This study was presented at the American College of Veterinary Internal Medicine Forum, New Orleans, LA, June 2012
Corresponding author: T. Watari, Laboratory of Comprehensive Veterinary Clinical Studies, Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan; e-mail: email@example.com.
Nucleotide Oligomerization Domain Two (NOD2) is suggested to be an intracellular pathogen-associated molecular pattern recognition molecule. NOD2, plays a key role against bacteria by triggering a host defense response through activation of the transcription factor NFkappaB and subsequent proinflammatory cytokine production. NOD2 recently was reported to be overproduced in inflamed colonic mucosa in Crohn's disease, and to be accompanied by a significant increase in NFkappaB activity. However, few studies to date have investigated intercellular signaling molecules in dogs with lymphocytic plasmacytic colitis (LPC).
NOD2 mRNA expression and NFkappaB activation are increased in mucosal biopsies of LPC dogs as compared with control dogs.
Five healthy dogs and 19 dogs with LPC.
Descending colon biopsies were obtained endoscopically. Expression of NOD2 mRNA was evaluated by semiquantitative RT-PCR in the colonic mucosa. NFkappaB binding activity was assessed by electrophoretic mobility shift assay.
NOD2 mRNA expression was approximately 63% greater in LPC dogs than in healthy controls (P =.019). NFkappaB binding activity was approximately 45% higher in the inflamed colonic mucosa of the LPC dogs, as compared with healthy controls (P =.011). No correlations were observed among NOD2 mRNA expression levels, NFkappaB binding activity, and CIBDAI in LPC dogs.
Conclusions and Clinical Importance
NOD2 mRNA and NFkappaB activity were significantly higher in the inflamed colon of dogs with LPC, as compared with healthy controls. Our data suggest that NOD2 and NFkappaB play an important role in the pathogenesis of LPC.
Idiopathic lymphocytic plasmacytic colitis (LPC) is a common form of inflammatory bowel disease (IBD) affecting the canine large intestine.[1, 2] The molecular basis of the pathogenesis is unclear, although it has been suggested that the disease occurs in genetically susceptible hosts as a consequence of a dysregulated response of the mucosal immune system toward commensal enteric flora and dietary components.[3-6]
Crohn's disease and ulcerative colitis are the 2 major forms of IBD in humans, and were previously thought to be sustained by an altered adaptive immune response. However, recently it has been hypothesized that the primary defect is impaired innate immune function, which relies on specific sensing of conserved pathogen-associated molecular patterns (PAMPs).[7, 8] Pattern recognition receptors (PRRs) are a class of innate immune response-expressed proteins that respond to PAMPs.[9, 10] These receptors are expressed by a wide range of immune and nonimmune cells, such as intestinal epithelial cells.
The 2 best-characterized classes of PRRs are the Toll-like receptors (TLRs), which sense the presence of microbial and viral molecules on the cell surface and in endosomes, and the nucleotide binding oligomerization domain (NOD)-like receptors (NLRs), which recognize microbes in the cytosol.[12, 13] NOD1 and NOD2 are the first NLRs reported to act as PRRs and are composed of an N-terminal caspase recruitment domain, a centrally located nucleotide binding oligomerization domain, and C-terminal leucine-rich repeats (LRRs). NOD proteins have been shown to recognize bacterial components, including bacterial lipopolysaccharides (LPS), peptidoglycan, or both through their LRRs, and this interaction leads to the activation of NFkappaB, a transcription factor that plays a central role in innate immunity, which is necessary for clearance of infectious pathogens from the host.[15, 16] Recently, a frameshift mutation and 3 nucleotide polymorphisms in the coding region of NOD2 have been found to be associated with susceptibility to Crohn's disease.[17-20] It also has been reported that NOD2 mRNA and protein are overexpressed, and that NFkappaB activity is upregulated in the intestinal mucosa in humans with IBD.[21-23]
In dogs, there have been studies into NOD2 expression in canine primary colonic epithelial cells and anal furunuclosis,[25, 26] but to our knowledge, there have been no reports on NOD2 in canine LPC. A previous immunopathological study, has shown that NFkappaB activity is upregulated in macrophages of the lamina propria and in epithelial cells in the small intestine of dogs with chronic enteropathies (CE). However, there have been no reports on NFkappaB activity in dogs with LPC.
