Equine gastric ulcer syndrome (EGUS) is a highly prevalent disease with a high importance due to its negative effects on the welfare and performance of horses [1
]. According to the European College of Equine Internal Medicine (ECEIM) Consensus Statement, EGUS can be distinguished into two different diseases, depending on which anatomical region is affected: equine squamous gastric disease (ESGD), in which there is a lesion in the non-glandular stomach; and equine glandular gastric disease (EGGD), in which the alteration appears in the glandular stomach [1
]. EGUS has nonspecific clinical signs and its mechanism has not been totally elicited. The most common clinical signs that can be observed include poor appetite, loss of weight, yawning, bruxism, anorexia, salivation, abdominal discomfort, and reduced performance [2
]. The only antemortem diagnostic method currently validated and considered as a gold standard is the evaluation of the entire stomach using gastroscopy, in order to visualize the specific lesions and their locations [4
]. Due to the current lack of specific haematological or biochemical markers to diagnose EGUS, it would be of interest to discover analytes that could potentially serve as biomarkers of this syndrome [2
Proteomics allows the identification of a high number of proteins simultaneously, thus being useful in the identification of proteins that can change in a selected disease [7
]. Tandem mass tag (TMT) is a labelling procedure that allows the quantification of different peptides, which are marked and identified [8
]. This technique has been used previously to investigate the salivary proteome in horses with acute abdominal syndrome [9
]. In a previous study, an evaluation of changes in the serum proteins of horses with ESGD using gel electrophoresis and mass spectrometry has been performed [3
]. In this report, 10 serum proteins were found to be possible biomarkers for ESGD. However, to the best of the current authors’ knowledge, there are no proteomic studies that evaluate changes in the proteome of saliva in EGUS, and the possible differences between ESGD and EGGD.
The hypothesis of this study is that ESGD and EGGD could produce changes in the proteins in saliva that could be detected by gel-free proteomics. Therefore, the objective was to evaluate the changes produced in saliva proteins of horses with ESGD, EGGD, and healthy horses through proteomic analysis using TMT. Serum was also analysed for comparative purposes. This data can contribute to a better understanding of the pathophysiological changes that occur in these diseases and to identify new potential biomarkers of these conditions.
Our results provide preliminary evidence of EGUS-induced changes in the salivary protein profile, which could further differentiate between the two EGUS clinical forms, EGGD and ESGD. The number of differentially expressed proteins was more than three times higher in ESGD than in EGGD. Furthermore, the changes were not uniform when the horses with EGGD and ESGD were independently compared with the healthy horses. Therefore, it could be a better approach to assess the changes in saliva in EGGD and ESGD separately, instead of in all horses with EGUS.
Many of the proteins with the increased abundance of saliva in EGGD (e.g., TMPRSS11D, S100-A9, joining (J) chain, and adenosylhomocysteinase) share a common feature as they are involved in the regulation and activation of the immune system. This is in line with a recent report indicating the involvement of immune-mediated mechanisms and lymphoplasmacytic infiltration of the glandular mucosal inflammation in EGGD [5
TMPRSS11D is a transmembrane serine protease and, as such, it is deeply involved in the inflammation and immune system reaction due to its ability to cleave the peptide bonds. Serine protease activation has been associated with an exacerbation of the immune response [20
]. In addition, it has been reported in humans that Helicobacter pylori, a bacterium that could be involved in EGGD pathogenesis [1
], can activate serine proteases, impairing the cellular repair and leading to connective tissue and extracellular matrix degradation [20
]. However, to date, there remains conflict in the literature as to the role of bacteria in EGGD and there could be other factors that could activate serine proteases in this disease. For example, vascular and circulatory diseases can activate proteases [21
]. This could potentially occur in EGGD, where circulatory disorders within the stomach leading to a reduced gastric glandular blood flow wall have been described as a possible cause [22
S100-A9, also known as MRP14, is a Ca2 + binding protein belonging to the S100 family that forms a complex with S100-A8 called calprotectin. The upregulation of S100-A9 occurs in multiple immune system dysfunction diseases that result in excessive immune responses leading to autoimmune diseases and hypersensitivity reactions [23
The joining (J) chain is a small polypeptide expressed by mucosal and glandular plasma cells, which regulates polymer formation of immunoglobulin (Ig) A and IgM. J chain shows a high affinity for the polymeric Ig receptor (pIgR) also known as transmembrane secretory component (SC). This epithelial glycoprotein mediates active external transfer of IgA and pentameric IgM to exocrine secretions. Therefore, it is a key protein in mucosal immunity [24
The adenosylhomocysteinase, also named S-adenosylhomocysteine hydrolase, is an enzyme that converts S-adenosylhomocysteine into homocysteine and adenosine [25
]. The in vivo activity of this enzyme depends on the function of another enzyme, adenosine deaminase (ADA). Both are located in the same chromosome and it seems that S-adenosylhomocysteine hydrolase, a eukaryotic enzyme, evolutionarily appeared later than ADA, which occurs in prokaryotes as well as eukaryotes [26
]. Therefore, it could be postulated that the increase in adenosylhomocysteinase could also imply an increase in ADA, which is an enzyme related to immune function. This would be in line with the recent report on the increase in ADA in saliva in EGGD [28
In the GO analysis of EGGD there was an activation of the pathway of the serine-type endopeptidase activity, which is in line with the increase found in the serine protease TMPRSS11D that is directly related to this GO term.
