Mouse Model Of Chronic Colitis Research Articles

The efficacy of conditioned medium released by tonsil-derived mesenchymal stem cells in a chronic murine colitis model.

Tonsil-derived mesenchymal stem cells (TMSC) have characteristics of MSC and have many advantages. In our previous studies, intraperitoneal (IP) injection of TMSC in acute and chronic colitis mouse models improved the disease activity index, colon length, and the expression levels of proinflammatory cytokines. However, TMSC were not observed to migrate to the inflammation site in the intestine. The aim of this study was to verify the therapeutic effect of conditioned medium (CM) released by TMSC (TMSC-CM) in a mouse model of dextran sulfate sodium (DSS)-induced chronic colitis. TMSC-CM was used after seeding 5×105 cells onto a 100 mm dish and culturing for 5-7 days. TMSC-CM was concentrated (TMSC-CM-conc) by three times using a 100 kDa cut-off centrifugal filter. Seven-week-old C57BL/6 mice were randomly assigned to the following 5 groups: 1) normal, 2) colitis, 3) TMSC, 4) TMSC-CM, and 5) TMSC-CM-conc. Chronic colitis was induced by continuous oral administration of 1.5% dextran sulfate sodium (DSS) for 5 days, followed by 5 additional days of tap water feeding. This cycle was repeated two more times (total 30 days). Phosphate buffered saline (in the colitis group), TMSC, TMSC-CM, and TMSC-CM-conc were injected via IP route 4, 4, 12, and 4 times, respectively. Reduction of disease activity index, weight gain, recovery of colon length, and decreased in the expression level of the proinflammatory cytokines, interleukin (IL)-1β, IL-6, and IL-17 were observed at day 30 in the treatment groups, compared to control. However, histological colitis scoring and the expression level of tumor necrosis factor α and IL-10 did not differ significantly between each group. TMSC-CM showed an equivalent effect to TMSC related to the improvement of inflammation in the chronic colitis mouse model. The data obtained support the use of TMSC-CM to treat inflammatory bowel disease without any cell transplantation.

Ion Transport Basis of Diarrhea in a Mouse Model of Adoptive T Cell Transfer Colitis.

Diarrhea, a major pathological hallmark of inflammatory bowel disease, is characterized by a significant reduction in the expression and function of key intestinal ion transporters. The adoptive naïve CD4+ T cell transfer colitis is an immune-based, chronic colitis mouse model which resembles human Crohn's disease. Although mice with T cell transfer colitis demonstrate diarrhea, the ion transporter basis of this phenotype has not been explored. In the current studies, we aimed to determine the mRNA and protein levels of the key NaCl transporters DRA and NHE3 along with the mRNA expression of other transporters in the inflamed intestine. Naïve CD4+ T cells, transferred to Rag2 knockout mice, induced severe colonic inflammation characterized by histological damage and increased mRNA levels of cytokines in the colon with no effect in the ileum. Diarrheal phenotype was a key feature of the excised colons of mice where loose stools were evident. Our results demonstrated that the key chloride transporter DRA, mRNA, and protein levels were significantly reduced in the inflamed colon. However, expression of the key sodium hydrogen exchanger NHE3 was unaffected. The mRNA expression of other important transporters was also determined; in this regard, the sodium channel ENACα and the monocarboxylate transporters MCT1 and SMCT1 mRNA levels were also significantly lower compared to control mice. However, CFTR mRNA was not altered in the colon or ileum. The studies conducted herein for the first time demonstrate the downregulation of important intestinal ion transporters in proximal and distal colon in T cell transfer colitis mouse model, providing valuable evidence for the ion transporter basis of diarrhea in this chronic model of inflammation.

The commensal Escherichia coli CEC15 reinforces intestinal defences in gnotobiotic mice and is protective in a chronic colitis mouse model

Escherichia coli is a regular inhabitant of the gut microbiota throughout life. However, its role in gut health is controversial. Here, we investigated the relationship between the commensal E. coli strain CEC15 (CEC), which we previously isolated, and the intestine in homeostatic and disease-prone settings. The impact of CEC was compared to that of the probiotic E. coli Nissle 1917 (Nissle) strain. The expression of ileal and colonic genes that play a key role in intestinal homeostasis was higher in CEC- and Nissle-mono-associated wild-type mice than in germfree mice. This included genes involved in the turnover of reactive oxygen species, antimicrobial peptide synthesis, and immune responses. The impact of CEC and Nissle on such gene expression was stronger in a disease-prone setting, i.e. in gnotobiotic IL10-deficient mice. In a chronic colitis model, CEC more strongly decreased signs of colitis severity (myeloperoxidase activity and CD3+ immune-cell infiltration) than Nissle. Thus, our study shows that CEC and Nissle contribute to increased expression of genes involved in the maintenance of gut homeostasis in homeostatic and inflammatory settings. We show that these E. coli strains, in particular CEC, can have a beneficial effect in a chronic colitis mouse model.

