Mouse Model Of Crohn's Disease Research Articles

α-Methyl-Tryptophan Inhibits SLC6A14 Expression and Exhibits Immunomodulatory Effects in Crohn's Disease.

Crohn's disease (CD) is a chronic inflammatory condition of the intestines with a rising global incidence. Traditional diagnostic and therapeutic methods have limitations, necessitating the exploration of more effective strategies. In this study, we employed the Gene Expression Omnibus database to identify genes that are differentially expressed in CD. RT-PCR and immunohistochemical analysis were used to SLC6A14 RNA and protein expression in the colons of CD mice and CD tissues from patients. The mouse model of CD was induced by dextran sodium sulfate (DSS). Infiltrating immune cells in mouse model were screened by flow cytometry. We discovered that SLC6A14 is significantly overexpressed in CD samples, and its expression is positively correlated with the degree of infiltration by CD4+ and CD8+ T cells. The elevated levels of SLC6A14 RNA and protein were confirmed in clinical CD tissues. The SLC6A14 inhibitor α-methyl-tryptophan (α-MT) significantly decreased the expression of SLC6A14 RNA and protein in the colons of CD mice. The α-MT treatment group also exhibited reduced levels of cytokines involved in T cell differentiation (IFN-γ and TNF-α) and the expression of immune cell surface markers CXCR-3 and LAG-3. Flow cytometry analysis revealed a significant increase in the infiltration of CD4+ and CD8+ T cells in the DSS-treated group compared to the control group. Conversely, the α-MT treatment group showed a significant reduction in CD4+ and CD8+ T cell infiltration and the restoration of intestinal parameters in CD mice. These findings underscore the role of SLC6A14 in regulating intestinal immune cell infiltration during CD progression. Our findings suggest that SLC6A14 could serve as a potential diagnostic biomarker and therapeutic target for CD. Furthermore, α-MT offers a novel approach for the clinical diagnosis and treatment of CD by targeting SLC6A14 for therapeutic intervention.

A New Probiotic Formulation Promotes Resolution of Inflammation in a Crohn's Disease Mouse Model by Inducing Apoptosis in Mucosal Innate Immune Cells.

The interaction between gut-residing microorganisms plays a critical role in the pathogenesis of Crohn's disease (CD), where microbiome dysregulation can alter immune responses, leading to unresolved local inflammation. The aim of this study is to analyze the immunomodulatory properties of a recently developed probiotic + amylase blend in the SAMP1/YitFc (SAMP) mouse model of CD-like ileitis. Four groups of SAMP mice were gavaged for 56 days with the following treatments: 1) probiotic strains + amylase (0.25 mg/100 µL PBS); 2) only probiotics; 3) only amylase; PBS-treated controls. Ilea were collected for GeoMx Digital Spatial Profiler (DSP) analysis and histological evaluation. Histology assessment for inflammation indicated a significantly reduced level of ileitis in mice administered the probiotics + amylase blend. DSP analysis showed decreased abundance of neutrophils and increased abundance of dendritic cells, regulatory T cells, and macrophages, with a significant enrichment of five intracellular pathways related to apoptosis, in probiotics + amylase-treated mice. Increased apoptosis occurrence was confirmed by (TdT)- deoxyuridine triphosphate (dUTP)-biotin nick end labeling assay. Our data demonstrate a beneficial role of the probiotic and amylase blend, highlighting an increased apoptosis of innate immunity-associated cell subsets, thus promoting the resolution of inflammation. Hence, we suggest that the developed probiotic enzyme blend may be a therapeutic tool to manage CD and therefore is a candidate formulation to be tested in clinical trials.

Targeting NCAPD2 as a Therapeutic Strategy for Crohn's Disease: Implications for Autophagy and Inflammation.

