Disturbances in metabolic, transport and structural genes in experimental colonic inflammation in the rat: a longitudinal genomic analysis.
ABSTRACT: BACKGROUND: Trinitrobenzenesulphonic acid (TNBS) induced rat colitis is one of the most widely used models of inflammatory bowel disease (IBD), a condition whose aetiology and pathophysiology are incompletely understood. We have characterized this model at the genomic level using a longitudinal approach. Six control rats were compared with colitic animals at 2, 5, 7 and 14 days after TNBS administration (n = 3). The Affymetrix Rat Expression Array 230 2.0 system was used. RESULTS: TNBS-induced colitis had a profound impact on the gene expression profile, which was maximal 5 and 7 days post-induction. Most genes were affected at more than one time point. They were related to a number of biological functions, not only inflammation/immunity but also transport, metabolism, signal transduction, tissue remodeling and angiogenesis. Gene changes generally correlated with the severity of colitis. The results were successfully validated in a subset of genes by real-time PCR. CONCLUSION: The TNBS model of rat colitis has been described in detail at the transcriptome level. The changes observed correlate with pathophysiological disturbances such as tissue remodelling and alterations in ion transport, which are characteristic of both this model and IBD.
Project description:BACKGROUND:Inflammatory bowel diseases (IBD) are chronic relapsing inflammatory conditions of unknown cause and likely result from the loss of immunological tolerance, which leads to over-activation of the gut immune system. Gut macrophages and dendritic cells (DCs) are essential for maintaining tolerance, but can also contribute to the inflammatory response in conditions such as IBD. Current therapies for IBD are limited by high costs and unwanted toxicities and side effects. The possibility of reducing intestinal inflammation with DCs genetically engineered to over-express the apoptosis-inducing FasL (FasL-DCs) has not yet been explored. OBJECTIVE:Investigate the immunomodulatory effect of administering FasL-DCs in the rat trinitrobenzene sulfonic acid (TNBS) model of acute colitis. METHODS:Expression of FasL on DCs isolated from the mesenteric lymph nodes (MLNs) of normal and TNBS-colitis rats was determined by flow cytometry. Primary rat bone marrow DCs were transfected with rat FasL plasmid (FasL-DCs) or empty vector (EV-DCs). The effect of these DCs on T cell IFN? secretion and apoptosis was determined by ELISPOT and flow cytometry for Annexin V, respectively. Rats received FasL-DCs or EV-DCs intraperitoneally 96 and 48 hours prior to colitis induction with TNBS. Colonic T cell and neutrophil infiltration was determined by immunohistochemistry for CD3 and myeloperoxidase activity assay, respectively. Macrophage number and phenotype was measured by double immunofluorescence for CD68 and inducible Nitric Oxide Synthase. RESULTS:MLN dendritic cells from normal rats expressed more FasL than those from colitic rats. Compared to EV-DCs, FasL-DCs reduced T cell IFN? secretion and increased T cell apoptosis in vitro. Adoptive transfer of FasL-DCs decreased macroscopic and microscopic damage scores and reduced colonic T cells, neutrophils, and proinflammatory macrophages when compared to EV-DC adoptive transfer. CONCLUSION:FasL-DCs are effective at treating colonic inflammation in this model of IBD and represent a possible new treatment for patients with IBD.
Project description:<h4>Background</h4>Rectal instillation of trinitrobenzene sulphonic acid (TNBS) in ethanol is an established model for inflammatory bowel disease (IBD). We aimed to 1) set up a TNBS-colitis protocol resulting in an endoscopic and histologic picture resembling IBD, 2) study the correlation between endoscopic, histologic and gene expression alterations at different time points after colitis induction, and 3) compare rat and human IBD mucosal transcriptomic data to evaluate whether TNBS-colitis is an appropriate model of IBD.<h4>Methodology/principal findings</h4>Five female Sprague Daley rats received TNBS diluted in 50% ethanol (18 mg/0.6 ml) rectally. The rats underwent colonoscopy with biopsy at different time points. RNA was extracted from rat biopsies and microarray was performed. PCR and in situ hybridization (ISH) were done for validation of microarray results. Rat microarray profiles were compared to human IBD expression profiles (25 ulcerative colitis Endoscopic score demonstrated mild to moderate colitis after three and seven days, but declined after twelve days. Histologic changes corresponded with the endoscopic appearance. Over-represented Gene Ontology Biological Processes included: Cell Adhesion, Immune Response, Lipid Metabolic Process, and Tissue Regeneration. IL-1?, IL-1?, TLR2, TLR4, PRNP were all significantly up-regulated, while PPAR? was significantly down-regulated. Among genes with highest fold change (FC) were SPINK4, LBP, ADA, RETNLB and IL-1?. The highest concordance in differential expression between TNBS and IBD transcriptomes was three days after colitis induction. ISH and PCR results corresponded with the microarray data. The most concordantly expressed biologically relevant pathways included TNF signaling, Cell junction organization, and Interleukin-1 processing.<h4>Conclusions/significance</h4>Endoscopy with biopsies in TNBS-colitis is useful to follow temporal changes of inflammation visually and histologically, and to acquire tissue for gene expression analyses. TNBS-colitis is an appropriate model to study specific biological processes in IBD.
