Mouse Model Of Dextran Sulfate Sodium Research Articles

Fibroblast growth factor receptor 4 deficiency in macrophages aggravates experimental colitis by promoting M1-polarization.

Compelling evidence indicates that dysregulated macrophages may play a key role in driving inflammation in inflammatory bowel disease (IBD). Fibroblast growth factor (FGF)-19, which is secreted by ileal enterocytes in response to bile acids, has been found to be significantly lower in IBD patients compared to healthy individuals, and is negatively correlated with the severity of diarrhea. This study aims to explore the potential impact of FGF19 signaling on macrophage polarization and its involvement in the pathogenesis of IBD. The dextran sulfate sodium (DSS)-induced mouse colitis model was utilized to replicate the pathology of human IBD. Mice were created with a conditional knockout of FGFR4 (a specific receptor of FGF19) in myeloid cells, as well as mice that overexpressing FGF19 specifically in the liver. The severity of colitis was measured using the disease activity index (DAI) and histopathological staining. Various techniques such as Western Blotting, quantitative PCR, flow cytometry, and ELISA were employed to assess polarization and the expression of inflammatory genes. Myeloid-specific FGFR4 deficiency exacerbated colitis in the DSS mouse model. Deletion or inhibition of FGFR4 in bone marrow-derived macrophages (BMDMs) skewed macrophages towards M1 polarization. Analysis of transcriptome sequencing data revealed that FGFR4 deletion in macrophages significantly increased the activity of the complement pathway, leading to an enhanced inflammatory response triggered by LPS. Mechanistically, FGFR4-knockout in macrophages promoted complement activation and inflammatory response by upregulating the nuclear factor-κB (NF-κB)-pentraxin3 (PTX3) pathway. Additionally, FGF19 suppressed these pathways and reduced inflammatory response by activating FGFR4 in inflammatory macrophages. Liver-specific overexpression of FGF19 also mitigated inflammatory responses induced by DSS in vivo. Our study highlights the significance of FGF19-FGFR4 signaling in macrophage polarization and the pathogenesis of IBD, offering a potential new therapeutic target for IBD.

Fibroblast growth factor receptor 4 deficiency in macrophages aggravates experimental colitis by promoting M1-polarization.

Compelling evidence indicates that dysregulated macrophages may play a key role in driving inflammation in inflammatory bowel disease (IBD). Fibroblast growth factor (FGF)-19, which is secreted by ileal enterocytes in response to bile acids, has been found to be significantly lower in IBD patients compared to healthy individuals, and is negatively correlated with the severity of diarrhea. This study aims to explore the potential impact of FGF19 signaling on macrophage polarization and its involvement in the pathogenesis of IBD. The dextran sulfate sodium (DSS)-induced mouse colitis model was utilized to replicate the pathology of human IBD. Mice were created with a conditional knockout of FGFR4 (a specific receptor of FGF19) in myeloid cells, as well as mice that overexpressing FGF19 specifically in the liver. The severity of colitis was measured using the disease activity index (DAI) and histopathological staining. Various techniques such as Western Blotting, quantitative PCR, flow cytometry, and ELISA were employed to assess polarization and the expression of inflammatory genes. Myeloid-specific FGFR4 deficiency exacerbated colitis in the DSS mouse model. Deletion or inhibition of FGFR4 in bone marrow-derived macrophages (BMDMs) skewed macrophages towards M1 polarization. Analysis of transcriptome sequencing data revealed that FGFR4 deletion in macrophages significantly increased the activity of the complement pathway, leading to an enhanced inflammatory response triggered by LPS. Mechanistically, FGFR4-knockout in macrophages promoted complement activation and inflammatory response by upregulating the nuclear factor-κB (NF-κB)-pentraxin3 (PTX3) pathway. Additionally, FGF19 suppressed these pathways and reduced inflammatory response by activating FGFR4 in inflammatory macrophages. Liver-specific overexpression of FGF19 also mitigated inflammatory responses induced by DSS in vivo. Our study highlights the significance of FGF19-FGFR4 signaling in macrophage polarization and the pathogenesis of IBD, offering a potential new therapeutic target for IBD.

Steamed broccoli sprouts alleviate DSS-induced inflammation and retain gut microbial biogeography in mice.