Our earlier studies identified that non-SMC condensin I complex subunit D2 (NCAPD2) induces inflammation through the IKK/NF-κB pathway in ulcerative colitis. However, its role in the development of Crohn's disease (CD) and the specific molecular mechanism still need to be further studied. NCAPD2 expression in clinical ileal CD mucosa vs normal mucosa was examined, alongside its correlation with CD patients' clinical characteristics via their medical records. The biological function and molecular mechanism of NCAPD2 in CD were explored using a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced CD mouse model, along with immunofluorescence, western blot, quantitative real-time PCR, immunohistochemistry, hematoxylin and eosin staining, and cell functional analysis. NCAPD2 was overexpressed in CD tissues and significantly correlated with disease activity in CD patients (P = .016). In a TNBS-induced CD mouse model, NCAPD2 knockdown inhibited the development of TNBS-induced intestinal inflammation in mice. In addition, we found that NCAPD2 inhibited autophagy. Mechanistically, NCAPD2 promoted the phosphorylation of mammalian target of the rapamycin (mTOR) and its direct effector S6K and downregulated the expression of autophagy-related proteins Beclin1, LC3II, and Atg5. In addition, NCAPD2 activates the NF-κB signaling pathway, and the downstream inflammatory factors are continuously released, leading to the persistence of inflammation. Our results show that NCAPD2 suppresses autophagy and worsens intestinal inflammation by modulating mTOR signaling and impacting the NF-κB pathway, suggesting a critical role in CD progression. Targeting NCAPD2 could be a promising therapeutic approach to stop CD advancement.

Targeting AQP9 enhanced the anti-TNF therapy response in Crohn's disease by inhibiting LPA-hippo pathway

Although anti-TNF antibodies are extensively used to treat Crohn's disease (CD), a significant proportion of patients, up to 40%, exhibit an inadequate response to this therapy. Our objective was to identify potential targets that could improve the effectiveness of anti-TNF therapy in CD. Through the integration and analysis of transcriptomic data from various CD databases, we found that the expression of AQP9 was significantly increased in anti-TNF therapy-resistant specimens. The response to anti-TNF therapy in the CD mouse model was significantly enhanced by specifically inhibiting AQP9. Further experiments found that the blockade of AQP9, which is dominantly expressed in macrophages, decreased inflamed macrophage functions and cytokine expression. Mechanistic studies revealed that AQP9 transported glycerol into macrophages, where it was metabolized to LPA, which was further metabolized to LPA, resulting in the activation of the LPAR2 receptor and downstream hippo pathway, finally promoting the expression of cytokines, especially IL23 and IL1β⊡ Taken together, the expansion of AQP9+ macrophages is associated with resistance to anti-TNF therapy in Crohn’s disease. These findings indicated that AQP9 could be a potential target for enhancing anti-TNF therapy in Crohn’s disease.

Genetic and Small-Molecule Modulation of Stat3 in a Mouse Model of Crohn's Disease.

Crohn's disease (CD), is an inflammatory bowel disease that can affect any part of the gastro-intestinal tract (GI) and is associated with an increased risk of gastro-intestinal cancer. In the current study, we determined the role of genetic and small-molecule modulation of STAT3 in a mouse model of CD. STAT3 has 2 isoforms (α, β) which are expressed in most cells in a 4:1 ratio (α: β). STAT3α has pro-inflammatory and anti-apoptotic functions, while STAT3β has contrasting roles. We used an animal model of CD consisting of intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid and examined the severity of CD in transgenic-mice that express only STAT3α (∆β/∆β), as well as in wild-type (WT) mice administered TTI-101 (formerly C188-9), a small molecule STAT3 inhibitor. We determined that clinical manifestations of CD, such as mortality, rectal-bleeding, colonic bleeding, diarrhea, and colon shortening, were exacerbated in ∆β/∆β transgenic versus cage-control WT mice, while they were markedly decreased by TTI-101 treatment of WT mice. TTI-101 treatment also increased apoptosis of pathogenic CD4+ T cells and reduced colon levels of IL-17-positive cells. Our results indicate that STAT3 contributes to CD and that targeting of STAT3 with TTI-101 may be a useful approach to treating CD.