The purpose of this study, was to assess the expression of NOD2 mRNA and activation of NFkappaB in the colonic mucosa of dogs with LPC.
Materials and Methods
Tissue samples were obtained from 19 dogs referred to the Animal Medical Center of Nihon University from June 2011 to May 2012 for investigation of large intestinal disease (diarrhea, tenesmus, hematochezia, and increased frequency of defecation) of more than 3 weeks' duration. Cases during this period included lymphoma, leiomyosarcoma, adenocarcinoma eosinophilic enteritis, inflammatory polyp, ulcerative colitis, colorectal enteritis with infiltration of neutrophils, and LPC. Moreover, the LPC cases included concomitant illness (eg, pancreatitis). We excluded all diseases other than LPC, leaving only 19 dogs with LPC. All 19 dogs, were assessed and given a clinical score by the canine inflammatory bowel disease activity index (CIBDAI) scoring system. Scoring criteria included attitude, activity, appetite, vomiting, stool consistency, stool frequency, and weight loss. By this system, 6 prominent clinical signs were scored from 0 to 3 based on the magnitude of their change. All dogs underwent a thorough investigation, including CBC, urinalysis, serum biochemistry profile, parasitological and bacteriological examination of feces, radiographic examination, abdominal ultrasound examination and endoscopy with intestinal biopsy sampling to exclude any other causes of gastrointestinal signs. Mucosal biopsy specimens from the colon, were obtained by the same endoscopist and were examined histologically by a board-certified veterinary pathologist.
As healthy control dogs, 5 laboratory Beagles (females) were used. The median age of the dogs was 5 years (range, 3–9 years). No clinical signs of gastrointestinal disease were observed in these dogs. There were no abnormalities on in laboratory tests, including CBC and urinalysis, parasitological and bacteriological examination of feces, abdominal ultrasound examination, endoscopic findings and histology of biopsies.
The use of dogs in this study was approved by the Animal Experimentation Committee of the College of Bioresource Sciences, Nihon University.
Tissue Sampling and Treatment of Biopsy Specimens
Under general anesthesia, tissue sampling was performed by colonoscopy1 according to World Small Animal Veterinary Association (WSAVA) guidelines. Multiple mucosal biopsies (6 pieces) of the descending colon were taken in each dog by means of biopsy forceps,2 and 2 samples each were used for DNA, nuclear extract (NFkappaB binding activity), and histopathological analyses in all dogs. Colonic specimens for total RNA extraction and electrophoretic mobility shift assay analysis were immediately immersed in liquid nitrogen and stored at −80°C until use. Samples for histopathology were immediately placed in 10% formalin, and haematoxylin and eosin-stained sections were prepared.