Tu1765 – Histone 3 Lysine 27 Acetylation Profiling Reveals Its Role in the Chronic Dss-Induced Colitis Mouse Model

Tu1765 – Histone 3 Lysine 27 Acetylation Profiling Reveals Its Role in the Chronic Dss-Induced Colitis Mouse Model

The effects and mechanisms of myeloid differentiation protein 2 on intestinal mucosal permeability in mice with chronic colitis.

The present study was designed to investigate the mechanism of myeloid differentiation protein 2 (MD2) on intestinal mucosa destruction in mice with chronic colitis. Briefly, a chronic colitis mouse model was established by the administration of dextran sulfate sodium (DSS) in transgenic mice of MD2 overexpression (Transgenic, MD2-Tg) and C57BL/6 wild-type mice (MD2-WT). In addition, Caco-2 cells were cultured to form a monolayer cell model in vitro. The small interfering RNA was utilized to silence the MD2 gene in Caco-2 cells, and tumor necrosis factor-α (TNF-α) was used to establish the model of intestinal mucosal inflammation. After DSS induction, the intestinal mucosal tissue inflammation was more severe in MD2-Tg mice than MD2-WT. In addition, the intestinal mucosa was severely damaged, the intestinal mucosal permeability was increased, bacterial translocation was obvious, and the expression levels of MD2, MyD88, Toll-like receptor 4 (TLR4), and HMGB1 in mucosal tissues were significantly increased, while the expression levels of tight junction proteins, occludin, and claudin-1 were significantly lower in MD2-Tg mice compared with those in MD2-WT mice. TNF-α could induce inflammatory apoptosis in Caco-2 cell models. After MD2 silencing, the apoptotic level was decreased, the value of transepithelial electrical resistance was increased, the permeability of intestinal mucosa was decreased, the cellular expression levels of MD2, MyD88, TLR4, and HMGB1 were decreased, while the expression levels of tight junction proteins, occludin and claudin-1 were increased. MD2 could aggravate the destruction of intestinal mucosa in chronic colitis through the HMGB1-TLR4-MyD88 pathway.

Emu Oil reduces disease severity in a mouse model of chronic ulcerative colitis

Objective: Ulcerative colitis (UC) is characterized by mucosal inflammation and ulceration of the large intestine. Emu Oil (EO) has been reported to protect the intestine against mucositis, NSAID-enteropathy, UC-associated colorectal cancer and acute UC. We aimed to determine whether EO could reduce the severity chronic UC in mice.Methods: Female C57BL/6 mice (n = 10/group) were orally administered (gavage) water (Groups 1–2) or EO (Groups 3: low dose-80 µl and 4: high dose-160 µl), thrice weekly. Group 1 mice consumed plain drinking water throughout the trial. Groups 2–4 mice underwent two cycles [each consisting of seven days dextran sulfate sodium (DSS; 2% w/v) and 14 days water], followed by a third DSS week. All mice were euthanized two days later (day 51). Bodyweight, disease activity index (DAI), burrowing activity, myeloperoxidase activity, crypt depth and histologically assessed damage severity were assessed. p < .05 was considered significant.Results: DSS decreased bodyweight and increased DAI compared to normal controls (p < .05), which was partially attenuated by both EO doses (p < .05). Burrowing activity was impaired in DSS-controls compared to normal controls (days 27 and 40); an effect prevented by both EO doses (p < .05). DSS increased colonic myeloperoxidase activity and crypt depth compared to controls (p < .05), with no significant EO effect. Moreover, DSS increased colonic damage severity compared to normal controls (p < .001). Importantly, both EO doses decreased distal colonic damage severity compared to DSS-controls (p < .001).Conclusions: Emu Oil attenuated clinically- and histologically-assessed disease severity in a mouse model of chronic UC. Emu Oil demonstrates promise as an adjunct to conventional treatment options for UC management.

