including the horse), showed the type I IFN response to positively bias the sensitivity of RNA virus surveillance initiatives, recognising the problems associated with differentiating between “self” and “exogenous” cytoplasmic RNA (39). Indeed, it is feasible that the type 1 IFN signature identified during training may be attributable to sub-clinical viral infection in this cohort of horses. Further work on a more geographically diverse population would be required to address this possibility. Despite the transcriptomic and proteomic evidence of altered immunity during the training period, the causal factors which underpin these changes remain speculative. Such causal factors may act in isolation or in concert and may include the following: repetitive periods of intense exercise, changes in airborne environment and subclinical infection. Indeed, both infectious and environmental causes have been proposed for the increased risk of airway inflammation within the early training period, potentially reflecting the co-mingling of horses from diverse locations and increased housing associated with the transition into this period (6, 64–66).
Previous studies have identified an association between the bacterial load within the trachea and both cytological and endoscopic evidence of airway inflammation (6, 64, 66). Furthermore, in addition to the early training period, the detection of bacteria (streptococcal species) within the trachea was associated with evidence of airway inflammation; however, this latter association was not confirmed as causal, potentially reflecting a more indirect association (e.g. increased colonisation of mucus following compromised clearance). In our study, the timing of the second sample collection was such that this “early training” association should have subsided, which, together with the limited cytological or endoscopic evidence of significant airway inflammation offers more assurance that the transcriptomic and proteomic data was not solely reflective of the early transition to the training environment. Viral infections should also be considered as a potential explanation for the association between training and airway inflammation, particularly in light of the specific IFN pathways altered by training. Indeed, previous studies have shown that viral infections are common among young horses after entry into training (67, 68). However, attempts to identify viral RNA based on proteomic analysis, failed to identify any appropriate candidates. If a viral cause was indeed present, it was not associated with overt clinical signs, nor did it appear to impact athletic performance.
Previous studies have demonstrated a clear association between housing and airway inflammation, reflective of the increased exposure to organic dust (69, 70). As horses were housed during the training period and largely at pasture during the T0 sampling, it is quite feasible that some of the immunological changes observed, at both the individual gene and protein level and the pathway level, were consistent with an increased exposure to organic dust; however, this was not universally or consistently reflected in the TW differential cytology data. The ventilation within the stable was considered to be good and all horses were bedded on low dust shavings and fed haylage from the ground. Additional data relating to dust exposures would have assisted the interpretation of the data in light of the change in housing; however, this was not possible within the constraints of the study design. Lastly, in line with previous studies (10), the immunological alterations associated with training may have been attributable to the repeatable episodes of high intensity exercise. Frellstedt et al (2014) previously demonstrated training-associated changes in immunity at the level of the lower airways in treadmill exercised horses (10).
Training induced cellular stress
As well as the potentially direct effect of training on airway immunity, various transcription modulation processes also seem to play an important role. Cappelli and others (2020) recently demonstrated the dominant role of transcription modulation in orchestrating the genomic response of equine PBMCs to exercise induced stress (71). Muscle ATP demand increases with increasing exercise intensity with the rate of production dependent on the availability of oxygen, carbon substrates, Ca2+.and other molecules including, nitric oxide and reactive oxygen and nitrogen species (RONS) (72). During intense exercise, increased RONS production may cause oxidative stress and damage to cellular structures and reduce mitochondrial efficiency, resulting in inflammation and transient immune dysfunction (73).
In the current study, pathway analysis detected several differentially expressed genes related to oxidative stress, oxidative phosphorylation, mitochondrial dysfunction and protein synthesis (Fig. 4, 5; Additional file 3, 4). Reactive oxygen species modulator 1 gene, responsible for increasing the level of reactive oxygen species (ROS) in cells, was significantly upregulated and superoxide dismutases (SOD), the first line of defence against superoxide radicals (73), were downregulated during the training period. At the protein level, both SOD1, with the capacity to limit the detrimental effects of ROS and apoptotic signalling, and SOD2 were significantly downregulated during the training period. Proteomic analysis also revealed downregulation of other antioxidants; these included thioredoxin (TXN) and various heat shock proteins (HSP90B1, HSPA5, ST13, HSPD1, HSPE1, HSPB1) (74). TXN has the capacity to directly interact with several transcription factors including NFKB (74), also regulated by ROS and which itself regulates the transcription of a host of acute phase, cytokine and cell surface receptor genes; in some systems, antioxidants have been shown to reduce or block NFKB activation (74). Our data showed the training-associated differential expression of several genes (NFKBIZ, CARD19, NKAP) involved in the NFKB pathway. ROS can also activate MAP kinases via a Ras-dependent mechanism (74) and several components of the MAPK/ERK1/2 signalling pathway were either upregulated (MCRIP2, LAMTOR1, LAMTOR2, LAMTOR4, MAPK13, MAPKAPK3) or down regulated (MAP3K7, GIMAP7, MAP3K8) during the training period. The unchecked production of ROS and nitrogen reactive species will ultimately jeopardise cell survival (72), a consequence consistent with the evidence of stress induced cell death detected in our dataset; namely, the training associated upregulation of members of the BCL2 family (BAD, BAX, FADD), CARD19, AIFM2 and the pro-apoptotic molecules (BAX, TMBIM1). Excessive ROS production will also result in impaired mitochondrial efficiency, as detected during fatiguing and intense prolonged exercise in humans (72). Canonical pathway analysis of both our transcriptomic and proteomic datasets revealed mitochondrial dysfunction in relation to training. The upregulated COX14 gene encodes for a core protein component of the mitochondrial translation regulation assembly intermediate of cytochrome c oxidase complex (MITRAC) which is essential for the regulation of complex IV assembly of the mitochondrial respiratory chain. Upregulation of genes encoding several components of the complex IV (COX5B, COX6A1, COX6B1, COX7A1, COX7A2, COX8A) were also detected during the training period.
