Dextran Sulfate Sodium Administration Research Articles

The expression of intestinal Cyp2c55 is regulated by the microbiota and inflammation.

Although the mutualistic relationship between the intestinal microbiota and the human host is crucial for maintaining health, the underlying mechanisms of this relationship remain unclear. In the present study, aiming to elucidate the regulatory mechanisms governing the Cyp2c55 expression, which is predominantly observed in colonic tissues, germ-free, antibiotic-administered and colitic mice, as well as mouse colonoids, were used as experimental models. RNA sequencing showed comparable decreases in the colonic Cyp2c55 expression in germ-free and antibiotic-administered mice, when compared with that in specific pathogen-free mice. Furthermore, administration of dextran sulfate sodium decreased the Cyp2c55 expression in colitic mice. For these mice, a Pearson correlation analysis also showed a positive correlation between the Cyp2c55 expression and unconjugated bile acids (BAs), including chenodeoxycholic, muricholic, deoxycholic, lithocholic, and ursodeoxycholic acids, as well as taurine (T)-conjugated secondary BAs, including deoxycholic acid. Moreover, bacterial genera, such as Muribaculaceae and unclassified Lachnospiraceae, also exhibited a positive correlation with these BAs. While administration of an agonist of the pregnane X receptor (PXR) increased the Cyp2c55 expression in mouse colonoids, inflammatory cytokines decreased it. In conclusion, Cyp2c55 was highly expressed in the colonic epithelial cells of mice in a microbiota-dependent manner. The underlying mechanism seemed to involve a BA-mediated PXR activation. In addition, the colonic expression of Cyp2c55 was regulated by the inflammatory response. Although the physiological function of Cyp2c55 remains largely unidentified, our findings suggested that Cyp2c55 may play a role in the mutualistic interaction between the intestinal microbiota and the intestinal homeostasis.

Establishment of an in-vitro inflammatory bowel disease model using immunological differentiation of Caco-2 cells

Studies on intestinal cell differentiation, particularly in dextran sodium sulfate (DSS)-induced inflammatory bowel disease (IBD), have predominantly focused on the disruption of intestinal crypts and suppressive effects on the intestinal microbiota; however, repeated administration of DSS is required to induce inflammatory pathology, and there is a lack of observation of early responses and consideration of differentiation stages. Although colonic adenocarcinoma (Caco-2) cells can be used as intestinal cell models, research on these cells in an immature state is limited. We, therefore, investigated the relationship between Caco-2 cell culture duration and immunological differentiation using α-defensin5 (DEFA5) as an indicator of intestinal immunity and differentiation. Changes in protein and gene expression levels in response to DSS were examined at each differentiation stage. Expression of immune- and differentiation-related proteins, including DEFA5 and lysozyme, was evident from Day 8 of culture. Immune responses to DSS varied with the differentiation stage, affecting cell viability and cytokine expression.•Caco-2 cell culture duration correlates with the differentiation stage of Paneth cells.•DSS exposure elicits different effects depending on the differentiation stage.•Our in-vitro model of IBD facilitates the characterization of the cell differentiation process and provides a methodology to help elucidate the causal mechanisms of IBD.

Yellow Teas Protect against DSS-Induced Ulcerative Colitis by Inhibiting TLR4/NF-κB/NLRP3 Inflammasome in Mice.

Yellow tea (YT), a slightly fermented tea with a unique yellowing process and mellow taste, is becoming widely popular. Currently, the YT includes bud yellow tea (BYT), small-leaf yellow tea (SYT), and large-leaf yellow tea (LYT) based on maturity of raw materials. Previous studies have shown that YT has outstanding potential in preventing metabolic syndrome. However, the distinct effects and mechanisms of different types of YT on ulcerative colitis (UC) are still unclear. This study investigated the effects and mechanisms of continuous or intermittent intervention of three yellow tea water extracts (YTEs) on dextran sulfate sodium (DSS)-induced ulcerative colitis in CD-1 mice. The results showed that YTE intervention significantly improves the syndrome of DSS-induced UC in mice. Mechanistic studies reveal that YTEs increase the expression levels of tight junction (TJ) proteins and reduce the levels of pro-inflammatory cytokines in the colon by inactivating TLR4/NF-κB/NLRP3. YTE treatment protected intestinal barrier integrity and reduced serum lipopolysaccharide (LPS) levels. Interestingly, our results indicate that large-leaf yellow tea (LYT) has a better alleviating effect than BYT and SYT. YTE intervention before DSS administration has a certain degree of preventive effect on ulcerative colitis, while continuous YTE intervention after DSS induction has a significant reversing effect on the damage caused by DSS. Our results indicated that drinking YT may have preventive and therapeutic effect on UC, especially drinking LYT.

