The Anti-Inflammatory Effect and Intestinal Barrier Protection of HU210 Differentially Depend on TLR4 Signaling in Dextran Sulfate Sodium-Induced Murine Colitis

Background

Ulcerative colitis (UC) is strongly associated with inflammation and intestinal barrier disorder. The nonselective cannabinoid receptor agonist HU210 has been shown to ameliorate inflamed colon in colitis, but its effects on intestinal barrier function and extraintestinal inflammation are unclear.

Aims

To investigate the effects and the underlying mechanism of HU210 action on the UC in relation to a role of TLR4 and MAP kinase signaling.

Methods

Wild-type (WT) and TLR4 knockout (Tlr4 ^?/?) mice were exposed to 4% dextran sulfate sodium (DSS) for 7 days. The effects of HU210 on inflammation and intestinal barrier were explored.

Results

Upon DSS challenge, mice suffered from bloody stool, colon shortening, intestinal mucosa edema, pro-inflammatory cytokine increase and intestinal barrier destruction with goblet cell depletion, increased intestinal microflora accompanied with elevated plasma lipopolysaccharide, reduced mRNA expression of the intestinal tight junction proteins, and abnormal ratio of CD4⁺/CD8⁺ T cells in the intestinal Peyer’s patches. Pro-inflammatory cytokines in the plasma and the lung, as well as pulmonary myeloperoxidase activity, indicators of extraintestinal inflammation were increased. Protein expression of p38α and pp38 was up-regulated in the colon of WT mice. Tlr4 ^?/? mice showed milder colitis. HU210 reversed the intestinal barrier changes in both strains of mice, but alleviated inflammation only in WT mice.

Conclusions

Our study indicates that in experimental colitis, HU210 displays a protective effect on the intestinal barrier function independently of the TLR4 signaling pathway; however, in the extraintestinal tissues, the anti-inflammatory action seems through affecting TLR4-mediated p38 mitogen-activated protein kinase pathway.

     

The Severity of Dextran Sodium Sulfate-Induced Colitis Can Differ Between Dextran Sodium Sulfate Preparations of the Same Molecular Weight Range

Background

We hypothesized that the severity of dextran sodium sulfate (DSS)-induced colitis could differ between DSS preparations of the same molecular weight, and that this difference may be affected by the sulfur content. To test this, we used three DSS preparations of similar molecular weights but with different sulfur contents.

Methods

Three DSS preparations with molecular weights of 40,000 to 50,000 were tested: MP Biomedicals (MP Bio), USB (USB), and The Lab Depot (The Lab). Epithelial cell lines were used to assess the levels of poly (ADP-ribose) polymerase (PARP) in the presence of 2.0% DSS in vitro. Eight-week-old female C57/B6 mice were fed 2.0% DSS in water for 1?week, and then sacrificed to investigate the effects of the DSS preparations in vivo.

Results

In vitro experiments using CaCo-2 and CMT-93 cells revealed decreased PARP levels from all DSS preparations. Notably, the PARP level was significantly decreased in CaCo-2 cells treated with DSS from USB as compared to The Lab Mice treated with The Lab DSS had significantly decreased body weight losses on day 7 as compared to mice receiving DSS from MP Bio and USB. This result was supported by their DAI score, colon weight/length ratio, and histological scores.

Conclusion

The severity of colitis can differ between similar DSS preparations of the same molecular weight range. This difference in colitogenic properties may be affected by the total sulfur content of each DSS preparation.     

Bolus injection of newly synthesized vitamin E derivative ETS-GS for the treatment of acute severe ulcerative colitis in a mouse model

Purpose

Vitamin E with its antioxidant action has therapeutic effects on ulcerative colitis (UC), but use of vitamin E is limited because of its insolubility in water. We developed ETS-GS (γ-l-glutamyl-S-[2-[[[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltri-decyl)-2?H-1-benzopyran-6-yl]oxy]carbonyl]-3-oxo-3-[(2-sulfoethyl)amino]propyl]-l-cysteinylglycine sodium salt), a newly synthesized soluble vitamin E derivative with strong antioxidant action. We evaluated the therapeutic effects of bolus injection of ETS-GS on acute severe UC in a mouse model.