Herbs-partitioned moxibustion improves intestinal epithelial tight junctions by upregulating A20 expression in a mouse model of Crohn’s disease

BackgroundTo investigate effects moxibustion exerts on A20 expression and regulation of intestinal epithelial tight junctions via the TNF-α-NF-κB-MLCK pathway in Crohn's disease (CD). MethodsC57BL/6 wild type (WT) and A20IEC−KO mice (48 each) were randomly divided into normal control (NC), model control (MC), mesalazine (MESA) and herbs-partitioned moxibustion (HPM) groups (12 mice per group). An experimental model of CD was established using 2, 4, 6 trinitrobenzene sulfonic acid. MESA and HPM mice were treated with MESA and HPM (at Tianshu (ST25) and Qihai (CV6)), respectively. In HPM group, moxa cones (0.5 cm in diameter and 0.3 cm in height) made of refined mugwort floss were placed on herbal cakes (medicinal formula dispensing [radix] Aconiti praeparata, [cortex] Cinnamomi, etc.) at Tianshu (ST25) and Qihai (CV6) and ignited. The moxa cones were ignited, and two moxa cones were used for each treatment once daily for 10 days. In MESA group, mice were fed MESA, which was prepared at a proportion of 1:0.0026, twice daily for 10 days. ResultsIntestinal epithelial ultrastructure of WT HPM mice improved more than A20IEC−KO HPM mice compared to MC mice. WT HPM mice exhibited greater expression of A20 compared with MC mice (P < 0.01). TNF-α, NF-kB p65, MLCK, MLC, TRAF6 and RIP1 levels in A20IEC−KO and WT HPM mice were all decreased compared to MC mice (Pall < 0.01). NF-κB p65、MLCK and TRAF6 levels were increased in A20IEC−KO HPM mice as compared to WT HPM mice (Pall < 0.05). Intestinal epithelial levels of occludin, claudin-1, ZO-1 and F-actin increased in all HPM mice (Pall < 0.01–0.05), while occludin, claudin-1, and ZO-1 levels were lower in A20 IEC-KO HPM mice (P < 0.05, P < 0.01, P < 0.01). ConclusionHPM downregulates abnormal activation of the TNF-α-NF-κB-MLCK pathway by upregulating expression of A20 in a mouse model of CD, thereby protecting intestinal epithelial tight junctions and repairing the damage CD causes to the intestinal epithelial barrier.

Dietary Nondigestible Polysaccharides Ameliorate Colitis by Improving Gut Microbiota and CD4+ Differentiation, as Well as Facilitating M2 Macrophage Polarization.

The aim of this study was to investigate the therapeutic mechanism of a specific multifiber mix diet (MF) designed to match the fiber content of a healthy diet in interleukin-10 knockout (IL-10-/- ) mice with spontaneous chronic colitis displaying similar characteristics to those of human Crohn's disease (CD). Sixteen-week-old IL-10-/- mice were used for the experiments with MF diet for 4 weeks. Severity of colitis, the composition of the fecal microbiota, expression of Th1/Th17 cells, myeloperoxidase (MPO) concentrations, and inflammatory cytokines and chemokines (tumor necrosis factor-α [TNF-α], IL-6, macrophage inflammatory protein [MIP]-2, monocyte chemoattractant protein-1 [MCP-1], and MIP-1α), as well as arginase 1 (Arg1) and signal transducers and activators of transcription 6 (STAT6) proteins, were measured at the end of the experiment. In addition, the corresponding metabolites (short-chain fatty acids) of MF on CD4+ CD25+ Foxp3+ regulatory T cells (Tregs) were also detected in vivo and in vitro. MF treatment significantly ameliorated colitis associated with decreased lamina propria frequency of Th1/Th17 cells, MPO concentrations, and inflammatory cytokines and chemokines (TNF-α, IL-6, MIP-2, MCP-1, and MIP-1α). An increase in gut microbial diversity was observed after MF treatment compared with IL-10-/- mice, including a significant increase in bacteria belonging to the Firmicutes phylum and a significant decrease in bacteria belonging to the Proteobacteria phylum. Moreover, MF treatment increased the differentiation of CD4+ CD25+ Foxp3+ Tregs mainly by microbial metabolites butyrate. In addition, Arg1 and STAT6 proteins were also significantly increased after MF treatment. These results shed light on the contribution of MF treatment to the CD mouse model and suggest that MF has potential as a therapeutic strategy for enhancing efficacy in inducing remission in patients with active CD.