3375 Chronic inflammation promotes intestinal macrophages to become modulators of the Notch pathway

OBJECTIVES/SPECIFIC AIMS: The purpose of this research was to investigate how chronic inflammation promotes the generation of proinflammatory intestinal macrophages and if macrophages contribute to intestinal inflammation through Notch activation. METHODS/STUDY POPULATION: We utilized two animal models of chronic colitis, the chronic DSS-induced colitis mouse model and the spontaneous enterocolitis development in IL-10-deficient mice to investigate the role of chronic inflammation in the generation of proinflammatory intestinal macrophages and its influence in notch signaling. Bone marrow-derived monocytes were collected from each group and differentiated into macrophages (BMM) for gene and protein analysis. Ex vivo phenotypical and functional analysis of colonic macrophages was assessed as was the presence of goblet cells and mucosal T cells. In addition, we analyzed the development of goblet cell differentiation in colonoids in a co-culture system with proinflammatory macrophages. RESULTS/ANTICIPATED RESULTS: Our chronic inflammation models revealed an increase in proinflammatory macrophages present in the lamina propria and that these cells expressed significantly higher levels of notch ligand, Jagged1. Jagged 1 has been shown to enhance TH1 differentiation and T cells isolated from the mucosa of both chronic colitis models display strong TH1 skewing compared to controls. Chronic inflammation also contributes to intestinal barrier defects, enhanced permeability and bacterial translocation. We believe this enhanced intestinal permeability and subsequent bacterial translocation promote Jagged1 expression in intestinal macrophages. To support this concept, we show TLR stimulation induces the upregulation of Jagged1 in BMM. Additionally, the generation of BMM from our chronic DSS-induced colitis mice or age matched controls, revealed BMM derived from a host of chronic inflammation were skewed to a proinflammatory state prior to stimulation showing increased gene expression of several proinflammatory molecules including IL-1α, IL-1β, IL-12 and TNF-α. This would suggest monocytes migrating to the intestinal mucosa have more potential to become proinflammatory instead of traditional anti-inflammatory macrophages. Furthermore, proinflammatory notch ligand-positive macrophages co-cultured with colonoids, derived from unperturbed mice, significantly decreased the number of mucus producing goblet cells. In support of this observation, notch activation in intestinal stem cells promote absorptive (i.e. colonocytes) cell differentiation and prevents secretory cell (i.e. goblet cells) differentiation. DISCUSSION/SIGNIFICANCE OF IMPACT: Taken together, our results strongly suggest chronic inflammation modulates macrophages role in maintaining intestinal homeostasis through possible notch activation in both T cells and the intestinal epithelial barrier.

Profiling of histone 3 lysine 27 acetylation reveals its role in a chronic DSS-induced colitis mouse model.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract. In current dogma, pathogenesis of IBD is attributed to the dysregulated mucosal immune response to gut flora in genetically susceptible individuals, but the genetics evidence from GWAS studies so far is insufficient to explain the observed heritability in IBD. For this discordance, epigenetics has emerged to be one of the important causes. Recent studies have reported that histone acetylation is correlated with the development of IBD, whereas its role and underlying molecular mechanism in the disease still remain elusive. Here, we established a dextran sulfate sodium (DSS)-induced chronic colitis model and performed RNA-sequencing (RNA-seq) and Chromatin Immunoprecipitation followed by NGS sequencing (ChIP-seq) for H3K27ac in the mice colon tissues to investigate whether H3K27ac is involved in the development of intestinal inflammation. We found that the global H3K27ac level and distribution in colon tissue had no significant difference after DSS treatment, while H3K27ac signals were significantly enriched in the typical-enhancers of the DSS group compared with the control. By combining with RNA-seq data (fold change >2), we identified 56 candidate genes as potential target genes for H3K27ac change upon DSS treatment. We further predicted transcription factors (TFs) involved in DSS-induced colitis according to the enhancers with increased H3K27ac. H3K27ac increase in special typical-enhancers in the DSS group is possibly related to the development of intestinal inflammation by up-regulating adjacent gene expression and shifting TF networks, which will provide new insight into the pathogenesis and therapy of IBD.

