Chronic Dextran Sulfate Sodium Research Articles

Embryonic stem cell-derived mesenchymal stem cells promote colon epithelial integrity and regeneration by elevating circulating IGF-1 in colitis mice.

Rationale: Mesenchymal stem cells (MSCs) show promising therapeutic potential in treating inflammatory bowel disease (IBD) due to their immunomodulatory and trophic functions. However, their efficacy is influenced by tissue origin, donator condition, isolation, and expansion methods. Here, we generated phenotypically uniform MSCs from human embryonic stem cells (T-MSCs) and explored the molecular mechanisms involved in promoting mucosal integrity and regeneration in colitis mice.Methods: T-MSCs were injected intravenously into mice with dextran sulfate sodium (DSS)-induced colitis, and the in vivo distribution and therapeutic efficacy were evaluated. We performed serum cytokine antibody microarrays to screen potentially effective proteins and examined the therapeutic effect of insulin-like growth factor-1 (IGF-1). Colon epithelial regeneration potential was evaluated, and RNA sequencing was employed to determine the underlying molecular mechanisms. Finally, in vitro IGF-1 stimulation was performed to assess its effect on cell functions and organoid growth.Results: Intravenous administration of T-MSCs alleviated colitis in both acute and chronic DSS mouse models. Labeled T-MSCs were mainly distributed in the lungs, liver, and spleen after systemic infusion. The antibody array analysis of serum cytokines indicated that the IGF-1 level was increased in the treatment group, and serum ELISA further confirmed its elevation in the regeneration stage. Intraperitoneal injection of IGF-1 receptor inhibitors abrogated the anti-inflammatory activity of T-MSCs. The colonic epithelium of the treatment group showed greater regenerative potency than the controls and the IGF1R-PI3K-AKT pathway was up-regulated. RNA sequencing showed that T-MSC treatment contributed to colonic cell integrity and promoted xenobiotic metabolism. In vitro IGF-1 stimulation promoted the growth and proliferation of colon cells and organoids.Conclusions: Intravenous infusion of T-MSCs alleviated colitis in mice by elevating the circulating IGF-1 level. Increased IGF-1 maintained the integrity of epithelial cells and contributed to their repair and regeneration. Our study has identified T- MSCs as a potential cell resource for IBD treatment.

Fibrogenesis in Chronic DSS Colitis is Not Influenced by Neutralisation of Regulatory T Cells, of Major T Helper Cytokines or Absence of IL-13

Mechanisms underlying fibrogenesis in chronic colitis are largely unknown. There is an urgent need for clinical markers and identification of targets to prevent, treat and limit intestinal fibrosis. This study investigated the contribution of major T cell cytokines and T regulatory cells (Tregs) to inflammation and fibrosis induced in a model of experimental colitis by oral intake of dextran sodium sulphate (DSS) in wild type and IL-13 knock-out C57Bl/6 mice. Inflammation and fibrosis were scored by macroscopic and histological examination and fibrosis was quantified by hydroxyproline. Numbers of Tregs and IFN-γ+, IL-13+ and IL-17A+ CD4+ T helper (Th) cells in mesenteric lymph nodes increased during chronic DSS administration and mRNA for IFN-γ and IL-17 in the inflamed colon tissue was upregulated. However, antibody-mediated neutralisation of IFN-γ or IL-17A/F in a therapeutic setting had no effect on chronic intestinal inflammation and fibrosis. Antibody-mediated depletion of Tregs did not enhance fibrosis, nor did IL-13 deficiency have an effect on the fibrotic disease. These data argue against an important contribution of Tregs and of the cytokines IFN-γ, IL-13, IL-17A, IL-17F in the induction and/or control of fibrosis in this Crohn’s disease like murine model.

Fecal Microbiota Transplantation Controls Murine Chronic Intestinal Inflammation by Modulating Immune Cell Functions and Gut Microbiota Composition.