The Role of Claudins in the Pathogenesis of Dextran Sulfate Sodium-Induced Experimental Colitis: The Effects of Nobiletin.

The pathogenesis of inflammatory bowel diseases such as ulcerative colitis and Crohn's disease is not well understood. This study investigated the roles and regulation of the claudin-1, -2, -3, and -4 isoforms in the pathogenesis of ulcerative colitis, and the potential therapeutic effects of nobiletin. Colitis was induced in rats by administering dextran sulfate sodium [DSS] in drinking water for seven days. Animals were treated daily with nobiletin [oral, 60 mg/Kg body weight] and studied in four groups, C [non-colitis control], D [DSS-induced colitis], CN [nobiletin-treated non-colitis control], and DN [nobiletin-treated DSS-induced colitis]. On day seven, the animals were sacrificed, and colonic tissues were collected and analyzed. Both macroscopic and microscopic findings suggest the progression of colitis. In the inflamed colon, claudin-1 and -4 proteins were decreased, claudin-2 increased, while the claudin-3 protein remained unchanged. Except for claudin-1, these changes were not paralleled by mRNA expression, indicating a complex regulatory mechanism. Uniform β-actin expression along with consistent quality and yield of total RNA indicated selectivity of these changes. Nobiletin treatment reversed these changes. Altered expression of the claudin isoforms -1, -2, and -4 disrupts tight junctions, exposing the lamina propria to microflora, leading to electrolyte disturbance and the development of ulcerative colitis. Nobiletin with its anti-inflammatory properties may be useful in IBD.

Establishment and Evaluation of a Mouse Model of Experimental Ulcerative Colitis Induced by the Gavage Administration of Dextran Sulfate Sodium.

Given the critical role of dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) mouse models in the appraisal of associated therapeutic drugs, the optimization of the administration method and dosages is of paramount importance. Therefore, UC was induced in mice through the gavage administration of a DSS solution instead of free drinking water. The effects of varying daily dosages (2, 4, 6, and 8 g/kg) and frequencies (once or twice) of administration on the body weight and survival rate of the model mice were evaluated. Concurrently, the inflammatory indicators and tissue sections of the model mice were thoroughly evaluated. The results revealed that when the daily dosage reached 8 g/kg, the dosage exhibited a high level of toxicity, resulting in a high mortality rate among the mice. The DSS administration of 6 g/kg*2 not only elicited conspicuous symptoms, significant weight loss, substantial shortening of the colon, and significant changes in various inflammatory indicators, such as myeloperoxidase (MPO), nitric oxide (NO), reactive oxygen species (ROS), and glutathione (GSH), but it also maintained a high survival rate in the UC mice. The findings from this experiment lay a solid experimental foundation for future research on drugs intended for the treatment of UC.

Sex hormone deprivation abolishes sex-specific differences in murine colon inflammation and related lipid mediator production.

Unresolved inflammation, due to unfavorable imbalances between pro-inflammatory and pro-resolving mediators, leads to chronic inflammatory pathologies that are often sex-biased and regulated by sex hormones, including inflammatory bowel disease. Lipid mediators (LM) produced from polyunsaturated fatty acids by various lipoxygenases (LOX) and cyclooxygenases govern all stages of inflammation, i.e., the initiation and progression by pro-inflammatory eicosanoids and its resolution by specialized pro-resolving mediators (SPM). Here, we reveal sex-specific differences in murine experimental colitis with male preponderance, which was abolished by sex hormone deprivation using gonadectomy, and this correlated to the levels of inflammation-relevant mediators in the colon. Oral dextran sodium sulfate administration caused more severe colon inflammation in male CD-1 mice than in female counterparts during the acute phase. Colitis in males yielded higher colonic cytokine/chemokine levels but lower 12-/15-LOX-derived LM including SPM compared to female animals in the resolving phase. Sex hormone deprivation in male mice by orchidectomy ameliorated colitis and impaired pro-inflammatory cytokine/chemokine levels but elevated 12-/15-LOX products including SPM, thus abolishing the observed sex differences. Conversely, ovariectomy impaired the levels of those LM that dominated in females and that were increased in males after gonadectomy. Our findings suggest that male sex hormones promote the development of colitis connected to the biosynthesis of inflammatory cytokines, chemokines, and certain LM, especially pro-resolving 12-/15-LOX products that appear to be suppressed in the male colon due to androgens.