PVAX1-A20 alleviates colitis in mice by promoting regulatory T cells

AimTo investigate whether the intrarectal administration of the ubiquitin E3 ligase A20 (A20) attenuates intestinal inflammation and influences regulatory T cells in experimental colitis. MethodsA dextran sulfate sodium induced chronic colitis mouse model was established. The symptoms and manifestations of colitis and the severity of colonic mucosal inflammation were evaluated. The protective role of A20 expression in the intestine was analyzed after the administration of a pVAX1-A20 recombinant eukaryotic vector, which was encapsulated into poly(L-lactide-co-glycolide) as a nanoparticle. ResultspVAX1-A20 administration markedly ameliorated colonic tissue damage and reduced intestinal inflammation via the suppression of the mucosal mitogen-activated protein kinase and nuclear factor (NF)-κB signaling cascade. Furthermore, pVAX1-A20 promoted the splenic regulatory T cell population and forkhead box P3 expression in colonic tissue. ConclusionA20 plays a key role in the regulation of intestinal inflammation and that the overexpression of A20 in the intestine protects mice from dextran sulfate sodium induced chronic colitis.

Chronic colitis induces meninges traffic of gut-derived T cells, unbalances M1 and M2 microglia/macrophage and increases ischemic brain injury in mice

Ischemic stroke is one of the most common diseases leading to death and is the primary cause of physical handicap. Recent studies have reported that chronic colitis increases the risk of ischemic stroke, but it is unknown whether chronic colitis participates in ischemic brain injury directly. A combined mouse model of chronic colitis induced by dextran sodium sulfate (DSS) and ischemic stroke induced by photochemical infarction was used in this study. We demonstrated that chronic colitis significantly increased the infarction volume, activated microglia/macrophage numbers, proliferation of M1 microglia/macrophage, non-gut-derived CD4+ T lymphocyte penetration and decreased neuron numbers in the peri-infarction at 7 d after stroke. Furthermore, gut-derived CD4+ T cell accumulation on the meninges was observed at 7 d after stroke. In addition, selective depletion of meningeal macrophages resulted in a reduction of infarction volume and the non-gut-derived CD4+ T lymphocyte penetration. We concluded that chronic colitis exacerbated ischemic stroke by promoting CD4+ T cell migration from the gut to the meninges and disequilibrium of M1 and M2 microglia/macrophages. We speculated that the gut-derived CD4+ T cells may interact with meningeal macrophages and result in non-gut-derived CD4+ T lymphocyte infiltration that aggravated brain injury in ischemic stroke.

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.

MCC950, a specific small molecule inhibitor of NLRP3 inflammasome attenuates colonic inflammation in spontaneous colitis mice

MCC950 a potent, highly specific small molecule inhibitor of canonical and noncanonical activation of NLRP3 inflammasome has been evaluated in a multitude of NLRP3 driven inflammatory diseases. However, the effect of MCC950 on colonic inflammation has not yet been reported. In the present study we investigated the effect of MCC950 in a spontaneous chronic colitis mouse model Winnie, which mimics human ulcerative colitis. Oral administration of 40 mg/kg MCC950 commencing at Winnie week seven for three weeks significantly improved body weight gain, colon length, colon weight to body weight ratio, disease activity index and histopathological scores. MCC950 significantly suppressed release of proinflammatory cytokines IL-1β, IL-18, IL1-α, IFNγ, TNF-α, IL6, IL17, chemokine MIP1a and Nitric Oxide in colonic explants. Moreover, MCC950 resulted in a significant decrease of IL-1β release and activation of caspase-1 in colonic explants and macrophage cells isolated from Winnie. Complete inhibition with MCC950 in Winnie colonic explants shows, for the first time, the contribution of inflammatory effects resulting exclusively from canonical and noncanonical NLRP3 inflammasome activation in colitis. Taken together, our results illustrate the efficacy of MCC950 in the treatment of murine ulcerative colitis and provides avenue for a potential novel therapeutic agent for human inflammatory bowel diseases.

The Onset and Progression of Chronic Colitis Parallels Increased Mucosal Serotonin Release via Enterochromaffin Cell Hyperplasia and Downregulation of the Serotonin Reuptake Transporter.