Different gastrointestinal disorders, including inflammatory bowel diseases (IBD), have been linked to alterations of the gut microbiota composition, namely dysbiosis. Fecal microbiota transplantation (FMT) is considered an encouraging therapeutic approach for ulcerative colitis patients, mostly as a consequence of normobiosis restoration. We recently showed that therapeutic effects of FMT during acute experimental colitis are linked to functional modulation of the mucosal immune system and of the gut microbiota composition. Here we analysed the effects of therapeutic FMT administration during chronic experimental colitis, a condition more similar to that of IBD patients, on immune-mediated mucosal inflammatory pathways. Mucus and feces from normobiotic donors were orally administered to mice with established chronic Dextran Sodium Sulphate (DSS)-induced colitis. Immunophenotypes and functions of infiltrating colonic immune cells were evaluated by cytofluorimetric analysis. Compositional differences in the intestinal microbiome were analyzed by 16S rRNA sequencing. Therapeutic FMT in mice undergoing chronic intestinal inflammation was capable to decrease colonic inflammation by modulating the expression of pro-inflammatory genes, antimicrobial peptides, and mucins. Innate and adaptive mucosal immune cells manifested a reduced pro-inflammatory profile in FMT-treated mice. Finally, restoration of a normobiotic core ecology contributed to the resolution of inflammation. Thus, FMT is capable of controlling chronic intestinal experimental colitis by inducing a concerted activation of anti-inflammatory immune pathways, mechanistically supporting the positive results of FMT treatment reported in ulcerative colitis patients.

Protein tyrosine phosphatase non-receptor type 22 modulates colitis in a microbiota-dependent manner.

The gut microbiota is crucial for our health, and well-balanced interactions between the host's immune system and the microbiota are essential to prevent chronic intestinal inflammation, as observed in inflammatory bowel diseases (IBD). A variant in protein tyrosine phosphatase non-receptor type 22 (PTPN22) is associated with reduced risk of developing IBD, but promotes the onset of autoimmune disorders. While the role of PTPN22 in modulating molecular pathways involved in IBD pathogenesis is well studied, its impact on shaping the intestinal microbiota has not been addressed in depth. Here, we demonstrate that mice carrying the PTPN22 variant (619W mice) were protected from acute dextran sulfate sodium (DSS) colitis, but suffered from pronounced inflammation upon chronic DSS treatment. The basal microbiota composition was distinct between genotypes, and DSS-induced dysbiosis was milder in 619W mice than in WT littermates. Transfer of microbiota from 619W mice after the first DSS cycle into treatment-naive 619W mice promoted colitis, indicating that changes in microbial composition enhanced chronic colitis in those animals. This indicates that presence of the PTPN22 variant affects intestinal inflammation by modulating the host's response to the intestinal microbiota.

Diet Rich in Animal Protein Promotes Pro-inflammatory Macrophage Response and Exacerbates Colitis in Mice.

Diet is a major factor determining gut microbiota composition and perturbances in this complex ecosystem are associated with the inflammatory bowel disease (IBD). Here, we used gnotobiotic approach to analyze, how interaction between diet rich in proteins and gut microbiota influences the sensitivity to intestinal inflammation in murine model of ulcerative colitis. We found that diet rich in animal protein (aHPD) exacerbates acute dextran sulfate sodium (DSS)-induced colitis while diet rich in plant protein (pHPD) does not. The deleterious effect of aHPD was also apparent in chronic DSS colitis and was associated with distinct changes in gut bacteria and fungi. Therefore, we induced acute DSS-colitis in germ-free mice and transferred gut microbiota from aCD or aHPD fed mice to find that this effect requires presence of microbes and aHPD at the same time. The aHPD did not change the number of regulatory T cells or Th17 cells and still worsened the colitis in immuno-deficient RAG2 knock-out mice suggesting that this effect was not dependent on adaptive immunity. The pro-inflammatory effect of aHPD was, however, abrogated when splenic macrophages were depleted with clodronate liposomes. This treatment prevented aHPD induced increase in colonic Ly-6Chigh pro-inflammatory monocytes, but the ratio of resident Ly-6C−/low macrophages was not changed. These data show that the interactions between dietary protein of animal origin and gut microbiota increase sensitivity to intestinal inflammation by promoting pro-inflammatory response of monocytes.

Fasting-Mimicking Diet Modulates Microbiota and Promotes Intestinal Regeneration to Reduce Inflammatory Bowel Disease Pathology

Dietary interventions are potentially effective therapies for inflammatory bowel diseases (IBDs). We tested the effect of 4-day fasting-mimicking diet (FMD) cycles on a chronic dextran sodium sulfate (DSS)-induced murine model resulting in symptoms and pathology associated with IBD. These FMD cycles reduced intestinal inflammation, increased stem cell number, stimulated protective gut microbiota, and reversed intestinal pathology caused by DSS, whereas water-only fasting increased regenerative and reduced inflammatory markers without reversing pathology. Transplants of Lactobacillus or fecal microbiota from DSS- and FMD-treated mice reversed DSS-induced colon shortening, reduced inflammation, and increased colonic stem cells. In a clinical trial, three FMD cycles reduced markers associated with systemic inflammation. The effect of FMD cycles on microbiota composition, immune cell profile, intestinal stem cell levels and the reversal of pathology associated with IBD in mice, and the anti-inflammatory effects demonstrated in a clinical trial show promise for FMD cycles to ameliorate IBD-associated inflammation in humans.