Protective effects of Amauroderma rugosum on dextran sulfate sodium-induced ulcerative colitis through the regulation of macrophage polarization and suppression of oxidative stress

BackgroundAmauroderma rugosum (AR) is a medicinal mushroom commonly used to treat inflammation, gastric disorders, epilepsy, and cancers due to its remarkable anti-inflammatory and anti-oxidative properties. This study was designed to evaluate the pharmacological effects of AR and its underlying mechanism of action against ulcerative colitis (UC) in vitro and in vivo. MethodsA UC mouse model was established by administration of dextran sulfate sodium (DSS). AR extract was administered intragastrically to mice for 7 days. At the end of the experiment, histopathology, macrophage phenotype, oxidative stress, and inflammatory status were examined in vivo. Furthermore, RAW 264.7, THP-1, and Caco-2 cells were used to elucidate the mechanism of action of AR in vitro. ResultsAR extract (0.5–2 mg/mL) significantly suppressed lipopolysaccharide (LPS) and interferon-gamma (IFN-γ)-induced M1 macrophage (pro-inflammatory) polarization in both RAW 264.7 and THP-1 cells. LPS-induced pro-inflammatory mediators (nitric oxide, TNF-α, IL-1β, MCP-1, and IL-6) were reduced by AR extract in a concentration-dependent manner. Similarly, AR extract downregulated MAPK signaling activity in LPS-stimulated RAW 264.7 cells. AR extract elicited a concentration-dependent increase in the mRNA expression of M2 (anti-inflammatory) phenotype markers (CD206, Arg-1, Fizz-1, and Ym-1) in RAW 264.7 cells. Moreover, AR extract suppressed DSS-induced ROS generation and mitochondrial dysfunction in Caco-2 cells. The in vivo experiment revealed that AR extract (200 mg/kg) increased colon length compared to the DSS-treated group. In addition, disease activity index, spleen ratio, body weight, oxidative stress, and colonic inflammation were markedly improved by AR treatment in DSS-induced UC mice. Finally, AR suppressed M1 and promoted M2 macrophage polarization in UC mice. ConclusionThe AR extract protected against DSS-induced UC by regulating macrophage polarization and suppressing oxidative stress. These valuable findings suggest that adequate intake of AR can prevent and/or treat UC.

Bacillus subtilis SF106 and Bacillus clausii SF174 spores reduce the inflammation and modulate the gut microbiota in a colitis model.

Chronic intestinal inflammation is associated with strong alterations of the microbial composition of the gut. Probiotic treatments and microbiota-targeting approaches have been considered to reduce the inflammation, improve both gut barrier function as well as overall gastrointestinal health. Here, a murine model of experimental colitis was used to assess the beneficial health effects of Bacillus subtilis SF106 and Bacillus clausii (recently renamed Shouchella clausii) SF174, two spore-forming strains previously characterised in vitro as potential probiotics. Experimental colitis was induced in BALB/c mice by the oral administration of dextran sodium sulphate (DSS) and groups of animals treated with spores of either strain. Spores of both strains reduced the DSS-induced inflammation with spores of B. clausii SF174 more effective than B. subtilis SF106. Spores of both strains remodelled the mouse gut microbiota favouring the presence of beneficial microbes such as members of the Bacteroidetes and Akkermansia genera.

HOXD10 regulates intestinal permeability and inhibits inflammation of dextran sulfate sodium-induced ulcerative colitis through the inactivation of the Rho/ROCK/MMPs axis

