Acute Dextran Sodium Sulfate Research Articles

Microbiota-inducible Innate Immune, Siderophore Binding Protein Lipocalin 2 is Critical for Intestinal Homeostasis.

Background & AimsLipocalin 2 (Lcn2) is a multifunctional innate immune protein whose expression closely correlates with the extent of intestinal inflammation. However, whether Lcn2 plays a role in the pathogenesis of gut inflammation is unknown. Herein, we investigated the extent to which Lcn2 regulates inflammation and gut bacterial dysbiosis in mouse models of IBD.MethodsLcn2 expression was monitored in murine colitis models and upon microbiota ablation/restoration. Wild-type (WT) and Lcn2 knockout (Lcn2KO) mice were analyzed for gut bacterial load, composition by 16S ribosomal RNA gene pyrosequencing, and their colitogenic potential by co-housing with interleukin (Il)10KO mice. Acute (dextran sodium sulfate) and chronic (IL10R neutralization and T-cell adoptive transfer) colitis were induced in WT and Lcn2KO mice with or without antibiotics.ResultsLcn2 expression was dramatically induced on inflammation and was dependent on the presence of a gut microbiota and MyD88 signaling. Use of bone marrow–chimeric mice showed that nonimmune cells are the major contributors of circulating Lcn2. Lcn2KO mice showed increased levels of entA-expressing gut bacteria burden, and, moreover, a broadly distinct bacterial community relative to WT littermates. Lcn2KO mice developed highly colitogenic T cells and showed exacerbated colitis on exposure to DSS or neutralization of IL10. Such exacerbated colitis could be prevented by antibiotic treatment. Moreover, exposure to the microbiota of Lcn2KO mice, via cohousing, resulted in severe colitis in Il10KO mice.ConclusionsLcn2 is a bacterially induced, MyD88-dependent protein that plays an important role in gut homeostasis and a pivotal role on challenge. Hence, therapeutic manipulation of Lcn2 levels may provide a strategy to help manage diseases driven by alteration of the gut microbiota.

IL-36R signalling activates intestinal epithelial cells and fibroblasts and promotes mucosal healing in vivo.

Interleukin (IL)-36R signalling plays a proinflammatory role in different organs including the skin, but the expression of IL-36R ligands and their molecular function in intestinal inflammation are largely unknown. We studied the characteristics of IL-36R ligand expression in IBDs and experimental colitis. The functional role of IL-36R signalling in the intestine was addressed in experimental colitis and wound healing models in vivo by using mice with defective IL-36R signalling (IL-36R-/-) or Myd88, neutralising anti-IL-36R antibodies, recombinant IL-36R ligands and RNA-seq genome expression analysis. Expression of IL-36α and IL-36γ was significantly elevated in active human IBD and experimental colitis. While IL-36γ was predominantly detected in nuclei of the intestinal epithelium, IL-36α was mainly found in the cytoplasm of CD14+ inflammatory macrophages. Functional studies showed that defective IL-36R signalling causes high susceptibility to acute dextran sodium sulfate colitis and impairs wound healing. Mechanistically, IL-36R ligands released upon mucosal damage activated IL-36R+ colonic fibroblasts via Myd88 thereby inducing expression of chemokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-6. Moreover, they induced proliferation of intestinal epithelial cells (IECs) and expression of the antimicrobial protein lipocalin 2. Finally, treatment of experimental intestinal wounds with IL-36R ligands significantly accelerated mucosal healing in vivo. IL-36R signalling is activated upon intestinal damage, stimulates IECs and fibroblasts and drives mucosal healing. Modulation of the IL-36R pathway emerges as a potential therapeutic strategy for induction of mucosal healing in IBD.

Decreased Frequency of Intestinal Regulatory CD5+ B Cells in Colonic Inflammation.

