Equine asthma is an inflammatory disease of the small airways secondary to hypersensitivity, most commonly due to dust and hay antigens.1 Weight loss of unknown origin can occur in asthmatic horses and could be secondary to small intestinal inflammation as a collateral lesion developing secondary to the systemic inflammation associated with asthma, as suggested recently in both the remission and exacerbation phases.2–6 Also, in human medicine, there is a possible association between inflammatory bowel diseases (IBD) and asthma.7–10 To date, there is no published study in horses that has evaluated the association between the pulmonary inflammation of severe equine asthma and gastrointestinal inflammation.
In human medicine, it has been demonstrated that T cells are important mediators of allergic airway inflammation of asthma11 and are abundant in the gastrointestinal mucosa.12 Data in human medicine also support the hypothesis that the whole common mucosal immune system may be involved as a cause or consequence of various T-lymphocyte–mediated diseases.13 In the intestine, 2 major lymphocyte population localizations exist: the intraepithelial lymphocytes and the lamina propria lymphocytes, which localize to the subepithelial lamina propria and are in contact with intraepithelial lymphocytes. Intraepithelial lymphocytes reside within the intestinal epithelium and consist of a heterogenous mix of natural and induced intraepithelial lymphocytes. The control of the immune response in the gut is critical for normal immune homeostasis in the host, and failure to control such responses has been proposed as 1 mechanism in the development of inflammatory bowel disease.14 A study15 on transmural jejunal mucosal biopsies highlighted a majority of lymphocytes and eosinophils in the lamina propria of healthy adult horses. A more recent study16 on leucocyte quantity within the intestinal segments of healthy horses revealed that lymphocytes were the most numerous cells in all segments analyzed.
Systemic corticosteroids are used to treat equine asthma17 as well as IBD. Also, inhaled corticosteroids are commonly used to treat asthmatic horses, but 70% to 90% of such inhaled drugs do not reach the bronchoalveolar space and thus could potentially go to the lower digestive tract mucosa, such as the small intestine.18–20 Fluticasone has poor oral bioavailability,21 but aerosol particles could potentially have a local action on the proximal intestine. Indeed, Crohn’s disease has already been treated successfully with inhaled corticosteroids.22
The objectives of this study were to characterize and quantify lymphocytes and eosinophils in the duodenal and rectal mucosa in symptomatic asthmatic horses (with and without inhaled fluticasone). Our hypotheses were that (1) there is an elevated infiltration of T lymphocytes in the duodenal and rectal mucosa in asthmatic horses compared to control horses, (2) inhaled corticosteroids can decrease this immune cell population, and (3) there are differences between in the immune cells’ distributions in the duodenal and rectal mucosa in all groups.
Methods
Horses
Horses were owned by the equine respiratory research group of the University of Montreal and were regularly dewormed. None of the horses had received any medication for at least 14 days before the beginning of the experiment. The protocol and procedures used for this study were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Montreal, and the IACUC No. was 07-Rech-1356.
Eight mares (3 Standardbred, 2 Arabian, and 3 mixed breed), 10 to 16 years old and weighing between 450 and 550 kg, were used to evaluate duodenal and rectal histology in healthy horses (control group). Control horses were kept indoors and fed dry hay. Horses were determined to be clinically normal based on physical examination, PCV, total protein concentration, and CBC. Pulmonary function tests and bronchoalveolar cytology were not performed on the control horses. Ten asthmatic horses (7 mares and 3 geldings; 5 mixed-breed horses, 4 quarter horses, and 1 Standardbred), 10 to 20 years old and weighing between 410 and 535 kg, were also studied. These horses were part of a larger scale study on severe equine asthma that was previously reported.23,24 A diagnosis of severe equine asthma was based on history, respiratory signs (including nostril flaring, abdominal breathing effort at rest), maximal change in transpulmonary pressure (ΔPL) > 15 cm H2O, and > 25% neutrophils in bronchoalveolar lavage fluid cytology during exacerbation. At the beginning of the study, asthmatic horses were made symptomatic after being exposed to moldy hay for 6 weeks. Once in exacerbation, asthmatic horses were randomly separated into 2 groups. The first group (4 horses) was treated with environmental change (alfalfa pellets) only, and the second group (6 horses) was treated with inhaled corticosteroids (fluticasone). Environmental changes and inhaled corticosteroids are the 2 main therapies to focus on when treating equine asthma and, consequently, were the 2 treatments we chose to investigate. During the initial phase of this study (first 3 months of experimentations), all horses were maintained indoors in individual stalls and bedded on wood shavings. During the initial phase of the study, the first group was fed alfalfa pellets, was kept indoors, and was not receiving any medication. During the same period, the second group was fed dry hay, kept indoors, and treated with inhaled fluticasone. In the second phase of this study, the horses were maintained outdoors in a grass paddock as a protocol for another study performed at the same time on these horses. The first group was still fed alfalfa pellets and not receiving any medication, and the second group was still fed dry hay and treated with fluticasone on the same protocol as before.