Performance of behavioral assays: the Rat Grimace Scale, burrowing activity and a composite behavior score to identify visceral pain in an acute and chronic colitis model.

The Rat Grimace Scale (RGS), a facial expression scale, quantifies the affective component of pain in rats. The RGS was developed to identify acute and inflammatory pain, and applicability in acute and chronic visceral pain is unknown. The dextran sulfate sodium (DSS) colitis model is commonly used in rats, but pain is rarely assessed, instead, disease progression is monitored with the Disease Activity Index (DAI; assessing fecal blood, stool consistency, and weight loss). The aim of this study was to assess whether the RGS and 2 additional behavioral tools (composite behavior score [CBS] and burrowing) could identify pain in an acute and chronic DSS colitis model. Male and female Sprague-Dawley rats were block randomized to (1) acute colitis (4 days DSS in drinking water); (2) chronic colitis (4 days DSS, 7 days water, and 3 days DSS); or (3) control (14 days water). Disease Activity Index, RGS, CBS, and burrowing assessments were performed daily. Rat Grimace Scale scores increased as DAI scores increased during both acute and chronic phases. Burrowing only decreased during the acute phase. By contrast, CBS scores did not increase significantly during either colitis phase. These data show that the RGS and burrowing did not decrease in a sustained manner during chronic phase visceral pain, and that variables assessed in the DAI are indicative of pain. This suggests that the RGS can be applied to a wider range of pain types and chronicity than originally suggested. These findings increase the application of the RGS as a pain scale and welfare improvement tool.

Disease Development Is Accompanied by Changes in Bacterial Protein Abundance and Functions in a Refined Model of Dextran Sulfate Sodium (DSS)-Induced Colitis

Using the acute dextran sulfate sodium (DSS)-induced colitis model, studies have demonstrated that intestinal inflammation is accompanied by major changes in the composition of the intestinal microbiota. Only little is known about the microbial changes and more importantly their functional impact in the chronic DSS colitis model. We used a refined model of chronic DSS-induced colitis that reflects typical symptoms of the human disease without detrimental weight loss usually observed in DSS models. We sampled cecum and colon content as well as colon mucus from healthy and diseased mouse cohorts ( n = 12) and applied 16S rRNA gene sequencing and metaproteomics. An increase of Prevotella sp. in both colon content and mucus was observed. Functional differences were observed between sample types demonstrating the importance of separately sampling lumen content and mucus. The abundance of Desulfovibrio, a sulfate-reducing bacterium, was positively associated with the carbon metabolism. Lachnoclostridium was positively correlated to both vitamin B6 and tryptophan metabolism. In summary, functional changes in the distal colon caused by DSS treatment were more pronounced in the mucus-associated microbiota than in the microbiota present in the distal colon content.

The EBI2-oxysterol axis promotes the development of intestinal lymphoid structures and colitis

The gene encoding for Epstein-Barr virus-induced G-protein-coupled receptor 2 (EBI2) is a risk gene for inflammatory bowel disease (IBD). Together with its oxysterol ligand 7α,25-dihydroxycholesterol, EBI2 mediates migration and differentiation of immune cells. However, the role of EBI2 in the colonic immune system remains insufficiently studied. We found increased mRNA expression of EBI2 and oxysterol-synthesizing enzymes (CH25H, CYP7B1) in the inflamed colon of patients with ulcerative colitis and mice with acute or chronic dextran sulfate sodium (DSS) colitis. Accordingly, we detected elevated levels of 25-hydroxylated oxysterols, including 7α,25-dihydroxycholesterol in mice with acute colonic inflammation. Knockout of EBI2 or CH25H did not affect severity of DSS colitis; however, inflammation was decreased in male EBI2−/− mice in the IL-10 colitis model. The colonic immune system comprises mucosal lymphoid structures, which accumulate upon chronic inflammation in IL-10-deficient mice and in chronic DSS colitis. However, EBI2−/− mice formed significantly less colonic lymphoid structures at baseline and showed defects in inflammation-induced accumulation of lymphoid structures. In summary, we report induction of the EBI2-7α,25-dihydroxycholesterol axis in colitis and a role of EBI2 for the accumulation of lymphoid tissue during homeostasis and inflammation. These data implicate the EBI2-7α,25-dihydroxycholesterol axis in IBD pathogenesis.