Inflammatory bowel diseases (IBDs) are devastating conditions of the gastrointestinal tract with limited treatments, and dietary intervention may be effective and affordable for managing symptoms. Glucosinolate compounds are highly concentrated in broccoli sprouts, especially glucoraphanin (GLR), and can be metabolized by certain mammalian gut bacteria into anti-inflammatory isothiocyanates, such as sulforaphane. Gut microbiota exhibit biogeographic patterns, but it is unknown if colitis alters these or whether the location of glucoraphanin-metabolizing bacteria affects anti-inflammatory benefits. We fed specific pathogen-free C57BL/6 mice either a control diet or a 10% steamed broccoli sprout diet and gave a three-cycle regimen of 2.5% dextran sodium sulfate (DSS) in drinking water over a 34-day experiment to simulate chronic, relapsing ulcerative colitis (UC). We monitored body weight, fecal characteristics, lipocalin, serum cytokines, and bacterial communities from the luminal- and mucosal-associated populations in the jejunum, cecum, and colon. Mice fed the broccoli sprout diet with DSS treatment performed better than mice fed the control diet with DSS, and had significantly more weight gain, lower Disease Activity Index scores, lower plasma lipocalin and proinflammatory cytokines, and higher bacterial richness in all gut locations. Bacterial communities were assorted by gut location but were more homogenous across locations in the control diet + DSS mice. Importantly, our results showed that broccoli sprout feeding abrogated the effects of DSS on gut microbiota, as bacterial richness and biogeography were similar between mice receiving broccoli sprouts with and without DSS. Collectively, these results support the protective effect of steamed broccoli sprouts against dysbiosis and colitis induced by DSS. IMPORTANCE Evaluating bacterial communities across different locations in the gut provides a greater insight than fecal samples alone and provides an additional metric by which to evaluate beneficial host-microbe interactions. Here, we show that 10% steamed broccoli sprouts in the diet protects mice from the negative effects of dextran sodium sulfate-induced colitis, that colitis erases biogeographic patterns of bacterial communities in the gut, and that the cecum is not likely to be a significant contributor to colonic bacteria of interest in the DSS mouse model of ulcerative colitis. Mice fed the broccoli sprout diet during colitis performed better than mice fed the control diet while receiving DSS. The identification of accessible dietary components and concentrations that help maintain and correct the gut microbiome may provide universal and equitable approaches to IBD prevention and recovery, and broccoli sprouts represent a promising strategy.

Eliminating Pathobionts With Bacteriophages: A Novel Approach to Reduce Gut Inflammation in Inflammatory Bowel Diseases?

Federici, Kredo-Russo S, Valdés-Mas R, et al. Targeted suppression of human IBD-associated gut microbiota commensals by phage consortia for treatment of intestinal inflammation. Cell 2022;185:2879–2898.e24. Gut microbes have been repeatedly linked to inflammatory bowel diseases (IBD), with reproducible changes demonstrated in diversity, composition, and function. Modest efficacy of microbe-altering treatments, including probiotics, antibiotics, and fecal microbial transplantation, and the lack of an obvious microbial pathogen causing IBD, have raised the discussion on cause versus effect regarding the role of microbes in IBD pathogenesis. In fact, both are likely important because animal models of IBD point to a causative role and the inflamed gut environment in IBD clearly impacts microbes. A recent article by Federici et al, published in Cell, supports a clear causative role of a specific bacterium (Klebsiella pneumonia [Kp]) and shows that selective elimination of Kp could provide a novel treatment approach for IBD. Remarkably, to overcome challenges with selective bacterial suppression, the authors developed a consortium of 5 bacteriophages able to target this pathobiont. Kp and associated antibiotic resistance genes were linked to both Crohn disease and ulcerative colitis in 4 cohorts from France, Israel, Japan, and the United States (found in approximately 40% of patients with IBD); Kp presence also correlated with disease severity. Metagenomics allowed for strain-level analysis, which identified specific IBD-associated strains (termed Kp2). Inoculating Kp2 (but not other strains) into germ-free mice induced gut inflammation, with immune features similar to those seen in human IBD, including a suppression of interleukin-10. When germ-free interleukin-10–/– mice were infected with Kp2, they developed more severe bowel inflammation. After identifying this candidate pathobiont, the researchers next isolated and screened phages to develop a consortium that would suppress Kp2. After a complex iterative process, a consortium of 5 phages was selected and tested in another mouse model (antibiotic-treated, specific pathogen-free mice). Indeed, treating mice infected with Kp2 with the phage cocktail reduced Kp2 colonization and gut inflammation in a dextran sodium sulfate mouse model, as well as a longer term germ-free Rag1–/– IBD model. To test the ability of these phages to survive in the human gut, the phages were passaged through a human gut simulator and showed good survival, although some phages were impacted by the low gastric pH. In a placebo-controlled randomized controlled trial, 14 healthy volunteers received 2 phages for 3 days and 4 received the control vehicle (all with omeprazole). The phages were detected in the stool of all individuals receiving them, even up to 3 days after discontinuing treatment, suggesting phage survival and persistence in the human gut. Only minor adverse events were reported, but these were similar in both groups. This comprehensive, multilayered study introduces a few key concepts that could impact IBD patient care. The identification of strain-level pathobionts supports microbe-targeted treatments, and the use of combined phage consortia is a promising, and likely safe, option for targeted elimination of pathobionts. Some challenges remain, such as identifying individuals most likely to benefit from such therapy (using personal and disease features), impact of diet and other treatments, and risk of complex resistance over time, but this ground-breaking work promises to open exciting opportunities for future IBD care.