Asthmatic horses with or without inhaled fluticasone
Before the experiment, horses were fed dry hay ad libitum and 1 kg of sweet feed twice a day. On the first day after the 6-week period to get them in exacerbation, asthmatic horses in group 1 were transitioned to alfalfa pellets over 1 week, resulting in a diet of 4 kg of alfalfa pellets and 1 kg of sweet feed fed twice daily for 6 months. Asthmatic horses in group 2 were fed dry hay ad libitum and 1 kg of sweet feed twice daily. Group 2 horses were treated with inhaled fluticasone with the Aeromask spacer device with an initial dose of 2 mg every 12 hours for a month; dosing was adjusted as needed to keep horses asymptomatic (2 to 3 mg every 12 to 24 hours) for 6 months.23,24 CBC, PCV, total protein, albumin concentration, and glucose absorption tests, as well as endoscopic duodenal and rectal biopsies, were performed on day 0 and again when horses were asymptomatic at 3 months. One asthmatic horse, not present at the beginning of the study, was included in the group treated with fluticasone at 3 months. Another horse was excluded from the study at 7 months because of significant deterioration of his pulmonary clinical signs that rendered it unethical to continue the experimentations on him.
Oral glucose absorption test
The oral glucose absorption test is a test used to detect glucose malabsorption in horses by measuring blood glucose concentrations at different times after an initial bolus of a dextrose solution given via nasogastric intubation. We performed this test because horses with IBD may have altered intestinal absorption of nutrients. The oral glucose absorption test was performed in all horses at the same frequency as the endoscopic digestive biopsies protocol, using a standard protocol.25,26 In brief, horses were held off feed overnight (14 to 16 hours), and 1 g/kg of glucose (anhydrous glucose diluted as a 20% solution) was administered through a nasogastric tube. Blood samples were collected from direct venipunctures in heparinized tubes at 0, 30, 60, 90, 120, 150, 180, and 240 minutes after administration, and plasma glucose concentrations were measured (UniCel DxC 600; Beckman Coulter). A glucose concentration at 90 to 120 minutes showing a greater than 85% increase over the resting concentration was considered as normal absorption, a concentration with an increase of less than 85% but greater than 15% over the resting concentration was considered as suspected partial malabsorption, and an increase in glucose concentration of less than 15% was considered as suspected total malabsorption.27
Gastroduodenal endoscopies and duodenal biopsies
Following the completion of the glucose test, horses were restrained in stocks and sedated with 0.4 mg/kg of intravenous xylazine and 0.02 mg/kg of intravenous butorphanol. A 3-meter endoscope (Olympus) was passed into the stomach and the duodenum.28 The macroscopic appearance of all parts of the glandular stomach and the proximal duodenal mucosa was evaluated. When the endoscope was in the duodenum, a biopsy forceps with fenestrated cups was passed into the operating canal of the endoscope until the forceps were extended past the tip of the endoscope; then, they were opened and advanced until the open cups encountered the area to be biopsied. The biopsy areas were decided between the duodenal ampulla and papilla. After adequate pressure was applied to the tissue, the jaws were firmly closed, and the forceps were then retracted. Biopsies were placed in 10% neutral-buffered formalin for histopathologic interpretation. Four to 6 adequate-size biopsies were taken from different areas of the duodenum. For the second (t = 3 months) and third (t = 7 months) endoscopic biopsy sequences, taken from asthmatic horses only, no macroscopic evidence of a scar or sequela from the previous biopsy was observed.