10 A VARIANT OF CHOLERA TOXIN B SUBUNIT ENHANCES COLON EPITHELIAL WOUND HEALING VIA AN UNFOLDED PROTEIN RESPONSE

Cholera toxin B subunit (CTB) exhibits multifaceted biological activity in the mucosa via binding to GM1 ganglioside on intestinal epithelial cells. We recently found that a recombinant variant of CTB, which contains an artificial KDEL endoplasmic reticulum (ER) retention signal (CTB-KDEL), induces mucosal healing in a mouse dextran sodium sulfate (DSS) acute colitis model. The objectives of this study are to investigate CTB-KDEL’s epithelial wound repair mechanism and characterize its efficacy in preclinical models. We hypothesize that CTB-KDEL’s wound healing effect is mediated through ER retention within epithelial cells, thereby facilitating mucosal healing in IBD. The effects of CTB-KDEL, CTB and G33D-CTB-KDEL (a non-GM1-binding variant) were analyzed for cell migration, subcellular localization, an unfolded protein response (UPR) and TGFβ expression in Caco2 cells. In a DSS acute colitis model, C57BL/6J mice were gavaged with a single dose of CTB-KDEL, CTB or PBS at the end of 7-day DSS exposure. An optimal dosing regimen of CTB-KDEL was evaluated in mice exposed to repeated DSS cycles. Efficacy was measured on the basis of colon length, Disease Activity Index, histopathological scoring, E-cadherin immunohistochemistry and wound healing gene/cytokine expression in the colon. For ex vivohuman colonic explant experiments, colon tissues were obtained from consenting IBD patients who underwent total colectomy, which were cultured with CTB-KDEL, CTB or a PBS for 24 hours. Caco2 cell migration was enhanced by CTB-KDEL at concentrations of 0.3 – 3 μM, while CTB and G33D-CTB-KDEL failed to show such an effect. CTB-KDEL showed longer residence in the ER than CTB, leading to the activation of UPRs, particularly the inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1 (XBP1) arm, and subsequent TGFβ signaling in Caco2 cells. In the acute colitis model, CTB-KDEL, but not CTB, facilitated recovery from colonic damage as evident in macroscopic and histological evaluations, with significant upregulation of wound healing genes and E-cadherin positive staining in the colon (Fig. 1). Weekly oral administration of 3 μg CTB-KDEL showed significant efficacy against established colitis in the chronic DSS model. In human IBD colon explants, CTB-KDEL at 1 μM induced significant upregulation of the IRE1/Xbp1 pathway as well as wound healing genes including TGFB1and the E-cadherin gene CDH1, along with the retention of viable crypts in the colon. The results demonstrated CTB-KDEL’s unique colon epithelial wound healing activity via a UPR, providing implications for new IBD treatment strategies.

CCR2+ Monocyte-Derived Ly6C-F4/80+ Fibrocytes Inhibit Collagen Degradation and Contribute to the Development of Colon Fibrosis Via Production of TIMP-1