Novel probiotic yeast from Miso promotes regulatory dendritic cell IL-10 production and attenuates DSS-induced colitis in mice.

Yeasts are a type of fungi thought to have probiotic functions. In this study, we isolated a novel probiotic yeast (Zygosaccharomyces sapae strain I-6) from Miso (a traditional Japanese fermented food). We examined its effects on phenotypic changes in intestinal dendritic cells (DCs), and evaluated its anti-inflammatory effects in dextran sulfate sodium (DSS)-induced colitis. A single colony was selected from homogenized Miso, based on its ability to produce interleukin (IL)-10 in CD11c+ bone marrow DCs (BMDCs) in vitro. The anti-inflammatory effects of strain I-6 on CD11c+ BMDCs and CD11c+ CD103+ DCs were analyzed in mouse mesenteric lymph nodes in vitro and in a DSS mouse model. The IL-10 concentrations in the co-culture BMDC supernatants treated with I-6 were dramatically higher than in those treated with Saccharomyces cerevisiae (Sc). IL-10 production is mediated by both TLR2 and Dectin-1. β-Glucan extracted from I-6 also induced higher levels of IL-10 production in BMDCs than β-glucan from Sc. The number of mesenteric lymph node CD11c+ CD103+ DCs was significantly increased by I-6 administration, compared with Sc administration. Strain I-6 showed strong anti-inflammatory effects on DSS-induced colitis compared to Sc. Moreover, the adoptive transfer of I-6-treated BMDCs showed anti-inflammatory effects on DSS-induced colitis in mice without oral administration of I-6 cells. Strain I-6 induced phenotypic changes in intestinal CD11c+ DCs characterized by high IL-10 production and exerted strong anti-inflammatory effects on DSS-induced colitis. Traditional Japanese fermented foods may be a valuable source of probiotic yeasts for effective IBD therapy and treatment.

Extensive Histopathological Characterization of Inflamed Bowel in the Dextran Sulfate Sodium Mouse Model with Emphasis on Clinically Relevant Biomarkers and Targets for Drug Development.

This study aims to develop a reliable and reproducible inflammatory bowel disease (IBD) murine model based on a careful spatial–temporal histological characterization. Secondary aims included extensive preclinical studies focused on the in situ expression of clinically relevant biomarkers and targets involved in IBD. C57BL/6 female mice were used to establish the IBD model. Colitis was induced by the oral administration of 2% Dextran Sulfate Sodium (DSS) for 5 days, followed by 2, 4 or 9 days of water. Histological analysis was performed by sectioning the whole colon into rings of 5 mm each. Immunohistochemical analyses were performed for molecular targets of interest for monitoring disease activity, treatment response and predicting outcome. Data reported here allowed us to develop an original scoring method useful as a tool for the histological assessment of preclinical models of DSS-induced IBD. Immunohistochemical data showed a significant increase in TNF-α, α4β7, VEGFRII, GR-1, CD25, CD3 and IL-12p40 expression in DSS mice if compared to controls. No difference was observed for IL-17, IL-23R, IL-36R or F480. Knowledge of the spatial–temporal pattern distribution of the pathological lesions of a well-characterized disease model lays the foundation for the study of the tissue expression of meaningful predictive biomarkers, thereby improving translational success rates of preclinical studies for a personalized management of IBD patients.

Effects of Vitamin D3 on Intestinal Flora in a Mouse Model of Inflammatory Bowel Disease Treated with Rifaximin.