Abstract Ulcerative colitis (UC) has been identified as a severe inflammatory disease with significantly increased incidence across the world. The detailed role and mechanism of HOXD10 in UC remain unclear. In present study, we found that HOXD10 was lowly expressed in UC samples and was notably decreased by dextran sulfate sodium (DSS) administration. Overexpression of HOXD10 dramatically ameliorated DSS-induced UC symptoms, including the loss of weight, increased disease activity index values, and the shortened colon length. Additionally, terminal-deoxynucleoitidyl transferase mediated nick end labeling and immunohistochemistry staining assays showed that HOXD10 overexpression suppressed cell apoptosis and facilitated proliferation of colon tissues after DSS treatment. Moreover, HOXD10 overexpression obviously suppressed DSS-triggered inflammatory response by decreasing the expression level of TNF-α, IL-6, and IL-1β. Furthermore, overexpression of HOXD10 effectively restored the intestinal permeability, thereby alleviating DSS-induced intestinal barrier dysfunction. Mechanistic study demonstrated that HOXD10 significantly reduced the activities of Rho/ROCK/MMPs axis in colon tissues of mice with UC. In conclusion, this study revealed that HOXD10 might effectively improve DSS-induced UC symptoms by suppressing the activation of Rho/ROCK/MMPs pathway.

Curcumin-mediated alleviation of dextran-induced leaky gut in Drosophila melanogaster.

Aging is commonly characterized by a decline in the physiological functioning of the body organs, with one hallmark being the impairment of intestinal function, leading to increased intestinal permeability known as leaky gut. The aim of this study was to investigate the potential of curcumin to prevent the development of leaky gut in Drosophila melanogaster utilizing the smurf fly method. In this study, flies aged 3-5 days underwent a 10-day dextran sulfate sodium (DSS) treatment to induce intestinal permeability, followed by a smurf assay using brilliant blue dye and locomotor testing the next day. Flies displaying the smurf phenotype were divided into four groups: untreated control and curcumin-treated (10 μM, 50 μM, and 250 μM). After 21 days of treatment, flies were reassessed for the smurf phenotype and underwent locomotor testing. On day 23, flies were subjected to RT-qPCR analysis. By inducing increased intestinal permeability through the administration of DSS, a higher proportion of flies exhibiting the smurf phenotype and a reduced survival rate in the DSS-treated group were observed. Such phenotypes were reversed, decreased number of flies displaying the smurf phenotype and improved fly survival, upon the incorporation of curcumin in the fly food at concentrations of 10, 50, and 250 μM. Subsequent molecular analysis revealed upregulated expression of sod1, cat, and pepck genes, while no significant changes were observed in the expression of sod2, indy, and srl genes following treatment with curcumin at high concentration. Overall, our findings provide insight into the potential effect of curcumin to alleviate the phenotypical features associated with DSS-induced leaky gut, possibly via the selective regulation of aging-related genes.

Myeloid Cell-Derived IL-1 Signaling Damps Neuregulin-1 from Fibroblasts to Suppress Colitis-Induced Early Repair of the Intestinal Epithelium.

Neuregulin-1 (Nrg1, gene symbol: Nrg1), a ligand of the ErbB receptor family, promotes intestinal epithelial cell proliferation and repair. However, the dynamics and accurate derivation of Nrg1 expression during colitis remain unclear. By analyzing the public single-cell RNA-sequencing datasets and employing a dextran sulfate sodium (DSS)-induced colitis model, we investigated the cell source of Nrg1 expression and its potential regulator in the process of epithelial healing. Nrg1 was majorly expressed in stem-like fibroblasts arising early in mouse colon after DSS administration, and Nrg1-Erbb3 signaling was identified as a potential mediator of interaction between stem-like fibroblasts and colonic epithelial cells. During the ongoing colitis phase, a significant infiltration of macrophages and neutrophils secreting IL-1β emerged, accompanied by the rise in stem-like fibroblasts that co-expressed Nrg1 and IL-1 receptor 1. By stimulating intestinal or lung fibroblasts with IL-1β in the context of inflammation, we observed a downregulation of Nrg1 expression. Patients with inflammatory bowel disease also exhibited an increase in NRG1+IL1R1+ fibroblasts and an interaction of NRG1-ERBB between IL1R1+ fibroblasts and colonic epithelial cells. This study reveals a novel potential mechanism for mucosal healing after inflammation-induced epithelial injury, in which inflammatory myeloid cell-derived IL-1β suppresses the early regeneration of intestinal tissue by interfering with the secretion of reparative neuregulin-1 by stem-like fibroblasts.