BackgroundCD5+ B cells are a type of regulatory immune cells, though the involvement of this B cell subset in intestinal inflammation and immune regulation is not fully understood.MethodsWe examined the distribution of CD5+ B cells in various mouse organs. Expression levels of CD11b, IgM, and toll-like receptor (TLR)-4 and -9 in B cells were evaluated. In vitro, TLR-stimulated IL-10 production by colonic lamina propria (LP) CD5+ and CD5- B cells was measured. In vivo, mice with acute or chronic dextran sulfate sodium (DSS)-induced colonic injury were examined, and the frequency of colonic LP CD5+ B cells in those was assessed by flow cytometry.ResultsThe expression level of TLR9 was higher in colonic LP CD5+ B cells as compared to CD5- B cells. Colonic LP CD5+ B cells produced greater amounts of IL-10 following stimulation with TLR ligands, especially TLR9, as compared with the LP CD5- B cells. Acute intestinal inflammation transiently decreased the frequency of colonic LP CD5+ B cells, while chronic inflammation induced a persistent decrease in colonic LP CD5+ B cells and led to a CD5- B cell-dominant condition.ConclusionA persistent altered mucosal B cell population caused by chronic gut inflammation may be involved in the pathogenesis of inflammatory bowel diseases.

Systemic IL-12 burst expands intestinal T-lymphocyte subsets bearing the α₄ β₇ integrin in mice.

The intestinal immune system is complex and displays unique anatomic and functional characteristics. Numerous immune cell subsets are located beneath the epithelial barrier and their activity is highly regulated. Using hydrodynamic shear of IL-12 cDNA to achieve systemic expression of IL-12 in mice, we evaluated the effect of a transient burst of this cytokine on the activation status of T cells from Peyer's patches (PPs), mesenteric lymph nodes (MLNs), and colonic lamina propria (LP). Following systemic IL-12 release, intestinal T lymphocytes became activated, exhibiting a CD44(high) CD62L(-) phenotype. After 5 days of the cytokine burst, the frequency of α4β7(+) CD4(+) and CD8(+) cells increased, and CD8(+) α4β7(+) cells mainly expressed T bet, a critical regulator of the Th1 differentiation program. The incremental increase in α4β7 expression involved the IL-12 receptor-signal transducer and activator of transcription (STAT)-4 axis, and occurred independently of IFN-γ, IL-4, IL-10, and TNF-α signaling. Moreover, IL-12 priming exacerbated the outcome of acute dextran sodium sulphate (DSS)-induced colitis with higher scores of weight loss, blood in stool, and diarrhea and lower hematocrit. Together, our findings demonstrate that systemic polarizing signals could effectively expand the number of effector cells able to home to the LP and contribute to local inflammation.

Regenerating Reputations: Are Wnt and Myc the Good Guys After All?

Inflammatory bowel disease (IBD) is one of the biggest challenges facing gastroenterologists today, due to the lack of efficacy of many of its treatments, its widespread morbidities, and its increasing incidence [1]. One of the etiological factors thought to contribute to IBD pathogenesis is diminished intestinal barrier function, which at least in theory permits luminal antigens such as bacterial products to enter the submucosa [2]. Hence, a major unmet need in IBD treatment is a strategy to reverse barrier breaches by directly promoting mucosal restitution and repair. While the importance of this endpoint has been widely acknowledged [3], no IBD therapies currently in use directly target regeneration of the intestinal or colonic epithelium. Rather, it is currently a case of ‘‘Mucosa, heal thyself!’’ in which repair is expected to occur secondarily to the blockade of inflammation. Nevertheless, a wounded mucosa and its attendant disrupted barrier are thought to contribute to ongoing inflammation. Thus, directly bolstering mucosal regeneration may provide an improved alternative or adjunct approach to IBD treatment. In this issue of Digestive Diseases and Sciences, Raup-Konsavage and colleagues report proof-of-concept data supporting pharmacologically targeting glycogen synthase kinase (GSK)-3b to promote intestinal repair and regeneration through a c-Myc-dependent pathway [4]. The Wnt signaling pathway and the Myc transcription factor are central regulators of cell division that for years have been theoretical clinical targets. They have typically been investigated as targets for inhibition, due to the strongly oncogenic effects of constitutively activating mutants. However, an emerging literature suggests that harnessing these pathways’ ability to promote cell division could be an effective therapeutic approach for the treatment of injury and inflammation, including in the gut. The Yochum laboratory has previously characterized a mouse with a deletion in the 30 Wnt responsive DNA element of the Myc gene [5]. This mouse displayed a moderate increase in Myc protein expression, heightened intestinal crypt proliferation, and accelerated recovery from dextran sulfate sodium (DSS)-induced experimental colitis. Thus, in the current study, they tested whether indirectly inducing Myc by pharmacologically activating Wnt signaling with LiCl promotes healing. The data show that LiCl does indeed stimulate recovery when given to mice with established acute DSS colitis, and that this protection is sensitive to pharmacological Myc inhibition. The authors also demonstrated direct effects of LiCl and Myc on epithelial cells in vitro by observing lithium-induced accelerated scratch assay restitution, which was reversed by the Myc inhibitor. They consequently outline a surprisingly specific pathway driving recovery from an inflammatory insult in the gut, using a drug related to compounds already widely used in human patients with psychiatric disorders, such as Li2CO3. This study is distinct from previous experiments with lithium or Myc activation in mouse colitis models in that the investigators gave lithium therapeutically rather than prophylactically. The therapeutic approach is an important & Mark R. Frey mfrey@usc.edu

