DSS Colitis Model Research Articles

Th17 Responses Are Not Induced in Dextran Sodium Sulfate Model of Acute Colitis

Dextran sodium sulfate (DSS) is a widely used chemical model for inflammatory bowel disease (IBD). It is thought that imbalances in the T helper (Th) cell subsets contribute to IBD. Recent studies suggest that the acute DSS-colitis model is polarized toward a Th1/Th17 profile based on RT-PCR analysis of colonic tissues. In the current study we determined whether colonic Th cells from DSS-colitis mice were skewed toward the Th17 profile. Mice were treated with 5% DSS for 7 days and colonic T cells isolated and examined for production of IFN-γ (Th1 cell), IL-4 (Th2 cell) and IL-17 (Th17 cell) by intracellular flow cytometry. We found that the percentage of colonic Th17 cells were similar to non-treated controls but the percentage of Th1 cells were elevated in DSS-colitis mice. These results suggest that in the acute DSS-colitis model the colonic Th cells exhibit a Th1 profile and not a Th17 profile.

Blockade of angiotensin II Type 1a receptor in a DSS model of colitis results in significant expansion of Foxp3 regulatory T cells

Blockade of angiotensin II Type 1a receptor in a DSS model of colitis results in significant expansion of Foxp3 regulatory T cells

Ikaros silences the ifnγ gene during T cell anergy and T helper polarization (88.14)

Abstract Ikaros is a zinc finger transcription factor required for lymphocyte development, but recent studies indicate that this factor also regulates peripheral T cell function. Our previous studies showed that Ikaros is a major repressor of il2 gene expression during the induction of T cell anergy, and in the present study, we have examined the role of Ikaros in ifnγ gene expression in CD4+ T cells. We find that Ikaros binds to the endogenous ifnγ and tbx21 loci, an event associated with increased DNA methylation at the ifnγ gene. Loss of Ikaros function in vitro resulted in increased IFNγ production by anergic T cells and Th2 cells, and was reinforced in Th2 cells by dysregulated expression of Tbet. Loss of Ikaros function in vivo led to an inappropriate Th1 response to the Th2 parasite S. mansoni, and also led to increased ifnγ gene expression and fatal intestinal immunopathology in a DSS model of colitis. Our results show that Ikaros is an important transcriptional repressor of Tbet and IFNγ expression, and further establish a role for Ikaros in T cell tolerance and T helper polarization.

Bioactive sphingolipids in inflammation and cancer

The bioactive sphingolipids ceramide, sphingosine, and sphingosine‐1‐phosphate (S1P) have recently emerged as important modulators of cell growth, cellular senescence, and programmed cell death. In order to study the roles of these lipids in cell regulation, it is important to understand their interconnected metabolic pathways. Ceramide, the backbone of sphingolipids is broken down by one of many ceramidases (CDAse) to generate sphingosine the precursor of S1P by the action of sphingosine kinase (SK). The enzymes involved in the breakdown of ceramide, and in the generation and breakdown of S1P all appear to be important regulated enzymes. Our laboratory has focused on studying the role of CDAse, SK, and its product S1P in inflammation and in cancer. We have recently implicated the SK1/ S1P pathway in mediating inflammatory responses in cells and most recently in vivo in a DSS model of colitis. In order to determine the mechanism of substrate generation for SK in inflammation we are utilizing a combination of CDAse inhibitors and KO mice. We demonstrate distinct roles for neutral and acid CDAses in regulation of ceramide/ S1P levels in the stromal and epithelial layers of intestine and consequently in the inflammatory response to DSS‐induced colitis. In addition, significant evidence has implicated bioactive sphingolipids in cancer progression and apoptosis. In probing the role of CDAses and SK in cancer we find distinct roles for the different CDAses and SK1 in colon cancer progression. These studies lend strong support for the role of bioactive lipids and their metabolizing enzymes in inflammation and cancer and make them attractive therapeutic targets for inflammation and cancer.