RT-PCR Amplification Assay
Total RNA was isolated from biopsy specimens by commercial reagent.3 For synthesis of first-strand cDNA, all extracted total RNA was added to a 20-μL reaction mixture (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol [DTT]) containing 1.3 μM oligo d(T) primer (15-mer), 2 mM dNTP mix, 40 U of Ribonuclease inhibitor4 and 15 U of Avian Myeloblastosis Virus (AMV) reverse transcriptase. Conditions for reverse transcription were an initial incubation at 42°C for 15 minutes, 95°C for 5 minutes and incubation at 4°C for 5 minutes. cDNA samples were amplified by PCR in a total reaction volume of 20 μL containing 0.1 μL of DNA polymerase,5 2 μL of 10× Ex taq buffer, 1.6 μL of dNTP Mixture, and 1 μL of each primer (10 μM). The following NOD2-specific primers were used for PCR: NOD2 forward 5′-CCTGAACTCATCAAAGCCATCG-3′ and reverse 5′-TGCTCACCATCCTACCTATT-3′, which amplify a fragment of 559 bp. To confirm the use of equal amounts of RNA in each experiment, all samples were checked in parallel for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA expression (primers for GAPDH were forward 5′-CTCATGACCACAGTCCATGC-3′ and reverse 5′-TGAGCTTGACAAAGTGGTCA-3′). The expected amplicon length was 412 bp. The annealing temperature was 58.6°C for NOD2 and 60°C for GAPDH. PCR products were visualized by electrophoresis on a 2% agarose gel followed by gel staining in ethidium bromide. Densitometric analysis of electrophoretic bands was performed by computer-assisted densitometry analysis by commercial software.6
Electrophoretic Mobility Shift Assay
For nuclear extracts, cells were lysed in a commercial extraction reagent7 in accordance with the manufacturer's protocols. Protein concentrations were determined with a commercial protein assay kit.8 DNA probes were prepared by annealing complementary sequences and treating them at 95°C for 5 minutes, followed by cooling at room temperature. The specific oligodeoxynucleotide duplexes used as DNA probes carry a Cy 5 label for consensus sequences for NFkappaB (3′-TCAACTCCCCTGAAAGGGTCCG-5′). Binding reactions were carried out for 30 minutes at 25°C in 20 μL of binding buffer (5 M NaCl, 1 M Tris-HCl, 0.5 M EDTA pH 8, 1 M DTT, 37.8% glycerol, 1.5% NP-40, 5 mg/mL bovine serum albumin [BSA]) containing 1 μg of poly (dI-dC), 10 μg of nuclear extract, and 17.5 fmol of fluoresceinated probe. Samples were electrophoresed on 4% polyacrylamide gels and were run at 100 V for 1 hour. Gel analysis was performed by an image analyzer9 and commercial software.6
A statistical software package was used for all calculations.10 A nonparametric Mann–Whitney U-test was used for comparisons of NOD2 mRNA expression and NFkappaB binding activity between the LPC dogs and control dogs. The relationships among NOD2 mRNA expression, NFkappaB, and clinical score were evaluated by Spearman's rank correlation coefficient. Statistical significance was set at P <.05 for all analyses.
All dogs were diagnosed histopathologically with LPC. The median age of the dogs was 8 years (range, 2–14 years) with 11 females (6 intact and 5 neutered) and 8 males (3 intact and 5 neutered). Dog breeds included Shiba Dog (3), Miniature Dachshund (3), Boston Terrier (1), Miniature Schnauzer (1), Chihuahua (1), Doberman Pinscher (1), Papillon (1), Rottweiler (1), Bernese Mountain Dog (1), Yorkshire Terrier (1), Pembroke Welsh Corgi (1), Jack Russell Terrier (1), Irish Setter (1), Wire-haired Fox Terrier (1), and mixed breed dog (1). The CIBDAI score range was 2–14 (median, 5) in all dogs.
Expression of NOD2 mRNA in Colonic Specimens of LPC and Control Dogs
NOD2 mRNA expression was analyzed by semiquantitative RT-PCR in endoscopic biopsies derived from the colons of 19 LPC dogs and 5 controls. Significantly higher NOD2 mRNA expression (63%) was seen in the colons of LPC dogs, as comparedwith control dogs (P =.019) (Figs 1, 2). We found no correlations between NOD2 mRNA expression levels and CIBDAI scores in LPC dogs (P =.918, r = −0.025).
DNA Binding Activity of NFkappaB in Colonic Specimens of LPC and Control Dogs
The DNA binding activity of NFkappaB was significantly higher (45%) in LPC dogs as compared with controls (P =.011) (Figs 3, 4). No correlations were seen between NFkappaB binding activity and CIBDAI (P =.105, r = 0.384) or NOD2 mRNA expression levels (P =.715, r = 0.090) between LPC and control dogs.