Effects of Dietary Plant Sterols and Stanol Esters with Low- and High-Fat Diets in Chronic and Acute Models for Experimental Colitis.

In this study, we evaluated the effects of dietary plant sterols and stanols as their fatty acid esters on the development of experimental colitis. The effects were studied both in high- and low-fat diet conditions in two models, one acute and another chronic model of experimental colitis that resembles gene expression in human inflammatory bowel disease (IBD). In the first experiments in the high fat diet (HFD), we did not observe a beneficial effect of the addition of plant sterols and stanols on the development of acute dextran sulphate sodium (DSS) colitis. In the chronic CD4CD45RB T cell transfer colitis model, we mainly observed an effect of the presence of high fat on the development of colitis. In this HFD condition, the presence of plant sterol or stanol did not result in any additional effect. In the second experiments with low fat, we could clearly observe a beneficial effect of the addition of plant sterols on colitis parameters in the T cell transfer model, but not in the DSS model. This positive effect was related to the gender of the mice and on Treg presence in the colon. This suggests that especially dietary plant sterol esters may improve intestinal inflammation in a T cell dependent manner.

Galectin-3 Modulates Experimental Colitis

Background: We recently identified galectin-3 (gal-3) as a new and strong fibroblast activator produced by colonic epithelial cells. Very little is known about the influence of gal-3 in inflammatory bowel disease. We, therefore, investigated the impact of gal-3 on dextran sodium sulfate (DSS)-induced colitis in a mouse model. Methods: Colonic lamina propria fibroblasts of healthy controls were used for co-incubation studies of gal-3 with gal-1, TGF-β1, IFNγ, IL-4 and IL-10. Acute and chronic DSS colitis was induced by 3% DSS in drinking water in female Balb/c mice weighing 20-22 g. Recombinant gal-3 was expressed by the pET vector system and used for a 5-day treatment in different concentrations intraperitoneally. The distal third of the colon was used for histologic analysis. Colonic cytokine expression was determined by quantitative RT-PCR. Results: In vitro, gal-3 induced IL-8 secretion was significantly reduced by co-incubation with IL-10 (5 ng/ml) and IL-4 (10 ng/ml). Acute DSS-induced colitis was ameliorated by gal-3 treatment as indicated by increased colonic length and reduced weight loss compared to that of controls. In acute and chronic colitis, gal-3 treatment resulted in a significant suppression of colonic IL-6. Conclusion: Gal-3 significantly reduces inflammation in acute and chronic DSS colitis in mice indicating a potential role in intestinal inflammation.

Interferon Gamma Counteracts the Angiogenic Switch and Induces Vascular Permeability in Dextran Sulfate Sodium Colitis in Mice.

Interferon (IFN)-γ is a central pathogenesis factor in inflammatory bowel disease (IBD) with pleiotropic effects on many different cell types. However, as yet, the immune modulatory functions of IFN-γ in IBD have been predominantly investigated. Based on previous studies showing that IFN-γ acts antiangiogenic in colorectal carcinoma, we investigated the effects of IFN-γ on the vascular system in IBD. Colon tissues of patients with IBD and dextran sulfate sodium-induced colitis in mice were subjected to immunohistochemistry, quantitative real-time polymerase chain reactions, and in situ hybridization to quantify cell activation, angiogenesis, and immune responses. Vascular structure and permeability in mice were analyzed by ultramicroscopy and in vivo confocal laser endomicroscopy. We showed a significantly increased blood vessel density in IBD and dextran sulfate sodium colitis. In mice, this was associated with a disorganized blood vessel structure and profound vascular leakage. As compared with genes associated with angiogenesis, genes associated with inflammatory cell activation including IFN-γ were more strongly upregulated in colitis tissues. IFN-γ exerted direct effects on endothelial cells in IBD tissues in vivo, as indicated by the expression of IFN-γ-induced guanylate binding protein 1 (GBP-1). Neutralization of IFN-γ in the acute dextran sulfate sodium colitis model demonstrated that this cytokine exerts endogenous angiostatic activity in IBD and contributes to increased vascular permeability. The dissection of the pleiotropic activities of IFN-γ in IBD provides new insights to the pathological functions of this cytokine and may be of high relevance for the optimization of combination therapy approaches.