Introduction: Fibrocytes are derived from a subset of monocytes and express mesenchymal markers such as collagen type I (Col I) and hematopoietic markers such as CD45 and CD11b. They also express several chemokine receptors such as CC chemokine receptor (CCR)-1, CCR2, CCR5, CCR7, and CXC chemokine receptor type 4 (CXCR4). They circulate in the peripheral blood (PB) and can be isolated from many fibrotic tissues. Fibrocytes participate in both physiological wound healing and various pathological fibrosis including myelofibrosis, hypertrophic scar, systemic sclerosis, idiopathic pulmonary fibrosis, liver cirrhosis, and progressive kidney disease. In murine and human colitis, fibrocytes are also reported to be associated with the colon fibrosis. It has been described that migration of fibrocytes to the injured sites involves CC chemokine ligand 2 (CCL2)/CCR2 axis in the liver and kidney and CXC chemokine ligand 12 (CXCL12)/CXCR4 axis in the lung. However, there are few reports concerning the role of fibrocytes and their expression of chemokine receptors related to the induction of colon fibrosis.Methods: We generated bone marrow (BM) chimeric mice by transplantation of BM total-nucleated cells, which were isolated from enhanced green fluorescent protein (EGFP)-transgenic mice or CCR2 knockout (KO) mice, into lethally irradiated C57BL/6J-Ly5.1 mice. Two months after BM transplantation, BM chimeric mice were treated with a single intraperitoneal injection of azoxymethane (10 mg/kg body weight) followed by 3 cycles of 1% dextran sulfate sodium (DSS) in the drinking water. We assessed the level of fibrosis in the colon using Sirius red staining and analyzed the presence of BM-derived CD45+CD11b+Col I+ fibrocytes in the colon lamina propria (LP) using immunofluorescence staining and flow cytometry. Furthermore, we investigated the expressions of Col I, transforming growth factor-ß (TGF- ß), matrix metalloproteinases (MMPs), and tissue inhibitor of MMPs (TIMP)-1 in the colon tissues and fibrocytes sorted from colon LP cells after chronic DSS treatment using quantitative real-time RT-PCR.Results: During chronic inflammation, infiltration of CCR2+ BM-derived monocytes and fibrocytes and production of CCL2 in the colon were particularly increased and colon fibrosis was developed in EGFP BM chimeric mice. Two types of fibrocytes, CCR2+CXCR4+Ly6C-F4/80+ fibrocytes and CCR2-CXCR4+Ly6ChighF4/80- fibrocytes, were identified in the colon LP, whereas only the latter fibrocytes were detected in the PB. Adoptive transferred CCR2+Ly6ChighCol I- monocytes migrated to the injured colon and a part of them differentiated into CCR2+Col I+ fibrocytes. In CCR2KO BM chimeric mice, the numbers of monocytes and fibrocytes in the colon LP were significantly decreased and colon fibrosis was attenuated. However, there was no difference in the mRNA expressions of Col I, TGF-ß, and MMPs (MMP-1a, MMP-8, and MMP-13, known as collagenases) in colon tissues between EGFP BM chimeric mice and CCR2KO BM chimeric mice. Improvement of colon fibrosis in CCR2KO BM chimeric mice was associated with the decreased expression of Timp1 mRNA in colon tissues. We analyzed the expression of Timp1 mRNA in CCR2+ cells and CCR2- cells sorted from colon LP cells and found a high expression of Timp1 in CCR2+ monocytes/macrophages and fibrocytes.Conclusions: Circulating CCR2+ monocytes migrate into the inflamed colon via CCL2/CCR2 axis and differentiate into CCR2+Ly6C-F4/80+ fibrocytes, which inhibit collagen degradation and contribute to the development of colon fibrosis by the production of TIMP-1. DisclosuresMasuya:Kyowa Hakko Kirin Co., Ltd.: Research Funding. Katayama:Ono Pharmaceutical: Research Funding; Novo Nordisk: Honoraria, Research Funding; Chugai Pharma: Honoraria, Research Funding; Toyama Chemical Co: Research Funding; Sysmex: Honoraria; Mochida Pharmaceutical Co. Ltd.,: Research Funding; Bristol-Myers Squibb: Honoraria; Astellas Pharma: Honoraria, Research Funding; Daiichi Sankyo: Research Funding; Takeda: Honoraria, Research Funding; Teijin Pharma: Research Funding; Eisai: Research Funding; Sumitomo Group: Honoraria, Research Funding; Nippon Shinyaku: Honoraria, Research Funding; Shire: Honoraria; Alexion Pharmaceuticals: Honoraria; Celgene: Honoraria; Taisho Toyama Pharma: Honoraria; Pfizer: Honoraria, Research Funding; Shionogi Pharmaceutical: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen: Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding.

Environmental Factors Modify the Severity of Acute DSS Colitis in Caspase-11-Deficient Mice.

Human and mouse studies implicate the inflammasome in the pathogenesis of inflammatory bowel diseases, though the effects in mice are variable. The noncanonical inflammasome activator caspase-11 (Casp11) reportedly attenuates acute dextran sodium sulfate (DSS) colitis in mice. However, the effects of Casp11 on chronic experimental colitis and factors that influence the impact of Casp11 on acute DSS colitis are unknown. We studied the role of Casp11 in Il10-/- mice and acute and chronic DSS colitis mouse models. We quantified colonic Casp11 mRNA using quantative polymerase chain reaction and colitis using weight loss, blinded histological scoring, IL-12/23p40 secretion by colonic explants, and fecal lipocalin-2. We determined fecal microbial composition using 16S amplicon sequencing. We detected increased colonic Casp11 mRNA in Il10-/- mice with chronic colitis, but not in mice with DSS colitis. The presence of Casp11 did not alter the severity of chronic colitis in DSS-treated or Il10-/- mice. Contrary to prior reports, we initially observed that Casp11 exacerbates acute DSS colitis. Subsequent experiments in the same animal facility revealed no effect of Casp11 on acute DSS colitis. There were pronounced stochastic changes in the fecal microbiome over this time. The majority of bacterial taxa that changed over time in wild-type vs Casp11-/- mice belong to the Clostridiales. Casp11 does not impact chronic experimental colitis, and its effects on acute DSS colitis vary with environmental factors including the microbiota, particularly Clostridiales. Stochastic drifts in intestinal microbiota composition, even in mice in the same housing facility, should be considered when interpreting studies of acute DSS colitis models.

