Dietary Selenium Deficiency Exacerbates DSS-Induced Epithelial Injury and AOM/DSS-Induced Tumorigenesis

Selenium (Se) is an essential micronutrient that exerts its functions via selenoproteins. Little is known about the role of Se in inflammatory bowel disease (IBD). Epidemiological studies have inversely correlated nutritional Se status with IBD severity and colon cancer risk. Moreover, molecular studies have revealed that Se deficiency activates WNT signaling, a pathway essential to intestinal stem cell programs and pivotal to injury recovery processes in IBD that is also activated in inflammatory neoplastic transformation. In order to better understand the role of Se in epithelial injury and tumorigenesis resulting from inflammatory stimuli, we examined colonic phenotypes in Se-deficient or -sufficient mice in response to dextran sodium sulfate (DSS)-induced colitis, and azoxymethane (AOM) followed by cyclical administration of DSS, respectively. In response to DSS alone, Se-deficient mice demonstrated increased morbidity, weight loss, stool scores, and colonic injury with a concomitant increase in DNA damage and increases in inflammation-related cytokines. As there was an increase in DNA damage as well as expression of several EGF and TGF-β pathway genes in response to inflammatory injury, we sought to determine if tumorigenesis was altered in the setting of inflammatory carcinogenesis. Se-deficient mice subjected to AOM/DSS treatment to model colitis-associated cancer (CAC) had increased tumor number, though not size, as well as increased incidence of high grade dysplasia. This increase in tumor initiation was likely due to a general increase in colonic DNA damage, as increased 8-OHdG staining was seen in Se-deficient tumors and adjacent, non-tumor mucosa. Taken together, our results indicate that Se deficiency worsens experimental colitis and promotes tumor development and progression in inflammatory carcinogenesis.     

GPR65 (TDAG8) inhibits intestinal inflammation and colitis-associated colorectal cancer development in experimental mouse models

GPR65 (TDAG8) is a proton-sensing G protein-coupled receptor predominantly expressed in immune cells. Genome-wide association studies (GWAS) have identified GPR65 gene polymorphisms as an emerging risk factor for the development of inflammatory bowel disease (IBD). Patients with IBD have an elevated risk of developing colorectal cancer when compared to the general population. To study the role of GPR65 in intestinal inflammation and colitis-associated colorectal cancer (CAC), colitis and CAC were induced in GPR65 knockout (KO) and wild-type (WT) mice using dextran sulfate sodium (DSS) and azoxymethane (AOM)/DSS, respectively. Disease severity parameters such as fecal score, colon shortening, histopathology, and mesenteric lymph node enlargement were aggravated in GPR65 KO mice compared to WT mice treated with DSS. Elevated leukocyte infiltration and fibrosis were observed in the inflamed colon of GPR65 KO when compared to WT mice which may represent a cellular mechanism for the observed exacerbation of intestinal inflammation. In line with high expression of GPR65 in infiltrated leukocytes, GPR65 gene expression was increased in inflamed intestinal tissue samples of IBD patients compared to normal intestinal tissues. Moreover, colitis-associated colorectal cancer development was higher in GPR65 KO mice than WT mice when treated with AOM/DSS. Altogether, our data demonstrate that GPR65 suppresses intestinal inflammation and colitis-associated tumor development in murine colitis and CAC models, suggesting potentiation of GPR65 with agonists may have an anti-inflammatory therapeutic effect in IBD and reduce the risk of developing colitis-associated colorectal cancer.     