Dietary fiber guar gum-induced shift in gut microbiota metabolism and intestinal immune activity enhances susceptibility to colonic inflammation

ABSTRACT With an increasing interest in dietary fibers (DFs) to promote intestinal health and the growth of beneficial gut bacteria, there is a continued rise in the incorporation of refined DFs in processed foods. It is still unclear how refined fibers, such as guar gum, affect the gut microbiota activity and pathogenesis of inflammatory bowel disease (IBD). Our study elucidated the effect and underlying mechanisms of guar gum, a fermentable DF (FDF) commonly present in a wide range of processed foods, on colitis development. We report that guar gum containing diet (GuD) increased the susceptibility to colonic inflammation. Specifically, GuD-fed group exhibited severe colitis upon dextran sulfate sodium (DSS) administration, as evidenced by reduced body weight, diarrhea, rectal bleeding, and shortening of colon length compared to cellulose-fed control mice. Elevated levels of pro-inflammatory markers in both serum [serum amyloid A (SAA), lipocalin 2 (Lcn2)] and colon (Lcn2) and extensive disruption of colonic architecture further affirmed that GuD-fed group exhibited more severe colitis than control group upon DSS intervention. Amelioration of colitis in GuD-fed group pre-treated with antibiotics suggest a vital role of intestinal microbiota in GuD-mediated exacerbation of intestinal inflammation. Gut microbiota composition and metabolite analysis in fecal and cecal contents, respectively, revealed that guar gum primarily enriches Actinobacteriota, specifically Bifidobacterium. Guar gum also altered multiple genera belonging to phyla Bacteroidota and Firmicutes. Such shift in gut microbiota composition favored luminal accumulation of intermediary metabolites succinate and lactate in the GuD-fed mice. Colonic IL-18 and tight junction markers were also decreased in the GuD-fed group. Importantly, GuD-fed mice pre-treated with recombinant IL-18 displayed attenuated colitis. Collectively, unfavorable changes in gut microbiota activity leading to luminal accumulation of lactate and succinate, reduced colonic IL-18, and compromised gut barrier function following guar gum feeding contributed to increased colitis susceptibility.

Effects of Potential Prebiotics from Codium fragile on Intestinal Diseases

This study examined the effects of an extract of the green algae Codium fragile (hereafter referred to as CFE) on dextran sulfate sodium (DSS)-induced colitis. As the administration of CFE increased, the proliferation of Akkermansia muciniphila, which is a key player in metabolic and gastrointestinal disorders, also increased. After CFE administration for 10 weeks, acetic acid was identified as the major metabolite in mouse cecum and β-glucuronidase activity in mouse fecesdecreased. Further, CFE significantly alleviated the acute intestinal injury induced by DSS administration, including DAI score, colon length, and histological score. The experimental group also displayed indications of significantly lower neutrophil activity and inflammation. In conclusion, the protective effect of CFE against DSS colitis suggests its clinical use by IBD patients.

Sedanolide alleviates DSS-induced colitis by modulating the intestinal FXR-SMPD3 pathway in mice

IntroductionInflammatory bowel disease (IBD) is a global disease with limited therapy. It is reported that sedanolide exerts anti-oxidative and anti-inflammatory effects as a natural phthalide, but its effects on IBD remain unclear. ObjectivesIn this study, we investigated the impacts of sedanolide on dextran sodium sulfate (DSS)-induced colitis in mice. MethodsThe mice were administered sedanolide or vehicle followed by DSS administration, after which colitis symptoms, inflammation levels, and intestinal barrier function were evaluated. Transcriptome analysis, 16S rRNA sequencing, and targeted metabolomics analysis of bile acids and lipids were performed. ResultsSedanolide protected mice from DSS-induced colitis, suppressed the inflammation, restored the weakened epithelial barrier, and modified the gut microbiota by decreasing bile salt hydrolase (BSH)-expressing bacteria. The downregulation of BSH activity by sedanolide increased the ratio of conjugated/unconjugated bile acids (BAs), thereby inhibiting the intestinal farnesoid X receptor (FXR) pathway. The roles of the FXR pathway and gut microbiota were verified using an intestinal FXR-specific agonist (fexaramine) and germ-free mice, respectively. Furthermore, we identified the key effector ceramide, which is regulated by sphingomyelin phosphodiesterase 3 (SMPD3). The protective effects of ceramide (d18:1/16:0) against inflammation and the gut barrier were demonstrated in vitro using the human cell line Caco-2. ConclusionSedanolide could reshape the intestinal flora and influence BA composition, thus inhibiting the FXR-SMPD3 pathway to stimulate the synthesis of ceramide, which ultimately alleviated DSS-induced colitis in mice. Overall, our research revealed the protective effects of sedanolide against DSS-induced colitis in mice, which indicated that sedanolide may be a clinical treatment for colitis. Additionally, the key lipid ceramide (d18:1/16:0) was shown to mediate the protective effects of sedanolide, providing new insight into the associations between colitis and lipid metabolites.