Rectal biopsies
Rectal endoscope-guided biopsies were taken following duodenal biopsies in standing horses restrained in stocks and still under adequate sedation. We used this rectal biopsy procedure to compare the results with those collected by the duodenal biopsy, with approximately the same sample size. The endoscope was inserted into the rectum until the rectal fold, about 15 to 20 cm proximal to the anal sphincter. The rectal fold was gently grasped, biopsy forceps were passed in the operating channel, and the biopsy technique was the same as for the duodenal biopsy procedure previously described. Biopsies were placed in 10% neutral-buffered formalin for histopathologic interpretation. Three to five biopsies were taken from different areas of the rectum.
Histopathology
All samples were kept in formalin for 24 hours, embedded in paraffin, and 5-μm-thick sections were stained with hematoxylin, eosin, phloxine, and saffron. Biopsies were assessed in a blind fashion by a board-certified anatomic pathologist (PH) at the end of the experiment. Morphology (villous stunting, epithelial injury, crypt distension, lacteal dilatation, and mucosal fibrosis) and inflammation (lymphocytes, eosinophils, plasma cells, macrophages, and neutrophils in epithelium and/or in lamina propria) were interpreted according to the criteria of the World Small Animal Veterinary Association (WSAVA) gastrointestinal standardization group for dogs and cats.29,30 The population of inflammatory cells in duodenal biopsies was graded according to modified WSAVA criteria as normal (grade 0), minimally increased (grade 1), mildly increased (grade 2), moderately increased (grade 3), or markedly increased (grade 4). A grade (minimal) was added because a preliminary work31 showed variable numbers of lymphocytes, plasma cells, and/or eosinophils (slight to moderate infiltration) in the duodenal biopsies of normal horses, and an intermediate grade permitted a refined interpretation. The immune cell population of rectal biopsies was graded according to the criteria of WSAVA’s standard as normal (grade 0), mildly increased (grade 1), moderately increased (grade 2), or markedly increased (grade 3).29,30 For both duodenal and rectal sequence biopsies, the samples of 1 horse/1 time/1 site were evaluated, and the most severe infiltration score was used for analysis.
Immunohistochemistry and histomorphometry
Immunohistochemistry was performed on all biopsies to quantify T lymphocytes (CD3) and B lymphocytes (CD20). Biopsy quality was evaluated for each horse/1 time/1 site sequence, and the best one was used for histomorphometry analysis. The immunohistochemistry using antisera specific for CD3 (T lymphocytes) and CD20 (B lymphocytes) was performed on formalin-fixed tissues. A murine monoclonal mouse anti-CD3 antibody (Dako; clone F7.2.38) and a polyclonal rabbit antibody anti-CD20 (Fisher; PA5-16701) were used (1:200 and 1:400, respectively). The antibodies were validated in the pathology service of Montreal University on several tissues and different species, including horses. The secondary antibody biotinylated horse anti-mouse/rabbit (BA-1400; Vector 38 Laboratories) was used at the same concentration as the primary antibody (1:200 or 1:400). Antigen retrieval for sections labeled with CD3 antibody (T-cell phenotype) was accomplished by microwave heating for 2 5-min sessions in boiling citrate buffer (pH 6.0), followed by standing in the hot solution for 20 min. Sections to be labeled with CD20 antibody (for B-cell phenotype) were treated with a pH 9.0 buffer for antigen retrieval. Endogenous peroxidase activity was blocked by incubation with a 70% hydrogen peroxide solution. The slides were loaded into an automated immunohistochemistry machine and incubated with a primary monoclonal antibody to CD3 (1:200) or a polyclonal antibody to CD20. An indirect method was used, with a secondary antibody conjugated to horseradish peroxidase. The immunoreaction was developed with 3,3'-diaminobenzidine substrates, and sections were counterstained with Mayer’s hematoxylin (Fisher Scientific).