Methods

An animal model of acute severe UC was induced by feeding mice 5 % dextran sulfate sodium (DSS) for 5 days, followed by 1 % DSS on days 5–8, the experimental period. ETS-GS or saline was administered by subcutaneous bolus injection during the experimental period. We examined disease activity index (DAI) score, histological score, colon length, colon weight, and serum cytokines in the mice.

Results

The following results at day 8 in the DSS + ETS-GS group were significantly lower than those in the DSS + Saline group: DAI score, 2.6?±?0.6 vs. 3.1?±?0.5; histological score, 2.1?±?1.0 vs. 3.1?±?0.8; serum interleukin (IL)-6, 15?±?9.4 vs. 39?±?23 pg/ml; and keratinocyte-derived chemokine (KC), 122?±?61 vs. 228?±?66 pg/ml (P?<?0.05). Colon length, colon weight, and serum IL-10 in the DSS + ETS-GS group were significantly higher than those in the DSS + Saline group (88?±?12 vs. 75?±?5.7 mm, 0.48?±?0.09 vs. 0.38?±?0.05 g, and 55?±?18 vs. 31?±?10 pg/ml, respectively; P?<?0.05).

Conclusions

Bolus injection of ETS-GS may be one therapeutic modality for acute severe UC. Its effects are associated with suppression of serum IL-6 and serum KC and promotion of serum IL-10.     

Reduced Diversity and Imbalance of Fecal Microbiota in Patients with Ulcerative Colitis

Background

Clinical observations and experimental colitis models have indicated the importance of intestinal bacteria in the etiology of ulcerative colitis (UC), but a causative bacterial agent has not been identified.

Aim

To determine how intestinal bacteria are associated with UC, fecal microbiota and other components were compared for UC patients and healthy adults.

Methods

Fresh feces were collected from 48 UC patients. Fecal microbiota were analyzed by use of terminal-restriction fragment length polymorphism (T-RFLP), real-time PCR, and culture. The concentrations of organic acids, indole, and ammonia, and pH and moisture, which are indicators of the intestinal environment, were measured and compared with healthy control data.

Results

T-RFLP data divided the UC patients into four clusters; one cluster was obtained for healthy subjects. The diversity of fecal microbiota was significantly lower in UC patients. There were significantly fewer Bacteroides and Clostridium subcluster XIVab, and the amount of Enterococcus was higher in UC patients than in healthy subjects. The fecal concentration of organic acids was significantly lower in UC patients who were in remission.

Conclusion

UC patients have imbalances in the intestinal environment—less diversity of fecal microbiota, lower levels of major anaerobic bacteria (Bacteroides and Clostridium subcluster XIVab), and a lower concentration of organic acids.     

Disruption of GPR35 Exacerbates Dextran Sulfate Sodium-Induced Colitis in Mice

Background

G protein-coupled receptor 35 (GPR35) is an orphan receptor and is vastly expressed in immune cells and gastrointestinal cells, suggesting the potential physiological importance of GPR35 in these cells. Here, we tested the hypothesis that the lack of GPR35 expression in the colon mucosa exacerbates the severity of dextran sulfate sodium (DSS)-induced experimental colitis in mice.

Methods

Colitis was induced in GPR35 wild-type (GPR35^+/+) and GPR35 knockout (GPR35^?/?) mice through the administration of DSS in drinking water for 5 days followed by regular facility water for 1 day. Induction of colitis was evaluated by measuring relative body weight loss, clinical illness scores, and morphological changes in the colon. Abolition of Gpr35 gene expression in the colon mucosa of GPR35^?/? mice was confirmed by quantitative real-time PCR (qPCR). Gene expressions of inflammatory and tissue remodeling cytokines were detected by qPCR. Human colorectal epithelial Caco cells were transfected with siRNA against GPR35 before treated with 1% DSS in vitro. Protein expressions were measured using Western blot.