Vitexin prevents colitis-associated carcinogenesis in mice through regulating macrophage polarization

BackgroundPatients with inflammatory bowel disease are at increased risks of developing ulcerative colitis-associated colorectal cancer (CAC). Vitexin can suppress the proliferation of colorectal carcinoma cells in vitro orin vivo. However, different from colorectal carcinoma, CAC is more consistent with the transformation from inflammation to cancer in clinical chronic IBD patients. Therefore, we aim to investigated that vitexin whether possess benefic effects on CAC mice.PurposeWe aimed to determine the beneficial effects of vitexin on CAC mice and reveal its underlying mechanism.MethodsThe mouse CAC model was induced by Azoxymethane and dextran sodium sulfate (AOM/DSS) and CAC mice were treated with vitexin. At the end of this study, inflammatory cytokines of IL-1β, IL-6, TNF-α, IL-10 as well as nitric oxide (NO) were detected by kits after long-term treatment of vitexin. Pathological changes and macrophage polarization were determined by H&E and immunofluorescence in adjacent noncancerous tissue and carcinomatous tissue respectively of CAC mice.ResultsOur results showed that oral administration of vitexin could significantly improve the clinical signs and symptoms of chronic colitis, relieve colon damage, regulate colonic inflammatory cytokines, as well as suppress tumor incidence and tumor burden. Interesting, vitexin caused a significant increase in serum level of NO and a higher content of NO in tumor tissue. In addition, vitexin significantly decreased M1 phenotype macrophages in the adjacent noncancerous tissue, while markedly up-regulated M1 macrophage polarization in the tumor tissue in the colon of CAC mice.ConclusionVitexin can attenuate chronic colitis-associated carcinogenesis induced by AOM/DSS in mice and its protective effects are partly associated with its alternations in macrophage polarization in the inflammatory and tumor microenvironment .     

Modulation of the Intestinal Microbiota Alters Colitis-Associated Colorectal Cancer Susceptibility

It is well established that the intestinal microbiota plays a key role in the pathogenesis of Crohn''s disease (CD) and ulcerative colitis (UC) collectively referred to as inflammatory bowel disease (IBD). Epidemiological studies have provided strong evidence that IBD patients bear increased risk for the development of colorectal cancer (CRC). However, the impact of the microbiota on the development of colitis-associated cancer (CAC) remains largely unknown. In this study, we established a new model of CAC using azoxymethane (AOM)-exposed, conventionalized-Il10^−/− mice and have explored the contribution of the host intestinal microbiota and MyD88 signaling to the development of CAC. We show that 8/13 (62%) of AOM-Il10^−/− mice developed colon tumors compared to only 3/15 (20%) of AOM- wild-type (WT) mice. Conventionalized AOM-Il10^−/− mice developed spontaneous colitis and colorectal carcinomas while AOM-WT mice were colitis-free and developed only rare adenomas. Importantly, tumor multiplicity directly correlated with the presence of colitis. Il10^−/− mice mono-associated with the mildly colitogenic bacterium Bacteroides vulgatus displayed significantly reduced colitis and colorectal tumor multiplicity compared to Il10^−/− mice. Germ-free AOM-treated Il10^−/− mice showed normal colon histology and were devoid of tumors. Il10^−/−; Myd88^−/− mice treated with AOM displayed reduced expression of Il12p40 and Tnfα mRNA and showed no signs of tumor development. We present the first direct demonstration that manipulation of the intestinal microbiota alters the development of CAC. The TLR/MyD88 pathway is essential for microbiota-induced development of CAC. Unlike findings obtained using the AOM/DSS model, we demonstrate that the severity of chronic colitis directly correlates to colorectal tumor development and that bacterial-induced inflammation drives progression from adenoma to invasive carcinoma.     

Therapeutic effects of lentinan on inflammatory bowel disease and colitis‐associated cancer

In this study, we investigated the therapeutic potential of lentinan in mouse models of inflammatory bowel disease (IBD) and colitis‐associated cancer (CAC). Lentinan decreased the disease activity index and macroscopic and microscopic colon tissue damage in dextran sulphate sodium (DSS)‐induced or TNBS‐induced models of colitis. High‐dose lentinan was more effective than salicylazosulfapyridine in the mouse models of colitis. Lentinan decreased the number of tumours, inflammatory cell infiltration, atypical hyperplasia and nuclear atypia in azoxymethane/DSS‐induced CAC model. It also decreased the expression of pro‐inflammatory cytokines, such as IL‐13 and CD30L, in IBD and CAC model mice possibly by inhibiting Toll‐like receptor 4 (TLR4)/NF‐κB signalling and the expression of colon cancer markers, such as carcinoembryonic antigen, cytokeratin 8, CK18 and p53, in CAC model mice. In addition, lentinan restored the intestinal bacterial microbiotal community structure in IBD model mice. Thus, it shows therapeutic potential in IBD and CAC model mice possibly by inhibiting TLR4/NF‐κB signalling‐mediated inflammatory responses and disruption of the intestinal microbiotal structure.     