Saliva from horses diagnosed with ESGD showed a higher number of proteins with significant expression changes than the horses with EGGD. Some of those, such as serpin B5, WDR1, phosphoglycerate kinase 1 (PGK1), and keratins 15 and 4, have as a common feature the regulation of the growth of squamous epithelial cells, which represent the cytological substrate for ESGD [1
Serpin B5 (also called maspin) is an intracellular serine protease inhibitor expressed in squamous epithelial cells, and increases in its expression have been described in ulcerative colitis in humans. This protein is involved in epithelial cell proliferation and resistance to apoptosis [29
]. In addition, serpin B5 expression could be a marker of disease activity, since it was shown to be increased in over 90% of patients with active inflammatory bowel disease, being correlated to the activity of the disease [30
]. This protein has been detected in saliva in humans associated to oral squamous cell carcinoma [31
]. This could indicate that this protein is involved in alterations of the normal function, regulation, and metabolism of squamous cells that can occur in ESGD.
WDR1 is a protein that is involved in epithelial development and membrane epithelial cell junctions [32
PGK1 has been found to increase in epithelial cells in the situation of anoxia and cellular damage and it has even been related to squamous cell neoplasm [33
KRT15 and KRT8 are present in basal keratinocytes of all stratified epithelia, being also involved in epithelial cell proliferation [34
]. In a previous study, other members of the keratin family, keratins 5 and 10, were found increased in the serum of horses with ESGD [3
Besides serpin B5, WDR1, PGK1, and keratins 15 and 4, our results identify additional proteins involved in epithelial regulation that have a higher abundance in ESGD than in EGGD, thus implicating the presence of a complex disturbance in the squamous cell regulation. One of these proteins was arginase, which is involved in the growth of squamous cells. The upregulation of arginase activity has been described in gastric, breast, renal cell, and head and neck squamous cell carcinomas, thus making the inhibition of this enzyme a possible target for the treatment of these neoplasms [35
]. On the other hand, arginase was lower in EGGD. Decreases in arginase activity in gastric ulcers have been reported in mice [36
]. In addition to this, arginase has a regulatory role in gastrointestinal inflammation and low values could be related to an impaired tissue repair since upregulation of arginase increases the level of polyamines, which play a significant role in wound healing [37
Overall, the changes in proteins in ESGD indicated an alteration of the squamous mucosa cells. This alteration has been found to be caused by acid and results in hyperkeratosis, erosions, and ulceration. Many of the risks for development of squamous disease relate to factors that allow or promote a more acidic gastric pH or increase exposure of the squamous mucosa to this acid [21
In our study, the GO terms altered in horses with ESGD showed an activation of the pathways related to epithelium metabolism. Overall, when ESGD and EGGD were compared, the GO analysis in ESGD showed a tendency for activation of the pathways related to epithelium development and activation, while serine-type endopeptidase activity was more related to EGGD, as indicated above.
Serum of horses with ESGD and EGGD, showed a lower number of proteins with significant changes than saliva. Additionally, the GO analysis showed that the changes present in saliva mirror a higher number of upregulated processes. In addition, there was no substantial match between the pattern of proteome changes in the saliva and serum. This discrepancy in the number and types of protein changing between saliva and serum has also been observed in other diseases in different species, such as canine pyometra or mammary tumours [38
], or in cows with mastitis [40
]. Those results would indicate that changes in the composition of both fluids show a different response to the disease and can provide complementary information. These differences could be explained in part because there are analytes present in saliva that are locally produced in the salivary glands [41
In a previous proteomic study analysing the saliva of horses with colic of intestinal aetiology and using the same technology, different proteins were identified compared to the current report [9
]. These proteins were mostly related to a protective effect against inflammation and an impaired immune defence and antimicrobial capacity of the mucosa. This would reflect a different pathophysiological mechanism of this disease in comparison to ESGD or EGGD.
It is important to point out that this should be considered a pilot study. The sine qua non for the large-scale validation of these results should be the development of high-throughput assays for the quantitation of the main proteins that had different abundance levels between the groups. This large-scale validation should include a larger number of horses with ESGD and EGGD, as well as horses with other diseases, in order to determine the clinical sensitivity and specificity of these proteins, and to evaluate their possible use as biomarkers for diagnostic or monitoring purposes. In addition, since the gastroscopy exam of the horses with EGUS required a period of fasting, it would be interesting to investigate the possible effect of fasting in salivary protein variations.