Inflammatory Bowel Disease Dogs Research Articles

Decreased Immunoglobulin A Concentrations in Feces, Duodenum, and Peripheral Blood Mononuclear Cells of Dogs with Inflammatory Bowel Disease

Although immunoglobulin A (IgA) plays a key role in regulating gut homeostasis, its role in canine inflammatory bowel disease (IBD) is unknown. IgA expression may be altered in dogs with IBD, unlike that observed in healthy dogs and dogs with other gastrointestinal diseases. Thirty-seven dogs with IBD, 10 dogs with intestinal lymphoma, and 20 healthy dogs. Prospective study. IgA and IgG concentrations in serum, feces, and duodenal samples were measured by ELISA. IgA(+) cells in duodenal lamina propria and IgA(+) CD21(+) peripheral blood mononuclear cells (PBMCs) were examined by immunohistochemistry and flow cytometry, respectively. Duodenal expression of the IgA-inducing cytokine transforming growth factor β (TGF-β), B cell activating factor (BAFF), and a proliferation-inducing ligand (APRIL) was quantified by real-time RT-PCR. Compared to healthy dogs, dogs with IBD had significantly decreased concentrations of IgA in fecal and duodenal samples. The number of IgA(+) CD21(+) PBMCs and IgA(+) cells in duodenal lamina propria was significantly lower in dogs with IBD than in healthy dogs or dogs with intestinal lymphoma. Duodenal BAFF and APRIL mRNA expression was significantly higher in IBD dogs than in the healthy controls. Duodenal TGF-β mRNA expression was significantly lower in dogs with IBD than in healthy dogs and dogs with intestinal lymphoma. IBD dogs have decreased IgA concentrations in feces and duodenum and fewer IgA(+) PBMCs, which might contribute to development of chronic enteritis in dogs with IBD.

Expression of trefoil factor genes in the duodenum and colon of dogs with inflammatory bowel disease and healthy dogs

Trefoil factors (TFF) are small peptides produced by goblet cells, which are crucial for epithelial restitution. In humans with inflammatory bowel disease (IBD), TFF expression is up-regulated as part of an unspecific repair mechanism. The goal of this study was to assess TFF gene expression in the gastrointestinal tract from dogs with IBD compared to healthy controls. Preliminary assessment by PCR revealed TFF1 and 3 expression in the small and large intestine, whereas TFF2 was amplified only in the stomach. Subsequent RT-qPCR (with relative quantification against 3 reference genes) on endoscopic duodenal (IBD n=22, healthy controls n=18) and colonic (IBD n=12, controls n=11) biopsies revealed that TFF1 expression was significantly up-regulated in the duodenum from IBD dogs (Mann–Whitney p=0.001), whereas TFF3 expression was significantly lower in IBD colon compared to controls (t-test p=0.018).This study demonstrates evidence for dysregulation of TFF gene expression in canine IBD. Up-regulation of TFF1 could signify ectopic expression as a compensatory repair-mechanism, whereas down-regulation of TFF3 could contribute to defective epithelial barrier function, respectively. Whether this is a cause or consequence of IBD could not be established.

Quantification of chemokine and chemokine receptor gene expression in duodenal mucosa of dogs with inflammatory bowel disease

Although chemokines and their receptors play an integral role in the regulation of the immune response, there is very little information about their involvement in canine inflammatory bowel disease (IBD). The objective of this study was to evaluate the mRNA expression of 9 selected chemokines and 6 chemokine receptors by real-time reverse transcription PCR in the duodenal mucosa from 21 dogs with IBD and 25 control dogs. The transcription levels of monocyte chemotactic protein-1 (MCP-1)/CCL2, macrophage inflammatory protein-3 alpha (MIP-3α)/CCL20, thymus-expressed chemokine (TECK)/CCL25, mucosae-associated epithelial chemokine (MEC)/CCL28 and IL-8/CXCL8 mRNA in IBD dogs were significantly higher than the corresponding levels in control dogs, but there was no significant difference in the mRNA levels of the chemokine receptors between the 2 groups. In addition, the CCL2 and CXCL8 mRNA levels were significantly higher in the high clinical severity score group than in the low clinical severity score group. However, there was no correlation between chemokine or chemokine receptor mRNA expressions and histopathological severity score. The present results suggest that several chemokines may play important roles in the pathogenesis of canine IBD.