IQ Motif-Containing GTPase-Activating Protein 2 (IQGAP2) Is a Novel Regulator of Colonic Inflammation in Mice

IQ motif-containing GTPase-activating protein 2 (IQGAP2) is a multidomain scaffolding protein that plays a role in cytoskeleton regulation by juxtaposing Rho GTPase and Ca²⁺/calmodulin signals. While IQGAP2 suppresses tumorigenesis in liver, its role in pathophysiology of the gastrointestinal tract remains unexplored. Here we report that IQGAP2 is required for the inflammatory response in colon. Mice lacking Iqgap2 gene (Iqgap2^-/- mice) were resistant to chemically-induced colitis. Unlike wild-type controls, Iqgap2^-/- mice treated with 3% dextran sulfate sodium (DSS) in water for 13 days displayed no injury to colonic epithelium. Mechanistically, resistance to colitis was associated with suppression of colonic NF-κB signaling and IL-6 synthesis, along with diminished neutrophil and macrophage production and recruitment in Iqgap2^-/- mice. Finally, alterations in IQGAP2 expression were found in colons of patients with inflammatory bowel disease (IBD). Our findings indicate that IQGAP2 promotes inflammatory response at two distinct levels; locally, in colonic epithelium through TLR4/NF-κB signaling pathway, and systemically, via control of maturation and recruitment of myeloid immune cells. This work identifies a novel mechanism of colonic inflammation mediated by signal transducing scaffolding protein IQGAP2. IQGAP2 domain-specific blocking agents may represent a conceptually novel strategy for therapy of IBD and other inflammation-associated disorders, including cancer.     

Intestinal anti-inflammatory effect of the probiotic Saccharomyces boulardii in DSS-induced colitis in mice: Impact on microRNAs expression and gut microbiota composition

The beneficial effects exerted by probiotics in inflammatory bowel disease (IBD) are well known, although their exact mechanisms have not been fully elucidated, and only few studies have focused on their impact on selected miRNAs and the gut microbiota composition. Therefore, our aim was to correlate the intestinal anti-inflammatory activity of the probiotic Saccharomyces boulardii in the dextran sodium sulphate (DSS) model of mouse colitis and the changes induced in miRNA expression and gut microbiota populations. Probiotic was given orally (5×10⁹ CFU) to C57BL/6 mice for 26 days. After 2 weeks, the colitis was induced adding DSS to the drinking water. Mice were scored daily using a Disease Activity Index (DAI). After sacrifice, the colonic specimens were evaluated by determining the expression of inflammatory markers and micro-RNAs by qRT-PCR. Moreover, changes in microbiota populations were evaluated by pyrosequencing. Probiotic ameliorated the colonic damage induced by DSS, as evidenced by lower DAI values and colonic weight/length compared with untreated mice. The treatment modified the colonic expression of different inflammatory markers and the epithelial integrity proteins, and induced changes in micro-RNAs expression. Moreover, microbiota characterization showed that probiotic treatment increased bacterial diversity, thus ameliorating the dysbiosis produced by DSS-colitis. Saccharomyces boulardii exerted intestinal anti-inflammatory effects in DSS-mouse colitis, through the modulation in the immune response, involving modification of altered miRNA expression, being associated to the improvement of the inflammation-associated dysbiosis in the intestinal lumen, which could be of great interest to control the complex pathogenesis of IBD.     

Genetic deletion of IL-25 (IL-17E) confers resistance to dextran sulfate sodium-induced colitis in mice

Background

IL-25 is emerging as a key regulator of inflammation in the intestinal mucosa because of its ability to promote type 2 while suppressing Th1 and Th17 responses. Several previous studies reported inconsistent results on the role of exogenous IL-25 in development of colonic inflammation and none were performed in animals with a genetic deletion of IL-25. We investigated the contribution of endogenous IL-25 to DSS-induced colitis using mice deficient in IL-25.