Results

GPR35^?/? mice receiving DSS showed a significantly worsened colitis disease with profound loss of body weight and a considerable amount of severe clinical illness compared to GPR35^+/+ mice that received DSS. The histology of colon sections from GPR35^?/? mice showed extensive pathological changes including submucosal edema, diffuse ulcerations, and evidence of complete loss of crypts compared to wild-type mice. The mean histopathological score was significantly higher in GPR35^?/? mice as compared to GPR35^+/+ mice. The qPCR data revealed significant expression of pro-inflammatory and tissue remodeling cytokines in GPR35^?/? colon mucosa, including IL-1β, CXCL1, CXCL2, CCL2, HMGB1, TGFβ1, TGFβ3, MMP1/9/12. The protein expressions of Zonula occludens-1, E-cadherin, Claudin1 were decreased upon knocking down GPR35 with or without 1% DSS treatment.

Conclusions

Our experimental data suggest that lack of GPR35 resulted in worsened disease outcome in DSS-induced experimental colitis, indicating that GPR35 could play a crucial role in protecting from colonic inflammation and serve as a therapeutic target.

     

A decrease in anaerobic bacteria promotes <Emphasis Type="Italic">Candida glabrata</Emphasis> overgrowth while β-glucan treatment restores the gut microbiota and attenuates colitis

Background

The intestinal microbiota plays a crucial role in the maintenance of gut homeostasis. Changes in crosstalk between the intestinal epithelial cells, immune cells and the microbiota are critically involved in the development of inflammatory bowel disease. In the experimental mouse model, the development of colitis induced by dextran sulfate sodium (DSS) promotes overgrowth of the opportunistic yeast pathogen Candida glabrata. Conversely, fungal colonization aggravates inflammatory parameters. In the present study, we explored the effect of C. glabrata colonization on the diversity of the gut microbiota in a DSS-induced colitis model, and determined the impact of soluble β-glucans on C. glabrata-host interactions.

Results

Mice were administered a single inoculum of C. glabrata and were exposed to DSS treatment for 2 weeks in order to induce acute colitis. For β-glucan treatment, mice were administered with soluble β-glucans purified from C. glabrata (3?mg per mouse), orally and daily, for 5 days, starting on day 1. The number of C. glabrata colonies and changes in microbiota diversity were assessed in freshly collected stool samples from each tagged mouse, using traditional culture methods based on agar plates. An increase in Escherichia coli and Enterococcus faecalis populations and a reduction in Lactobacillus johnsonii and Bacteroides thetaiotaomicron were observed during colitis development. This decrease in L. johnsonii was significantly accentuated by C. glabrata overgrowth. Oral administration of β-glucans to mice decreased the overgrowth of aerobic bacteria and IL-1β expression while L. johnsonii and B. thetaiotaomicron populations increased significantly. β-glucan treatment increased IL-10 production via PPARγ sensing, promoting the attenuation of colitis and C. glabrata elimination.

Conclusions

This study shows that the colonic inflammation alters the microbial balance, while β-glucan treatment increases the anaerobic bacteria and promotes colitis attenuation and C. glabrata elimination.

     

Dietary Red Meat Aggravates Dextran Sulfate Sodium-Induced Colitis in Mice Whereas Resistant Starch Attenuates Inflammation

Background

Although a genetic component has been identified as a risk factor for developing inflammatory bowel disease, there is evidence that dietary factors also play a role in the development of this disease.

Aims

The aim of this study was to determine the effects of feeding a red meat diet with and without resistant starch (RS) to mice with dextran sulfate sodium (DSS)-induced colitis.

Methods

Colonic experimental colitis was induced in Balb/c mice using DSS. The severity of colitis was evaluated based on a disease activity index (based on bodyweight loss, stool consistency, rectal bleeding, and overall condition of the animal) and a histological score. Estimations were made of numbers of a range of different bacteria in the treatment pools of cecal digesta using quantitative real-time PCR.

Results

Consumption of a diet high in red meat increased DSS-induced colitis as evidenced by higher disease activity and histopathological scores. Addition of RS to the red meat diet exerted a beneficial effect in acute DSS-induced colitis. Subjective analysis of numbers of a range of bacterial targets suggest changes in the gut microbiota abundance were induced by red meat and RS treatments and these changes could contribute to the reported outcomes.

Conclusions

A dietary intake of red meat aggravates DSS-induced colitis whereas co-consumption of resistant starch reduces the severity of colitis.     