Serotonin (5-hydroxytryptamine, 5-HT) has been linked with several inflammation-associated intestinal diseases, including ulcerative colitis (UC). The largest pool of 5-HT in the body is in enterochromaffin (EC) cells located throughout the intestinal tract. EC cells are mechanosensitive and detect noxious stimuli, inducing secretion of 5-HT, which plays an important role in enteric reflexes and immunomodulation. In this study, we evaluated intestinal 5-HT levels in the Winnie mouse model of spontaneous chronic colitis, which closely replicates UC. Real-time electrochemical recordings of 5-HT oxidation currents were obtained from ex vivo preparations of jejunum, ileum, proximal, and distal colon from Winnie (5-25 weeks old) and age matched C57BL/6 mice. EC cells were examined by immunohistochemistry, and the gene expression of tryptophan hydroxylase 1 (5-HT synthesis) and the serotonin reuptake transporter (SERT) were determined by quantitative Real-Time Polymerase Chain Reaction (RT-qPCR). Compression-evoked and basal 5-HT concentrations were elevated in the distal and proximal colon of Winnie mice. EC cell hyperplasia and downregulation of SERT on the transcriptional level were identified as mechanisms underlying increased levels of 5-HT. Increase in mucosal 5-HT release was observed at the onset of disease at 7-14 weeks, confirmed by disease activity scores. Furthermore, increases in 5-HT levels and progression of disease activity correlated linearly with age, but not sex. Our findings in the Winnie mouse model of spontaneous chronic colitis demonstrate for the first time that the onset and progression of chronic UC-like intestinal inflammation is associated with increased 5-HT levels in the colonic mucosa.

P091 Inflammation in relation to mucosal barrier function in chronic colitis: a study in time

The pathogenesis of inflammatory bowel diseases (IBD) is multifactorial. Midst in the complex interaction between genetic, environmental and immunological factors is the intestinal barrier. IBD patients have been observed to have increased intestinal permeability which can partially explain the exaggerated immune response towards luminal antigens. Inflammatory mediators perpetuate this barrier defect by causing further damage to the intestinal barrier. Our aim is to investigate the interaction between permeability and inflammation in a chronic colitis mouse model. Colitis was induced in immunodeficient SCID mice by the adoptive transfer of naive T cells, isolated from BALB/c mice. Mice were sacrificed at fixed time points to study disease progression resulting in five groups: control mice (Week 0); colitis mice in week 1, 2, 4 and 6 of the experimental protocol (Week 1; Week 2; Week 4, Week 6; all n = 10). FITC-dextran was used to assess intestinal permeability, next to the messenger RNA of the tight junction proteins Claudin-1 and Occludin. After sacrifice, colonic inflammation was assessed by macro- and microscopic scoring, myeloperoxidase (MPO) activity and cytometric bead array (CBA) for TNF- α and IL-1β . Messenger RNA of transcription factors that regulate T helper (Th) cell differentiation including T-bet (Th1), GATA-3 (Th2), ROR- γt (Th17) were quantified using RT-qPCR technique. Mucosal inflammation appeared gradually in the colon of the diseased mice. Macroscopically there was a significant increase starting from Week 2 on, gradually increasing until Week 6. The same was true for the MPO activity. Microscopically, however, the first significant signs of inflammation were observed at Week 1, which then also gradually increased with the highest score at Week 6. The colonic inflammation was mainly Th1/Th2 driven, since the transcription factors T-bet and GATA-3 were significantly upregulated from respectively Week 2 and Week 4 on. TNF-α and IL-1β were significantly elevated starting from Week 1. Intestinal permeability was significantly elevated at Week 1 and remained elevated at Week 2, 4 and 6. Data are presented as mean±SEM and analysed by one-way ANOVA with multiple comparisons SNK posthoc testing. *<0.05 vs. WEEK 0 # <0.05 vs. WEEK 1. Our results show a gradual increase of intestinal inflammation over time which, for most parameters, started at Week 2. However, we observed an unexpectedly early increase in intestinal permeability at Week 1. This shows that the mucosal barrier function is of great importance in disease onset and could be an interesting therapeutic target or biomarker predicting flares.

Regulation of epithelial cell expressed C3 in the intestine – Relevance for the pathophysiology of inflammatory bowel disease?