Expression of Toll-like receptor 2 in duodenal biopsies from dogs with inflammatory bowel disease is associated with severity of disease

There is growing evidence that aberrant innate immune responses towards the bacterial flora of the gut play a role in the pathogenesis of canine inflammatory bowel disease (IBD). Toll-like receptors (TLR) play an important role as primary sensors of invading pathogens and have gained significant attention in human IBD as differential expression and polymorphisms of certain TLR have been shown to occur in ulcerative colitis (UC) and Crohn's disease (CD).The aim of the current study was to evaluate the expression of two TLR important for recognition of commensals in the gut. TLR2 and TLR4 mRNA expression in duodenal biopsies from dogs with IBD was measured and correlated with clinical and histological disease severity. Endoscopic duodenal biopsies from 20 clinical cases and 7 healthy control dogs were used to extract mRNA. TLR2 and TLR4 mRNA expression was assessed using quantitative real-time PCR. TLR2 mRNA expression was significantly increased in the IBD dogs compared to controls, whereas TLR4 mRNA expression was similar in IBD and control cases. In addition, TLR2 mRNA expression was mildly correlated with clinical severity of disease, however, there was no correlation between TLR2 expression and histological severity of disease.

Molecular analysis of the bacterial microbiota in duodenal biopsies from dogs with idiopathic inflammatory bowel disease

An association between mucosa-adherent commensal bacteria and inflammatory bowel disease (IBD) has been proposed for humans. There are no reports characterizing the mucosa-adherent duodenal microbiota in dogs with idiopathic IBD using molecular methods. The aim of this study was to investigate differences in the mucosa-adherent duodenal microbiota between dogs with idiopathic IBD and healthy dogs. Duodenal biopsy samples were collected from seven dogs with IBD and seven healthy control dogs. DNA was extracted, 16S ribosomal RNA genes were amplified and 16S rRNA gene clone libraries were constructed and compared between groups. A total of 1035 clones were selected, and based on a 98% similarity criterion, 133 unique phylotypes were identified across all dogs. These phylotypes belonged to seven bacterial phyla: Proteobacteria (52.9%), Firmicutes (26.1%), Bacteroidetes (7.7%), Actinobacteria (8.6%), Fusobacteria (4.4%), Tenericutes (0.2%) and Verrucomicrobia (0.1%). Significant differences were identified in the relative abundance of several bacterial groups between dogs with IBD and healthy dogs (p<0.001). Healthy dogs and dogs with IBD clustered according to their disease status. Dogs with IBD had a significantly higher abundance of clones belonging to Alpha-, Beta-, and Gamma-proteobacteria (p<0.0001 for all classes), and a significantly lower abundance of Clostridia (p<0.0001). Bacteria of the genera Pseudomonas, Acinetobacter, Conchiformibious, Achromobacter, Brucella, and Brevundimonas, were significantly more abundant in dogs with IBD. In conclusion, significant differences of the mucosa-adherent duodenal microbiota were observed between dogs with idiopathic IBD and healthy dogs in this study. These results warrant further investigations into the role of the intestinal microbiota in the pathophysiology of canine IBD.