BackgroundRifaximin is an antimicrobial agent used to treat inflammatory bowel disease (IBD). Vitamin D3 can control IBD due to its effects on inflammatory cytokines. The purpose of this study was to assess the effect of vitamin D3 on the intestinal flora of a dextran sulfate sodium (DSS)-induced mouse model treated with rifaximin.Material/MethodsThe mouse model of IBD was developed using DSS (4%) administered via the drinking water. Twenty-four male C57BL6 mice were divided into the control group with a normal diet (N=6), the DSS group with a normal diet (N=6), the DSS group with a normal diet treated with rifaximin (N=6), and the DSS group with a normal diet treated with rifaximin and vitamin D3 (N=6). After 14 days, the colonic tissue was studied histologically. Serum levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) and enzyme-linked immunosorbent assay (ELISA) were used to measure the level of IL-6 and P65, and phospho-p65 was measured by western blot. 16S rRNA gene sequencing was used to analyze fecal samples.ResultsIn the DSS mouse model of IBD, rifaximin reduced the inflammation severity of the colon and reduced the expression of phospho-p65, p65, TNF-α, and IL-6. In the DSS+rifaximin+vitamin D3 group, the therapeutic influences of rifaximin, in terms of weight loss and colonic disease activity, were significantly reduced, and the gut microbiota of the mice were completely changed in composition and diversity.ConclusionsIn a mouse model of IBD, treatment with vitamin D3 significantly increased the metabolism of rifaximin and reduced its therapeutic effects.

Anatabine ameliorates intestinal inflammation and reduces the production of pro-inflammatory factors in a dextran sulfate sodium mouse model of colitis

BackgroundInflammatory bowel disease (IBD) is the collective term for chronic immune-mediated diseases of unknown, multifactorial etiology, arising from the interplay between genetic and environmental factors and including two main disease manifestations: ulcerative colitis (UC) and Crohn’s disease. In the last few decades, naturally occurring alkaloids have gained interest because of their substantial anti-inflammatory effects in several animal models of disease. Studies on mouse models of IBD have demonstrated the anti-inflammatory action of the main tobacco alkaloid, nicotine. In addition, anatabine, a minor tobacco alkaloid also present in peppers, tomato, and eggplant presents anti-inflammatory properties in vivo and in vitro. In this study, we aimed to evaluate the anti-inflammatory properties of nicotine and anatabine in a dextran sulfate sodium (DSS) mouse model of UC.ResultsOral administration of anatabine, but not nicotine, reduced the clinical symptoms of DSS-induced colitis. The result of gene expression analysis suggested that anatabine had a restorative effect on global DSS-induced gene expression profiles, while nicotine only had limited effects. Accordingly, MAP findings revealed that anatabine reduced the colonic abundance of DSS-associated cytokines and increased IL-10 abundance.ConclusionsOur results support the amelioration of inflammatory effects by anatabine in the DSS mouse model of UC, and suggest that anatabine constitutes a promising therapeutic agent for IBD treatment.

Fusobacterium nucleatum exacerbates colitis by damaging epithelial barriers and inducing aberrant inflammation.

Fusobacterium nucleatum (F. nucleatum) has been reported to be enriched in patients with inflammatory bowel disease (IBD). This study aimed to explore the role of F. nucleatum in IBD and its pathogenic mechanism. Several bacteria that have been reported to be associated with IBD or colorectal cancer were measured in the fecal samples of 91 patients with IBD and 43 healthy individuals. Mice with dextran sulfate sodium (DSS)-induced colitis and a Caco-2 cell line were used to explore the pathogenicity of F. nucleatum. Barrier damage was evaluated by a transmission electron microscope, the permeability of fluorescein isothiocyanate-dextran, transepithelial electrical resistance and immunofluorescence. Protein levels of the cell-cell junction and activation of the STAT3 signaling pathway were detected by immunohistochemistry and immunoblot. Cytokine secretion and T-cell differentiation were measured by quantitative real-time polymerase chain reaction and flow cytometry. F. nucleatum was significantly enriched in the feces of patients with IBD and its abundance correlated with disease activity. Administration of F. nucleatum markedly exacerbated colitis in a DSS mouse model. Mechanistically, F. nucleatum damaged epithelial integrity and increased permeability by regulating the expression and distribution of tight junction proteins zonula occludens-1 and occludin. Moreover, F. nucleatum promoted the secretion of cytokines (tumor necrosis factor-α, interferon-γ, interleukin [IL]-1β, IL-6, and IL-17), activated the STAT3 signaling pathway, and induced CD4+ T cell proliferation and Th1 and Th17 subset differentiations. F. nucleatum can damage the intestinal barrier and induce aberrant inflammation, which exacerbates colitis.

Panaxynol, a bioactive component of American ginseng, targets macrophages and suppresses colitis in mice.