Results

Mice were exposed to DSS in drinking water ad libitum either for seven days (acute) or for three cycles of seven days with DSS followed by 14 days without DSS (chronic) to induce colitis, respectively. The loss of body weight, appearance of diarrhea and bloody stools, and shortening of colon length were significantly less pronounced in IL-25^?/? mice compared to WT mice after exposure to acute DSS. Histological examination showed that DSS-treated IL-25^?/? mice had only mild inflammation in the colon, while severe inflammation developed in DSS-treated WT mice. A significant up-regulation of IL-33 was observed in acute DSS-treated WT but not in the IL-25^?/? mice. There was significantly lower expression of pro-inflammatory cytokines in the colon of acute DSS-treated IL-25^?/? compared to WT mice. IL-25^?/? mice were also partially protected from chronic DSS challenge especially during the first 2 cycles of DSS exposure. In contrast to IL-25^?/? mice, IL-13^?/? mice were more susceptible to DSS-induced colitis. Finally, stimulation of T84 colonic epithelial cells with IL-25 up-regulated the expression of IL-33 and several pro-inflammatory cytokines.

Conclusions

These data indicate that endogenous IL-25 acts as a pro-inflammatory factor in DSS-induced colitis, which is unlikely to be mediated by IL-13 but possibly the induction of IL-33 and other pro-inflammatory mediators from colonic epithelial cells. The present study suggests that IL-25 may contribute to the pathogenesis of inflammatory bowel disease in at least a subgroup of patients.

     

Antiinflammatory Effect of Phytosterols in Experimental Murine Colitis Model: Prevention,Induction, Remission Study

Phytosterols, besides hypocholesterolemic effect, present anti-inflammatory properties. Little information is available about their efficacy in Inflammatory Bowel Disease (IBD). Therefore, we have evaluated the effect of a mixture of phytosterols on prevention/induction/remission in a murine experimental model of colitis. Phytosterols were administered x os before, during and after colitis induction with Dextran Sodium Sulfate (DSS) in mice. Disease Activity Index (DAI), colon length, histopathology score, ¹⁸F-FDG microPET, oxidative stress in the intestinal tissue (ileum and colon) and gallbladder ileum and colon spontaneous and carbachol (CCh) induced motility, plasma lipids and plasma, liver and biliary bile acids (BA) were evaluated. A similar longitudinal study was performed in a DSS colitis control group. Mice treated with DSS developed severe colitis as shown by DAI, colon length, histopathology score, ¹⁸F-FDG microPET, oxidative stress. Both spontaneous and induced ileal and colonic motility were severely disturbed. The same was observed with gallbladder. DSS colitis resulted in an increase in plasma cholesterol, and a modification of the BA pattern. Phytosterols feeding did not prevent colitis onset but significantly reduced the severity of the disease and improved clinical and histological remission. It had strong antioxidant effects, almost restored colon, ileal and gallbladder motility. Plasmatic levels of cholesterol were also reduced. DSS induced a modification in the BA pattern consistent with an increase in the intestinal BA deconjugating bacteria, prevented by phytosterols. Phytosterols seem a potential nutraceutical tool for gastrointestinal inflammatory diseases, combining metabolic systematic and local anti-inflammatory effects.     