Lactulose Mediates Suppression of Dextran Sodium Sulfate-Induced Colon Inflammation by Increasing Hydrogen Production

Background

Molecular hydrogen (H₂) is a potent antioxidant and able to protect organs from oxidative stress injuries. Orally administered lactulose, a potent H₂ inducer, is digested by colon microflora and significantly increases H₂ production, indicating its potential anti-inflammatory action.

Objective

To evaluate the anti-inflammatory effects of lactulose on dextran sodium sulfate (DSS)-induced colitis in mice.

Methods

Mice were randomly assigned into seven groups, receiving regular distilled water, H₂-rich saline (peritoneal injection), DSS, oral lactulose (0.1, 0.15, 0.2 ml/10 g, respectively), and lactulose (0.2 ml/10 g) + oral antibiotics. The mouse model of human ulcerative colitis was established by supplying mice with water containing DSS. The H₂ breath test was used to determine the exhaled H₂ concentration. Body weight, colitis score, colon length, pathological features and tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), maleic dialdehyde (MDA) and marrow peroxidase (MPO) levels in colon lesions were evaluated.

Results

After 7 days, DSS-induced loss of body weight, increase of colitis score, shortening of colon length, pathological changes and elevated levels of TNF-α, IL-1β, MDA, and MPO in colon lesions, were significantly suppressed by oral lactulose administration and intraperitoneally injected H₂-rich saline. Ingestion of antibiotics significantly compromised the anti-inflammatory effects of lactulose. The H₂ breath test showed that lactulose administration significantly induced hydrogen production and that antibiotics administration could inhibit H₂ production.

Conclusion

Lactulose can prevent the development of DSS-induced colitis and alleviate oxidative stress in the colon, as measured by MDA and MPO, probably by increasing endogenous H₂ production.     

The Myc 3′ Wnt Responsive Element Regulates Neutrophil Recruitment After Acute Colonic Injury in Mice

Background

The Wnt/β-catenin pathway regulates intestinal development, homeostasis, and regeneration after injury. Wnt/β-catenin signaling drives intestinal proliferation by activating expression of the c-Myc proto-oncogene (Myc) through the Myc 3′ Wnt responsive DNA element (Myc 3′ WRE). In a previous study, we found that deletion of the Myc 3′ WRE in mice caused increased MYC expression and increased cellular proliferation in the colon. When damaged by dextran sodium sulfate (DSS), the increased proliferative capacity of Myc 3′ WRE^?/? colonocytes resulted in a more rapid recovery compared with wild-type (WT) mice. In that study, we did not examine involvement of the immune system in colonic regeneration.

Purpose

To characterize the innate immune response in Myc 3′ WRE^?/? and WT mice during and after DSS-induced colonic injury.

Methods

Mice were fed 2.5 % DSS in their drinking water for five days to induce colonic damage and were then returned to normal water for two or four days to recover. Colonic sections were prepared and neutrophils and macrophages were analyzed by immunohistochemistry. Cytokine and chemokine levels were analyzed by probing a cytokine array with colonic lysates.

Results

In comparison with WT mice, there was enhanced leukocyte infiltration into the colonic mucosal and submucosal layers of Myc 3′ WRE^?/? mice after DSS damage. Levels of activated neutrophils were substantially increased in damaged Myc 3′ WRE^?/? colons as were levels of the neutrophil chemoattractants C5/C5a, CXCL1, and CXCL2.

Conclusion

The Myc 3′ WRE regulates neutrophil infiltration into DSS-damaged colons.     

Variable Impact of CD39 in Experimental Murine Colitis

Background

Dysregulation of immune responses in inflammatory bowel diseases (IBD) results in intestinal inflammation and vascular injury while exacerbating systemic disease. CD39 is an ectonucleotidase, expressed by T regulatory cells and dendritic cells, that hydrolyzes extracellular nucleotides to modify those cellular immune responses implicated in IBD. Genetic polymorphisms of CD39 have been linked to Crohn??s disease while gene deletion in mice exacerbates dextran sodium sulphate-induced colitis.

Aim

The aim of this study was to test how global deletion of CD39 in mice impacts other models of experimental colitis.

Methods

Colitis was induced in CD39-null and -wt mice, using trinitrobenzene sulfonic acid (TNBS, 125 mg/kg) administered intrarectally. Oxazolone colitis (1.5% oxazolone in 50% alcohol) was induced in comparable groups. Morphology, clinical and molecular parameters, and FACS analyses of lamina propria mononuclear cells (LPMC) were examined in CD39-null mice. CD39 expression was analyzed in human IBD biopsies.

