Intestinal immune regulation including development of oral tolerance is of great importance for the maintenance of gut homeostasis . Concerning this, Tregs occupy a pivotal role in cell-mediated immunosuppression [5, 6]. Therefore, the aim of the study was to investigate the expression of Foxp3 in canine GALT of healthy and gastrointestinal diseased dogs.
Animals lacking clinical and pathomorphological signs of gastrointestinal diseases were used as healthy controls. Interestingly, minimal histopathological findings were present in the gastrointestinal tract of these dogs. Due to the absence of clinical and morphological correlates, these findings were interpreted as background variances of the canine gut. Although several reports on age-related changes and immunosenescence in dogs  would suggest an increasing inflammatory background in the canine elderly, no differences between the WSAVA scores of different age groups were observed. In contrast, WSAVA scores were significantly increased in dogs with gastrointestinal diseases compared to controls.
Immunohistochemically, application of anti-Foxp3 antibodies produced clear nuclear staining of cells revealing distinct lymphocyte morphology. Although Foxp3 has been established as a specific marker for classical Tregs further characterised by the expression of CD4 and CD25, heterogeneity of this cell population has been described . In dogs, recent analyses on canine Tregs illustrate CD4-positive cells with intermediate Foxp3 expression and authors postulate that they are activated heterogeneous conventional T cells instead of Tregs . In our study, differences in staining intensity for Foxp3 were noticed immunohistochemically. This suggests differences in Foxp3 expression in situ supporting the concept of canine Treg heterogeneity. However, this result could also be due to the localisation of Tregs in different tissue layers and needs to be further investigated.
Structured lymphoid tissue in the intestine plays a crucial role in mucosal immunology especially in induction and gut homing of intestinal Tregs and therefore for the establishment of oral tolerance [13, 93]. The high density of Foxp3-expressing lymphocytes in those areas suggests similar roles of these anatomical compartments in dogs.
Additionally, Foxp3 was expressed by intraepithelial lymphocytes (IEL). It is well established that canine IEL are mainly characterised by the expression of CD8. However, less than 15 percent of these cells are CD4-positive . Although most canine Tregs are known to express CD4, CD25, and Foxp3, a minor subpopulation of CD8-positive lymphocytes with strong Foxp3 expression is described . In spite of their correct phenotype, our results confirm the immunoregulatory phenotype of canine IEL, which has recently been observed in vitro .
Furthermore, Foxp3-positive lymphocytes were present in the lamina propria. Concerning different compartments (vertical distribution), Tregs were significantly increased in the villus lamina propria compared to basal crypt area and villus-crypt junction. Previous studies about intestinal T cells in dogs report similar differences in their distribution [95–97]. Referring to this, a stronger exposition of the intestinal apical surface to luminal antigens is discussed. Analogously, increasing numbers of Tregs in the villus area may be related to the great importance of immunoregulation in areas characterised by high antigenic load, antigen sampling by dendritic cells, and priming of immune responses.
Additionally, the numbers of Foxp3-positive cells were significantly higher in the basal crypt area than in the villus-crypt junction. This finding was in contrast to previous observations of a constant increase in CD3-positive cells towards the villus tip [95–97]. Possibly, the increase in Tregs in the basal crypt area compared to the villus-crypt junction is due to increased numbers of IgA-secreting plasma cells towards the crypt  requiring a more intense local tuning of immunological tolerance in basal areas.
Interestingly, a vertical decline in Tregs was found in the colonic mucosa since the numbers of Foxp3-positive lymphocytes were significantly lower in the apical crypt area than in the basal part. Potentially, this is related to general differences of colonic Tregs in comparison to those located in the small intestine highlighting the heterogeneity of regulatory T cells. Otherwise, these differences may be due to the individual anatomy and physiology of the small and large intestines. However, further studies are needed to confirm possible differences in the vertical distribution of lymphocytes in the colonic mucosa and to evaluate its relevance.
Concerning the horizontal distribution of Tregs along the small intestinal villus, numbers of Foxp3-positive lymphocytes were significantly decreased from duodenal to jejunal as well as to ileal mucosa. In contrast to this, no horizontal differences in the expression of CD3, CD4, or CD8 have been reported [95, 97]. The horizontal decline in expression of Foxp3 may be explained by decreasing amounts of food antigens progressively destroyed along the small intestine, although the numbers of bacteria progressively increase from the duodenum to the large bowel . Regarding the lower frequencies of Tregs at the luminal site of the colonic mucosa compared to those in the small intestine, it is possible that food proteins are more potent generators of intestinal Tregs than commensal bacteria. In mice, it has recently been shown that T cell receptors of colonic Tregs differ from those used by Tregs in other locations . Therefore, canine colonic Tregs may generally differ from those in other areas including the small intestine.