Previous studies showed that NOD2 mRNA was overproduced in colonic mucosa with inflammation in humans and rats.[10, 22, 31, 32] We found that the mRNA expression of NOD2 was significantly higher in LPC dogs, as compared with control dogs. Swerdlow et al. reported that NOD2 mRNA was expressed at lower levels in nonstimulated canine primary colonic epithelial cells in healthy dogs, when compared with cells stimulated with peptidoglycan. Similarly, Gutierrez et al reported that unstimulated epithelial cells from normal human colon express low levels of Nod2 mRNA, as compared with cells simulated by TNFα. In our study, it is unknown whether NOD2 mRNA expression levels in healthy dogs are low, because we did not perform analysis of the expression levels before and after stimulation by peptidoglycan and TNFα. However, we also observed low expression of NOD2 mRNA in healthy dogs, as compared with LPC dogs. This indicates that epithelial and macrophage NOD2 receptors conventionally exhibit low responsiveness to comensal microflora, and this condition may be beneficial to the host in the maintenance of cooperation with commensal microflora in the mucosa of normal intestine. In humans with Blau syndrome, a systemic auto-immune disease, NOD2 hyperfunction and NFkappaB activation are seen because of gene mutations in the regulatory site for NOD2 mRNA expression. Similarly, inflammation may be induced in the intestines of LPC dogs. A previous study has indicated that mice lacking NOD2 do not exhibit intestinal inflammation, despite the presence of numerous enteric bacteria. This suggests that NOD2 mRNA plays an important role in the initiation of enteritis and is a critical regulator of bacterial immunity within the mucosal region. Although we did not investigate the relationship between NOD2 and enteric bacteria in this study, we believe that the phenomenon in the mucosa of LPC dogs is as follows: 1) overproduced NOD2 receptors increase peptidoglycan recognition initiating inflammation; 2) the intestine is no longer able to tolerate the products of lumen bacteria; 3) this initiates an intestinal disorder, allowing more bacteria to invade the intestinal region; and 4) further overexpression of NOD2 mRNA is induced. In this study, we assessed NOD2 mRNA levels, but not protein levels or genetic abnormalities. As higher detection of mRNA is not direct proof of the functional activity of a gene, future protein and gene analysis is necessary. In humans, there have been some studies of NOD2 in the large intestine, in which numerous bacteria are present.[10, 21, 22, 32] However, in dogs, the importance of inflammation in the small intestine must be acknowledged. Here, we only studied expression in the colon mucosa. Thus, further studies in the small intestine are necessary.
We found significantly higher NFkappaB activation in colonic mucosa from dogs with LPC, as compared with healthy control dogs. Luckschander et al previously reported that NFkappaB is activated in the duodenal mucosa of dogs with CE, as compared with control dogs. Similarly, we also found activation of NFkappaB in the colonic mucosa. Interestingly, our study indicated that NFkappaB activation was higher in LPC dogs with significantly higher NOD2 mRNA expression, as compared with control dogs. Generally, NFkappaB is activated that as a result of proinflammatory stimuli derived from several mechanisms, including NOD2, induce inflammation. A recent study of Crohn's disease indicated that NFkappaB can regulate NOD2 transcription by binding to a DNA element included in the NOD2 promoter site showing a high homology to transcription factor consensus sequence.[32, 36] In dogs, expression of mRNA encoding TLR2, 4 and 9, which all may activate NFkappaB, is higher in the duodenal and colonic mucosa of dogs with IBD than in control dogs. In addition, a recent study reported that single nucleotide polymorphisms in the TLR4 and TLR5 genes are significantly associated with IBD in German shepherd dogs, and the TLR5 risk-associated haplotype for canine IBD induces hyper-responsiveness to flagellin and activates NFkappaB. It is not clear whether LPC is caused by activation of NOD2 and NFkappaB in the mucosa or by recruitment of inflammatory cells expressing NOD2. However, we believe that in LPC dogs, proinflammatory irritation because of enteropathogens or other pathogenic bacteria may activate NFkappaB by various mechanisms, including NOD2, TLR, or TNFα receptor signaling. Under these conditions, activation of NFkappaB may induce additional expression of NOD2, which in turn activates NFkappaB, establishing a positive feedback loop that may contribute to the secretion of proinflammatory cytokines and chemokines at epithelial sites. Proinflammatory cytokines such as TNFα and IL1β, the expression of TLR, and adhesion molecules such as intercellular introduction adhesion molecule-1,[39, 40] induced by NFkappaB were not analyzed in our study. In the future, correlations among NOD2, TLR, proinflammatory cytokines, chemokines, and adhesion molecules should be investigated.