Topical antisense oligonucleotide therapy against LIX, an enterocyte-expressed CXC chemokine, reduces murine colitis

Epithelial neutrophil-activating peptide-78 (ENA-78), a member of the CXC chemokine subfamily, is induced by inflammatory cytokines in human colonic enterocyte cell lines and increased in the colon of patients with inflammatory bowel disease (IBD). Lipopolysaccharide-induced CXC-chemokine (LIX) was recently identified as the murine homolog of ENA-78. Here we show that, similar to ENA-78, inflammatory cytokine stimulation of a murine colonic epithelial cell line, MODE-K, results in increased LIX expression. Consistent with the expression pattern of ENA-78 in IBD, LIX expression is significantly increased in mice with colitis induced by the ingestion of dextran sodium sulfate (DSS). Treating mice with antisense oligonucleotides to LIX via rectal enema delivery before DSS treatment results in colonic enterocyte uptake and a significant reduction in neutrophil infiltration and severity of colitis. These findings indicate that LIX plays an integral role in the pathogenesis of DSS-induced colitis. Similarly, enterocyte-derived CXC chemokines may play a key role in regulating neutrophil recruitment and intestinal injury in IBD. The intracolonic administration of ENA-78 antisense oligonucleotides may be effective in treating distal ulcerative colitis in humans.     

Investigating Intestinal Inflammation in DSS-induced Model of IBD

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn''s Disease (CD). Both UC and CD, when present in the colon, generate a similar symptom profile which can include diarrhea, rectal bleeding, abdominal pain, and weight loss.¹ Although the pathogenesis of IBD remains unknown, it is described as a multifactorial disease that involves both genetic and environmental components.²There are numerous and variable animal models of colonic inflammation that resemble several features of IBD. Animal models of colitis range from those arising spontaneously in susceptible strains of certain species to those requiring administration of specific concentrations of colitis-inducing chemicals, such as dextran sulphate sodium (DSS). Chemical-induced models of gut inflammation are the most commonly used and best described models of IBD. Administration of DSS in drinking water produces acute or chronic colitis depending on the administration protocol.³ Animals given DSS exhibit weight loss and signs of loose stool or diarrhea, sometimes with evidence of rectal bleeding.^4,5 Here, we describe the methods by which colitis development and the resulting inflammatory response can be characterized following administration of DSS. These methods include histological analysis of hematoxylin/eosin stained colon sections, measurement of pro-inflammatory cytokines, and determination of myeloperoxidase (MPO) activity, which can be used as a surrogate marker of inflammation.⁶The extent of the inflammatory response in disease state can be assessed by the presence of clinical symptoms or by alteration in histology in mucosal tissue. Colonic histological damage is assessed by using a scoring system that considers loss of crypt architecture, inflammatory cell infiltration, muscle thickening, goblet cell depletion, and crypt abscess.⁷ Quantitatively, levels of pro-inflammatory cytokines with acute inflammatory properties, such as interleukin (IL)-1β, IL-6 and tumour necrosis factor (TNF)-α,can be determined using conventional ELISA methods. In addition, MPO activity can be measured using a colorimetric assay and used as an index of inflammation.⁸In experimental colitis, disease severity is often correlated with an increase in MPO activity and higher levels of pro-inflammatory cytokines. Colitis severity and inflammation-associated damage can be assessed by examining stool consistency and bleeding, in addition to assessing the histopathological state of the intestine using hematoxylin/eosin stained colonic tissue sections. Colonic tissue fragments can be used to determine MPO activity and cytokine production. Taken together, these measures can be used to evaluate the intestinal inflammatory response in animal models of experimental colitis.     

Increased colonic apelin production in rodents with experimental colitis and in humans with IBD

Apelin and its receptor, the APJ receptor, are expressed in the gastrointestinal tract. The aims of this study were to examine the effects of sodium dextran sulfate (DSS)-induced experimental colitis in rats and mice and inflammatory bowel disease (IBD) in humans on intestinal apelin production, and the influence of exogenous apelin on colonic epithelial cell proliferation in mice. In rodents with experimental colitis, colonic apelin mRNA levels were elevated during the inflammatory reaction as well as during the tissue repair phase that ensues after DSS withdrawal. Fluctuations in colonic apelin expression were paralleled by similar changes in apelin immunostaining. Apelin immunostaining was increased in the surface epithelium, in epithelial cells along the length of the tubular gland and in the stem cell region at the gland base. In ulcerative colitis (UC) and Crohn's disease patients, apelin immunostaining revealed a pattern of increased intestinal apelin content similar to that observed in rodents with experimental colitis. Administration of synthetic apelin to mice during the recovery phase of DSS-induced colitis stimulated colonic epithelial cell proliferation significantly. Our observations that colonic apelin production is increased during and after DSS exposure indicate that apelin plays multiple roles during the different stages of colitis. Additionally, the stimulatory action of exogenous apelin on colonic epithelial proliferation suggests that the increased apelin production during intestinal recovery stage may contribute to the repair of the intestinal epithelium in experimental rodent models of colitis and in IBD patients.     