Ulcerative colitis has a significant impact on the quality of life for the patients, and can substantially increase the risk of colon cancer in patients suffering long-term. Conventional treatments provide only modest relief paired with a high risk of side effects, while complementary and alternative medicines can offer safe and effective options. Over the past decade, we have shown that both American ginseng and its hexane fraction (HAG) have anti-oxidant and anti-inflammatory properties that can suppress mouse colitis and prevent colitis-associated colon cancer. With the goal of isolating a single active compound, we further fractionated HAG, and found the most abundant molecule in this fraction was the polyacetylene, panaxynol (PA). After isolating and characterizing PA, we tested the efficacy of PA in the treatment and prevention of colitis in mice and studied the mechanism of action. We demonstrate here that PA effectively treats colitis in a Dextran Sulfate Sodium mouse model by targeting macrophages for DNA damage and apoptosis. This study provides additional mechanistic evidence that American ginseng can be used for conventional treatment of colitis and other diseases associated with macrophage dysfunction.

Dextran sulfate sodium mouse model of inflammatory bowel disease evaluated for systemic genotoxicity via blood micronucleus and Pig-a gene mutation assays

Inflammatory bowel disease (IBD) is an important risk factor for gastrointestinal cancers. Inflammation and other carcinogenesis-related effects at distal, tissue-specific sites require further study. In order to better understand if systemic genotoxicity is associated with IBD, we exposed mice to dextran sulfate sodium salt (DSS) and measured the incidence of micronucleated cells (MN) and Pig-a mutant phenotype cells in blood erythrocyte populations. In one study, 8-week-old male CD-1 mice were exposed to 0, 1, 2, 3 or 4% w/v DSS in drinking water. The 4-week in-life period was divided into four 1-week intervals-alternately on then off DSS treatment. Low volume blood samples were collected for MN analysis at the end of each week, and cardiac blood samples were collected at the end of the 4-week period for Pig-a analyses. The two highest doses of DSS were observed to induce significant increases in reticulocyte frequencies. Even so, no statistically significant treatment-related effects on the genotoxicity biomarkers were evident. While one high-dose mouse showed modestly elevated MN frequencies during the DSS treatment cycles, it also exhibited exceptionally high reticulocyte frequencies (e.g. 18.7% at the end of the second DSS cycle). In a second study, mice were treated with 0 or 4% DSS for 9-18 consecutive days. Exposure was continued until rectal bleeding or morbidity was evident, at which point the treatment was terminated and blood was collected for MN analysis. The Pig-a assay was conducted on samples collected 29 days after the start of treatment. The initial blood specimens showed highly elevated reticulocyte frequencies in DSS-exposed mice (mean ± SEM = 1.75 ± 0.10% vs. 13.04 ± 3.66% for 0 vs. 4% mice, respectively). Statistical analyses showed no treatment-related effect on MN or Pig-a mutant frequencies. Even so, the incidence of MN versus reticulocytes in the DSS-exposed mice were positively correlated (linear fit R2 = 0.657, P = 0.0044). Collectively, these results suggest that in the case of the DSS CD-1 mouse model, systemic effects include stress erythropoiesis but not remarkable genotoxicity. To the extent MN may have been slightly elevated in a minority of individual mice, these effects appear to be secondary, likely attributable to stimulated erythropoiesis.

ACUTE TOXICITY OF CHITOSAN NANOPARTICLES CONTAINING MAHKOTA DEWA (PHALERIA MACROCARPA) LEAF EXTRACT AND ANTI-INFLAMMATORY EFFECTS IN A DEXTRAN SODIUM SULFATE-INDUCED MOUSE MODEL OF ULCERATIVE COLITIS

Objective: The plant mahkota dewa (Phaleria macrocarpa) is known to have anti-inflammatory effects. This study aimed to determine whetherchitosan nanoparticles containing mahkota dewa leaf extract would yield superior anti-inflammatory effects in the colon of a mouse model of dextransodium sulfate (DSS)-induced ulcerative colitis, compared with ethanol extract alone after testing the acute toxicities (lethal dose) of both preparations.Methods: For acute toxicity testing, 10 Sprague-Dawley rats were administered 6000 mg/kg body weight (BW) of leaf extract alone or with nanoparticles.Subsequently, mice were divided into the following six groups to determine the anti-inflammatory effects: Untreated, negative control (DSS 2% w/v), leafextract at 12.5 or 25 mg/kg BW, and leaf extract in chitosan nanoparticles at 6.25 or 12.5 mg/kg BW. To induce colitis, DSS (2% w/v) was administeredthrough drinking water for 6 weeks. The anti-inflammatory effect was observed histopathologically by imaging the inflammatory cells of the mice colonwith hematoxylin-eosin (HE) staining.Results: For acute toxicity testing, 10 Sprague-Dawley rats were administered 6000 mg/kg BW of leaf extract alone or with nanoparticles. Subsequently,mice were divided into the following six groups to determine the anti-inflammatory effects: Untreated, negative control (DSS 2% w/v), leaf extract at12.5 or 25 mg/kg BW, and leaf extract in chitosan nanoparticles at 6.25 or 12.5 mg/kg BW. To induce colitis, DSS (1% w/v) was administered throughdrinking water for 6 weeks. The anti-inflammatory effect was observed histopathologically by imaging the inflammatory cells of the mice colon withHE staining.Conclusion: Chitosan nanoparticles containing mahkota dewa leaf extract can be included in the practically non-toxic class of materials. However, anethanol extract of mahkota dewa leaf effectively inhibited DSS-induced inflammation in the mouse colon, regardless of delivery vehicle.