The complement system not only plays a critical role in efficient detection and clearance of bacteria, but also in intestinal immune homeostasis as mice deficient for key complement components display enhanced intestinal inflammation upon experimental colitis. Because underlying molecular mechanisms for this observation are unclear, we investigated the crosstalk between intestinal epithelial cells (IEC), bacteria and the complement system in the course of chronic colitis.Surprisingly, mouse intestinal epithelial cell lines constitutively express high mRNA levels of complement component 3 (C3), Toll-like receptor 2 (Tlr2) and Tlr4. Stimulation of these cells with lipopolysaccharide (LPS), but not with flagellin, LD-muramyldipeptide or peptidoglycan, triggered increased C3 expression, secretion and activation. Stimulation of the C3aR on these cell lines with C3a resulted in an increase of LPS-triggered pro-inflammatory response. Tissue biopsies from C57BL/6J mice revealed higher expression of C3, Tlr1, Tlr2 and Tlr4 in colonic primary IECs (pIECs) compared to ileal pIECs, while in germ-free mice no differences in C3 protein expression was observed. In DSS-induced chronic colitis mouse models, C3 mRNA expression was upregulated in colonic biopsies and ileal pIECs with elevated C3 protein in the lamina propria, IECs and the mucus. Notably, increased C3b opsonization of mucosa-attached bacteria and decreased fecal full-length C3 protein was observed in DSS-treated compared to untreated mice. Of significant interest, non-inflamed and inflamed colonic biopsy samples from CD but not UC patients displayed exacerbated C3 expression compared to controls.These findings suggest that a novel TLR4-C3 axis could control the intestinal immune response during chronic colitis.

Colon tumorigenesis mediated by Tollip in neutrophils.

Abstract Innate neutrophils have ability to confer either pro- or anti-tumor immune responses. However, molecular mechanisms responsible for the cell polarization are not well understood. Here, we aim to identify and characterize the neutrophil phenotypes in chronic colitis (CC) mouse model with use of AOM and DSS. We identify that Toll-interacting-protein (Tollip) may play a key role in modulating the long-term polarization and function of neutrophils as well as pro-tumorigenesis. We observed that Tollip deficient mice had improved clinical score and reduced generation of colon polyps when subjected to prolonged challenges with AOM and DSS. At the cellular level, Tollip deficient mice challenged with AOM-DSS had distinct neutrophil activation profiles as compared to wild type mice, including the differential expression of cell surface co-stimulatory molecules, migratory behaviors, as well as cytokine expression profiles. Adoptive transfer of neutrophils from Tollip deficient mice to wild type mice recapitulated the outcome of Tollip deficient mice. Based on the in vivo and in vitro studies, we conclude that Tollip may play a key role during the polarization of innate neutrophils involved in the modulation of chronic colitic tumor environment. Further studies with regard to the molecular mechanisms may enable a better understanding of neutrophil modulation in colon tumorigenesis.

Emu Oil Reduces Colonic Damage Severity in a Mouse Model of Chronic Ulcerative Colitis

Background/AimsUlcerative colitis (UC) is characterised by large intestinal inflammation and ulceration. Previously, Emu Oil (EO) has been reported to protect the intestine in models of NSAID‐enteropathy, mucositis and acute UC. It was hypothesized that EO would reduce disease and colonic damage severity in a mouse model of dextran sulphate sodium (DSS)‐induced chronic UC.MethodsMice (n=10/group) were gavaged with water or EO (80microL [low dose] or 160microL [high dose]) thrice weekly. Mice were subjected to two cycles each consisting of water or DSS (2% w/v) ad libitum for one week and two weeks water recovery. Mice were euthanized two days after a third week of DSS. Disease activity index (DAI), burrowing activity and histologically‐assessed damage were quantified. Data were statistically analysed using ANOVA with LSD and Kruskal Wallis with Mann Whitney U tests. P&lt;0.05 was considered significant.ResultsDAI was significantly greater in DSS controls compared to controls (P&lt;0.05). EO‐low dose (days 6–9, 26–35) and EO‐high dose (days 6–16, 25–26, 33–35, 45) reduced DAI compared to DSS controls (P&lt;0.05). DSS reduced burrowing activity in mice compared to controls; an effect significantly improved by both doses of EO (days 27, 40; P&lt;0.05). In DSS‐treated mice, colonic damage severity increased compared to controls (P&lt;0.001). Importantly, both doses of EO significantly decreased damage severity in the distal colon compared to DSS controls (P&lt;0.001).ConclusionsEmu Oil reduced colonic damage severity and disease activity and increased burrowing activity in experimentally‐induced chronic UC, suggesting potential as a treatment adjunct for UC.Support or Funding InformationN/A

P044 Therapeutic effects of mesenchymal stem cells induced regulatory dendritic cells on chronic colitis model in mice