We found no correlations among levels of NOD2 mRNA expression, activation of NFkappaB, and CIBDAI score in LPC dogs. Previous studies reported that TLR2 mRNA expression is weakly correlated with, or not correlated with, clinical severity of disease in CE dogs.[5, 6] Similar to our study, Luckschander et al reported that no significant correlations were present between CIBDAI group scores and number of cells with activated NFkappaB in CE. There may be no correlations between the severity of clinical manifestations and NFkappaB activity.
In a report by Luckschander et al all dogs with CE showed decreased NFkappaB activation in the duodenal mucosa after treatment. Our study did not investigate NOD2 mRNA and NFkappaB activity after treatment. In the study of humans, NOD2 mRNA expression and NFkappaB activity in intestinal specimens of IBD patients was significantly down-regulated in treated patients, as compared with untreated patients.[22, 41] Therefore, suppressing activation of these factors may be important in the assessment of therapeutic efficacy in canine IBD. Moreover, therapeutic targeting of NFkappaB is considered to be important as a future approach to IBD treatment. Further investigation into NOD2, TLR, and NFkappaB before and after treatment in canine IBD is necessary.
In this study, the number of cases was insufficient to draw definitive conclusions. Additional studies with a larger number of cases are necessary to validate our observations in LPC dogs.
This study also is limited by the fact that the laboratory Beagles we used as control dogs all were female. In addition, we were unable to assess breed differences. There may be specific breeds that have higher or lower levels of NOD2 expression and NFkappaB activation. For example, breeds such as German Shepherds are more likely to be affected by chronic enteritis and may have higher expression levels. Differences in the respective environments of the dogs and controls also require further study. Dietary and environmental factors can alter intestinal flora, which could have an impact on PRRs and intracellular signaling. In the future, more extensive studies are necessary to resolve these issues.
In conclusion, we found that significantly higher NOD2 mRNA and NFkappaB activation are present in the colonic mucosa of dogs with LPC, as compared with control dogs. Understanding the mechanisms by which the innate response is induced in dogs will be useful for highlighting the potential role of molecules involved in the inflammatory cascades of IBD.
The authors thank Drs Soichi Maruyama, Hidenori Kabeya, Shinya Oda, and Shingo Sato for technical advice, and the residents and interns of the Animal Medical Center of Nihon University for sample collection.
Funding: This study was funded by the Laboratory of Comprehensive Veterinary Clinical Studies, Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University.
Conflict of Interest Declaration: Authors disclose no conflict of interest.
VQ-8142A flexible video endoscope; Olympus Medical System Corp, Tokyo, Japan
VH-143-B25; Olympus Medical System Corp
TRIzol Reagent; Life Technologies Corp, Tokyo, Japan
RNasin; Promega Corp, Madison, WI
VersaDoc Imaging System Model 5000; Bio-Rad Laboratories, Tokyo, Japan
Software Quantity One; Bio-Rad Laboratories, Hercules, CA
NE-PER Nuclear and Cytoplasmic Extraction Reagents; Pierce, Rockford, IL
BCA Protein Assay Kit-Reducing Agent Compatible; Pierce
Thyphoon 9410 high performance imager; GE Healthcare, Tokyo, Japan
GraphPad Prism; GraphPad Software Inc, San Diego, CA