The effect of a4β7 blockade on T-lymphocyte trafficking to intestinal compartments in the normal and inflamed intestine

Blockade of the integrin α4β7 has demonstrated promise as an organ specific therapy for the treatment of inflammatory bowel disease (IBD). It is believed that the therapeutic effect of α4β7 blockade is mediated by inhibiting T-cell trafficking into the intestine, however the specific T-lymphocyte compartment affected by α4β7 blockade and the effects of α4β7 blockade on normal T-lymphocyte trafficking and during acute injury are not well understood. In this study we investigated the effect of blocking α4β7 function on the presence of intestinal T-lymphocytes in the diffuse lamina propria and the lymphoid aggregates in the uninflamed intestine, in the acute injury model DSS colitis and the chronic splenocyte transfer model. The presence of intestinal T-lymphocytes in the diffuse lamina propria and lymphoid aggregates was evaluated in wildtype mice, β7-/- mice, and wildtype mice given blocking antibodies to α4β7 or control antibodies using flow cytometry and immunohistochemistry. To evaluate the effect of α4β7 blockade in acute injury, the onset and regression of inflammation, lymphoid aggregate formation and regression and the presence of T-lymphocytes were evaluated in the acute injury model of DSS colitis during α4β7 blockade. To evaluate the effect of α4β7 blockade on T-cell mediated colitis, the course of colitis and the presence of T-lymphocytes in the diffuse lamina propria and lymphoid aggregates was evaluated in the splenocyte transfer model of colitis. In the unchallenged animals, the intestinal T-lymphocyte population in the diffuse lamina propria was normal in the absence of α4β7 function. The T-lymphocyte population in the lymphoid aggregates was also normal with the exception that the Foxp-3 T-lymphocyte population was significantly decreased in the absence of β7. DSS-induced colitis peaked at day 3 after the removal of as DSS demonstrated by increased histological score, MPO activity, and began to regress at day 7 after removal of DSS. The number of B-cell containing lymphoid aggregates peaked at 7 days after the removal of DSS and remained elevated at 14 days after the removal of DSS even though clinical inflammation had regressed. Anti-α4β7 blockade or loss of β7 function dramatically decreased the number B-cell containing lymphoid aggregates, but did not show any other effects, including changes in T-lymphocyte trafficking into the aggregates, during DSS-induced colitis. In the T-cell mediated splenocyte transfer model of colitis, the population of T-lymphocytes in the diffuse lamina propria and the lymphoid aggregates were significantly decreased in β7-/- mice and the wildtype mice receiving α4β7 blockade. This reduction corresponded to the decreased inflammation as evidenced by significant lower expression of TNF-α, IFN-γ, IL-6, MPO and IL-17. The presence of Foxp3+ T-lymphocytes in lymphoid aggregates is dependent upon α4β7, however the trafficking of other T-lymphocytes into the diffuse lamina propria and lymphoid aggregates in the basal state and during acute injury is not dependent upon α4β7. α4β7 blockade can block pathogenic T-lymphocyte trafficking to the intestine in a T-cell mediated model of colitis and accordingly is effective at ameliorating inflammation in this model. These observations suggest that α4β7 blockade may have fewer effects than anticipated on normal T-lymphocyte trafficking and T-lymphocyte trafficking during acute injury and may be a more specific therapy for T-cell mediated inflammatory diseases of the intestine.

P4.9 Molecular changes underlying increases in 5-HT availability in a mouse model of DSS-colitis

Serotonin (5-HT) containing enterochromaffin (EC) cells of the intestine detect chemical and mechanical stimuli in the lumen and respond by releasing 5-HT on to afferent nerve terminals. Our recent electrochemical studies have shown that the real-time release of 5-HT is increased during inflammation, but the molecular changes are unknown. Our aim was to characterise the molecular basis of increased 5-HT availability in a mouse model of colitis.