Effect of Eucommia ulmoides Leaf Extract on Chronic Dextran Sodium Sulfate-Induced Colitis in Mice.

Ulcerative colitis (UC) is a refractory disease that causes chronic inflammation or ulceration in the mucosa of the large intestine with multiple relapses. Although several drugs, including 5-aminosalicylic acid, steroids, immunosuppressants, and infliximab, are used for UC therapy, patients suffer from side effects of these drugs, and a new safer therapeutic agent is desired. Eucommia ulmoides OLIV. leaf extract (ELE) has an anti-inflammatory effect. Therefore, we examined the effect of ELE on UC using a chronic dextran sulfate sodium (DSS)-induced colitis model in mice. Chronic DSS-induced colitis was triggered by alternately repeating 5 days' DSS and 7 days' water administration in mice for 29 d. The severity of DSS-induced colitis was evaluated by daily body weight and bloody stool score, and colon length and myeloperoxidase (MPO) activity in colon tissue on day 29. ELE (3 or 9%) was administered in combination by feeding for 29 d, and the effect on colitis was evaluated. The mice given DSS exhibited chronic colitis symptoms with body weight loss, increased bloody stool score and MPO activity, and shortened colon length. Administration of 3 or 9% ELE suppressed the body weight loss, bloody stool score, colon shortening, and MPO activity in a dose-dependent manner. Histological analysis showed that the ELE-treated mice had less damages and leukocyte infiltration in the mucosal layer of the large intestine compared to DSS alone group. These results suggested that ELE has the potential to prevent the development of DSS-induced colitis and a therapeutic effect on UC in a safe manner.

Role of MAdCAM-1-Expressing High Endothelial Venule-Like Vessels in Colitis Induced in Mice Lacking Sulfotransferases Catalyzing L-Selectin Ligand Biosynthesis.

Ulcerative colitis (UC) is a chronic inflammatory disease histologically characterized by diffuse mononuclear cell infiltrates in colonic mucosa. These inflammatory cells are considered to be recruited via high endothelial venule (HEV)-like vessels displaying mucosal addressin cell adhesion molecule 1 (MAdCAM-1), the ligand for α4β7 integrin, and/or peripheral lymph node addressin (PNAd), an L-selectin ligand. 6- O-sulfation of N-acetylglucosamine in the carbohydrate moiety of PNAd is catalyzed exclusively by N-acetylglucosamine-6- O-sulfotransferase 1 (GlcNAc6ST-1) and GlcNAc6ST-2. To determine the role of 6- O-sulfation of N-acetylglucosamine on HEV-like vessels in UC, we used a chronic dextran sulfate sodium-induced colitis model using mice deficient in both GlcNAc6ST-1 and GlcNAc6ST-2. We found that more inflammatory cells, with expression of tumor necrosis factor α, were infiltrated in double knockout mouse colitis compared with that in wild-type mice. Moreover, the number of MAdCAM-1-positive vessels was increased in double knockout mouse colitis, and these vessels were bound by E-selectin•IgM chimeras that bind to unsulfated sialyl Lewis X (sLeX). These findings suggest that interactions between MAdCAM-1 and α4β7 integrin and/or unsulfated sLeX and L-selectin may become a dominant mechanism for inflammatory cell recruitment in the absence of 6-sulfo sLeX and contribute to more severe colitis phenotypes seen in double knockout mice.

A refined and translationally relevant model of chronic DSS colitis in BALB/c mice.