Activation of nuclear factor-κB in dogs with chronic enteropathies

Homeostasis in the intestinal microenvironment between the immune system and luminal antigens appears disturbed in chronic enteropathies. Pro-inflammatory cytokines likely play a role in the pathogenesis of intestinal inflammation. Several inflammatory and immunoregulatory genes have associated nuclear factor-κB (NF-κB) binding sites, which allow NF-κB to regulate gene transcription. The purpose of this study was to investigate (1) the occurrence of NF-κB activation during mucosal inflammation in situ, (2) the mucosal distribution pattern of cells expressing activated NF-κB within treatment groups, and (3) the effect of specific therapy on NF-κB activation. Dogs with chronic enteropathy were studied ( n = 26) and compared with 13 healthy dogs. Ten dogs had food responsive disease (FRD) and 16 had inflammatory bowel disease (IBD). NF-κB activation was detected in duodenal mucosal biopsies using a mouse monoclonal antibody (MAB 3026) that selectively binds the nuclear localization sequence of activated NF-κB. To identify macrophages, biopsies were stained using the MAC 387 antibody. Macrophages in the lamina propria double-stained for MAC 387 and NF-κB were quantitated; epithelial cell expression of activated NF-κB was determined semi-quantitatively. Results showed that more macrophages positive for activated NF-κB were present in lamina propria of dogs with chronic enteropathy compared to control dogs ( p < 0.01). More NF-κB positive epithelial cells were observed in FRD dogs compared to IBD dogs ( p < 0.05). After therapy, the number of macrophages and epithelial cells staining positive for activated NF-κB decreased ( p < 0.01) in chronic enteropathy dogs. In conclusion, activation of NF-κB is closely associated with the pathophysiology of canine chronic enteropathy. Down-regulation follows successful therapy.

Evaluation of lymphocyte apoptosis in dogs with inflammatory bowel disease

To determine whether lymphocyte apoptosis in intestinal mucosae is more common in healthy dogs than dogs with inflammatory bowel disease (IBD) and whether numbers of apoptotic cells increase after successful treatment of affected dogs. 8 dogs with IBD (IBD dogs) and 8 healthy control dogs. Biopsy specimens of the duodenum and colon were obtained via endoscopy from dogs with IBD before and after 10 weeks of standard treatment and compared with specimens obtained from control dogs. Expression of activated caspase 3 (Casp3), caspase-cleaved fragment p85 from poly-ADP-ribose polymerase (PARP), and B-cell leukemia/lymphoma 2 (Bcl-2) was measured in the duodenal (villous tip and base) and colonic mucosae. Expression of Casp3 was greater in the duodenal villous tips of control dogs, compared with expression in similar tissues from dogs with IBD before or after treatment. Despite clinical improvement of dogs with IBD, expression of Casp3 did not increase after treatment. Expression of PARP did not differ between groups at any time point. Expression of Bcl-2 was greater at all 3 tissue sites in control dogs, compared with expression at the same sites in dogs with IBD. Furthermore, Bcl-2 expression in duodenal villous tips was higher in dogs with IBD after treatment but was not higher elsewhere. In control dogs, expression patterns for all 3 markers were similar between sites (villous tip > villous base > colon). Expression of Casp3 in lymphocytes in duodenal villous tips was significantly reduced in dogs with IBD, compared with expression in healthy dogs, but no increase was detected following successful treatment of IBD. Increased expression of Bcl-2 may be a potential marker of the success of treatment.

Molecular-phylogenetic characterization of microbial communities imbalances in the small intestine of dogs with inflammatory bowel disease

An association between luminal commensal bacteria and inflammatory bowel disease (IBD) has been suggested in humans, but studies investigating the intestinal microbial communities of dogs with IBD have not been published. The aim of this study was to characterize differences of the small intestinal microbial communities between dogs with IBD and healthy control dogs. Duodenal brush cytology samples were endoscopically collected from 10 dogs with IBD and nine healthy control dogs. DNA was extracted and 16S rRNA gene was amplified using universal bacterial primers. Constructed 16S rRNA gene clone libraries were compared between groups. From a total of 1240 selected clones, 156 unique 16S rRNA gene sequences were identified, belonging to six phyla: Firmicutes (53.4%), Proteobacteria (28.4%), Bacteroidetes (7.0%), Spirochaetes (5.2%), Fusobacteria (3.4%), Actinobacteria (1.1%), and Incertae sedis (1.5%). Species richness was significantly lower in the IBD group (P=0.038). Principal component analysis indicated that the small intestinal microbial communities of IBD and control dogs are composed of distinct microbial communities. The most profound difference involved enrichment of the IBD dogs with members of the Enterobacteriaceae family. However, differences involving members of other families, such as Clostridiaceae, Bacteroidetes and Spirochaetes, were also identified. In conclusion, canine IBD is associated with altered duodenal microbial communities compared with healthy controls.