A lactic acid bacterium isolated from kimchi ameliorates intestinal inflammation in DSS-induced colitis

Some species of lactic acid bacteria have been shown to be beneficial in inflammatory bowel disease (IBD). In the present study, a strain of lactic acid bacterium (Lactobacillus paracasei LS2) was isolated from the Korean food, kimchi, and was shown to inhibit the development of experimental colitis induced by dextran sulfate sodium (DSS). To investigate the role of LS2 in IBD, mice were fed DSS in drinking water for seven days along with LS2 bacteria which were administered intragastrically to some of the mice, while phosphate-buffered saline (PBS) was administered to others (the controls). The administration of LS2 reduced body weight loss and increased survival, and disease activity indexes (DAI) and histological scores indicated that the severity of colitis was significantly reduced. The production of inflammatory cytokines and myeloperoxidase (MPO) activity also decreased. Flow cytometry analysis showed that the number of Th1 (IFN-γ) population cells was significantly reduced in the LS2-administered mice compared with the controls. The administration of LS2 induced the increase of CD4⁺FOXP3⁺ Treg cells, which are responsible for IL-10. Numbers of macrophages (CD11b⁺ F4/80⁺), and neutrophils (CD11b⁺ Gr-1⁺) among lamina propria lymphocytes (LPL) were also reduced. These results indicate that LS2 has an anti-inflammatory effect and ameliorates DSS-induced colitis.     

EP4 Receptor–Associated Protein in Macrophages Ameliorates Colitis and Colitis-Associated Tumorigenesis

Prostaglandin E₂ plays important roles in the maintenance of colonic homeostasis. The recently identified prostaglandin E receptor (EP) 4–associated protein (EPRAP) is essential for an anti-inflammatory function of EP4 signaling in macrophages in vitro. To investigate the in vivo roles of EPRAP, we examined the effects of EPRAP on colitis and colitis-associated tumorigenesis. In mice, EPRAP deficiency exacerbated colitis induced by dextran sodium sulfate (DSS) treatment. Wild-type (WT) or EPRAP-deficient recipients transplanted with EPRAP-deficient bone marrow developed more severe DSS-induced colitis than WT or EPRAP-deficient recipients of WT bone marrow. In the context of colitis-associated tumorigenesis, both systemic EPRAP null mutation and EPRAP-deficiency in the bone marrow enhanced intestinal polyp formation induced by azoxymethane (AOM)/DSS treatment. Administration of an EP4-selective agonist, ONO-AE1-329, ameliorated DSS-induced colitis in WT, but not in EPRAP-deficient mice. EPRAP deficiency increased the levels of the phosphorylated forms of p105, MEK, and ERK, resulting in activation of stromal macrophages in DSS-induced colitis. Macrophages of DSS-treated EPRAP-deficient mice exhibited a marked increase in the expression of pro-inflammatory genes, relative to WT mice. By contrast, forced expression of EPRAP in macrophages ameliorated DSS-induced colitis and AOM/DSS-induced intestinal polyp formation. These data suggest that EPRAP in macrophages functions crucially in suppressing colonic inflammation. Consistently, EPRAP-positive macrophages were also accumulated in the colonic stroma of ulcerative colitis patients. Thus, EPRAP may be a potential therapeutic target for inflammatory bowel disease and associated intestinal tumorigenesis.     