Intestinal barrier tightening by a cell-penetrating antibody to Bin1, a candidate target for immunotherapy of ulcerative colitis.

Patients afflicted with ulcerative colitis (UC) are at increased risk of colorectal cancer. While its causes are not fully understood, UC is associated with defects in colonic epithelial barriers that sustain inflammation of the colon mucosa caused by recruitment of lymphocytes and neutrophils into the lamina propria. Based on genetic evidence that attenuation of the bridging integrator 1 (Bin1) gene can limit UC pathogenicity in animals, we have explored Bin1 targeting as a therapeutic option. Early feasibility studies in the dextran sodium sulfate mouse model of experimental colitis showed that administration of a cell-penetrating Bin1 monoclonal antibody (Bin1 mAb 99D) could prevent lesion formation in the colon mucosa in part by preventing rupture of lymphoid follicles. In vivo administration of Bin1 mAb altered tight junction protein expression and cecal barrier function. Strikingly, electrophysiology studies in organ cultures showed that Bin1 mAb could elevate resistance and lower 14 C-mannitol leakage across the cecal mucosa, consistent with a direct strengthening of colonic barrier function. Transcriptomic analyses of colitis tissues highlighted altered expression of genes involved in circadian rhythm, lipid metabolism, and inflammation, with a correction of the alterations by Bin1 mAb treatment to patterns characteristic of normal tissues. Overall, our results suggest that Bin1 mAb protects against UC by directly improving colonic epithelial barrier function to limit gene expression and cytokine programs associated with colonic inflammation.

Immuno-PET in Inflammatory Bowel Disease: Imaging CD4-Positive T Cells in a Murine Model of Colitis.

Inflammatory bowel diseases (IBDs) in humans are characterized in part by aberrant CD4-positive (CD4+) T-cell responses. Currently, identification of foci of inflammation within the gut requires invasive procedures such as colonoscopy and biopsy. Molecular imaging with antibody fragment probes could be used to noninvasively monitor cell subsets causing intestinal inflammation. Here, GK1.5 cys-diabody (cDb), an antimouse CD4 antibody fragment derived from the GK1.5 hybridoma, was used as a PET probe for CD4+ T cells in the dextran sulfate sodium (DSS) mouse model of IBD. Methods: The DSS mouse model of IBD was validated by assessing changes in CD4+ T cells in the spleen and mesenteric lymph nodes (MLNs) using flow cytometry. Furthermore, CD4+ T cell infiltration in the colons of colitic mice was evaluated using immunohistochemistry. 89Zr-labeled GK1.5 cDb was used to image distribution of CD4+ T cells in the abdominal region and lymphoid organs of mice with DSS-induced colitis. Region-of-interest analysis was performed on specific regions of the gut to quantify probe uptake. Colons, ceca, and MLNs were removed and imaged ex vivo by PET. Imaging results were confirmed by ex vivo biodistribution analysis. Results: An increased number of CD4+ T cells in the colons of colitic mice was confirmed by anti-CD4 immunohistochemistry. Increased uptake of 89Zr-maleimide-deferoxamine (malDFO)-GK1.5 cDb in the distal colon of colitic mice was visible in vivo in PET scans, and region-of-interest analysis of the distal colon confirmed increased activity in DSS mice. MLNs from colitic mice were enlarged and visible in PET images. Ex vivo scans and biodistribution confirmed higher uptake in DSS-treated colons (DSS, 1.8 ± 0.40; control, 0.45 ± 0.12 percentage injected dose [%ID] per organ, respectively), ceca (DSS, 1.1 ± 0.38; control, 0.35 ± 0.09 %ID per organ), and MLNs (DSS, 1.1 ± 0.58; control, 0.37 ± 0.25 %ID per organ). Conclusion:89Zr-malDFO-GK1.5 cDb detected CD4+ T cells in the colons, ceca, and MLNs of colitic mice and may prove useful for further investigations of CD4+ T cells in preclinical models of IBD, with potential to guide development of antibody-based imaging in human IBD.