T1769 Mucosal Cytokine Levels Normalize Despite Continued Clinical and Histopathologic Evidence of Chronic Colitis in a Murine DSS Model of Colitis

T1769 Mucosal Cytokine Levels Normalize Despite Continued Clinical and Histopathologic Evidence of Chronic Colitis in a Murine DSS Model of Colitis

Alterations in Mucosal Cytokine levels reflect acute inflammation but not chronic inflammation in the Murine DSS model of Colitis

Alterations in Mucosal Cytokine levels reflect acute inflammation but not chronic inflammation in the Murine DSS model of Colitis

Alterations in Mucosal Cytokine levels reflect acute inflammation but not chronic inflammation in the Murine DSS model of Colitis

Alterations in Mucosal Cytokine levels reflect acute inflammation but not chronic inflammation in the Murine DSS model of Colitis

Inflammatory bowel disease, a dysregulated host‐microbiota interaction: Are probiotics a new therapeutic option?

Inflammatory bowel disease, a dysregulated host‐microbiota interaction: Are probiotics a new therapeutic option?

A Novel mTOR Inhibitor Is Efficacious in a Murine Model of Colitis

Purpose: Ulcerative colitis is an autoimmune-inflammatory disease characterized by increased proliferation of colonic epithelial cells, dysregulation of signal transduction pathways, elevated mucosal T-cell activation, increased production of pro-inflammatory cytokines (e.g., TNF-α, IFN-γ), and enhanced leukocyte infiltration into colonic interstitium. Several compounds that possess anti-proliferative activities (e.g., rapamycin and its analog everolimus; mammalian target of rapamycin (mTOR) inhibitors) and/or inhibit IFN-γ production (NCX-1015, a nitric oxide derivative of prednisolone) exhibit a therapeutic effect in murine models of colitis. In this study, we report that a novel aminopyridine based molecule (P2281) is a mTOR inhibitor and is efficacious in a murine model [the dextran sulfate sodium (DSS) model] of human colitis. Methods: Cell-based ELISAs, Western blot analysis and DSS-model of colitis were employed. Results: In vitro studies using western blot analysis and cell-based ELISA assays each showed that P2281 inhibits mTOR activity (69% inhibition of mTOR phosphorylation at 10 μM in ELISA assays). In vitro and in vivo assays of pro-inflammatory cytokine production revealed that P2281 diminishes induced IFN-γ synthesis/release (IC50: ∼20 μM) but not TNF-α synthesis/release (IC50: > 100 μM). In the disease model of colitis (i) macroscopic colon observations revealed that P2281, administered intraperitoneally, significantly inhibited DSS-induced weight loss (5.70 ± 2.4% for P2281-treated mice vs. 17.75 ± 3.07% for control mice), improved rectal bleeding index (0.42 ± 0.08 for P2281-treated mice vs. 0.67 ± 0.17 for control mice), decreased disease activity index (3.92 ± 0.8 for P2281-treated mice vs. 6.83 ± 0.76 for control mice) and reversed DSS-induced shortening of the colon (colon lengths: 11.15 ± 0.3, 6.85 ± 0.39, and 7.88 ± 0.3 cms for naïve, control and P2281-treated mice, respectively); (ii) histological analyses of colonic tissues revealed that P2281 attenuated DSS-induced edema, prominently diminished the leukocyte infiltration in the colonic mucosa and resulted in protection against DSS-induced crypt damage. Conclusion: These results provide direct evidence that P2281, a novel mTOR inhibitor, suppresses DSS-induced colitis by inhibiting synthesis/release of pro-inflammatory mediators (e.g, IFN-γ) and leukocyte infiltration, and is a potentially attractive therapeutic for colitis.