Computer-aided morphometric analysis and cell counts were performed on immunohistochemically stained slides for all digestive tissues. In brief, all sections were blinded and coded, then analyzed using a light microscope and video camera. All digestive tissue biopsies at a magnification of X400 were digitally imaged with the Aperio ImageScope program. Digitized images were transferred to an IBM-compatible computer with a Leica DM4000B microscope attached to an Allied Vision Prosilica GT1920C camera and the Panoptiq software. Calibration was performed using a micrometer slide to permit accurate measurements. Immunostained biopsies were scored by 2 observers (CL, DJ) using a modified WSAVA scoring system, and a final score was attributed based on consensus between the 2. For each set of biopsies (1 marker for 1 horse at 1 moment of the study), only the best-quality biopsy was chosen for the counting. The criteria considered for evaluating biopsy quality were the orientation of the tissue, the preservation of cellular structures, the number of villosities, and the integrity of the crypts. Immunopositive cell counts were performed by 1 blinded observer (CL) using a computer image analysis system (Aperio ImageScope). Eight biopsies were randomly chosen, and 3 counts were performed on each biopsy. The intraclass correlation coefficient was calculated in rectal biopsies to assess the variability in the immunologically stained cell counts. The intraclass correlation coefficient was 0.98 with F = 180 and P = 1.3e-12.
All well-oriented lamina propria villi and crypts in longitudinally oriented sections were separately delineated on the computer monitor (excluding the epithelium, blood vessels, and lymphatics). In the duodenum, the lamina propria was divided into lamina propria of villi and deep lamina propria (Figure 1), positive-stained cells were counted within each area (villi and under-crypts), and the results were expressed as the total number per 10,000 µm2. The total lamina propria was the sum of the lamina propria of villi plus the deep lamina propria. Also, the positive-stained cells in the lamina propria of villi in a transversal section were separately counted. Where solitary lymphoid follicles were observed in the deep lamina propria, their areas were measured after manual outlining. Sections containing large aggregates of lymphoid follicles in the lamina propria and submucosa (Peyer’s patches) were excluded from the analysis. For rectal mucosa lamina propria, the region inside the crypts was excluded and the lamina propria was divided into regions of approximatively equal area: apical and basal areas. Positive-stained cell count data were exported from Aperio ImageScope to a statistical analysis program. For the intraepithelial lymphocyte population, the immunopositive cells, having the morphology of an intraepithelial lymphocyte, in the villous area were counted and localized (basal, nuclear, and apical areas), and the numbers were expressed as total intraepithelial lymphocytes per 100 enterocytes.
The different regions of duodenum (A) and rectum (B) mucosa histomorphometric analysis with a CD3 immunostaining, X40.
Citation: American Journal of Veterinary Research 84, 10; 10.2460/ajvr.23.04.0083
Statistical analysis
A t test with unequal variances was used to compare age between healthy horses and asthmatic horses. Peak concentrations of oral glucose absorption tests were assessed with the increased percentage of glucose over the resting time. The total protein, albumin concentration, peak concentration, peak time, and AUC of the oral glucose absorption test were compared with a repeated measures linear model with the groups of asthmatic horses (pellet/fluticasone) as a within-subject factor and illness (symptomatic/asymptomatic) as a between-subjects factor in asthmatic horses, and a priori contrasts were done using the sequential Bonferroni procedure. Histological duodenal and rectal biopsy scores were compared with the Cochran-Mantel-Haenszel test. The peak concentration of the oral glucose absorption test and the biopsy score were compared with Spearman’s nonparametric correlation.
An intraclass correlation for T- and B-cell lymphocyte counts in duodenal and rectal biopsies was used to determine the intraobserver agreement for the immunohistochemistry analysis. For the duodenal and rectal biopsies, the differences between the mean concentrations of CD3 and CD20 in the lamina propria and the epithelium as well as the ratio of the concentrations counts between healthy horses, symptomatic asthmatic horses, and treated horses (fluticasone and alfalfa pellets) were performed with a mixed linear design. The group was considered the fixed effect, and the individual was considered the random effect. When the statistical model indicated a statistically significant difference, which was set at P < .05, a Tukey’s post hoc test or a Benjamini-Hochberg procedure with reduced alpha level was applied. A paired t test was used for the comparisons between duodenal and rectal biopsies for lymphocyte counts for each group at each time. All the statistical analyses were performed using the software R (R Foundation for Statistical Computing). The packages used were lmerTest (A Kuznetsova, PB Brockhoff, RHB Christensen, 2017).