Abstract A03: Determining the role of Vitamin D Receptor (VDR) and Retinoid X Receptor Alpha (RXRα) in colitis

Abstract A03: Determining the role of Vitamin D Receptor (VDR) and Retinoid X Receptor Alpha (RXRα) in colitis

Abstract A06: Exploring mechanisms of colorectal tumor radioresistance in organoids

Abstract Colorectal cancer is the third most common cancer in men and the second in women worldwide. It is currently proposed that a population of tumor stem cells that are resistant to radiotherapy leads to tumor recurrence. Accumulating evidence indicates that a transformed lgr5+ [leucine-rich-repeat-containing G-protein-coupled receptor 5] cell at the base of the colonic crypt represents the cell of origin of colorectal cancer. However, mechanisms underlying colorectal cancer radioresistance are not completely understood. The purpose of this study is to elucidate mechanisms of resistance to radiation of normal and tumor stem cells. Using lgr5-lacZ transgenic mice we show that lgr5+ stem cells in the distal part of the large intestine are significantly more radiation resistant than their counterpart in the small intestine. In agreement with in vivo data, isolated colonic crypts are also significantly more resistant to radiation than small intestinal crypts in vitro. Additionally, using a mouse model of colitis-associated colon adenoma (azoxymethane - dextran sulfate sodium mouse model) we determined that crypt-like structures derived from mouse adenoma are even more radioresistant than normal colon. These results validate intestinal organoids as a model for in vivo radiation responsiveness. The next step is to elucidate mechanisms of radiation resistance of human colorectal stem cell organoids derived from normal tissues and tumors. The findings from this work will shed light on properties of lgr5+ tumor stem cells that may be relevant when devising strategies to reduce tumor burden. Citation Format: Maria Laura Martin, Guoqiang Hua, Zhaoshi Zeng, Philip B. Paty. Exploring mechanisms of colorectal tumor radioresistance in organoids. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A06.

Alternatively Spliced EDA Domain of Fibronectin Is a Target for Pharmacodelivery Applications in Inflammatory Bowel Disease.

The antibody-based pharmacodelivery of cytokines to sites of disease has been extensively studied for various indications but not for the treatment of inflammatory bowel diseases. Here, we report that the alternatively spliced EDA domain of fibronectin, a marker of angiogenesis and of tissue remodeling, is expressed in the dextran sodium sulfate mouse model of colitis and in patients with inflammatory bowel conditions, while being virtually undetectable in most normal adult tissues. Radiolabeled preparations of the F8 antibody, specific to the EDA domain of fibronectin, were shown to selectively localize to sites of inflammation in mice with colitis, as revealed by autoradiographic analysis. Fusion proteins of the F8 antibody with various murine payloads (interleukin-4, the p40 subunit of interleukin-12, interleukin-13) were administered to mice with colitis. IL12p40-F8 mediated an anti-inflammatory activity, which was comparable with the one of cyclosporine, whereas F8-IL4 did not inhibit colitis and F8-IL13 worsened the inflammatory conditions.

Abstract 2472: Acute colitis and colitis-associated cancer are exacerbated in mice deficient N-deacetylase/N-sulfotransferase-4

Abstract NDST4 is one member of N-deacetylase/N-sulfotransferase (heparan glucosaminyl) (NDST) family, which are responsible for heparan sulfate (HS) biosynthesis on a core protein to form heparan sulfate proteoglycans (HSPGs). The HS chains of HSPGs bind to and regulate their functions of various growth factors, cytokines and chemokines. HSPGs ubiquitously reside on cell surface, inside the cell, and in the extracellular matrix. Importantly, the content and distribution of HSPGs are altered during tumorigenesis. In the study, 30 (57.7%) of 52 primary colorectal carcinomas showed a dramatic reduction in NDST4 RNA transcripts, and genetic loss of NDST4 was significantly associated with poor survival of patients with colorectal cancer. We further generated an Ndst4-knockout mouse strain, which could develop and reproduce normally. Notably, using the dextran sodium sulfate mouse model of acute colitis, Ndst4 deficiency resulted in a significantly higher disease activity index and obviously hyperemic appearance in the cecum. In the development of colitis-associated cancer, the size of colonic tumors induced by azoxymethane/dextran sodium sulfate was significantly increased in Ndst4-deficient mice compared with wild-type mice. Taken together, loss-of-function of NDST4 might impair the modification of HS chains of specific HSPGs leading to tumor-promoting inflammation in the colon. The full spectrum of signaling pathways contributed by NDST4 to colitis and tumor progression remains to be elucidated. Citation Format: Tzu-Ming Jao, Ming-Hong Tsai, Chi-Yen Huang, Sheng-Tai Tzeng, Jing-Xing Lee, Wei-Chen Lo, Che-Wei Chang, Ya-Chien Yang. Acute colitis and colitis-associated cancer are exacerbated in mice deficient N-deacetylase/N-sulfotransferase-4. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2472. doi:10.1158/1538-7445.AM2014-2472