Project description:Inflammatory Bowel Disease (IBD) is an autoimmune ailment of the gastrointestinal (GI) tract, which is characterized by enhanced activation of proinflammatory cytokines. It is suggested that the sigma-1 receptor (?1R) confers anti-inflammatory effects. As the exact pathogenesis of IBD is still unknown and treatment options are limited, we aimed to investigate the effects of ?1R in 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced experimental colitis. To this end, male Wistar-Harlan rats were used to model colitic inflammation through the administration of TNBS. To investigate the effects of ?1R, Fluvoxamine (FLV, ?1R agonist) and BD1063 (?1R antagonist) were applied via intracolonic administration to the animals once a day for three days. Our radioligand binding studies indicated the existence of ?1Rs as [3H](+)-pentazocine binding sites, and FLV treatment increased the reduced ?1R maximum binding capacity in TNBS-induced colitis. Furthermore, FLV significantly attenuated the colonic damage, the effect of which was abolished by the administration of BD1063. Additionally, FLV potentially increased the expression of ubiquitin C-terminal hydrolase ligase-1 (UCHL-1) and the levels of endothelial nitric oxide synthase (eNOS), and decreased the levels of interleukin-6 (IL-6) and inducible NOS (iNOS) expression. In summary, our study offers evidence for the anti-inflammatory potential of FLV and ?1R in experimental colitis, and our results present a promising approach to the development of new ?1R-targeted treatment options against IBD.
Project description:<h4>Background</h4>The Na-H exchanger [NHE] performs an electroneutral uptake of NaCl and water from the lumen of the gastrointestinal tract. There are several distinct NHE isoforms, some of which show an altered expression in the inflammatory bowel diseases (IBD). In this study, we examined a role of NHE-2 in experimental colitis.<h4>Methods</h4>Colitis was induced in male Sprague-Dawley rats by intra-rectal administration of trinitrobenzenesulphonic acid (TNBS). On day 6 post-TNBS, the animals were sacrificed, colonic and ileal segments were taken out, cleaned with phosphate buffered saline and used in this study.<h4>Results</h4>There was a significant decrease in the level of NHE-2 protein as measured by ECL western blot analysis and confocal immunofluorescence microscopy. The levels of NHE-2 mRNA and heteronuclear RNA measured by an end-point RT-PCR and a real time PCR were also decreased significantly in the inflamed colon. However, there was no change in the level of NHE-2 protein in response to in vitro TNF-? treatment of uninflamed rat colonic segment. These changes were selective and localized to the colon as actin, an internal control, remained unchanged. Confocal immunofluorescence microscopy revealed co-localization of NHE-2 and NHE-3 in the brush borders of colonic epithelial cells. Inflamed colon showed a significant increase in myeloperoxidase activity and colon hypertrophy. In addition, there was a significant decrease in body weight and goblet cells' mucin staining in the TNBS treated colon. These changes were not conspicuous in the non-inflamed ileum.<h4>Conclusions</h4>These findings demonstrate suppression of NHE-2 expression on the brush borders in the colonic epithelial cells which is regulated transcriptionally. However a role of TNF-? in the regulation of NHE-2 is discounted in the present model of colitis. This decrease in the NHE-2 expression will lead to a loss of electrolyte and water uptake thus contributing to the symptoms associated with IBD.