Murine Colitis Modeling using Dextran Sulfate Sodium (DSS)

Colitis can occur from viral or bacterial infections, ischemic insult, or autoimmune disorders; most notably Ulcerative Colitis and the colonic variant of Crohn’s Disease - Crohn’s Colitis. Acute colitis may present with abdominal pain and distention, malabsorption, diarrhea, hematochezia and mucus in the stool. We are beginning to understand the complex interactions between the environment, genetics, and epithelial barrier dysfunction in Inflammatory Bowel Disease and animal models of colitis have been essential in advancing our understanding of this disease. One popular model involves supplementing the drinking water of mice with low-molecular weight Dextran Sodium Sulfate (DSS), resulting in epithelial damage and a robust inflammatory response in the colon lasting several days ¹.Variations of this approach can be used to model acute injury, acute injury followed by repair, and repeated cycles of DSS interspersed with recovery modeling chronic inflammatory diseases ². After a single four-day treatment of 3% DSS in drinking water, mice show signs of acute colitis including weight loss, bloody stools, and diarrhea. Mice are euthanized at the conclusion of the treatment course and at necropsy dissected colons are processed and can be ''Swiss rolled" ³ to allow microscopic analysis of the entire colon or infused with formalin as "sausages" to allow macroscopic analysis. Tissue is then embedded in paraffin, sectioned, and stained for histologic review.Open in a separate windowClick here to view.^{(49M, flv)}     

Over-Expression of CD200 Protects Mice from Dextran Sodium Sulfate Induced Colitis

Background and aim

CD200:CD200 receptor (CD200R) interactions lead to potent immunosuppression and inhibition of autoimmune inflammation. We investigated the effect of "knockout"of CD200 or CD200R, or over-expression of CD200, on susceptibility to dextran sodium sulfate (DSS)—induced colitis, a mouse model of inflammatory bowel disease (IBD).

Methods

Acute or chronic colitis was induced by administration of dextran sodium sulfate (DSS) in four groups of age-matched C57BL/6 female mice: (1) CD200-transgenic mice (CD200^tg); (2) wild-type (WT) mice; (3) CD200 receptor 1-deficient (CD200R1KO) mice; and (4) CD200-deficient (CD200KO) mice. The extent of colitis was determined using a histological scoring system. Colon tissues were collected for quantitative RT-PCR and Immunohistochemical staining. Supernatants from colonic explant cultures and mononuclear cells isolated from colonic tissue were used for ELISA.

Results

CD200KO and CD200R1KO mice showed greater sensitivity to acute colitis than WT mice, with accelerated loss of body weight, significantly higher histological scores, more severe infiltration of macrophages, neutrophils and CD3⁺ cells, and greater expression of macrophage-derived inflammatory cytokines, whose production was inhibited in vitro (in WT/CD200KO mouse cells) by CD200. In contrast, CD200^tg mice showed less sensitivity to DSS compared with WT mice, with attenuation of all of the features seen in other groups. In a chronic colitis model, greater infiltration of Foxp3⁺ regulatory T (Treg) cells was seen in the colon of CD200^tg mice compared to WT mice, and anti-CD25 mAb given to these mice attenuated protection.

Conclusions

The CD200:CD200R axis plays an immunoregulatory role in control of DSS induced colitis in mice.     

Temporal and Spatial Analysis of Clinical and Molecular Parameters in Dextran Sodium Sulfate Induced Colitis

Background

Inflammatory bowel diseases (IBD), including mainly ulcerative colitis (UC) and Crohn''s disease (CD), are inflammatory disorders of the gastrointestinal tract caused by an interplay of genetic and environmental factors. Murine colitis model induced by Dextran Sulfate Sodium (DSS) is an animal model of IBD that is commonly used to address the pathogenesis of IBD as well as to test efficacy of therapies. In this study we systematically analyzed clinical parameters, histological changes, intestinal barrier properties and cytokine profile during the colitic and recovery phase.

Methods

C57BL/6 mice were administered with 3.5% of DSS in drinking water for various times. Clinical and histological features were determined using standard criteria. Myeloperoxidase (MPO) activity, transepithelial permeability and proinflammatory mediators were determined in whole colon or proximal and distal parts of colon.