Results

Paradoxically, TNBS colitis in CD39-null mice was characterized by improved survival, favorable clinical scores, and decreased MPO activity, when compared to wt mice (P < 0.05). LPMC from TNBS colitis contained significantly increased amounts of T-cells (CD3⁺ and CD4⁺) and TNF-?? mRNA expression were increased over those in CD39 null mice (P < 0.05). In contrast, oxazolone treated CD39-null and wt mice had comparable outcomes. In both ulcerative colitis and Crohn??s disease, CD39 is present at high levels in intestinal tissue biopsies.

Conclusions

TNBS colitis was attenuated in CD39-null mice whereas oxazolone-induced colitis was not impacted. Impaired adaptive cellular immune reactivity in the CD39-null environment appears protective in hapten-mediated Th1-type colitis. CD39 is expressed at high levels in clinical IBD tissues.     

Randomized clinical trial: Atorvastatin versus placebo in patients with acute exacerbation of mild to moderate ulcerative colitis

Background

Statins are known to possess pleiotropic anti-inflammatory properties which have been evaluated for clinical benefits in a number of disorders. Studies have demonstrated beneficial actions of statins in experimental models of colitis. Clinical evidence in acute exacerbation of ulcerative colitis (UC) is lacking.

Aim

This study aims to assess the efficacy and safety of add-on atorvastatin in mild to moderately severe acute exacerbation of UC.

Methods

Patients with acute exacerbation of UC were randomized to receive either atorvastatin (20 mg) or matching placebo once daily orally for 8 weeks in addition to the standard therapy. Clinical efficacy was assessed by using partial Mayo score (PMS).

Results

Previously diagnosed 64 cases of UC presenting with mild to moderately severe acute exacerbation were randomized to receive either atorvastatin of 20 mg or placebo. Mean PMS increased by 1.5 points and decreased by 0.31 points in atorvastatin and placebo groups, respectively, at 8 weeks compared to the baseline values (p?=?0.04). Eight (25 %) and 13 (40.6 %) patients attained the primary outcome criteria for clinical improvement in the atorvastatin and placebo arms, respectively (p?=?0.18). Fifteen (46.8 %) patients in the atorvastatin group and no patient in the placebo group had ≥2 point increase in PMS after 8 weeks (p?<?0.001).

Conclusion

Atorvastatin therapy in acute exacerbation of UC may not be associated with beneficial effects. Paradoxical increase in disease activity may be seen in some patients. However, these findings need to be substantiated in larger studies.     

STW 5 is effective in dextran sulfate sodium-induced colitis in rats

Purpose

An herbal preparation, STW 5, used clinically in functional dyspepsia and irritable bowel syndrome, has been shown to possess properties that may render it useful in inflammatory bowel disease (IBD). The present work was conducted to study its effectiveness in a rat model of IBD.

Methods

An experimental model reflecting ulcerative colitis in man was adopted, whereby colitis was induced in Wistar rats by feeding them 5?% dextran sulfate sodium (DSS) in drinking water for one week. STW 5 and sulfasalazine (as a reference standard) were administered orally daily for 1?week before colitis induction and continued during DSS feeding. The animals were then sacrificed, and the severity of colitis was evaluated macroscopically and microscopically. Colon samples were homogenized for determination of reduced glutathione, tumor necrosis factor-α, and cytokine-induced neutrophil chemoattractant-3 as well as myeloperoxidase, glutathione peroxidase, and superoxide dismutase. In addition, colon segments were suspended in an organ bath to test their reactivity towards carbachol, KCl, and trypsin.

Results

STW 5 and sulfasalazine were both effective in preventing the shortening of colon length and the increase in both colon mass index and total histology score as well as the changes in biochemical parameters measured except changes in dismutase activity. DSS-induced colitis led to marked depression in colonic responsiveness to the agents tested ex vivo, an effect which was normalized by both drugs.

Conclusions

The findings point to a potential usefulness of STW 5 in the clinical setting of ulcerative colitis.     