Human umbilical cord/placenta mesenchymal stem cell conditioned medium attenuates intestinal fibrosis in vivo and in vitro

BackgroundA significant unmet need in inflammatory bowel disease is the lack of anti-fibrotic agents targeting intestinal fibrosis. This study aimed to investigate the anti-fibrogenic properties and mechanisms of the conditioned medium (CM) from human umbilical cord/placenta-derived mesenchymal stem cells (UC/PL-MSC-CM) in a murine intestinal fibrosis model and human primary intestinal myofibroblasts (HIMFs).MethodsUC/PL-MSC-CM was concentrated 15-fold using a 3 kDa cut-off filter. C57BL/6 mice aged 7 weeks old were randomly assigned to one of four groups: (1) control, (2) dextran sulfate sodium (DSS), (3) DSS + CM (late-phase treatment), and (4) DSS + CM (early-phase treatment). Chronic DSS colitis and intestinal fibrosis was induced by three cycles of DSS administration. One DSS cycle consisted of 7 days of oral DSS administration (1.75%, 2%, and 2.5% DSS), followed by 14 days of drinking water. UC/PL-MSC-CM was intraperitoneally administered in the late phase (from day 50, 10 times) or early phase (from day 29, 10 times) of DSS cycles. HIMFs were treated with TGF-β1 and co-treated with UC/PL-MSC-CM (10% of culture media) in the cellular model.ResultsIn the animal study, UC/PL-MSC-CM reduced submucosa/muscularis propria thickness and collagen deposition, which improved intestinal fibrosis in chronic DSS colitis. The UC/PL-MSC-CM significantly reduced the expressions of procollagen1A1 and α-smooth muscle actin, which DSS significantly elevated. The anti-fibrogenic effect was more apparent in the UC-MSC-CM or early-phase treatment model. The UC/PL-MSC-CM reduced procollagen1A1, fibronectin, and α-smooth muscle actin expression in HIMFs in the cellular model. The UC/PL-MSC-CM downregulated fibrogenesis by suppressing RhoA, MRTF-A, and SRF expression.ConclusionsHuman UC/PL-MSC-CM inhibits TGF-β1-induced fibrogenic activation in HIMFs by blocking the Rho/MRTF/SRF pathway and chronic DSS colitis-induced intestinal fibrosis. Thus, it may be regarded as a novel candidate for stem cell-based therapy of intestinal fibrosis.

An inhibitory immunoreceptor Allergin-1 regulates the intestinal dysbiosis and barrier function in mice.

The intestinal barrier consists of mucosal, epithelial, and immunological barriers and serves as a dynamic interface between the host and its environment. Disruption of the intestinal barrier integrity is a leading cause of various gastrointestinal diseases, such as inflammatory bowel disease. The homeostasis of the intestinal barrier is tightly regulated by crosstalk between gut microbes and the immune system; however, the implication of the immune system on the imbalance of gut microbes that disrupts barrier integrity remains to be fully elucidated. An inhibitory immunoglobulin-like receptor, Allergin-1, is expressed on mast cells and dendritic cells and inhibits Toll-like receptor (TLR)-2 and TLR-4 signaling in these cells. Since TLRs are major sensors of microbiota and are involved in local epithelial homeostasis, we investigated the role of Allergin-1 in maintaining intestinal homeostasis. Allergin-1-deficient (Milr1-/-) mice exhibited more severe dextran sulfate sodium (DSS)-induced colitis than did wild-type (WT) mice. Milr1-/- mice showed an enhanced intestinal permeability than did WT mice even before DSS administration. Treatment of Milr1-/- mice with neomycin, but not ampicillin, restored intestinal barrier integrity. The 16S rRNA gene sequencing analysis demonstrated that Bifidobacterium pseudolongum was the dominant bacteria in Milr1-/- mice after treatment with ampicillin. Although the transfer of B. pseudolongum to germ-free WT mice had no effect on intestinal permeability, its transfer into ampicillin-treated WT mice enhanced intestinal permeability. These results demonstrated that Allergin-1 deficiency enhanced intestinal dysbiosis with expanded B. pseudolongum, which contributes to intestinal barrier dysfunction in collaboration with neomycin-sensitive and ampicillin-resistant microbiota.