TNFΔARE Mice Display Abnormal Lymphatics and Develop Tertiary Lymphoid Organs in the Mesentery

Chronic inflammatory diseases are associated with a persistent and enhanced response to environmental antigens. As an adaptive response to this exaggerated immune state, affected tissue typically develops tertiary lymphoid organs. Studies of Crohn disease (CD), a chronic inflammatory disease of the intestinal tract, report tertiary lymphoid organs present within the mucosal wall, along with other lymphatic diseases, such as lymphangiogenesis and obstructed lymphatic vessels. These observations suggest that downstream mesenteric lymphatic vessels and lymph drainage into mesenteric lymph nodes may be compromised. However, information is lacking on the morphologic features and functional status of mesenteric lymphatics in CD. Using confocal imaging, PCR, flow cytometry, and functional strategies, we addressed these questions in the established TNFΔARE mouse model of CD and found that this mouse model had many lymphatic abnormalities reminiscent of human CD. These abnormalities include intestinal lymphangiectasia, mesenteric lymph node lymphadenopathy, and lymphangiogenesis in both the mesentery and mucosa. Critically, TNFΔARE mice also present mesenteric tertiary lymphoid organs and have altered lymphatic transport of dendritic cells to mesenteric lymph nodes, two features likely to actively modulate immunity. Our findings provide key insights into lymphatic remodeling in the TNFΔARE mouse model. They shed light on the involvement of these lymphatic changes in immune dysfunctions observed in CD and suggest the lymphatic system as new target for therapeutic options.

The Antiadhesive Strategy in Crohn's Disease: Orally Active Mannosides to Decolonize Pathogenic Escherichia coli from the Gut.

Blocking the adherence of bacteria to cells is an attractive complementary approach to current antibiotic treatments, which are faced with increasing resistance. This strategy has been particularly studied in the context of urinary tract infections (UTIs), in which the adhesion of pathogenic Escherichia coli strains to uroepithelial cells is prevented by blocking the FimH adhesin expressed at the tips of bacteria organelles called fimbriae. Recently, we extended the antiadhesive concept, showing that potent FimH antagonists can block the attachment of adherent-invasive E. coli (AIEC) colonizing the intestinal mucosa of patients with Crohn's disease (CD). In this work, we designed a small library of analogues of heptyl mannoside (HM), a previously identified nanomolar FimH inhibitor, but one that displays poor antiadhesive effects in vivo. The anomeric oxygen atom was replaced by a sulfur or a methylene group to prevent hydrolysis by intestinal glycosidases, and chemical groups were attached at the end of the alkyl tail. Importantly, a lead compound was shown to reduce AIEC levels in the feces and in the colonic and ileal mucosa after oral administration (10 mg kg(-1) ) in a transgenic mouse model of CD. The compound showed a low bioavailability, preferable in this instance, thus suggesting the possibility of setting up an innovative antiadhesive therapy, based on the water-soluble and non-cytotoxic FimH antagonists developed here, for the CD subpopulation in which AIEC plays a key role.

Mycophenolate Mofetil Modulates Differentiation of Th1/Th2 and the Secretion of Cytokines in an Active Crohn's Disease Mouse Model.