Results

As expected after administration of DSS, mice manifest loss of body weight, shortening of colon length and bloody feces. Histological manifestations included shortening and loss of crypts, infiltration of lymphocytes and neutrophil, symptoms attenuated after DSS withdrawal. The MPO value, as inflammation indicator, also increases significantly at all periods of DSS treatment, and even after DSS withdrawal, it still held at very high levels. Trans-mucosal permeability increased during DSS treatment, but recovered to almost control level after DSS withdrawal. The production of proinflammatory mediators by colonic mucosa were enhanced during DSS treatment, and then recovered to pre-treated level after DSS withdrawal. Finally, enhanced expression of proinflammatory mediators also revealed a different profile feature in proximal and distal parts of the colon.

Conclusion

Experimental colitis induced by DSS is a good animal model to study the mechanisms underlying the pathogenesis and intervention against IBD, especially UC.     

Autotaxin (ATX) inhibits autophagy leading to exaggerated disruption of intestinal epithelial barrier in colitis

Inflammatory bowel disease (IBD) is an immune-mediated disease. Autotaxin (ATX) is associated with increased inflammatory molecules, however, its effect on IBD is not well understood. Autophagy plays an important role in IBD, whether ATX and autophagy act in concert in IBD remains unknown. This study is to explore the possible mechanisms of ATX affecting autophagy leading to the disruption of intestinal epithelial barrier, thereby exacerbating colitis. The expression of ATX was upregulated in UC patients and dextran sulfate sodium (DSS)-induced colitis mice. Here, we described that providing an ATX inhibitor during DSS colitis increased autophagy and ameliorated colonic inflammation. Conversely, intrarectal administration with recombinant (r)ATX increased colitis and decreased autophagy. This pro-colitic effect was attenuated in mice treated with rapamycin, resulting in increased autophagy activity and mild colitis. Moreover, the inhibitory effect of rATX on autophagy was confirmed in vitro and was reversed by the addition of rapamycin. The damaging effects of ATX on epithelial barrier function were reversed by ATX inhibitor or rapamycin treatment. In sum, our results show that ATX can inhibit autophagy through the mTOR pathway, resulting in exaggerated damage to the intestinal epithelial barrier during colitis. These findings suggest that ATX may be a key pro-colitic factor, and represent a potential therapeutic target for treating IBD in the future.     

Crucial Involvement of Tumor-Associated Neutrophils in the Regulation of Chronic Colitis-Associated Carcinogenesis in Mice

Ulcerative colitis (UC) is a major form of chronic inflammation that can frequently progress to colon cancer. Several studies have demonstrated massive infiltration of neutrophils and macrophages into the lamina propria and submucosa in the progression of UC-associated colon carcinogenesis. Macrophages contribute to the development of colitis-associated colon cancer (CAC). However, the role of neutrophils is not well understood. To better understand the involvement of tumor-associated neutrophils (TANs) in the regulation of CAC, we used a mouse CAC model produced by administering azoxymethane (AOM), followed by repeated dextran sulfate sodium (DSS) ingestion. This causes severe colonic inflammation and subsequent development of multiple tumors in mice colon. We observed that colorectal mucosal inflammation became increasingly severe with AOM and DSS treatment. Macrophages infiltrated the lamina propria and submucosa, together with a marked increase in neutrophil infiltration. The chemokine CXCL2 increased in the lamina propria and submucosal regions of the colons of the treated mice, together with the infiltration of neutrophils expressing CXCR2, a specific receptor for CXCL2. This process was followed by neoplastic transformation. After AOM and DSS treatment, the mice showed enhanced production of metalloproteinase (MMP)-9 and neutrophil elastase (NE), accompanied by excessive vessel generation and cell proliferation. Moreover, CXCL2 promoted neutrophil recruitment and induced neutrophils to express MMP-9 and NE in vitro. Furthermore, administration of neutrophil-neutralizing antibodies after the last DSS cycle markedly reduced the number and size of tumors and decreased the expression of CXCR2, CXCL2, MMP-9, and NE. These observations indicate a crucial role for TANs in the initiation and progression of CAC and suggest that the CXCL2–CXCR2 axis might be useful in reducing the risk of UC-associated colon cancer.