Melanocortin-derived tripeptide KPV has anti-inflammatory potential in murine models of inflammatory bowel disease

Despite some progress in recent years, the options for treating inflammatory bowel disease (IBD) are still dissatisfying, and surgery rates are still high. The anti-inflammatory effects of melanocortin peptides such as alpha-melanocyte-stimulating hormone (alpha-MSH) have been described recently in, for example, dextran sodium sulfate (DSS) colitis in mice. The aim of this study was to investigate the therapeutic potential of the melanocortin-derived tripeptide alpha-MSH(11-13) (KPV) and its mode of action in 2 models of intestinal inflammation. The anti-inflammatory activity of KPV was analyzed in 2 well-described models of IBD: DSS colitis, and CD45RB(hi) transfer colitis. Furthermore, animals expressing a nonfunctional melanocortin-1 receptor (MC1Re/e) received DSS for induction of colitis and were treated with KPV. The course of inflammation was monitored by weight loss and histological changes in the colon as well as by myeloperoxidase (MPO) activity. In the DSS-colitis model, treatment with KPV led to earlier recovery and significantly stronger regain of body weight. Histologically, inflammatory infiltrates were significantly reduced in KPV-treated mice, which was confirmed by the significant reduction of MPO activity in colonic tissue after KPV treatment. Supporting these findings, KPV treatment of transfer colitis led to recovery, regain of body weight, and reduced inflammatory changes histologically. In MC1Re/e mice, KPV treatment rescued all animals in the treatment group from death during DSS colitis. The melanocortin-derived tripeptide KPV showed significant anti-inflammatory effects in 2 murine models of colitis. These effects seem to be at least partially independent of MC1R signaling. In conclusion, our data suggest KPV as an interesting therapeutic option for the treatment of IBD.

An angiotensin II receptor antagonist reduces inflammatory parameters in two models of colitis

Little is known about the effects of the pro-inflammatory hormone Angiotensin II (Ang II) in inflammatory bowel disease. The aim of this study was to evaluate the effect of valsartan (Diovan), an Ang II receptor antagonist, in two models of colitis. Methods Colitis was induced in Sprague–Dawley rats by administration of trinitrobenzene sulfonic acid (TNBS; 30 mg in 50% ETOH ic) or 5% Dextran Sulphate Sodium (DSS) in drinking water ad libitum for 5days. Valsartan was administered orally in drinking water (160 mg/L) during thirty days prior to the induction of the colitis, and for 5days after. All animals were evaluated for weight change, diarrhea, myeloperoxidase activity, macroscopic and microscopic damage. Cytokine levels in the colon were measured by ELISA, real-time RT-PCR and immunohistochemistry. Results In the TNBS model, valsartan reduced the macroscopic damage score, significantly decreased the microscopic damage ( p < 0.01), and accelerated weight gain after colitis. In the DSS-colitis model, valsartan-treated animals had less diarrhea and microscopic damage. Valsartan reduced the protein levels of TGFβ ( p < 0.05), and IL-18 in the TNBS model, and led to over expression of IL-10 mRNA in the DSS model. Conclusion These data demonstrate a possible anti-inflammatory effect for valsartan in colitis.

Characteristic of milk whey culture with Propionibacterium freudenreichii ET-3 and its application to the inflammatory bowel disease therapy

After the screening of microorganism culture, the culture of Propionibacterium freudenreichii ET-3 in the milk whey (milk whey culture) was found to stimulate the growth of our own Bifidobacteria in the colon but not the growth of other microorganisms. One of the active substances was identified as 1,4-dihydroxy-2-naphthoic acid (DHNA).In healthy volunteers, the ingestion containing milk whey culture significantly increased the population of Bifidobacteria to total fecal bacterium. In the TNBS-induced colitis model of rats, milk whey culture significantly accelerated the healing of the colitis in a dose-dependent manner. It has been reported that DHNA inhibited the lymphocyte infiltration through reduction of MAdCAM-1 in DSS colitis model of mice and that the ingestion of milk whey culture was effective in the treatment of ulcerative colitis in human pilot study. These findings suggest that milk whey culture is a useful prebiotic for the therapy of inflammatory bowel disease.