Project description:Mycophenolate mofetil (MMF) is an alternative immunosuppressive agent that has been reported to be effective and well tolerated for the treatment of refractory inflammatory bowel disease (IBD). The aim of this study was to investigate the therapeutic effect of MMF on intestinal injury and tissue inflammation, which were caused by Crohn's disease (CD). Here, trinitrobenzene sulfonic acid-relapsing (TNBS) colitis was induced in mice; then, we measured the differentiation of Th1/Th2 cells in mouse splenocytes by flow cytometry and the secretion of cytokines in mice with TNBS-induced colitis by real-time polymerase chain reaction and/or enzyme-linked immunosorbent assay (RT-PCR/ELISA). The results show that MMF significantly inhibited mRNA expression of pro-inflammatory cytokines IFN-?, TNF-?, IL-12, IL-6, and IL-1? in mice with TNBS-induced colitis; however, MMF did not inhibit the expression of IL-10 mRNA. Additionally, ELISA showed that the serum levels of IFN-?, TNF-?, IL-12, IL-6, and IL-1? were down-regulated in a TNBS model of colitis. Flow cytometric analysis showed MMF markedly reduced the percentages of Th1 and Th2 splenocytes in the CD mouse model. Mycophenolic acid (MPA) also significantly decreased the percentages of splenic Th1 and Th2 cells in vitro. Furthermore, MMF treatment not only significantly ameliorated diarrhea, and loss of body weight but also abrogated the histopathologic severity and inflammatory response of inflammatory colitis, and increased the survival rate of TNBS-induced colitic mice. These results suggest that treatment with MMF may improve experimental colitis and induce inflammatory response remission of CD by down-regulation of pro-inflammatory cytokines via modulation of the differentiation of Th1/Th2 cells.
Project description:Pro-inflammatory cytokines like macrophage migration inhibitory factor (MIF), IL-1? and TNF-? predominate in inflammatory bowel diseases (IBD) and TNBS colitis. Increased levels of serine proteases activating protease-activated receptor 2 (PAR-2) are found in the lumen and colonic tissue of IBD patients. PAR-2 activity and pro-inflammatory cytokines impair epithelial barrier, facilitating the uptake of luminal aggressors that perpetuate inflammation and visceral pain. Soy extracts contain phytoestrogens (isoflavones) and serine protease inhibitors namely Bowman-Birk Inhibitors (BBI). Since estrogens exhibit anti-inflammatory and epithelial barrier enhancing properties, and that a BBI concentrate improves ulcerative colitis, we aimed to evaluate if a fermented soy germ extract (FSG) with standardized isoflavone profile and stable BBI content exert cumulative or synergistic protection based on protease inhibition and estrogen receptor (ER)-ligand activity in colitic rats. Female rats received orally for 15 d either vehicle or FSG with or without an ER antagonist ICI 182.780 before TNBS intracolonic instillation. Macroscopic and microscopic damages, myeloperoxidase activity, cytokine levels, intestinal paracellular permeability, visceral sensitivity, faecal proteolytic activity and PAR-2 expression were assessed 24 h, 3 d and 5 d post-TNBS. FSG treatment improved the severity of colitis, by decreasing the TNBS-induced rise in gut permeability, visceral sensitivity, faecal proteolytic activity and PAR-2 expression at all post-TNBS points. All FSG effects were reversed by the ICI 182.780 except the decrease in faecal proteolytic activity and PAR-2 expression. In conclusion, the anti-inflammatory properties of FSG treatment result from two distinct but synergic pathways i.e an ER-ligand and a PAR-2 mediated pathway, providing rationale for potential use as adjuvant therapy in IBD.
Project description:BACKGROUND:Inflammatory Bowel Disease (IBD) is characterized by overt inflammation of the intestine and is typically accompanied by symptoms of bloody diarrhea, abdominal pain and cramping. The Colonic Migrating Motor Complex (CMMC) directs the movement of colonic luminal contents over long distances. The tri-nitrobenzene sulphonic acid (TNBS) model of colitis causes inflammatory damage to enteric nerves, however it remains to be determined whether these changes translate to functional outcomes in CMMC activity. We aimed to visualize innate immune cell infiltration into the colon using two-photon laser scanning intra-vital microscopy, and to determine whether CMMC activity is altered in the tri-nitro benzene sulphonic (TNBS) model of colitis. METHODS:Epithelial barrier permeability was compared between TNBS treated and healthy control mice in-vitro and in-vivo. Innate immune activation was determined by ELISA, flow cytometry and by 2-photon intravital microscopy. The effects of TNBS treatment and IL-1? on CMMC function were determined using a specialized organ bath. RESULTS:TNBS colitis increased epithelial barrier permeability in-vitro and in-vivo. Colonic IL-1? concentrations, colonic and systemic CD11b+ cell infiltration, and the number of migrating CD11b+ cells on colonic blood vessels were all increased in TNBS treated mice relative to controls. CMMC frequency and amplitude were inhibited in the distal and mid colon of TNBS treated mice. CMMC activity was not altered by superfusion with IL-1?. CONCLUSIONS:TNBS colitis damages the epithelial barrier and increases innate immune cell activation in the colon and systemically. Innate cell migration into the colon is readily identifiable by two-photon intra-vital microscopy. CMMC are inhibited by inflammation, but this is not due to direct effects of IL-1?.