The precipitation of behavioral defects due to inflammation in the DSS mouse model of colitis (LB748)

Background: Anxiety, depression, and altered memory have been previously associated with intestinal diseases, including inflammatory bowel disease (IBD). Understanding the association between these behavioral changes and IBD may provide clinical significance seeing as concomitant mood disorders often increase a patient’s risk of developing secondary functional gastrointestinal diseases (FGIDs).Aims: The aim of this study was to determine whether active intestinal inflammation precipitates behavioral defects in memory and anxiety and to determine if these changes persist through the resolution of inflammation.Methods: Dextran Sodium Sulfate (DSS) was administered for 5 days via drinking water followed by either 3 days for active inflammation studies or 8 days for resolution of inflammation studies. Mice were weighed daily and colon lengths were measure at the time of sacrifice in order to assess the degree of sickness. Anxiety‐like behavior (light/dark box) and memory (novel object test) were assessed in the DSS mice vs. controls (WT;C57BL/6).Results: DSS mice at the peak of inflammation demonstrated impairments in non‐spatial memory with an exploration ratio of 54.06 +/‐ 1.84 while controls had a mean exploration ratio of 57.91 +/‐ 1.29 (p<.01; n=13). Anxiety‐like behavior was also evident in the DSS mice, which spent 133.91 +/‐10.17 seconds in the light area of the light/dark box while the controls spent 174 +/‐ 14.51 seconds in the light area (p<.04; n=11). These behavioral defects did not persist at the resolution of intestinal inflammation. However, physiological changes, such as a shortening of the colon, were equally evident at the peak of inflammation as well as at the resolution phase.Conclusions: At the peak of inflammation, the DSS mouse model of colitis displays altered baseline behaviors in regards to memory and anxiety. Although physiological changes persist at the resolution of colitis as seen with persistent shortening of the colon, behavioral defects are not evident post inflammation. Taken together, these findings indicate and underlying connection between the brain and the gut relevant to the context of Inflammatory Bowel Disease.

Involvement of autotaxin/lysophospholipase D expression in intestinal vessels in aggravation of intestinal damage through lymphocyte migration

Lysophosphatidic acid (LPA) has a critical role in lymphocyte migration to secondary lymphoid organs. Autotaxin (ATX)/lysophospholipase D, in the vascular endothelium, is the main enzyme involved in LPA production. Whether ATX is involved in pathological lymphocyte migration to the inflamed mucosa has not been studied. We investigated the involvement of ATX in inflammatory bowel disease patients and two murine models of colitis. Tissue samples were obtained by intestinal biopsies from patients with Crohn's disease and those with ulcerative colitis with informed consent. ATX immunoreactivity was colocalized with MAdCAM-1-positive high-endothelial-like vessels, close to sites of lymphocyte infiltration. Enhanced expression of ATX mRNA was observed in the inflamed mucosa from Crohn's disease and ulcerative colitis patients. ATX mRNA expression level was remarkably higher in the actively inflamed mucosa than in the quiescent mucosa in the same patient. In the T-cell-transferred mouse model, ATX mRNA expression level gradually increased as colitis developed. In the dextran sodium sulfate mouse model, the expression level was considerably higher in colonic mucosa of chronically developed colitis than in colonic mucosa of acute colitis. Administration of an ATX inhibitor, bithionol, remarkably decreased lymphocyte migration to the intestine and ameliorated both dextran sodium sulfate-induced colitis and CD4-induced ileocolitis. In transwell assays, administration of bithionol or 1-bromo-3(s)-hydroxy-4-(palmitoyloxy) butylphosphonate (BrP-LPA) significantly decreased transmigration of splenocytes through high-endothelial-like vessels induced by TNF-α. We conclude that enhanced expression of ATX in the active mucosa has been implicated in the pathophysiology of inflammatory bowel disease through enhancing aberrant lymphocyte migration to the inflamed mucosa.