Haemopoietic abnormalities during training
In light of the very high prevalence of EIPH in racing Thoroughbreds (approaching 100%) (13), it is unsurprisingly that most of the horses (~ 69%, 11 out of 16) developed cytological evidence of EIPH during the training period. The pulmonary pathology associated with EIPH reflects changes in the pulmonary interstitium and vasculature, resulting in interstitial oedema and fibrosis (75, 76); yet, to date, none of the EIPH studies have examined the transcriptomics of airway derived cells harvested from cases. Analysis of gene expression data derived from the current study revealed minimal differences between EIPH affected (n = 11) and unaffected (n = 5) horses during the training period (data not shown); however, this has reflected the small group sizes following subdivision of the T1 cohort; however, several genes related to haemopoietic abnormalities were detected in both sub-groups, suggesting that bleeding may have occurred even in those cases with no clear cytological evidence of such. Our data does however support the potential value in expanding the transcriptomic analyses to a larger sample population in order to more critically assess the gene expression profile of EIPH cases.
Transcriptomic profile of racehorses with high neutrophil count on TW samples
Although only a small number of horses developed a significant airway neutrophilia during the training period, supplementary comparative analysis was applied to the data derived from the three horses with the greatest magnitude of TW neutrophilia, compared with the other horses at time point T1. This revealed the differential expression of 57 genes, a number similar to that reported in circulating leukocytes derived from both Standardbred and endurance horses and BALF samples from horses with airway inflammation (28, 77). Also consistent with previous reports, almost all differentially expressed genes were upregulated, with only three being downregulated. The differential expression of IL1RN in the group of horses with airway inflammation (Additional file 7), was consistent with the findings of Hansen et al (2020) who reported an association between IL1RN expression in BALF derived cells and the neutrophilic form of MMEA (78). Similarly, genes cysteine rich secretory protein LCCL domain containing 2 (CRISPLD2) and transglutaminase 3 (TGM3) were also present in BALF cells derived from horses with neutrophilic form of MMEA (28). Interestingly, the neutrophilic group also showed increased expression of the mucin gene MUC5AC, shown previously to contribute to mucus hypersecretion in severe equine asthma (79) and also highly expressed in bronchial biopsies from asthmatic humans (80). Overall, the transcriptomic changes in this small group of horses were consistent with the airway cytological profiles observed, reflecting pathways associated with neutrophil chemotaxis, response to stress and immune defence (49). Interestingly, prior comparative transcriptomic analyses of endotracheal biopsies derived from asthmatic horses also revealed the differential expression (compared with non-asthmatic horses) of a similar gene list to that identified in the current study. This gene set included CSF3R, CXCR2, PLEK, IL1RN, RETN, VSTM1, TREM1 and PLAUR, and is related to neutrophil chemotaxis and immune responses. Colony stimulating factor 3 is responsible for neutrophil production and proliferation and promotes neutrophil trafficking by modulating chemokine and adhesion receptors, such as CXCR2 that mediates neutrophil migration to sites of inflammation. Although difficult to detect in blood, CSF3 is secreted to a greater extent during infectious or inflammatory conditions (81–83) including human asthma where it was positively associated with BALF neutrophil numbers and sputum from asthmatic patients (80). Moreover, CSF3 is considered as a potential target against pathological inflammation and tissue remodelling occurring in human asthma (80). Other genes detected in this group, such as TREM1 and PLAUR have also been reported in human asthma (59, 84).
A number of Th1 immune response agents were also enriched in the neutrophilic group of horses (CXCR2, IDO1, CSF3R, ISG20, IL18RAP and TREM1). Thus, it appears that the equine airway neutrophilia detected in these cases was associated with activation of the innate immune system, with a likely Th1 polarised response. Similar observations have been previously reported, although, within the context of MMEA, suggesting that immunological pathways vary according to the cytological profile of the airway inflammatory response and potentially the phase of the disease (85).
Despite limitations of the current study, this is the first whole genome sequencing study performed on equine TW derived cells. Results shared important similarities with previous equine and human studies and although the aim of the study was focussed on the impact of training, transcriptomic analysis detected a number of novel genes potentially related to disease pathogenesis which may provide new insights for future studies. It remains feasible that the assessment of gene expression in TW-derived cells may represent a relatively non-invasive method to identify molecular endotypes of equine asthma in larger studies. However, detailed phenotypic characterisation of the horses included in these studies would be crucial.