Cytokine mRNA abundance in intestinal biopsies from dogs with chronic diarrhea

The differentiation between inflammatory bowel disease (IBD) and food responsive diarrhea (FRD) is difficult and no objective markers are available. We postulated that patterns of selected key cytokines would help to objectively differentiate between the two subcategories of chronic enteropathies in dogs. We studied mRNA patterns of selected cytokines in dogs with chronic enteropathies. Ten dogs with FRD (= group FRDbef) and seven dogs with IBD (= group IBDbef) were presented for endoscopy at the Small Animal Clinic, University of Bern. A control endoscopy was performed in both groups after treatment with an elimination diet for four weeks (FRDaft) or with an elimination diet combined with prednisolone for 10 weeks (IBDaft). Intestinal control samples of gastrointestinally healthy dogs from an independent study were additionally available. Dogs were clinically examined and scored using the canine IBD activity index (CIBDAI). mRNA abundance of interleukin (IL)-5, -10, -12p40, and -13, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β1, and interferon (IFN)-γ were analyzed in intestinal samples by reverse transcription and real time polymerase chain reaction. Median CIBDAI decreased in FRDaft (&lt;i&gt;P&lt;/i&gt; &lt; 0.01) and IBDaft (&lt;i&gt;P&lt;/i&gt; = 0.07) during treatment. In duodenum, IL-12p40 mRNA levels tended to be lower in FRDbef than in IBDbef (&lt;i&gt;P&lt;/i&gt; = 0.07). The abundance of TNF-α mRNA was higher in IBDbef than in control dogs (&lt;i&gt;P&lt;/i&gt; &lt; 0.05). IL-5 mRNA levels decreased in FRD dogs during treatment (&lt;i&gt;P&lt;/i&gt; = 0.06), and IL-10 mRNA levels decreased in IBD dogs (&lt;i&gt;P&lt;/i&gt; &lt; 0.05). In colon, IL-5 and IL-12p40 mRNA levels were lower in FRDbef than in IBDbef (&lt;i&gt;P&lt;/i&gt; &lt; 0.05) and control dogs (&lt;i&gt;P&lt;/i&gt; &lt; 0.01). IL-13 mRNA abundance was lower in FRDbef than in control dogs (&lt;i&gt;P&lt;/i&gt; &lt; 0.05) and IFN-γ mRNA abundance was lower in FRD and IBD dogs than in control dogs (&lt;i&gt;P&lt;/i&gt; &lt; 0.01). Feeding the elimination diet additionally reduced IFN-γ mRNA levels (&lt;i&gt;P&lt;/i&gt; &lt; 0.01), but increased TNF-α mRNA levels (&lt;i&gt;P&lt;/i&gt; &lt; 0.05) in FRD dogs. In conclusion, mRNA levels of the selected cytokines before treatment did not show clear differences between FRD and IBD dogs.

Perinuclear Antineutrophilic Cytoplasmic Antibody and Response to Treatment in Diarrheic Dogs with Food Responsive Disease or Inflammatory Bowel Disease

The goal of this study was to investigate the correlation between perinuclear antineutrophilic cytoplasmic antibody (pANCA) and clinical scores before and after treatment in diarrheic dogs with food‐responsive disease (FRD) or inflammatory bowel disease (IBD). pANCA serology was evaluated prospectively by indirect immunofluorescence in 65 dogs with signs of gastrointestinal disease, and if positive, pANCA antibody titers were determined. Thirty‐nine dogs with FRD responded to a novel diet, and 26 dogs with IBD were treated with corticosteroids. The severity of clinical signs was scored by means of a canine IBD activity index (CIBDAI). At initial examination, a significantly (P= .002) higher percentage of dogs were pANCA‐positive in the FRD group (62%) compared with the IBD group (23%). pANCA titers were significantly higher (P=.003) before treatment in the FRD group (median titer 100) compared with the IBD group (median titer 1). However, there was no difference in pANCA titers between the groups after respective treatments because dogs in the IBD group had a significant increase in pANCA titer after treatment. The CIBDAI score decreased significantly (P &lt;.001) after treatment in both groups (74% moderate to severe in FRD dogs before versus 8% after treatment; 85% moderate to severe in IBD dogs before versus 32% after treatment). There was no correlation between pANCA status in FRD or IBD dogs before treatment and scores for CIBDAI, endoscopy, or histopathology before or after treatment, except for the endoscopic duodenal score in dogs with FRD after treatment (P= .03). A positive pANCA test before therapy may aid in the diagnosis of FRD.

