Pathogenesis of Canine IBD: The Interplay of Mucosal Immune System with Microbiota
The gut epithelium detects the presence of both commensal and pathogenic bacteria via Pattern Recognition Receptors (PRRs).4 This group contains Toll-like receptors (TLRs) and Nucleotide Binding Oligomerisation Domain-like receptors (NODs).5 These receptors recognize specific molecules termed Pathogen-Associated Molecular Patterns (PAMPs), which are conserved molecules found on bacteria and other infectious agents. Recognition of PAMPs by TLRs is a crucial part of the innate immune response to invading bacteria in the gut and initiates a complex intracellular signaling pathway culminating in the activation of the transcription factor NF-κB.6 This results in the transcription and secretion of a variety of pro- and anti-inflammatory cytokines and chemokines from the cell bearing the TLR. If successful, immune cells activated by this response clear offending pathogens. Despite the fact that commensal and pathogenic bacteria share PAMPs, the immune system remains unresponsive towards commensal organisms present in the intestinal lumen in the normal case scenario.7
The inflammatory response which is normally only seen as a reaction towards pathogenic bacteria breaching the intestinal barrier is similar to the response seen in the mucosa of dogs affected with IBD. However, the response in IBD occurs in the absence of pathogens. It is believed that the innate immune system reacts to normal commensals in the intestinal lumen as if they were pathogens.
Evidence of Innate Immunity Hyper-Responsiveness in Canine IBD
TLRs have been shown to be upregulated in the intestine of human beings with Crohn's disease and ulcerative colitis. This may be either a consequence of the ongoing stimulation of TLRs by an altered microbiota or it may be a causal factor contributing to the pathogenesis of disease. Most human studies show that the mRNA and protein expression of TLR2 as well as TLR4 are increased in the intestine of people with active IBD.8,9 In a recent clinical study at the Royal Veterinary College, London, we were able to show that dogs of any breed with clinically severe, active IBD express higher levels of TLR2 receptors in the duodenum compared to healthy dogs when measured by real-time PCR in whole endoscopic biopsies.10 In addition, TLR2 expression was correlated with the clinical severity of IBD using the Canine Chronic Enteropathy Clinical Activity Index (CCECAI). However, TLR4 expression levels were similar to those in healthy canine intestine. Other studies have found that only a subgroup of dogs with IBD (the ones responding only to steroid administration) showed an increased expression of TLR2, TLR4 and TLR9 compared to healthy intestines when expression was measured by real-time PCR.11 In further studies looking at German Shepherd Dogs (GSD) with IBD, we have found that TLR4 expression was 60-fold higher in the duodenum, ileum and colon of dogs with IBD compared to samples from healthy dogs, however, TLR2 and TLR9 were similarly expressed as in healthy dogs.12 These data show that it is important to look at similar phenotypes of dogs when choosing cases for such studies, as the results will vary depending on the severity of disease, the treatment response in the cases and the specific breed of dogs. In addition, care must be taken to compare studies using real-time PCR as the standardization method used depends on the reference genes, which need to be carefully chosen for each study. TLR2 has recently been shown to be overexpressed in the diseased intestine in mouse models of IBD.13 TLR2 in this context is implicated in the homeostasis and repair of intestinal tissue after injury. It is therefore possible that the high expression of TLR2 found in dogs with IBD in the studies mentioned above could be a marker of intestinal inflammation and its physiological action is to downregulate ongoing inflammation.
TLR5 expression was seen to be consistently downregulated in the intestine of GSD with IBD as compared to healthy dogs.12 In mice and human beings, TLR5 is highly expressed in the healthy small intestine, with CD11c+ dendritic cells (DCs) in the lamina propria mucosae expressing most TLR5.14 It is believed that this tolerogenic phenotype of DCs induces T regulatory cells and stimulates the production of anti-inflammatory cytokines such as IL-10 in response to flagellin.15 In contrast, in intestinal inflammation characterized by the upregulation of Th1- and Th17 cytokines, CD11c- DCs express low levels of TLR5 but instead high levels of TLR4.15 In this context, TLR4 is thought to be upregulated to compensate for the low TLR5 expression. It could be speculated that the differentially low expression of TLR5 and very high expression of TLR4 seen in the intestine in the GSD of our study indicates a similar compensatory role of TLR4, as gram-negative flagellated bacteria can also be recognized through binding of LPS by TLR4.
Dysbalance of the Intestinal Microbiota in Canine IBD
Molecular studies on the intestinal microbion in dogs of different breeds with IBD have found that members of the families Enterobacteriaceae and Clostridiaceae were enriched in the diseased intestine.16 These bacteria are thought to contribute to the pathogenesis of disease in dogs as well as human beings with IBD.17 However, in the duodenum of GSD with IBD, bacterial clones within the order Lactobacillales were significantly more frequently found than in the duodenum of healthy dogs.12 It appears that GSD with chronic enteropathies have a distinctly different microbion from healthy dogs as well as from other breeds of dogs presenting with IBD, with over-representation of certain, traditionally labelled "beneficial" bacteria in the duodenum, specifically sequences of the order of Lactobacillales.
Genetic Predisposition in GSD with IBD
Over the last decade, numerous genes have been found to be associated with an increased risk of development of IBD in human beings, many of them implicated in the innate immune response in the intestine.18,19 Mutations in PRRs, such as NOD2, TLR4, IL-23 receptor and others have all been associated with IBD. In dogs, it has always been obvious to clinicians that IBD seems to have a genetic component. This is particularly evident in breeds like the Boxer, who is predisposed to histiocytic ulcerative colitis. Another example of a breed disposition is the German Shepherd Dog, which seems to be predisposed to antibiotic-responsive diarrhoea. We recently performed a mutational analysis of the canine genes for TLR2, TLR4, TLR5 and NOD-2 in GSD with IBD. One of the three polymorphisms that we identified in the TLR5 gene (G22A) of GSD was evaluated further in a case-control study with over 50 cases and breed controls and was found to be significantly associated with IBD. In addition, 4 non-synonymous single nucleotide polymorphisms (SNPs) were identified in exon 4 of the canine NOD2 gene. The heterozygote genotype for all four NOD2 SNPs was significantly more frequently found in the IBD population (p=0.04, OR=2.34, CI=1.03-5.28) than in controls. These results were also mirrored in non-GSD breeds: The heterozygote genotype for all four SNPs was significantly more frequently found a population of 96 dogs of different breeds with IBD compared to the non-GSD control population (p=0.0009, OR=3.06, CI=1.55-6.05).
Where To Go From Here?
We are still a long way from our goal to try and find better ways of diagnosing and treating chronic enteropathies in dogs. The next step will involve investigations into the functional aspects of canine TLR and NOD2. In order to achieve this, current studies at the Royal Veterinary College are investigating signalling through canine TLR on a cellular level. These investigations will allow insights into the functional consequences of TLR polymorphisms observed in German Shepherd Dogs, and will help to elucidate the possible pathogenesis of antibiotic responsive diarrhoea and IBD in this breed.
If bacteria or bacterial products are identified which either stimulate the appropriate TLR response or inhibit an aberrant TLR-dependent NF-κB activation, then this knowledge can be used in prospective treatment studies in dogs with chronic enteropathies. Similar to the human system, it is possible that certain probiotic cocktails could be used as prophylactic treatments in dogs which are genetically predisposed to developing IBD, food allergy or antibiotic responsive diarrhoea. The identification of a specific bacterial product that reduces inflammation in the gut could serve as a treatment supplement or even as an adjuvant for potential vaccines against IBD in dogs.
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