Inflammatory bowel diseases (IBD) are chronic relapsing disorders of the gastrointestinal tract. Several mouse models for IBD are available, but the acute dextran sulfate sodium (DSS)-induced colitis model is mostly used for preclinical studies. However, this model lacks chronicity and often leads to significant loss of mice. The aim of this study was to establish a refined and translationally relevant model of DSS chronic colitis in BALB/c mice. In the first part, we compared several standard therapeutic (ST) treatments for IBD in the acute DSS colitis model to identify the optimal treatment control for a DSS colitis model as compared to literature data. In the second part, we tested the two most effective ST treatments in a refined model of chronic DSS colitis. Cyclosporine A (CsA) and 6-thioguanine (6-TG) caused considerable reduction of clinical scores in acute DSS colitis. The clinical outcome was confirmed by the results for colon length and by histopathological evaluation. Moreover, CsA and 6-TG considerably reduced mRNA expression of several pro-inflammatory cytokines in spleen and colon. Both compounds also showed a substantial therapeutic effect in the refined model of chronic DSS colitis with regard to clinical scores and histopathology as well as the expression of inflammatory markers. The refined model of chronic DSS colitis reflects important features of IBD and is well suited to test potential IBD therapeutics.

The Proton-activated Receptor GPR4 Modulates Intestinal Inflammation.

During active inflammation, intraluminal intestinal pH is decreased in patients with inflammatory bowel disease [IBD]. Acidic pH may play a role in IBD pathophysiology. Recently, proton-sensing G-protein coupled receptors were identified, including GPR4, OGR1 [GPR68], and TDAG8 [GPR65]. We investigated whether GPR4 is involved in intestinal inflammation. The role of GPR4 was assessed in murine colitis models by chronic dextran sulphate sodium [DSS] administration and by cross-breeding into an IL-10 deficient background for development of spontaneous colitis. Colitis severity was assessed by body weight, colonoscopy, colon length, histological score, cytokine mRNA expression, and myeloperoxidase [MPO] activity. In the spontaneous Il-10-/- colitis model, the incidence of rectal prolapse and characteristics of lamina propria leukocytes [LPLs] were analysed. Gpr4-/- mice showed reduced body weight loss and histology score after induction of chronic DSS colitis. In Gpr4-/-/Il-10-/- double knock-outs, the onset and progression of rectal prolapse were significantly delayed and mitigated compared with Gpr4+/+/Il-10-/- mice. Double knock-out mice showed lower histology scores, MPO activity, CD4+ T helper cell infiltration, IFN-γ, iNOS, MCP-1 [CCL2], CXCL1, and CXCL2 expression compared with controls. In colon, GPR4 mRNA was detected in endothelial cells, some smooth muscle cells, and some macrophages. Absence of GPR4 ameliorates colitis in IBD animal models, indicating an important regulatory role in mucosal inflammation, thus providing a new link between tissue pH and the immune system. Therapeutic inhibition of GPR4 may be beneficial for the treatment of IBD.

Therapeutic efficacy of a combined sage and bitter apple phytopharmaceutical in chronic DSS-induced colitis

Inflammatory bowel diseases are multifactorial disorders of the gastrointestinal tract with rising incidence worldwide. Current standard therapies are only partially effective and often show severe adverse effects. Thus, novel, more efficient and well-tolerated therapeutic options are urgently needed. We have studied the therapeutic potential of a phytopharmaceutical combining sage and bitter apple (SBA) in the mouse model of chronic dextran sulfate sodium (DSS) colitis. SBA represents a traditional medicine against diarrhea and was shown to exhibit anti-inflammatory effects in vitro. In the chronic DSS colitis model SBA treatment significantly reduced clinical symptoms in a dose-dependent manner. The positive therapeutic effect of SBA was characterized by a decreased histopathological score indicating tissue healing. Moreover, the number of neutrophils as well as the expression of the neutrophil-recruiting chemokine CXCL-1/KC in the colon tissue was significantly reduced, whereas the recruitment of macrophages was induced. Also, the expression of inflammatory markers was significantly suppressed, while the expression of the anti-inflammatory cytokine interleukin-10 was induced in colon tissue following treatment with SBA. Phytopharmaceuticals are increasingly recognized as potential therapeutics in IBD. Thus, based on the results from this study, SBA can be considered as an alternative or supplementary option for IBD therapy.

Epithelial TNF Receptor Signaling Promotes Mucosal Repair in Inflammatory Bowel Disease.