Downregulation of CX<Subscript>3</Subscript>CR1 ameliorates experimental colitis: evidence for CX<Subscript>3</Subscript>CL1-CX<Subscript>3</Subscript>CR1-mediated immune cell recruitment

Purpose

Inflammatory conditions like inflammatory bowel diseases (IBD) are characterized by increased immune cell infiltration. The chemokine ligand CX₃CL1 and its receptor CX₃CR1 have been shown to be involved in leukocyte adhesion, transendothelial recruitment, and chemotaxis. Therefore, the objective of this study was to describe CX3CL1-CX3CR1-mediated signaling in the induction of immune cell recruitment during experimental murine colitis.

Methods

Acute colitis was induced by dextran sodium sulfate (DSS), and sepsis was induced by injection of lipopolysaccharide (LPS). Serum concentrations of CX₃CR1 and CX₃CL1 were measured by ELISA. Wild-type and CX₃CR1^-/- mice were challenged with DSS, and on day 6, intravital microscopy was performed to monitor colonic leukocyte and platelet recruitment. Intestinal inflammation was assessed by disease activity, histopathology, and neutrophil infiltration.

Results

CX₃CR1 was upregulated in DSS colitis and LPS-induced sepsis. CX₃CR1^-/- mice were protected from disease severity and intestinal injury in DSS colitis, and CX₃CR1 deficiency resulted in reduced rolling of leukocytes and platelets.

Conclusions

In the present study, we provide evidence for a crucial role of CX₃CL1-CX₃CR1 in experimental colitis, in particular for intestinal leukocyte recruitment during murine colitis. Our findings suggest that CX₃CR1 blockade represents a potential therapeutic strategy for treatment of IBD.

     

Regulation of anergy-related ubiquitin E3 ligase,GRAIL, in murine models of colitis and patients with Crohn’s disease

Background

Abrogating tolerance is a critical step in the pathogenesis of Crohn’s disease (CD). T cell-anergy is one of the main mechanisms of tolerance and is regulated by the gene related to anergy in lymphocytes (GRAIL). This study investigated the expressions and regulation of GRAIL in CD and murine colitis models.

Methods

Expressions of GRAIL mRNA and protein in CD4⁺ T cells were investigated in the peripheral blood and mucosal tissues of patients with CD, mice with dextran sodium salt (DSS)-induced colitis, and Il-10-deficient mice. MicroRNAs responsible for the regulation of GRAIL were examined by miRNA microarray. GRAIL-overexpressing T cells were intravenously injected in mice with DSS-induced colitis.

Results

The GRAIL expression was higher in the lamina propria (LP) CD4⁺ T cells of CD patients than of the control subjects, while it was lower in the peripheral blood CD4⁺ T cells of the CD patients than of the control subjects. The GRAIL mRNA expression was lower, but the GRAIL protein expression was higher in the LP of colitic mice than that of non-colitic mice. The miRNA microarray identified miR-290-5p as an miRNA that inhibits expression of the GRAIL protein and that is highly expressed in the LP of non-colitic mice. GRAIL-expressing T cells expressed regulatory T cell markers and showed suppressive effects in murine DSS-induced colitis.

Conclusions

Our results show that expression of GRAIL is uniquely regulated by the specific miRNA in the intestinal mucosa, and suggest that GRAIL may associate with the pathophysiology of CD.

     

Partial normalization of pubertal timing in female mice with DSS colitis treated with anti-TNF-α antibody

Background

Inflammatory bowel disease (IBD) and resultant colitis occurring prior to puberty are frequently associated with delayed puberty and losses of growth and bone mineralization. Some of this delay may be due to colonic inflammation and associated systemic inflammation. To date no treatments for IBD have been shown to normalize the timing of puberty. Our objective in this study was to determine whether there is a normalization of the timing of puberty during treatment of colitis using monoclonal antibodies (abs) to tumor necrosis factor (TNF)-α.

Methods

We induced colitis in 23-day-old C57Bl6 female mice using 3% dextran sodium sulfate (DSS) for 7?days, followed by removal of DSS for an additional 3?days, resulting in 10?days of worsening colitis. DSS-treated mice received either TNF-α ab or Control ab on days 4 and 8 of colitis, while non-colitic Control mice received injections of TNF-α ab (Control?+?TNF-α ab). All groups were followed for the timing of vaginal opening until day of life 33, when they were euthanized for serum and colon collection.