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-γ, TNF-α, IL-12, IL-6, and IL-1β 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-γ, TNF-α, IL-12, IL-6, and IL-1β 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.

Behavioral despair associated with a mouse model of Crohn's disease: Role of nitric oxide pathway.

Crohn's disease (CD) is associated with increased psychiatric co-morbidities. Nitric oxide (NO) is implicated in inflammation and tissue injury in CD, and it may also play a central role in pathogenesis of the accompanying behavioral despair. This study investigated the role of the NO pathway in behavioral despair associated with a mouse model of CD. Colitis was induced by intrarectal (i.r.) injection of 2,4,6-trinitrobenzenesulfonic acid (10mg TNBS in 50% ethanol). Forced swimming test (FST), pharmacological studies and tissues collection were performed 72 h following TNBS administration. To address a possible inflammatory origin for the behavioral despair following colitis induction, tumor necrosis factor-alpha (TNF-α) level was measured in both the hippocampal and colonic tissue samples. In parallel, hippocampal inducible nitric oxide synthase (iNOS) and nitrite level were evaluated. Pharmacological studies targeting the NO pathway were performed 30-60 min before behavioral test. Colitis was confirmed by increased colonic TNF-α level and microscopic score. Colitic mice demonstrated a significantly higher immobility time in the FST associated to a significant increase of hippocampal TNF-α, iNOS expression and nitrite content. Acute NOS inhibition using either Nω-nitro-l-arginine methyl ester (a non-specific NOS inhibitor) or aminoguanidine hydrochloride (a specific iNOS inhibitor) decreased the immobility time in colitic groups. Moreover, acute treatment with both NOS inhibitors decreased the TNF-α level and nitrite content in the hippocampal samples. This study suggests that the NO pathway may be involved in the behavioral effects in the mouse TNBS model of CD. These findings endow new insights into the gut-brain communication during the development of colonic inflammation, which may ultimately lead to improved therapeutic strategies to combat behavior changes associated with gastrointestinal disorders.

Tu1852 Resveratrol Ameliorates Oxidative Stress-Induced Intestinal Epithelial Barrier Dysfunction by Upregulating Heme Oxygenase-1 Expression

Crohn's disease (CD) is a chronic, relapsing inflammatory disease, which affects nearly 1.4 million Americans. Genome-wide association studies (GWAS) have identified several genetic risk loci for CD including IRGM (immunity-related GTPase family M). IRGM functions in the innate immune control of intracellular pathogens. IRGM localizes to the mitochondria and induces mitochondrial fission which is linked to the autophagic elimination of intracellular bacteria. Surprisingly, IRGM expression is not lost but stronger in the epithelium of CD patients carrying the IRGM risk allele and more unexpectedly it is associated with an increase in intracellular bacteria. So how IRGM signaling contributes to the pathogenesis of CD remains unresolved. Our laboratory has generated a unique mouse model (DKO) of CD with a high mucosal expression of IRGM which duplicates the functional, histological and clinical features of human CD. The DKO mice were generated by genetic deletion of two anti-apoptotic proteins (villin/gelsolin) that regulate mitochondrial homeostasis and mucosal healing. Our studies with DKO mice and human CD patients demonstrate that by acting on the mitochondria, IRGM confers autophagic protection when expressed in moderation but induces cell death when over-expressed, explaining IRGM's action both in defense against bacteria and in damaging inflammation in CD. Furthermore, IRGM induced cell death is accompanied by the release of HMGB1 (high mobility group protein B1), a major proinflammatory alarmin/DAMP (danger-associated molecular pattern). This we demonstrate is the molecular basis of IRGM's role in the pathogenesis of CD. Our innovative study uses a novel mouse model of CD, provides a novel paradigm about the role of IRGM in CD and includes translational studies to diagnose, treat and monitor CD patients.