TNF plays an integral role in inflammatory bowel disease (IBD), as evidenced by the dramatic therapeutic responses in Crohn's disease (CD) patients induced by chimeric anti-TNF mAbs. However, treatment of CD patients with etanercept, a decoy receptor that binds soluble TNF, fails to improve disease. To explore this discrepancy, we investigated the role of TNF signaling in Wnt/β-catenin-mediated intestinal stem cell and progenitor cell expansion in CD patients, human cells, and preclinical mouse models. We hypothesized that TNF exerts beneficial effects on intestinal epithelial cell (IEC) responses to injury. In CD patients, intestinal stem cell and progenitor cell Wnt/β-catenin signaling correlates with inflammation status. TNF-deficient (Tnf-/-) mice exhibited increased apoptosis, less IEC proliferation, and less Wnt signaling when stimulated with anti-CD3 mAb. Bone marrow (BM) chimera mice revealed that mucosal repair depended on TNF production by BM-derived cells and TNFR expression by radioresistant IECs. Wild-type→Tnfr1/2-/- BM chimera mice with chronic dextran sodium sulfate colitis exhibited delayed ulcer healing, more mucosal inflammation, and impaired Wnt/β-catenin signaling, consistent with the hypothesis that epithelial TNFR signaling participates in mucosal healing. The direct effect of TNF on stem cells was demonstrated by studies of TNF-induced Wnt/β-catenin target gene expression in murine enteroids and colonoid cultures and TNF-induced β-catenin activation in nontransformed human NCM460 cells (TOPFlash) and mice (TOP-GAL). Together, these data support the hypothesis that TNF plays a beneficial role in enhancing Wnt/β-catenin signaling during ulcer healing in IBD. These novel findings will inform clinicians and therapeutic chemists alike as they strive to develop novel therapies for IBD patients.

Treatment of Intestinal Fibrosis in Experimental Inflammatory Bowel Disease by the Pleiotropic Actions of a Local Rho Kinase Inhibitor

Intestinal fibrosis resulting in (sub)obstruction is a common complication of Crohn's disease (CD). Rho kinases (ROCKs) play multiple roles in TGFβ-induced myofibroblast activation that could be therapeutic targets. Because systemic ROCK inhibition causes cardiovascular side effects, we evaluated the effects of a locally acting ROCK inhibitor (AMA0825) on intestinal fibrosis. Fibrosis was assessed in mouse models using dextran sulfate sodium (DSS) and adoptive T-cell transfer. The invitro and exvivo effects of AMA0825 were studied in different cell types and in CD biopsy cultures. ROCK is expressed in fibroblastic, epithelial, endothelial, and muscle cells of the human intestinal tract and is activated in inflamed and fibrotic tissue. Prophylactic treatment with AMA0825 inhibited myofibroblast accumulation, expression of pro-fibrotic factors, and accumulation of fibrotic tissue without affecting clinical disease activity and histologic inflammation in 2 models of fibrosis. ROCK inhibition reversed established fibrosis in a chronic DSS model and impeded exvivo pro-fibrotic protein secretion from stenotic CD biopsies. AMA0825 reduced TGFβ1-induced activation of myocardin-related transcription factor (MRTF) and p38 mitogen-activated protein kinase (MAPK), down-regulating matrix metalloproteinases, collagen, and IL6 secretion from fibroblasts. In these cells, ROCK inhibition potentiated autophagy, which was required for the observed reduction in collagen and IL6 production. AMA0825 did not affect pro-inflammatory cytokine secretion from other ROCK-positive cell types, corroborating the selective invivo effect on fibrosis. Local ROCK inhibition prevents and reverses intestinal fibrosis by diminishing MRTF and p38 MAPK activation and increasing autophagy in fibroblasts. Overall, our results show that local ROCK inhibition is promising for counteracting fibrosis as an add-on therapy for CD.

Inhibition of gelatinase B/MMP-9 does not attenuate colitis in murine models of inflammatory bowel disease

One third of patients with inflammatory bowel disease (IBD) inadequately respond to anti-TNF treatment and preclinical data suggest that matrix metalloproteinase-9 (MMP-9) is a novel therapeutic target. Here we show that IBD clinical and histopathological parameters found in wild type mice challenged with three different models of colitis, acute and chronic dextran sodium sulphate (DSS), and acute 2,4,6-trinitrobenzenesulfonic acid-induced colitis are not attenuated in MMP-9 knockout mice. We find similar colonic gene expression profiles in wild type and MMP-9 knockout mice in control and acute DSS conditions with the exception of eleven genes involved in antimicrobial response during colitis. Parameters of chronic colitis are similar in wild type and MMP-9 knockout mice. Pharmacological inhibition of MMP-9 with bio-active peptides does not improve DSS colitis. We suggest that MMP-9 upregulation is a consequence rather than a cause of intestinal inflammation and we question whether MMP-9 represents a disease target in IBD.