Results
Mean ages were significantly higher in asthmatic horses (17 years old, 10 to 20 years) compared with healthy horses (13 years, 10 to 16 years) (P = .05). CBC and total protein values, including albumin and globulin concentrations, were within the normal range for all horses and were not different between groups.
Oral glucose absorption test
Based on the results of the oral glucose absorption test showing an increase greater than 85% over the resting concentration, all horses had normal small intestinal absorption. However, some horses (50% of asthmatic horses) presented a delayed peak time at 150 minutes, without a significant difference between the peak time at 120 minutes or at 150 minutes. There were no differences within or between groups in peak concentration, peak time, and AUC between healthy horses and asthmatic horses nor with fluticasone treatment in asthmatic horses. The oral glucose absorption tests were not performed at 7 months.
Endoscopic digestive tract biopsies
Biopsies’ quality—No complications were detected following biopsy sample collection. The mucosa and portions of the submucosa were present in all samples. In the duodenum and rectum, of 38 sequence biopsies, 37 were considered adequate, and 1 was considered too superficial. Macroscopically, no differences in the aspect of duodenal mucosa or scars from endoscopy were observed a few weeks after the first and the second biopsy procedures. No specific abnormalities of duodenal or rectal morphology were noted in any biopsies.
Histological digestive biopsy interpretation (HE staining)—Immune cell infiltration and duodenal and rectal biopsy scores are reported in healthy and asthmatic horses (Table 1). Most biopsies were scored from grade 0 to 2. Despite some individual variation over time, there was no significant difference in duodenal histologic scores on HE staining for lymphocytes and eosinophils between control and asthmatic horses at different times. There was no difference between asthmatic horses treated with fluticasone or environmental change at any time. Thirty-six rectal biopsies were scored as grade 0, and 2 biopsies were scored as grade 1 with mild eosinophilic and lymphoplasmacytic infiltration, respectively. No significant difference was found in asthmatic horses’ rectal histology or between treatment groups (pellet/fluticasone).
HE evaluation of duodenal and rectal biopsies.
| Duodenal biopsies | Rectal biopsies | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Fluticasone | Pellets | Fluticasone | Pellets | |||||||||
| Type/score | T0 | T3M | T7M | T0 | T3M | T7M | T0 | T3M | T7M | T0 | T3M | T7M |
| Type of inflammatory cell infiltration | ||||||||||||
| Normal | 2/6 | 1/6 | 1/6 | 2/4 | 0/4 | 1/3 | 5/6 | 6/6 | 6/6 | 4/4 | 4/4 | 3/4 |
| Lymphocytes/plasma cells | 2/6 | 2/6 | 3/6 | 1/4 | 2/4 | 0/3 | 0/6 | 0/6 | 0/6 | 0/4 | 0/4 | 1/4 |
| Eosinophils | 2/6 | 0/6 | 1/6 | 0/4 | 0/4 | 0/3 | 1/6 | 0/6 | 0/6 | 0/4 | 0/4 | 0/4 |
| Lymphocytes/plasma cells/eosinophils | 0/6 | 3/6 | 1/6 | 1/4 | 2/4 | 2/3 | 0/6 | 0/6 | 0/6 | 0/4 | 0/4 | 0/4 |
| Biopsy score | ||||||||||||
| 0 | 2/6 | 1/6 | 0/6 | 2/4 | 0/4 | 1/3 | 5/6 | 6/6 | 6/6 | 4/4 | 4/4 | 3/4 |
| 1 | 4/6 | 5/6 | 5/6 | 2/4 | 3/4 | 1/3 | 1/6 | 0/6 | 0/6 | 0/4 | 0/4 | 1/4 |
| 2 | 0/6 | 0/6 | 0/6 | 0/4 | 1/4 | 1/3 | 0/6 | 0/6 | 0/6 | 0/4 | 0/4 | 0/4 |
Immunohistochemistry and histomorphometry—The duodenal and rectal epithelium of asthmatic and control horses contained exclusively T lymphocytes (CD3). Symptomatic asthmatic horses, compared to controls, had a significantly higher number of T lymphocytes (CD3) in the duodenal epithelium (P = .02; Figure 2) and the adjacent lamina propria of villi (P = .04; Figure 3) but not in the deep lamina propria.