Tu1849 Defining and Targeting IRGM's Mechanism of Action in Crohn's Disease

Crohn's disease (CD) is a chronic, relapsing inflammatory disease, which affects nearly 1.4 million Americans. Genome-wide association studies (GWAS) have identified several genetic risk loci for CD including IRGM (immunity-related GTPase family M). IRGM functions in the innate immune control of intracellular pathogens. IRGM localizes to the mitochondria and induces mitochondrial fission which is linked to the autophagic elimination of intracellular bacteria. Surprisingly, IRGM expression is not lost but stronger in the epithelium of CD patients carrying the IRGM risk allele and more unexpectedly it is associated with an increase in intracellular bacteria. So how IRGM signaling contributes to the pathogenesis of CD remains unresolved. Our laboratory has generated a unique mouse model (DKO) of CD with a high mucosal expression of IRGM which duplicates the functional, histological and clinical features of human CD. The DKO mice were generated by genetic deletion of two anti-apoptotic proteins (villin/gelsolin) that regulate mitochondrial homeostasis and mucosal healing. Our studies with DKO mice and human CD patients demonstrate that by acting on the mitochondria, IRGM confers autophagic protection when expressed in moderation but induces cell death when over-expressed, explaining IRGM's action both in defense against bacteria and in damaging inflammation in CD. Furthermore, IRGM induced cell death is accompanied by the release of HMGB1 (high mobility group protein B1), a major proinflammatory alarmin/DAMP (danger-associated molecular pattern). This we demonstrate is the molecular basis of IRGM's role in the pathogenesis of CD. Our innovative study uses a novel mouse model of CD, provides a novel paradigm about the role of IRGM in CD and includes translational studies to diagnose, treat and monitor CD patients.

Experimental study of adipose-derived mesenchymal stem cells in the treatment of Crohn's disease

To explore the efficacy of adipose-derived mesenchymal stem cells (ADMSCs) in a murine model of inflammatory bowel disease, and its potential mechanism. Murine colitis mouse model of Crohn's disease(CD) was created by trinitrobenzene sulfonic acid(TNBS)-induced colitis. Seventy-five 6-8 weeks female BALB/c mice were randomly divided into 3 groups: control group, TNBS group and ADMSC group. To verify the therapeutic effect of ADMSC, real-time PCR and immunohistochemical staining were performed to measure inflammatory cytokines levels in colon tissues. The 10-day survival statuses were recorded after the infusion of ADMSCs. Intraperitoneal injection of ADMSCs alleviated the clinical and histopathologic severity of intestinal inflammation, and increased survival(60% vs. 30%, P<0.05) in the TNBS-induced mouse model of CD. Compared with TNBS group, proinflammatory cytokines, including TNF-α, IL-12 and VEGF of ADMSC group were significantly reduced, with significant increase of IL-10 expression. ADMSCs can effectively repair the injury of colonitis through down-regulation of proinflammatory cytokines TNF-α, IL-12 and VEGF expression, and up-regulation of anti-inflammatory cytokine IL-10 expression, which may be a potential new alternative of cell-based therapy for CD.

Mitigation of Colitis with NovaSil Clay Therapy

Five million people currently live with Crohn's disease (CD) or ulcerative colitis, the two major forms of inflammatory bowel disease. Available treatments frequently result in side effects that compromise the immune health of the patient. Consequently, alternative therapies that cause fewer systemic effects are needed. Dioctahedral smectite clays have been utilized to treat medical conditions, including diarrheal and enteric disease. Herein, we report the ability of a refined dioctahedral smectite (NovaSil, NS) to sorb inflammatory proteins and reduce inflammation in a TNBS (2,4,6-trinitrobenzenesulfonic acid) mouse model of CD. We also investigated whether NS could rescue gut microbial diversity in TNBS-induced mice. ELISA, X-ray diffraction, and transmission electron microscopy were employed to characterize the NS-cytokine interaction in vitro. A TNBS mouse colitis model was utilized to study the efficacy of NS supplementation for 4weeks. The three treatment groups included control, TNBS, and TNBS+NS. DNA was extracted from feces and sorted for bacterial phylogenetic analysis. Results suggest that NS binds TNFα in vitro. In TNBS-treated mice, supplementation with NS significantly reduced weight loss, and serum proinflammatory cytokine levels (IL-2, IL-6, and IL-12, TNFα, IFNγ) compared with the TNBS group. TNBS-treated mice demonstrated a significant reduction in gut microbiota species richness when compared with the TNBS+NS group and control group. NovaSil mitigated the effects of TNBS-induced colitis based on reduction in systemic markers of inflammation, significant improvement in weight gain, and intestinal microbial profile.