A4 DNA HYPOMETHYLATION INHIBITS COLITIS-ASSOCIATED CANCER

Abstract Background Colorectal cancer is the second leading cause of cancer death in Canada. A major risk factor for the development of colorectal cancer is chronic inflammation leading to colitis-associated cancer (CAC). We previously described a CAC mouse model in which tumors arise from DCLK1+ cells following loss of the tumor suppressor adenomatous polyposis coli (APC) and induction of colitis. Interestingly, both colitis and CAC are associated with DNA methylation changes that lead to altered gene expression. Moreover, inhibition of DNA methylation has recently been shown to lead to a viral mimicry response. The effects of DNA methylation on colonic tumorigenesis, however, is not known. Aims Thus, we aim to investigate whether inhibition of DNA methylation leads to a viral mimicry response that inhibits colitis-associated tumorigenesis. Methods We first examined the effects of DNA hypomethylation on CAC, by crossing Dclk1-CreERT2/Apcf/f mice to Dnmt1f/f mice which allowed for knock-out of the DNA methyltransferase enzyme DNMT1 in DCLK1+ cells. Dclk1/Apcf/f/Dnmt1f/f and Dclk1/Apcf/f (control group) mice were administered three doses of tamoxifen followed by 2.5% dextran sodium sulfate (DSS) for five days to induce colitis. Fourteen weeks later, we assessed colonic tumor number and size. In a separate cohort of Dclk1/Apcf/f mice treated with DSS, we compared colonic tumor number in mice treated with six doses of 5-AZA-2’-deoxycytidine (5-AZA) versus vehicle. The effects of 5-AZA and DSS on DNA methylation were assessed using the Infinium Mouse Methylation BeadChip Array on colonic epithelial cells isolated from mice treated with vehicle versus 5-AZA in the presence or absence of colitis. RNA expression of viral transposable elements and type I interferon response related genes were measured as a readout of a viral mimicry response. Lastly, we investigated the mechanism by which DNA hypomethylation affects tumorigenesis by crossing Dclk1/Apcf/f/Dnmt1f/f mice to MAVS knockout mice and examining tumor number following tamoxifen and DSS administration as above. Results Deletion of DNMT1 in DCLK1+ cells or treatment with 5-AZA significantly decreased colonic tumor number and size. Treatment with 5-AZA or DNMT1 loss led to DNA hypomethylation and subsequent upregulation of endogenous retroviral expression. Both 5-AZA treatment and DNMT1 knockout in DCLK1+ cells led to increased expression of type I interferon response genes. Knockout of the type I interferon response related gene, MAVS, reversed the anti-tumor effect observed with DNMT1 loss. Conclusions Our findings demonstrate that DNA hypomethylation by 5-AZA or loss of DNMT1 reduces colitis-associated tumorigenesis through activation of a viral mimicry response. Funding Agencies CAG, CIHRCancer Research Society, Canada Foundation for Innovation, Lawson Health Research Institute

A199 ALKAL2/ALK AXIS PROMOTE PERSISTENT VISCERAL PAIN

Abstract Background Long-lasting changes in neural pain circuits precipitate the transition from acute to chronic pain in patients living with inflammatory bowel diseases (IBDs). While significant improvement in IBD therapy has been made to reduce inflammation, a large subset of patients continues to suffer throughout quiescent phases of the disease. Peripheral and central mechanisms contribute to the transition from acute to chronic pain during clinical remission. In the search of new treatments, we previously found that targeting the anaplastic lymphoma kinase (ALK) receptor could have therapeutic value for persistent pain conditions. Aims Here we investigated whether ALKAL2 (ALK ligand) /ALK signaling axis regulates chronic visceral pain. Methods We used the post-inflammatory dextran sodium sulfate (DSS) mouse model of colitis as a well-established rodent model of chronic visceral pain, in which animals display increased VHS 5 weeks after DSS administration in the absence of inflammation as described in IBD patients. To assess the role of ALKAL2/ALK signalling in chronic visceral pain, we pharmacologically blocked ALK receptor using clinically available compound Lorlatinib in the postcolitis model. Moreover we genetically depleted ALKAL2 in gut-innervating nociceptors that express the heat sensitive transient receptor potential vanilloid type 1 (TRPV1) channel, using shRNA vectorized in cell type specific Adeno-associated Viruses. Results We showed that ALKAL2 expression was significatively enhanced in lumbosacral (LS) dorsal root ganglia (DRG) sensory neurons in the post-colitis model. In naïve mice, ALKAL2 administration induced visceral hypersensitivity in an ALK dependant manner. In addition, depletion of ALKAL2 in TRPV1 nociceptors or blocking ALK with the clinically available compound Lorlatinib, reversed visceral hypersensitivity in the post-colitis model. Accordingly, phosphorylation of ALK receptor occurring at the LS of the spinal dorsal horn was reduced. Conclusions Overall, these findings provide novel insights into the mechanisms underlying the transition from acute to chronic visceral pain in IBD. Our work could generate a portfolio of clinically available anti-cancer drugs, including Lorlatinib, as novel pain therapeutics. Repurposed ALK inhibitor could facilitate pain management in in individuals with IBD. Funding Agencies CIHR