T lymphocytes (CD3) in duodenum epithelium in control and asthmatic horses. EC = Enterocytes. Env = Environment. Fluti = Fluticasone. T0 = Time 0. T3M = Time 3 months. T7M = Time 7 months.
Citation: American Journal of Veterinary Research 84, 10; 10.2460/ajvr.23.04.0083
T lymphocytes (CD3) in lamina propria of villi (A) and deep lamina propria (B) of duodenum in control and asthmatic horses. DLP = Deep lamina propria. VLP = Villi lamina propria.
Citation: American Journal of Veterinary Research 84, 10; 10.2460/ajvr.23.04.0083
Symptomatic asthmatic horses had a significantly higher number of T lymphocytes (CD3) in the rectal apical and total lamina propria compared to horses treated with fluticasone at 3 months (P < .01 for both; Figure 4) and 7 months (P < .01 for both) and horses treated with (exposed to) environmental change at 7 months (P < .01 for both). Also, symptomatic asthmatic horses had a significantly higher number of T lymphocytes (CD3) in the rectal basal lamina propria compared to horses treated with fluticasone at 7 months (P < .01) and those treated with environmental change at 7 months (P < .01). In addition, the concentration of T lymphocytes in the rectal total lamina propria was significantly higher in normal horses compared to asthmatic horses exposed to environmental changes (diet) at 7 months (P < .01). The rectal lamina propria contained B lymphocytes in asthmatic and control horses without significant differences. The total concentration of lymphocytes (B and T lymphocytes) in the rectal lamina propria was significantly lower in asthmatic horses exposed to environmental changes (diet) compared to symptomatic asthmatic horses (P < .01; Supplementary Table S1).
T lymphocytes (CD3) in apical rectal lamina propria (A), total rectal lamina propria (B), and basal rectal lamina propria (C). ALP = Apical lamina propria. BLP = Basal lamina propria. TLP = total lamina propria.
Citation: American Journal of Veterinary Research 84, 10; 10.2460/ajvr.23.04.0083
Comparison between duodenal and rectal biopsy data
There was no correlation between duodenal and rectal histology (P = 1.0) in all groups.
However, there were intragroup variations of T and B lymphocytes in the duodenal and rectal mucosal in both groups. When groups were compared for lymphocyte counts in duodenal versus rectal biopsies, no significant difference was found for the T and B lymphocytes in the lamina propria. The T-lymphoepithelial count was significantly different between duodenal and rectal biopsies in control and asymptomatic asthmatic horses as well as symptomatic asthmatic horses treated with fluticasone at 3 months.
Discussion
The present study was conducted to evaluate the presence of intestinal mucosa immune cell infiltrates in horses affected with inflammatory disease (severe equine asthma) and the influence of inhaled corticosteroid therapy and environmental modification on immune cell infiltration. The biopsy procedures were well tolerated by horses, and no macroscopic changes of the duodenal and rectal mucosa were observed during all the sequences of endoscopic digestive biopsies. Our results suggest an increased infiltration of T lymphocytes in the duodenal epithelium and lamina propria of villi in symptomatic asthmatic horses compared to control horses. Moreover, the T-lymphocyte infiltration of the rectal lamina propria in asthmatic horses, although not significantly different from normal horses, is reduced after treatment with fluticasone for 3 and 7 months or environmental changes (diet) for 7 months. These results suggest a certain degree of duodenal and rectal infiltration and reinforce the hypothesis of a multisystemic, T-cell–mediated inflammatory process involved in severe equine asthma and enhance a possible link between equine asthma and digestive immune infiltration in horses.