Thiazolylaminomannosides As Potent Antiadhesives of Type 1 Piliated Escherichia coli Isolated from Crohn’s Disease Patients

Adherent-invasive Escherichia coli (AIEC) have previously been shown to induce gut inflammation in patients with Crohn's disease (CD). We developed a set of mannosides to prevent AIEC attachment to the gut by blocking the FimH bacterial adhesin. The crystal structure of the FimH lectin domain in complex with a lead thiazolylaminomannoside highlighted the preferential position for pharmacomodulations. A small library of analogues showing nanomolar affinity for FimH was then developed. Notably, AIEC attachment to intestinal cells was efficiently prevented by the most active compound and at around 10000-fold and 100-fold lower concentrations than mannose and the potent FimH inhibitor heptylmannoside, respectively. An ex vivo assay performed on the colonic tissue of a transgenic mouse model of CD confirmed this antiadhesive potential. Given the key role of AIEC in the chronic intestinal inflammation of CD patients, these results suggest a potential antiadhesive treatment with the FimH inhibitors developed.

Identification of Epithelial to Mesenchymal Transition as a Novel Source of Fibroblasts in Intestinal Fibrosis

Intestinal fibrosis is a major complication of Crohn disease (CD), but the precise mechanism by which it occurs is incompletely understood. As a result, specific therapies to halt or even reverse fibrosis have not been explored. Here, we evaluated the contribution of epithelial to mesenchymal transition (EMT) to intestinal fibrosis associated with a mouse model of CD and also human inflammatory bowel disease. Mice administered intrarectal 2,4,6-trinitrobenzene sulfonic acid (TNBS) develop inflammation and fibrosis that resembles CD both histologically and by immunologic profile. We utilized this model to molecularly probe the contribution of EMT to intestinal fibrosis. Additionally, we utilized double-transgenic VillinCre;R26Rosa-lox-STOP-lox-LacZ mice, in which removal of the STOP cassette by Cre recombinase in villin(+) intestinal epithelial cells activates permanent LacZ expression, to lineage trace epithelial cells that might undergo EMT upon TNBS administration. TNBS-induced fibrosis is associated with the presence of a significant number of cells that express both epithelial and mesenchymal markers. In the lineage tagged transgenic mice, the appearance of LacZ(+) cells that also express the fibroblast marker FSP1 unequivocally demonstrates EMT. Transforming growth factor (TGF)-beta1, a known inducer of EMT in epithelial cells, induces EMT in rat intestinal epithelial cells in vitro, and bone morphogenic protein-7, an antagonist of TGF-beta1, inhibits EMT and fibrosis both in vitro and in the TNBS-treated mice. Our study demonstrates that EMT contributes to intestinal fibrosis associated with the TNBS-induced model of Crohn colitis and that inhibition of TGF-beta1 with recombinant human bone morphogenic protein-7 prevents this process and prevents fibrosis.