Project description:We investigated the relationship between chronic inflammation and fibrosis in a mouse model of chronic colitis. Six weekly Trinitrobenzenesulphonic acid (TNBS) enemas were given to establish colitis and temporal changes in gene expression was elucidated over the next six weeks. Keywords: disease state analysis Overall design: After the six TNBS enemas, mice were sacrificed at 3 days (termed as TNBS-w6, n=6), 2 weeks (TNBS-w8, n=4), 4 weeks (TNBS-w10, n=5), or 6 weeks (TNBS-w12, n=4). The saline control groups were also sacrificed at week 6 (Saline-w6, n=5), week 8 (n=2) and week 10 (n=2). The last two sets were pooled as Saline-w10 (n=4). Two replicates of saline-w6 were analyzed. We compared Saline-w6 with TNBS-w6, and Saline-w10 with TNBS-w8, TNBS-w10, TNBS-w12, respectively.
Project description:Inflammatory bowel diseases (IBD) are a growing health problem worldwide, severely affecting patients' life qualities and life expectancies. Therapeutic options, which are rare and focus on symptoms associated with the disease, suffer from increasing numbers of patients refractory to the established strategies. Thus, in order to generate new therapeutic regimens, the detailed understanding of the pathogenic mechanisms causing IBD is necessary. Histamine is an inflammatory mediator associated with IBD. Four histamine receptors are currently known of which the histamine H<sub>4</sub>-receptor (H<sub>4</sub>R) has been shown to possess a pro-inflammatory function in several experimental models of inflammatory diseases, including dextran sodium sulfate (DSS)-induced colitis in mice. No single model reflects the complexity of human IBD, but each model provides valuable information on specific aspects of IBD pathogenesis. While DSS-induced colitis mostly relies on innate immune mechanisms, trinitrobenzene sulfonic acid (TNBS)-induced colitis rather reflects T-cell mechanisms. Consequently, an observation made in a single model has to be verified in at least one other model. Therefore, in the present study we investigated the effect of genetic blockade of H<sub>4</sub>R-signaling in mice subjected to the model of TNBS-induced acute colitis. We analyzed severity and progression of clinical signs of colitis, as well as histopathologic alterations in the colon and local cytokine production. Genetic ablation of H<sub>4</sub>R expression worsened clinical signs of acute colitis and histological appearance of colon inflammation after TNBS application. Moreover, TNBS instillation enhanced local synthesis of inflammatory mediators associated with a neutrophilic response, i.e., CXCL1, CXCL2, and interleukin-6. Lastly, also myeloperoxidase concentration, indicative for the presence of neutrophils, was elevated in cola of TNBS-treated mice due to the absence of H<sub>4</sub>R expression. Our results indicate an anti-inflammatory role of histamine via H<sub>4</sub>R in TNBS-induced acute neutrophilic colitis in mice, thus questioning the strategy of pharmacological H<sub>4</sub>R blocked as new therapeutic option for patients suffering from IBD.
Project description:BACKGROUND AND PURPOSE: Leukotriene B(4) (LTB(4)), formed by the sequential actions of the 5-lipoxygenase (5-LO) and leukotriene A(4) hydrolase (LTA(4)H), is a pro-inflammatory mediator implicated in the pathogenesis of inflammatory bowel disease. However, inhibitors of 5-LO have not proved to be consistent in their therapeutic efficacy in colitis. Another approach to inhibiting LTB(4) synthesis is through the use of inhibitors of LTA(4)H, such as the novel, potent and selective compound, JNJ 26993135. EXPERIMENTAL APPROACH: The effect of oral administration of JNJ 26993135 has been evaluated in a rat model of colitis provoked by colonic instillation of trinitrobenzenesulphonic acid (TNBS). The extent and severity of the macroscopic inflammatory response, the colonic levels of myeloperoxidase (MPO) and LTB(4) and of the pro-inflammatory cytokines, tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) were measured. KEY RESULTS: Oral administration of JNJ 26993135 (5, 15 and 30 mg kg(-1), twice a day) dose-dependently reduced both the extent and intensity of the colonic inflammatory damage observed 3 days after TNBS challenge. JNJ 26993135 also dose-dependently reduced the elevated colonic levels of LTB(4), as well as the inflammatory biomarkers, MPO, IL-6 and TNF-alpha. This dosing regimen was supported by the pharmacokinetic profile of JNJ 26993135, along with the demonstration of the inhibition of ex vivo production of LTB(4) in whole blood following oral administration. CONCLUSIONS AND IMPLICATIONS: These results with JNJ 26993135 in the rat TNBS model support the role of LTB(4) in colitis and the potential value of targeting LTA(4)H for the treatment of inflammatory bowel diseases.