The S1P receptor modulator FTY720 prevents the development of experimental colitis in mice

To evaluate the therapeutic effects of the new synthetic sphingosine-1-phosphate (S1P) receptor modulator, FTY720, we investigated how FTY720 affects the development of dextran sulfate sodium (DSS)-induced colitis and CD4+CD62L+ T cell transfer colitis. BALB/c mice were fed a chow containing 3.5% (wt/wt) DSS to induce colitis. The CD4+CD62L+ T cell transfer colitis was induced by an intraperitoneal injection of CD4+CD62L+ spleen T cells into recipient CB17 SCID mice. The FTY720 was administered by lavage at a dose of 0.3 mg/kg/day. FTY720 was effective in preventing the body weight loss in the DSS-colitis model and the CD4+CD62L+ T cell transfer model. The disease activity index, histological colitis score, and MPO activity were all significantly lower in FTY720-treated mice than in the non-treated mice. Microscopically, mucosal edema, cellular infiltration and epithelial disruption were much more moderate in the FTY720-treated mice than in the non-treated mice. In both colitis models, FTY720 prevented the infiltration of CD4+ T cells into the inflamed colonic lamina propria. In conclusion, the development of DSS-colitis and CD4+CD62L+ T cell transfer colitis were significantly attenuated by FTY720. Since FTY720 is an immunosuppressive product that does not modulate T cell functions, it could be useful in the treatment of IBD patients.

Synthesis and Biological Evaluation of Novel Pyridazinone-Based α4Integrin Receptor Antagonists

A novel series of pyridazinone-functionalized phenylalanine analogues was prepared and evaluated for inhibition of cellular adhesion mediated by alpha4beta1/VCAM-1 and alpha4beta7/MAdCAM-1 interactions. Concise syntheses were developed and applied for exploration of structure-activity relationships pertaining to the pyridazinone ring as well as the N-acyl phenylalanine scaffold. Potent dual antagonists of alpha4beta1 and alpha4beta7 were generated from an amide subseries; antagonists selective for alpha4beta7 were identified from urea and carbamate-based subseries. The pharmacokinetic properties of selected members of the series have been determined in rats and demonstrate that the use of ester prodrugs and alterations to the amide linkage can lead to improved oral bioavailability in this series. An alpha4beta7-selective member of the carbamate subseries (36c), upon oral administration, demonstrated in vivo efficacy in the mouse DSS colitis model.

NF-κB Activation Precedes Increases in mRNA Encoding Neurokinin-1 Receptor, Proinflammatory Cytokines, and Adhesion Molecules in Dextran Sulfate Sodium–Induced Colitis in Rats

Nuclear factor kappa B (NF-kappa B) plays a key role in initiating inflammation associated with colitis. A systematic study was conducted in the rat DSS colitis model to determine the temporal relationship between NF-kappa B activation and expression of substance P (SP), neurokinin-1 receptor (NK-1R), proinflammatory cytokines, and adhesion molecules. Rats were given 5% DSS in their water and sacrificed daily for 6 days. Colon tissue was collected for assessment of histological changes, NF-kappa B activation, myeloperoxidase (MPO) activity, and expression of NK-1R, SP, TNFalpha, IL-1beta, VCAM-1, ICAM-1, E-selectin, CINC-1, MIP-1alpha, and iNOS. NF-kappa B activation increased, biphasically, on Day 1 and again on Days 4-6. The mRNA levels for ICAM-1, CINC-1, IL-1beta, TNFalpha, VCAM-1, and NK-1R rose significantly (P < 0.05) by 2-4 days. Increased iNOS mRNA levels, MPO activity, and mucosal damage occurred on Day 6. These data demonstrate that NF-kappa B activation substantially precedes the onset of physical disease signs and active inflammation.