Results

The DSS?+?TNF-α ab group had lower levels of systemic interleukin (IL)-6 and a partial normalization of the timing of vaginal opening compared to the DSS?+?Control ab group. There were no differences in weight gain, growth, or colon histological inflammatory scores between the DSS?+?TNFα ab and DSS?+?Control ab groups over the course of the experiment.

Conclusions

We conclude that anti-TNF-α ab treatment causes a partial normalization of pubertal timing coincident with decreased systemic inflammation in DSS colitis. These data may have implications regarding growth and bone mineralization outcomes in pediatric IBD.     

A Role for Intestinal Alkaline Phosphatase in the Maintenance of Local Gut Immunity

Background and Aims

Intestinal alkaline phosphatase (IAP) is a gut mucosal defense factor known to dephosphorylate lipopolysaccharide (LPS); however, the role of IAP in the gut response to luminal bacteria remains poorly defined. We investigated immune responses of wild-type (WT) and IAP-knockout (IAP-KO) mice to LPS and Salmonella typhimurium challenges.

Methods

Cryostat sectioning and standard indirect immunohistochemical staining for major histocompatibility complex (MHC) class II molecules were performed on liver tissue from WT and IAP-KO mice. WT and IAP-KO mice were orally gavaged with S. typhimurium; bacterial translocation to mesenteric nodes, liver, and spleen was determined by tissue homogenization and plating. In other experiments, WT and IAP-KO mice received intraperitoneal injections of LPS, with subsequent quantification of complete blood counts and serum interleukin (IL)-6 by enzyme-linked immunosorbent assay (ELISA). WT and IAP-KO whole blood were plated and stimulated with LPS and Pam-3-Cys, followed by cytokine assays.

Results

Immunohistologic liver examinations showed increased expression of MHC class II molecules in IAP-KO mice. Following S. typhimurium challenge, WT mice appeared moribund compared with IAP-KO mice, with increased bacterial translocation. WT mice had >50% decrease (P < .005) in platelets and 1.8-fold (P < .05) increased serum IL-6 compared with IAP-KO mice in response to LPS injections. IAP-KO whole-blood stimulation with LPS and Pam-3-Cys resulted in increased IL-6 and tumor necrosis factor (TNF)-alpha secretion compared with WT.

Conclusions

IAP-KO mice exhibit characteristics consistent with local LPS tolerance. Whole-blood response of IAP-KO mice did not reflect systemic tolerance. These data suggest that IAP is a local immunomodulating factor, perhaps regulating LPS?Ctoll-like receptor 4 (TLR4) interaction between commensal microflora and intestinal epithelium.     

Serum Metabolic Profiling in Inflammatory Bowel Disease

Background

The inflammatory bowel diseases (IBD), Crohn’s disease (CD), and ulcerative colitis (UC), are chronic inflammatory conditions of the gastrointestinal tract whose pathogenesis is not completely understood. ¹H nuclear magnetic resonance (NMR) spectroscopy of serum generates comprehensive metabolic profiles, reflecting systemic metabolism, which may be altered in disease states.

Aim

The aim of this study was to use ¹H NMR-based serum metabolic profiling in the investigation of CD patients, UC patients, and controls, potentially to provide insights into disordered metabolism in IBD, and into underlying mechanisms of disease.

Methods

Serum metabolic profiles were acquired from 67 individuals (24 CD patients, 20 UC patients, and 23 healthy controls). The multivariate pattern-recognition techniques of principal components analysis (PCA) and partial least squares discriminant analysis with orthogonal signal correction (OSC-PLS-DA) were used to investigate differences between cohorts.

Results

OSC-PLS-DA distinguished CD and UC cohorts with significant predictive accuracy, highlighting differences in lipid and choline metabolism. Metabolic profiles of both CD and UC cohorts, and the combined IBD cohort, differed significantly from controls: metabolites of importance in the OSC-PLS-DA models included lipoproteins (especially HDL cholesterol), choline, N-acetylglycoprotein, and amino acids.

Conclusions

¹H NMR-based metabolic profiling has identified distinct differences in serum metabolic phenotype between CD and UC patients, as well as between IBD patients and controls.