Choosing the optimal concentration of dextran sulfate sodium for reproducing chemically induced colitis in rabbits

Background. Due to the fact that inflammatory bowel diseases (IBD) are widespread among the population, the development of new drugs for the treatment of IBD is a public health priority. Preclinical drug testing is inextricably linked with the modeling of IBD in laboratory animals. This study purpose was to develop a model of chemically induced colitis in rabbits. Methods. The study was performed on male rabbits of the “Soviet Chinchilla” breed. Colitis was simulated by oral administration of dextran sulfate sodium (DSS) in a concentration of 0.1% to 2% solution for five consecutive days. Euthanasia of laboratory animals was carried out on the seventh day, after the end of taking the DSS solution. A histological examination of the intestine was performed. Results. It was found that using 0.1% DSS solution allows to obtain a model of mild erosive colitis. The use of 0.5% DSS solution gave the intermediate severity of erosive ulcerative colitis. DSS concentration of more than 1% led to the development of severe ulcerative colitis in rabbits and the death rate of 71.4% among them. Conclusion. The obtained results indicate that the use of 0.5% DSS solution makes it possible to create a model of erosive ulcerative colitis in rabbits suitable for studying human IBD.

Transient gestational hypothyroxinemia accelerates and enhances ulcerative colitis-like disorder in the male offspring.

Gestational hypothyroxinemia (HTX) is a condition that occurs frequently at the beginning of pregnancy, and it correlates with cognitive impairment, autism, and attentional deficit in the offspring. Evidence in animal models suggests that gestational HTX can increase the susceptibility of the offspring to develop strong inflammation in immune-mediated inflammatory diseases. Ulcerative colitis (UC) is a frequent inflammatory bowel disease with unknown causes. Therefore, the intensity of ulcerative colitis-like disorder (UCLD) and the cellular and molecular factors involved in proinflammatory or anti-inflammatory responses were analyzed in the offspring gestated in HTX (HTX-offspring) and compared with the offspring gestated in euthyroidism (Control-offspring). Gestational HTX was induced by the administration of 2-mercapto-1-methylimidazole in drinking water to pregnant mice during E10-E14. The HTX-offspring were induced with UCLD by the acute administration of dextran sodium sulfate (DSS). The score of UCLD symptomatology was registered every day, and colon histopathology, immune cells, and molecular factors involved in the inflammatory or anti-inflammatory response were analyzed on day 6 of DSS treatment. The HTX-offspring displayed earlier UCLD pathological symptoms compared with the Control-offspring. After 6 days of DSS treatment, the HTX-offspring almost doubled the score of the Control-offspring. The histopathological analyses of the colon samples showed signs of inflammation at the distal and medial colon for both the HTX-offspring and Control-offspring. However, significantly more inflammatory features were detected in the proximal colon of the HTX-offspring induced with UCLD compared with the Control-offspring induced with UCLD. Significantly reduced mRNA contents encoding for protective molecules like glutamate-cysteine ligase catalytic subunit (GCLC) and mucin-2 (MUC-2) were found in the colon of the HTX-offspring as compared with the Control-offspring. Higher percentages of Th17 lymphocytes were detected in the colon tissues of the HTX-offspring induced or not with UCLD as compared with the Control-offspring. Gestational HTX accelerates the onset and increases the intensity of UCLD in the offspring. The low expression of MUC-2 and GCLC together with high levels of Th17 Lymphocytes in the colon tissue suggests that the HTX-offspring has molecular and cellular features that favor inflammation and tissue damage. These results are important evidence to be aware of the impact of gestational HTX as a risk factor for UCLD development in offspring.