Increased disease activity in eNOS-deficient mice in experimental colitis

Oral dextran sodium sulfate (DSS, 3%) produces experimental colitis with many features of human inflammatory bowel disease (IBD), (leukocyte extravasation, cachexia, and histopathology). Previous studies suggest that the inducible nitric oxide synthase (iNOS) in blood cells or in the endothelium contribute to this injury. However, until now no study has been performed to directly evaluate the role of endothelial nitric oxide synthase (eNOS) in IBD. We compared disease activity in wild-type (eNOS +/+) and eNOS-deficient (eNOS −/−) mice in the DSS model of colitis. Administration of DSS induced weight loss, stool blood, and overt histopathology in both mouse strains. Disease activity was dramatically increased in eNOS −/− mice compared to wild types. Histologically, eNOS-deficient mice had greater leukocyte infiltration, gut injury, and expressed higher levels of the mucosal addressin, MAdCAM-1. These results demonstrate that eNOS plays an important role in limiting injury to the intestine during experimental colitis and altered eNOS content and/or activity may contribute to human IBD.

The janus of cytokines: IL-18 displays differential roles during acute and chronic phases of intestnal inflammation

4 mice. In addition, DSStreated ct~ + mice exhibited signLficantly less leukocyte infiltration and mucosal injury as assessed by histopathology. When RAG-14mice were injected with a~ + CD4+CD45RB ~gh Tcells, we observed a significant decrease in chronic inflammation as assessed by histopatbology scores (14.0 + 2.5 vs. 8.5 -+ 1.5 for wl vs. tx~ + T-cell injected mice, respectively; p<0.01). Although colonic cytokine mRNA levels for TNF-a and IEN-'y were significantly elevated in RAG1 /mice injected with wt CD4 § CD45RB h'gh T-cells compared to RAG-1 ~ reconstituted with wt CD4+CD45RB I~ T-cells (2.4 and 15.3 fold respectively), there was no difference in colonic expression of these two cytokines compared with mRNA isolated from colons of RAG-14 mice injected with cq 4 CD4+CD45RB h~gh T-cells. Additionally, no difference was evident in the TGF-~ mRNA levels between the groups. Conclusion: These data suggest that engagement of T-cell associated a ~ with the ECM plays an important role in attenuating inflammatory tissue injury in the acute, non-immune model of DSS colitis. In addition, our data suggest that T-ceil associated a~ / engagement mediates colonic inflammation and injury via a cytokine-independent mechanism (Supported by the Broad Medical Research Program).

A novel human fibroblast growth factor treats experimental intestinal inflammation

Background & Aims: We recently identified a novel member of the human fibroblast growth factor (FGF) family of signaling molecules, designated FGF-20. In the present study, we examined the activity of this protein in 2 animal models of acute intestinal inflammation and in mechanistic studies in vitro. Methods: In vivo experiments consisted of a murine dextran sulfate sodium (DSS) model of colitis and a rat indomethacin model of small intestinal ulceration/inflammation. Cell growth, restitution, gene expression (cyclooxygenase-2 [COX-2] and intestinal trefoil factor [ITF]), and prostaglandin E2 (PGE2) levels were examined in vitro. Results: In the DSS-colitis model, prophylactic administration of FGF-20 significantly reduced the severity and extent of mucosal damage as indicated by a 55%–93% reduction in luminal blood loss, distal colonic edema, histologic inflammation, and epithelial cell loss relative to animals administered vehicle control. No toxicity was noted during administration of FGF-20 to normal controls. In addition, therapeutic administration of FGF-20 enhanced survival in this model. In the indomethacin–small bowel ulceration/inflammation model, administration of FGF-20 reduced small intestinal weight gain, necrosis, inflammation, and weight loss (36%–53% relative to vehicle control). In vitro studies demonstrated that FGF-20 stimulates growth, restitution, mRNA expression of COX-2 and ITF, and PGE2 levels in human intestinal epithelial cells and enhances the growth of human intestinal fibroblasts. Conclusions: FGF-20, having demonstrated therapeutic activity in 2 experimental models of intestinal inflammation, represents a promising new candidate for the treatment of human inflammatory bowel disease.GASTROENTEROLOGY 2002;123:1151-1162