Perinuclear Antineutrophilic Cytoplasmic Antibody and Response to Treatment in Diarrheic Dogs with Food Responsive Disease or Inflammatory Bowel Disease

The goal of this study was to investigate the correlation between perinuclear antineutrophilic cytoplasmic antibody (pANCA) and clinical scores before and after treatment in diarrheic dogs with food-responsive disease (FRD) or inflammatory bowel disease (IBD). pANCA serology was evaluated prospectively by indirect immunofluorescence in 65 dogs with signs of gastrointestinal disease, and if positive, pANCA antibody titers were determined. Thirty-nine dogs with FRD responded to a novel diet, and 26 dogs with IBD were treated with corticosteroids. The severity of clinical signs was scored by means of a canine IBD activity index (CIBDAI). At initial examination, a significantly (P = .002) higher percentage of dogs were pANCA-positive in the FRD group (62%) compared with the IBD group (23%). pANCA titers were significantly higher (P = .003) before treatment in the FRD group (median titer 100) compared with the IBD group (median titer 1). However, there was no difference in pANCA titers between the groups after respective treatments because dogs in the IBD group had a significant increase in pANCA titer after treatment. The CIBDAI score decreased significantly (P < .001) after treatment in both groups (74% moderate to severe in FRD dogs before versus 8% after treatment; 85% moderate to severe in IBD dogs before versus 32% after treatment). There was no correlation between pANCA status in FRD or IBD dogs before treatment and scores for CIBDAI, endoscopy, or histopathology before or after treatment, except for the endoscopic duodenal score in dogs with FRD after treatment (P = .03). A positive pANCA test before therapy may aid in the diagnosis of FRD.

Nuclear receptor and target gene mRNA abundance in duodenum and colon of dogs with chronic enteropathies

Nuclear receptors (NR), such as constitutive androstane receptor (CAR), pregnane X receptor (PXR) and peroxisome proliferator-associated receptors alpha and gamma (PPARα, PPARγ) are mediators of inflammation and may be involved in inflammatory bowel disease (IBD) and food responsive diarrhea (FRD) of dogs. The present study compared mRNA abundance of NR and NR target genes [multi drug-resistance gene-1 (MDR1), multiple drug-resistance-associated proteins (MRD2, MRD3), cytochrome P450 (CYP3A12), phenol-sulfating phenol sulfotransferase (SULT1A1) and glutathione- S-transferase (GST A3-3)] in biopsies obtained from duodenum and colon of dogs with IBD and FRD and healthy control dogs (CON; n = 7 per group). Upon first presentation of dogs, mRNA levels of PPARα, PPARγ, CAR, PXR and RXRα in duodenum as well as PPARγ, CAR, PXR and RXRα in colon were not different among groups ( P > 0.10). Although mRNA abundance of PPARα in colon of dogs with FRD was similar in both IBD and CON ( P > 0.10), PPARα mRNA abundance was higher in IBD than CON ( P < 0.05). Levels of mRNA of MDR1 in duodenum were higher in FRD than IBD ( P < 0.05) or CON ( P < 0.001). Compared with CON, abundances of mRNA for MRP2, CYP3A12 and SULT1A1 were higher in both FRD and IBD than CON ( P < 0.05). Differences in mRNA levels of PPARα and MRP2 in colon and MDR1, MRP2, CYP3A12 and SULT1A1 in duodenum may be indicative for enteropathy in FRD and (or) IBD dogs relative to healthy dogs. More importantly, increased expression of MDR1 in FRD relative to IBD in duodenum may be a useful diagnostic marker to distinguish dogs with FRD from dogs with IBD.