Background: Mesenchymal stem cells (MSCs), emerging cell therapeutics for inflammatory bolwel disease (IBD), have demonstrated immunomodulatory effects by interactions between T cells and dendritic cells (DCs). In this study, we investigated whether MSCs could differentiate mature DCs (mDCs) into a distinct regulatory DCs and these DCs could alleviate inflammation through a T cell modulation in DSS induced chronic colitis mouse model. Methods: DCs were derived from mouse bone marrow and different subsets of DCs were characterized by fluorescence-activated cell sorting (FACS). Quantitative polymerase chain reaction (q-PCR) for anti-inflammatory (interleukin (IL)-10 and transforming growth factor (TGF)–β) and pro-inflammatory cytokines (interferon (IFN)-γ, tumor necrosis factor (TNF)-α and IL-6) were performed in both in vitro DC subsets and in vivo. Using splenocytes co-cultured with MSC induced DCs, q-PCRs for CD4, CD8, CD25 and Foxp3, and Western blotting for Foxp3 were performed to measure the expression of regulatory T (Treg) cell markers in vitro. In vivo dextran sulfate sodium (DSS) chronic colitis model, Expression of IL-10, TGF–β, STAT3 and Foxp3 were compared among 5 groups (control, DSS-colitis, immature DCs only, MSCs, and MSC-induced DCs). Results: MSC-induced DCs expressed CD11c+ CD80lo CD86locell surface marker. These DCs secreted high levels of anti-inflammatory cytokines, whereas production of pro-inflammatory cytokines were diminished in vitro. Also, the levels of mRNA and protein of Foxp3+, CD4+ and CD25+ were increased in splenocytes co-cultured with MSC-induced DCs. In addition, intraperitoneal injection of MSC induced DCs significantly increased anti-inflammatory cytokines at both m-RNA and protein level and enhanced differentiation of FOXP3+ Treg cells, resulting in considerabe amelioration of chronic colitis and improvement of survival in DSS treated mice. Conclusions: These results showed that MSCs induced DCs alliviated chronic colitis by promotin g Treg cells differentiation and the secretion of anti-inflammatory cytokines, providing potential application of MSC-induced DCs in the treatment of IBD.

Effect of TGF-β1 neutralizing antibody on intestinal fibrosis in a mouse model of chronic colitis induced with trinitrobenzene sulfonic acid

Effect of TGF-β1 neutralizing antibody on intestinal fibrosis in a mouse model of chronic colitis induced with trinitrobenzene sulfonic acid

Inflammatory hypoxia induces syndecan-2 expression through IL-1β-mediated FOXO3a activation in colonic epithelia.

Chronic inflammation is known to be a key causative factor in tumor progression, but we do not yet fully understand the molecular mechanism through which inflammation leads to cancer. Here, we report that the dextran sulfate sodium (DSS)-induced mouse model of chronic colitis is associated with increases in the serum level of IL-1β and the colonic epithelial expression of the cell-surface heparan sulfate proteoglycan, syndecan-2. We further show that IL-1β stimulated the transcription of syndecan-2 via NF-κB-dependent FOXO3a activation in CCD841CoN normal colonic epithelial cells and early-stage HT29 colon cancer cells. Inflammatory hypoxia was observed in the colonic epithelia of mice with chronic colitis, suggesting that hypoxic stress is involved in the regulation of syndecan-2 expression. Consistently, experimental inflammatory hypoxia induced hypoxia inducible factor-1α-dependent FOXO3a expression and the p38 MAPK-mediated nuclear localization of FOXO3a. FOXO3a directly mediated syndecan-2 expression in both cell lines and the colonic epithelia of mice with DSS-induced colitis. Moreover, syndecan-2 expression was detected in azoxymethane/DSS-induced colon tumors. Together, these data demonstrate that inflammatory hypoxia up-regulates syndecan-2 via the IL-1β-NF-κB-FOXO3a pathway. These findings provide new mechanistic insights into inflammatory hypoxia-mediated syndecan-2 expression to connect chronic inflammation and the development of colon cancer.-Choi, S., Chung, H., Hong, H., Kim, S. Y., Kim, S.-E., Seoh, J.-Y., Moon, C. M., Yang, E. G., Oh, E.-S. Inflammatory hypoxia induces syndecan-2 expression through IL-1β-mediated FOXO3a activation in colonic epithelia.