PTU-072 The influence of iron supplementation on the severity of a murine model of colitis

Introduction Iron deficiency anaemia is a common complication in inflammatory bowel disease (IBD). There is evidence that iron supplementation induces inflammation both in normal rats and in rodent models of IBD (Carrier J et al , Aliment Pharmacol Ther. 2001 Dec; 15(12): 1989–99.). Changes in the gut microbiota [dysbiosis] are common in IBD patients, but the mechanism is unclear. Iron may be a factor, as it is a growth factor for some bacteria. We have therefore investigated the effect of dietary iron intake upon a murine model of colitis. Method Animal studies were performed on 6 groups of female C57BL/6 mice. Acute colitis was induced with 2% Dextran Sodium Sulphate (DSS) for 5 days in drinking water before go back on a normal water for a another 5 days. Two modifications of diet (normal chow 200 ppm iron) were used: low iron (100 ppm) and high iron (400 ppm). The groups were 1 controls; 2 acute DSS normal diet; 3 acute DSS with low iron diet for 10 days; 4 acute DSS with high iron diet for 10 days; 5 low iron diet for 4 weeks; 6 high iron diet for 4 weeks. Daily weight and clinical features were logged. Histology was performed at day 10 for groups 1–4 and day 28 for the rest. Colitis was scored using the Bauer score (Bauer et al , Gut 2010; 59:9 1192–1199). Faecal Calprotectin measured by ELISA (Biohit Healthcare Ltd.), and faecal iron by immunoassay. One ANOVA and t tests were performed to compare groups or pairs of samples. Results Weight loss: DSS induced colitis in groups 2–4; it was most severe in the low iron group. Mean changes in weight were +4% in control, -5.3% in group 2, -9.2% in group 3, and -1% in group 4. Post-hoc testing showed p values of 0.0005, 0.16 and 0.01 for low iron diet vs groups 1, 2 and 4, respectively. Histology: mean colitis scores were; 0, 1.4, 1.9, 1.4, 0, 0 for groups 1–6, respectively. The score to low iron DSS mice was significantly worse than that in untreated mice and marginally worse than those on normal or high iron diets. Calprotectin: calprotectin concentrations were greatest in DSS mice on the low iron diet. Faecal iron: this was increased in a dose-dependent manner within 4 weeks in the groups of mice that did not receive DSS. In DSS treated mice, the low iron diet mice resulted in the highest faecal iron results p = 0.010, 0.0024, and 0.0019 for groups 1, 2 and 4 respectively. Conclusion Clinical observation and histological scoring showed that a low iron diet exacerbated DSS colitis. Mice consuming 50% of the normal amount of iron lost more weight and developed more severe colitis than other mice given DSS. The high iron diet had no measureable effect on the severity of DSS colitis. We will now investigate the effects of iron on the intestinal microbiota in the 6 groups. Disclosure of interest None Declared.

Effect of acute and chronic DSS induced colitis on plasma eicosanoid and oxylipin levels in the rat

Eicosanoids and oxylipins are potent lipid mediators involved in the regulation of inflammation. In order to evaluate their role and suitability as biomarkers in colitis, we analyzed their systemic levels in the acute and chronic phase of dextran sulfate sodium (DSS) induced colitis. Male Fischer 344 rats were treated in three cycles with 4% DSS in the drinking water (4 days followed by 10 days recovery) and blood was drawn 3 days prior to the first DSS treatment and on days 4, 11, 32 and 39. Histopathological evaluation of the colon tissue after 42 days showed that the animals developed a mild to severe chronic colitis. Consistently, prostaglandin levels were massively (twofold) elevated in the colonic tissue. LC–MS based targeted metabolomics was used to determine plasma oxylipin levels at the different time points. In the acute phase of inflammation directly after DSS treatment, epoxy-fatty acid (FA), dihydroxy-FA and hydroxy-FA plasma concentrations were uniformly elevated. With each treatment cycle the increase in these oxylipin levels was more pronounced. Our data suggest that in the acute phase of colitis release of polyunsaturated FAs from membranes in the inflamed tissue is reflected by a uniform increase of oylipins formed in different branches of the arachidonic acid cascade. However, during the recovery phases the systemic oxylipin pattern is not or only moderately altered and does not allow to evaluate the onset of chronic inflammation in the colon.