The duodenal and rectal epithelium of asthmatic and control horses contained exclusively T lymphocytes (CD3). Symptomatic asthmatic horses, compared to controls, had a significantly higher number of intraepithelial T lymphocytes (CD3) (P = .02) within the duodenal mucosa demonstrating that T lymphocytes are the predominant lymphocytes in this first digestive barrier. There are no data in the literature on the normal reference range for lymphocyte count in the duodenal epithelium of healthy horses, but in human medicine, the normal limit is under 35 lymphocytes/100 epithelial cells.32 Symptomatic horses with severe equine asthma in our study had an average T-lymphocyte count of 41.7/100 epithelial cells, with 6/8 horses having an average T-lymphocyte count of over 35 lymphocytes/100 epithelial cells. Control horses had an average T-lymphocyte count of only 19.7/100 epithelial cells. Even though rectal intraepithelial T-lymphocyte counts were not significantly different, the count was higher in symptomatic asthmatic horses and could also suggest a certain degree of infiltration. The intestine is a major site for exposure to potential pathogens, and the recognition of foreign antigens allows the intestinal mucosa to rapidly generate a robust adaptive immune response. Therefore, after contact between hay and the digestive mucosa, induced intraepithelial lymphocytes could be called to the mucosal epithelium, explaining this increased number of intraepithelial T lymphocytes in the duodenum mucosa. In humans, intraepithelial lymphocytes are almost exclusively T lymphocytes and can be divided into conventional and unconventional, with both playing a role in defense against infections.33 T lymphocytes can protect the host by clearing infected cells through the production of cytokines that strengthen the barrier function or recruitment of other immune-protective and immune-regulatory cells. In horses, the exact role of epithelial intestinal T lymphocytes is unknown.
In the same way, symptomatic asthmatic horses have a significantly higher count of T lymphocytes in the lamina propria of villi adjacent to the epithelium (P = .04), but not in the deep lamina propria (adjacent to crypts), when compared to control horses. These data could be explained by a migration of lymphocytes toward the villi and the epithelium, suggesting a potential immune response to digestive allergens and/or a more systemic inflammatory response. There is no reference for the normal count of both B and T lymphocytes in the duodenal and rectal lamina propria in horses, but 1 study by Rocchigiani et al16 evaluated the leukocyte numbers in the digestive mucosa of horses with no clinical intestinal disease. In the current study, lymphocyte counts in the lamina propria of villi of control horses were lower than those reported in normal young thoroughbreds (12.8 ± 6.2 lymphocytes/10,000 µm2 vs 33 ± 13 lymphocytes/20 000 µm2, respectively) and with a majority of T lymphocytes (8.7 ± 5.0 T lymphocytes/10 000 µm2). These differences could be explained by several factors, including the different staining used (IHC in our study and HE evaluation in the study of Rocchigiani et al) and the clinical context of both groups. Our control group horses were housed in the same barn environment and fed hay and sweet feed for several weeks, whereas in the study of Rocchigiani et al, the daily environmental context was not documented. Besides, the majority of lymphocytes in both control and symptomatic asthmatic horses were T lymphocytes, reinforcing the idea that T lymphocytes are the predominant cell type in villous lamina propria compared to B lymphocytes.
In the present study including control and asthmatic horses (symptomatic and asymptomatic status) without clinical malabsorption documented (normal clinical examination, protein concentration, and glucose absorption test), no significant abnormality of duodenal or rectal morphology was identified. Histological evaluation of duodenal biopsies showed normal evaluation to moderate infiltration with lymphocyte-plasma cells, eosinophils, and mixed lymphocyte-plasma cells and eosinophils, but contrary to what we expected, the disease and the treatment had no influence on the duodenal biopsy histology. Therefore, the semiquantitative method used for histological evaluation may have limited sensitivity compared to IHC counting, although this approach was found to be adequate in small animals (WSAVA).29,30 Immune cell infiltration between horses (interhorse variability) receiving the same treatment varied from normal evaluation to mild infiltrates. This interhorse variability was also observed in a previous study.31 Rectal biopsies of appropriate quality and size for histological evaluation were obtained. The majority were classified as normal, with only 1 biopsy scored as grade 1 with eosinophilic infiltration. The low variability of our results contrasts with previous reports30,34,35 evaluating rectal biopsies, in which normal horses could have mild to moderate infiltrates of lymphocytes, plasma cells, and eosinophils. The comparison of immunohistochemistry data with standard histologic interpretation outlines that the first technique is probably more sensitive than semiquantitative evaluation and allowed us to characterize the immune cell infiltration of asthmatic horses’ digestive biopsies more accurately in this study.