In healthy dogs, age-related changes in Foxp3 expression were observed since Tregs increased in middle-aged animals compared to juvenile as well as older individuals. Age-dependent alterations of the immune system are well-known in companion animals [91, 100] and several reports on alterations of the canine gastrointestinal tract concerning different age groups exist [97, 101, 102]. For instance, intestinal lamina propria CD3-positive T cells and macrophages decline in older animals, whereas IgA-containing plasma cells increase with age . Although age-related differences in Tregs are controversially discussed  and only age groups with small numbers of animals (5 to 7 dogs per age group) were examined in this study, our data suggest an increase of intestinal Tregs in middle-age dogs that may be an indication for an increase of oral tolerance in these dogs compared to juvenile individuals. Nevertheless, the numbers of Tregs may change in the canine gut during the first year of life since the canine immune system including the GALT undergoes maturation during this period. Additionally, the decline in Foxp3 expression in old animals could be a sign of immunosenescence and could possibly mean a loss of oral tolerance in the canine elderly.
Although age-related changes in Foxp3 expression were of statistical significance in this study, further investigations are needed to confirm these results using higher numbers of animals and to check the course of Tregs in juvenile dogs.
Dysregulation of mucosal immunology including a failure of oral tolerance against commensal bacteria and food proteins seems to play a crucial role in the pathogenesis of both human and canine IBD [36, 104]. As it is already known, depletion of regulatory T cells leads to chronic colitis in immunodeficient mice . Furthermore, intravenous application of Tregs can cure established intestinal inflammation in animal models for experimental colitis [105, 106]. In Long-Evans Cinnamon rats (deficient in thymocyte development) showing spontaneous development of IBD-like colitis, Tregs are significantly reduced in the colonic lamina propria . Interestingly, Foxp3-positive Tregs are present at higher densities in the lamina propria of human patients suffering from IBD than in healthy controls [107–109]. This indicates an impaired function of Tregs or a resistance of T effector cells to Treg-mediated suppression instead of a numerical aberrance in the Treg population. However, investigated dogs suffering form IBD revealed a significant reduction of Foxp3 expression in the duodenal villi in cases of both eosinophilic and lymphoplasmacytic inflammation compared to controls. Further studies using full-thickness intestinal samples are needed to check if similar differences in Tregs are also present in the jejunum and ileum in canine IBD. Interestingly, no differences in Treg numbers were present in the stomach and colon of dogs with IBD in comparison to controls. This may be an indication of different roles of small intestinal Tregs compared to those located in the stomach and the colon for canine IBD. Under physiological conditions, enrichment of Tregs in areas of inflammation is mediated by a negative feedback mechanism via IL-2 produced by T effector cells ensuring a balanced cooperation between protective immune responses and immunosuppression . Therefore, our results may be a sign of a primary defect in Treg homeostasis leading to chronic inflammation due to a lack of immune regulation as a pathogenetic factor in canine IBD. Further studies on Treg function are needed to confirm this possibility.
Unlike dogs with IBD, dogs with intestinal nematode infection revealed increased numbers of Tregs in the duodenal villus compared to controls. Interestingly, no differences in Treg numbers were present in the stomach and colon of dogs with nematode infection in comparison to controls. Therefore, further studies are needed to determine whether this is related to the nematode species and its localisation in the gut or due to general differences in canine Tregs in the small intestine compared to those located in the stomach and colon. Intestinal parasites are known to trigger Th2 dominated immune responses and cause down regulation of immune responses ensuring a better survival in their hosts [42, 111]. Particularly, suspension of Treg activity leads to exacerbated intestinal pathology and enhances parasite clearance [43, 44]. Furthermore, helminth secretions are able to induce Foxp3 expression and enhance the regulatory function of T cells . Therefore, our results suggest a parasite-related increase in intestinal immune regulation, although IL-2 mediated enrichment of Tregs may also be involved. Interestingly, parasites are discussed as potential strategies for the treatment of human IBD due to their ability to prevent intestinal pathology and their immunoregulatory properties [47, 48].
This study elucidates that regulatory T cells are expressed in the canine GALT with a decline of Tregs from the villus to the crypt and age-related changes suggesting a progressive establishment of oral tolerance and immunosenescence including decreased oral tolerance in the canine elderly. A reduced number of Tregs in canine IBD suggests a defect in Treg homeostasis leading to a loss of oral tolerance. In addition, Tregs were enriched in the duodenal villi of dogs with intestinal nematode infection indicating a parasite-induced enhancement of intestinal immune regulation.