Acute Dextran Sodium Sulfate Dose‐Dependently Induces Colitis in Formula‐Fed Piglets

Numerous factors are associated with the onset and development of inflammatory bowel disease and can lead to variations in nutritional status and growth during development. Acute dextran sodium sulfate (DSS) colitis is a model often used to study inflammatory mechanisms in the colon. The current study aimed to determine if increasing doses of DSS modulate the severity of colonic inflammation in suckling neonatal pigs. Day-old pigs were randomized among 4 treatments (0, 0.313, 0.625, or 1.25 g DSS/kg BW/d; n=5/trt) and fed 3 times per day for 21 days. Colitis was induced from d 17 -21 by DSS delivered in a small volume of milk before morning feeding. Pigs were euthanized and colon samples were collected on d 22. DSS dose did not affect feed intake and growth. There was a linear increase in diarrhea score with increasing dose of DSS on d17 through d21 (P<0.001). In addition, pathology assessments (means ± pooled SEM) showed a linear increase in inflammatory histological scores (ranging from 0 to 4, normal to severe, respectively) associated with DSS doseage. Epithelial damage (0.7, 1.2, 2.6, 3.1 ± 0.3, respectively) lamina propria inflammation (0, 1.2, 1.9, 3.5 ± 0.3), crypt abscess (0, 0.6, 1.4, 2.1 ± 0.4), crypt dropout (0, 0.5, 1.6, 2 ± 0.4), goblet cell depletion (0, 0.4, 1, 1.6 ± 0.3), and hyperplasia (1.1, 2, 2.6, 2.9 ± 0.3) were dose-dependently increased with increasing DSS (P<0.001). Colonic myeloperoxidase activity linearly increased dose-dependently (P < 0.05) as did overall colitis score (0.3, 1, 1.9, 2.4 ± 0.3; P<0.0001). In conclusion, severity of colitis is directly correlated with dose of DSS in the suckling piglet model. Funded by USDA-NIFA 2104-67017-21750.

Alterations of testosterone metabolism in microsomes from rats with experimental colitis induced by dextran sulfate sodium

Down-regulation of some hepatic cytochrome P450s (CYP450s) was observed in patients and animals with ulcerative colitis (UC). This study examined changes of CYP450s activities in microsomes of liver (RLMs), intestine (RIMs) and kidney (RRMs) from rats with experimental acute colitis induced by 5% dextran sulfate sodium (DSS) for 7days and those receiving DSS treatment followed by 7-d cessation through measuring 6α-(CYP1A1), 7α-(CYP2A1), 16α-(CYP2C11) and 2β-/6β-(CYP3A2) hydroxytestosterone (OHT) formed from testosterone. Both pro-(IL-1β, IL-6, TNF-α) and anti-(IL-4, IL-10) inflammatory cytokines were elevated in acute colitis, while the production of the former was enhanced and that of the latter declined by DSS withdrawal. In RLMs, the CYP2A1 activity was significantly increased at DSS stimulation and partially returned to normal level when DSS treatment was terminated. Activity of other CYP450s were decreased by acute colitis and remained after DSS withdrawal. In RRMs, formations of 6α-, 16α- and 2β-OHT significantly declined in acute colitis and DSS termination further potentiated the down-regulation, while 7α-OHT formation was suppressed at DSS stimulation and remained after DSS withdrawal. The formation of 6β-OHT only showed significant decrease after DSS withdrawal. Two metabolites (6α- and 6β-OHT) formed in RIMs and 6β-OHT formation was significantly decreased by DSS stimulation and continued after DSS treatment halted. These findings indicate that the alterations of CYP450s activities vary with organ, CYP isoforms and colitis status, which arouse cautions on efficacy and toxicity of drug therapy during disease progression.

Downregulation of FoxC2 Increased Susceptibility to Experimental Colitis: Influence of Lymphatic Drainage Function?