Asthmatic horses treated with fluticasone (at 3 and 7 months) and exposed to environmental changes (diet) (at 7 months) had significant decreases in T lymphocytes in the rectal lamina propria compared to symptomatic asthmatic horses. A first explanation of these results could be that treatment, either fluticasone or environmental change, has a direct impact on the lymphocyte infiltration of the mucosa, as described in humans.36 In human medicine, corticosteroid administration has been shown to induce transient lymphopenia,37 and T-cell lymphopenia is more pronounced than B-cell lymphopenia.38 Also, corticosteroids are used as a short-term treatment for Crohn’s disease because they reduce inflammation quickly,39 so the reduction in lymphocytes’ infiltration of the rectal mucosa may be a consequence of the systemic anti-inflammatory effect of fluticasone on systemic and digestive inflammation. These data could open the possibility of the use of inhaled corticosteroids in horses with significantly increased infiltration of immune cells when a systemic corticosteroid is contraindicated.
Some limitations of the present study are the lack of investigations in other digestive segments and the nature of the digestive endoscopic biopsies. These biopsies are smaller and less representative of the complete digestive tract compared to the full-thickness digestive biopsies used in other studies.15 The experimental design of several evaluations over time limited the possibility to use more invasive technics to obtain digestive tissues.
Other limitations are the limited number of cases, in the treatment groups of asthmatic horses particularly, and the representativity of the control group. We found a lack of homogeneity in the data for the control group, and besides, the total lymphocyte count of our control group was lower than the lymphocyte count in the study of Rocchigiani et al,16 which points out 1 limitation of our study.
Finally, in this study, we restrained our analysis of immune cells to T and B lymphocytes and eosinophils. It could have been of great interest to also perform counts on immune cells such as neutrophils, plasma cells, and macrophages, but we elected to study lymphocytes and eosinophils because they are in the majority in the digestive mucosa of horses,16 and these cells are most frequently observed in inflammatory bowel disease in horses.
Peripheral blood leucocyte activation, increased concentration of circulatory inflammatory mediators during disease exacerbation, and increased markers of inflammation in both the remission and exacerbation phases have been observed in asthmatic horses, suggesting that asthma might be a systemic inflammatory disease. Equine asthma shares many pathophysiological features with human asthma, which is considered a systemic disease, and systemic inflammation in these patients is thought to contribute to comorbidity. In this study, we found an increased infiltration of T lymphocytes in the duodenal and rectal lamina propria mucosa, indicating a certain degree of intestinal inflammation. This finding highlights a possible link between the 2 conditions and thus paves the way to further investigations.
In conclusion, these results suggest that symptomatic asthmatic horses have a greater degree of T-lymphocyte infiltration in the duodenal and rectal mucosa than control horses, which could be due to a systemic inflammatory effect and/or a local effect of ingested hay allergens in asthmatic horses. Treatment with fluticasone or environmental change seems to reduce the T-lymphocyte infiltration of the rectal mucosa and probably in the duodenum mucosa and opens the possibility of using inhaled corticosteroids in horses with a significant digestive lymphoplasmacytic infiltration. Studies with a larger number of cases are needed to better determine the real effect of corticosteroids on digestive mucosa immune cell infiltration.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org
Acknowledgments
The authors thank Jean-Pierre Lavoie for his contribution to the experimental protocol and Guy Beauchamp for assistance with the statistical analysis.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the generation of this manuscript.
Funding
The authors thank Pfizer Animal Health Foundation, Equine Health Fund, and Centenary Fund of Faculty of Veterinary Medicine of University of Montreal for funding.
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