Anti-colitis and -adhesion effects of daikenchuto via endogenous adrenomedullin enhancement in Crohn's disease mouse model

Background and aims Adrenomedullin (ADM) is a member of the calcitonin family of regulatory peptides, and is reported to have anti-inflammatory effects in animal models of Crohn's disease (CD). We investigated the therapeutic effects of daikenchuto (DKT), an extracted Japanese herbal medicine, on the regulation of endogenous ADM in the gastrointestinal tract in a CD mouse model. Methods Colitis was induced in mice by intrarectal instillation of 2,4,6-trinitrobenzenesulfonic acid (TNBS); afterwards, DKT was given orally. Colonic damage was assessed on day 3 by macroscopic and microscopic observation, enzyme immunoassays of proinflammatory cytokines in the colonic mucosa, and serum amyloid A (SAA), a hepatic acute-phase protein. To determine the involvement of ADM, an ADM antagonist was instilled intrarectally before DKT administration. The effect of DKT on ADM production by intestinal epithelial cells was evaluated by enzyme immunoassay and real-time PCR. Results DKT significantly attenuated mucosal damage and colonic inflammatory adhesions, and inhibited elevations of SAA in plasma and the proinflammatory cytokines TNFα and IFNγ in the colon. Small and large intestinal epithelial cells produced higher levels of ADM after DKT stimulation. A DKT-treated IEC-6 cell line also showed enhanced ADM production at protein and mRNA levels. Abolition of this effect by pretreatment with an ADM antagonist shows that DKT appears to exert its anti-colitis effect via up-regulation of endogenous ADM in the intestinal tract. Conclusion DKT exerts beneficial effects in a CD mouse model through endogenous release and production of ADM. Endogenous ADM may be a therapeutic target for CD.

Allantoin as A Biomarker of Inflammation in an Inflammatory Bowel Disease Mouse Model: NMR Analysis of Urine

Crohn's disease (CD) is an Inflammatory Bowel Disease (IBD) that is characterised by destructive inflamma- tion of the intestinal wall. Current methods for determining inflammation of the bowel are costly, time consuming and can cause discomfort to the patients. In order to address these problems, biomarker analysis of more accessible tissues is re- ceiving increasing attention. Oxidative stress has been implicated in the promotion of inflammation. Allantoin has re- cently been reported as a biomarker for oxidative stress in human serum and urine. This paper investigates allantoin as a biomarker of inflammation in a mouse model of CD. Proton nuclear magnetic resonance ( 1 H NMR) spectroscopy was used to analyse allantoin in urine from the mdr1a -/- mouse which is a model of CD. The data show that the levels of al- lantoin are strongly correlated with histological injury scores of mouse colonic tissue samples. Allantoin appears to be a useful biomarker of gut inflammation, involving oxidative stress, in a mouse model of CD and may be a potential bio- marker in human CD studies.

Interleukin-5 participates in the pathogenesis of ileitis in SAMP1/Yit mice.

SAMP1/Yit mice spontaneously develop ileitis resembling Crohn's disease (CD) without chemical or genetic manipulations. Since the focus of studies were Th1 cytokines, only Th1-type T cells were thought to be responsible for intestinal inflammation in these mice. To further characterize the pathogenesis of this ileitis, we investigated the implication of Th2 cytokines in ileitis of SAMP1/Yit mice. The expression of chemokine receptors (CCR) associated with both Th1 and Th2 lymphocytes, such as CCR2, CCR3, CCR4, CCR5, and CCR8, was increased. Among cytokines, IL-5 was remarkably increased in Peyer's patches, mesenteric lymph nodes, and mucosa involved in ileitis. Furthermore, infiltration of numerous eosinophils in ileitis was histologically evident. Severe combined immunodeficiency mice injected intraperitoneally with CD4(+) cells from SAMP1/Yit mice developed colitis and ileitis, with the infiltration of eosinophils. Administration of anti-IL-5 antibodies significantly attenuated ileitis in these mice. We suggest that IL-5 participates in the pathogenesis of ileitis and that anti-IL-5 antibodies are potentially useful for immunotherapy in CD patients. This is the first demonstration that IL-5 is crucial for the development of ileitis in this mouse model of CD.