Although inflammation-induced expansion of the intestinal lymphatic vasculature (lymphangiogenesis) is known to be a crucial event in limiting inflammatory processes, through clearance of interstitial fluid and immune cells, considerably less is known about the impact of an impaired lymphatic clearance function (as seen in inflammatory bowel diseases) on this cascade. We aimed to investigate whether the impaired intestinal lymphatic drainage function observed in FoxC2 mice would influence the course of disease in a model of experimental colitis. Acute dextran sodium sulfate colitis was induced in wild-type and haploinsufficient FoxC2 mice, and survival, disease activity, colonic histopathological injury, neutrophil, T-cell, and macrophage infiltration were evaluated. Functional and structural changes in the intestinal lymphatic vessel network were analyzed, including submucosal edema, vessel morphology, and lymphatic vessel density. We found that FoxC2 downregulation in FoxC2 mice significantly increased the severity and susceptibility to experimental colitis, as displayed by lower survival rates, increased disease activity, greater histopathological injury, and elevated colonic neutrophil, T-cell, and macrophage infiltration. These findings were accompanied by structural (dilated torturous lymphatic vessels) and functional (greater submucosal edema, higher immune cell burden) changes in the intestinal lymphatic vasculature. These results indicate that sufficient lymphatic clearance plays a crucial role in limiting the initiation and perpetuation of experimental colitis and those disturbances in the integrity of the intestinal lymphatic vessel network could intensify intestinal inflammation. Future therapies might be able to exploit these processes to restore and maintain adequate lymphatic clearance function in inflammatory bowel disease.

PTPN2 controls differentiation of CD4⁺ T cells and limits intestinal inflammation and intestinal dysbiosis.

Loss-of-function variants within the gene locus encoding protein tyrosine phosphatase non-receptor type 2 (PTPN2) are associated with increased risk for Crohn's disease (CD). A disturbed regulation of T helper (Th) cell responses causing loss of tolerance against self- or commensal-derived antigens and an altered intestinal microbiota plays a pivotal role in CD pathogenesis. Loss of PTPN2 in the T-cell compartment causes enhanced induction of Th1 and Th17 cells, but impaired induction of regulatory T cells (Tregs) in several mouse colitis models, namely acute and chronic dextran sodium sulfate colitis, and T-cell transfer colitis models. This results in increased susceptibility to intestinal inflammation and intestinal dysbiosis which is comparable with that observed in CD patients. We detected inflammatory infiltrates in liver, kidney, and skin and elevated autoantibody levels indicating systemic loss of tolerance in PTPN2-deficient animals. CD patients featuring a loss-of-function PTPN2 variant exhibit enhanced Th1 and Th17 cell, but reduced Treg markers when compared with PTPN2 wild-type patients in serum and intestinal tissue samples. Our data demonstrate that dysfunction of PTPN2 results in aberrant T-cell differentiation and intestinal dysbiosis similar to those observed in human CD. Our findings indicate a novel and crucial role for PTPN2 in chronic intestinal inflammation.

Abstract 3658: 90K attenuates the development of colitis-associated colorectal tumors through negative regulation of intestinal epithelial Toll-like receptor 4

Abstract Background: Inflammation is a risk factor for colon cancer. TLR4 expression is upregulated in almost all colitis-associated cancer and dysplasia. The molecular mechanisms linking inflammation and colon carcinogenesis are incompletely understood. We found that tumor associated antigen 90K antagonized TLR4/NF-kB signaling pathway in colon cancer cells. We hypothesized that 90K is involved in TLR4 expression in colitis-induced colon carcinogenesis. Methods: The effect of 90K against TLR4 was analysed by cell cultures, NF-kB reporter assay, and immunoprecipitation. C57BL/6 mice were treated with 3% dextran sodium sulfate (DSS) for acute colitis model. Colitis was assessed by disease activity index (DAI) including weight loss, stool consistency and rectal bleeding, and histopathology. Results: 90K interacts with TLR4, suppresses the NF-kB signal in the MYD88-dependent pathway that is major adaptor molecules in TLRs/NF-kB signal. Adenoviral 90K administration attenuated the severity of acute DSS-induced colitis as assessed by DAI and histopathologic scoring compared with the control group. 90K restored body weight loss and the severity of acute DSS colitis through the suppression of TLR4/MYD88/NF-kB signal. Conclusions: Tumor associated antigen 90K is involved in negative regulation of inflammatory signaling triggered by TLR4-induced NF-kB activation. The data suggest that 90K might thereby suppress proliferation and progression of colon cancers. Citation Format: Ik-Joo Chung, Jun-Eul Hwang, Ji-Hee Lee. 90K attenuates the development of colitis-associated colorectal tumors through negative regulation of intestinal epithelial Toll-like receptor 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 3658. doi:10.1158/1538-7445.AM2014-3658

Acute murine colitis reduces colonic 5-aminosalicylic acid metabolism by regulation of N-acetyltransferase-2

Pharmacotherapy based on 5-aminosalicylic acid (5-ASA) is a preferred treatment for ulcerative colitis, but variable patient response to this therapy is observed. Inflammation can affect therapeutic outcomes by regulating the expression and activity of drug-metabolizing enzymes; its effect on 5-ASA metabolism by the colonic arylamine N-acetyltransferase (NAT) enzyme isoforms is not firmly established. We examined if inflammation affects the capacity for colonic 5-ASA metabolism and NAT enzyme expression. 5-ASA metabolism by colonic mucosal homogenates was directly measured with a novel fluorimetric rate assay. 5-ASA metabolism reported by the assay was dependent on Ac-CoA, inhibited by alternative NAT substrates (isoniazid, p-aminobenzoylglutamate), and saturable with Km (5-ASA) = 5.8 μM. A mouse model of acute dextran sulfate sodium (DSS) colitis caused pronounced inflammation in central and distal colon, and modest inflammation of proximal colon, defined by myeloperoxidase activity and histology. DSS colitis reduced capacity for 5-ASA metabolism in central and distal colon segments by 52 and 51%, respectively. Use of selective substrates of NAT isoforms to inhibit 5-ASA metabolism suggested that mNAT2 mediated 5-ASA metabolism in normal and colitis conditions. Western blot and real-time RT-PCR identified that proximal and distal mucosa had a decreased mNAT2 protein-to-mRNA ratio after DSS. In conclusion, an acute colonic inflammation impairs the expression and function of mNAT2 enzyme, thereby diminishing the capacity for 5-ASA metabolism by colonic mucosa.

Role of metallothioneins as danger signals in the pathogenesis of colitis

Inflammatory bowel diseases (IBDs) are recurrent intestinal pathologies characterized by a compromised epithelial barrier and an exaggerated immune activation. Mediators of immune cell infiltration may represent new therapeutic opportunities. Metallothioneins (MTs) are stress-responsive proteins with immune-modulating functions. Metallothioneins have been linked to IBDs, but their role in intestinal inflammation is inconclusive. We investigated MT expression in colonic biopsies from IBDs and acute infectious colitis patients and healthy controls and evaluated MT's role in experimental colitis using MT knockout mice and anti-MT antibodies. Antibody potential to target extracellular MT and its mechanism was tested in vitro. Biopsies of patients with active colitis showed infiltration of MT-positive cells in a pattern that correlated with the grade of inflammation. MT knockout mice displayed less severe acute dextran sulphate sodium (DSS)-induced colitis compared to congenic wild-type mice based on survival, weight loss, colon length, histological inflammation and leukocyte infiltration. Chronic DSS-colitis confirmed that Mt1 and Mt2 gene disruption enhances clinical outcome. Blockade of extracellular MT with antibodies reduced F4/80-positive macrophage infiltration in DSS- and trinitrobenzene sulphonic acid-colitis, with a tendency towards a better outcome. Whole-body single-photon emission computer tomography of mice injected with radioactive anti-MT antibodies showed antibody accumulation in the colon during colitis and clearance during recovery. Necrotic and not apoptotic cell death resulted in western blot MT detection in HT29 cell supernatant. In a Boyden chamber migration assay, leukocyte attraction towards the necrotic cell supernatant could be abolished with anti-MT antibody, indicating the chemotactic potential of endogenous released MT. Our results show that human colitis is associated with infiltration of MT-positive inflammatory cells. Since antibody blockade of extracellular MT can reduce colitis in mice, MT may act as a danger signal and may represent a novel target for reducing leukocyte infiltration and inflammation in IBD patients.

Macrophages and Dendritic Cells Emerge in the Liver during Intestinal Inflammation and Predispose the Liver to Inflammation

The liver is a physiological site of immune tolerance, the breakdown of which induces immunity. Liver antigen-presenting cells may be involved in both immune tolerance and activation. Although inflammatory diseases of the liver are frequently associated with inflammatory bowel diseases, the underlying immunological mechanisms remain to be elucidated. Here we report two murine models of inflammatory bowel disease: RAG-2−/− mice adoptively transferred with CD4+CD45RBhigh T cells; and IL-10−/− mice, accompanied by the infiltration of mononuclear cells in the liver. Notably, CD11b−CD11clowPDCA-1+ plasmacytoid dendritic cells (DCs) abundantly residing in the liver of normal wild-type mice disappeared in colitic CD4+CD45RBhigh T cell-transferred RAG-2−/− mice and IL-10−/− mice in parallel with the emergence of macrophages (Mφs) and conventional DCs (cDCs). Furthermore, liver Mφ/cDCs emerging during intestinal inflammation not only promote the proliferation of naïve CD4+ T cells, but also instruct them to differentiate into IFN-γ-producing Th1 cells in vitro. The emergence of pathological Mφ/cDCs in the liver also occurred in a model of acute dextran sulfate sodium (DSS)-induced colitis under specific pathogen-free conditions, but was canceled in germ-free conditions. Last, the Mφ/cDCs that emerged in acute DSS colitis significantly exacerbated Fas-mediated hepatitis. Collectively, intestinal inflammation skews the composition of antigen-presenting cells in the liver through signaling from commensal bacteria and predisposes the liver to inflammation.

Acute DSS colitis alters EphB6 receptor expression in neurons of the spinal dorsal horn

The ephrin family of receptors (Eph) and their ephrin ligands are involved in pain associated hyperalgesia, but the underlying mechanisms involved have not been fully elucidated. The EphB6 receptor is a distinctive member of the EphB subclass in that its kinase domain contains several alterations in the conserved amino acids and thus lacks catalytic activity. We sought to identify a role for EphB6 in inflammatory pain, with the murine dextran sulfate sodium (DSS) colitis model of inflammatory bowel disease (IBD). Colitis, induced with the administration of 4% (wt./vol.) DSS in the drinking water, significantly decreased EphB6 protein expression levels in neurons of the lower thoracic superficial layers of spinal dorsal horns, the location of neurons that receive the majority of nociceptive information from the colon, via the primary afferents. A shift towards increased EphB/ephrinB forward signaling, mediated by EphB6 down-regulation in neurons of the dorsal horn, may play a role in inflammatory pain caused by IBD.

P-229 Muc5ac Expression Protects the Intestinal Mucosa During Acute Colitis

The mucus gel layer lining the intestinal epithelium is essential for maintaining mucosal homeostasis. Goblet cells within the epithelial barrier are responsible for the secretion of intestinal mucins. Goblet cell depletion and decreased mature mucin production is associated with enhanced mucosal inflammation in patients with ulcerative colitis (UC). Genetic loss of the secreted gel-forming mucin, Muc2 results in the spontaneous development of colitis in mice, suggestive of a key role for the mucus gel layer in colitis pathogenesis. Muc5ac is another member of this family of gel-forming mucins. Elevated Muc5ac expression has been detected in the mucosa of UC patients. However, the role of Muc5ac in UC is unknown. Previous studies demonstrate a beneficial role for gastrointestinal Muc5ac expression during nematode infection. We hypothesize that expression of colonic Muc5ac is a protective response during inflammation associated with ulcerative colitis. Muc5ac expression was studied during the course of acute dextran sulphate sodium (DSS) colitis in C57BL/6 mice. Mice with genetic loss of Muc5ac (Muc5ac-/-) and wildtype control mice (Muc5ac+/+) were subjected to DSS colitis followed by analysis of disease activity parameters. Muc5ac expression in colonic tissue was greatly increased throughout the timecourse of DSS colitis. Upon exposure to DSS, Muc5ac-/- mice demonstrated increased weight loss and colonic shortening in comparison to Muc5ac+/+ mice following 7 days of DSS. Blinded histological analysis revealed that Muc5ac-/- mice experienced dramatically elevated tissue inflammation and injury over that observed in Muc5ac+/+ mice during DSS. Finally, Muc5ac-/- mice exhibited significant mortality compared to wildtype controls as a result of DSS administration. Initial studies indicate that expression of the gel-forming mucin Muc5ac mediates a protective response during acute colitis. This is the first study to present evidence of a direct functional role for Muc5ac in a model of IBD.