Inflammatory Bowel Disease Mucosa Research Articles

PEG-functionalized microparticles selectively target inflamed mucosa in inflammatory bowel disease

IntroductionThe systemic therapy of inflammatory bowel diseases (IBD) by oral administration of anti-inflammatory and immunosuppressive agents is characterized by an increased probability of adverse drug reactions. A successful treatment with a simultaneous reduction in adverse events may be achieved by the administration of micro- and nanosized targeted drug delivery systems, which accumulate selectively in inflamed mucosal areas without systemic absorption. We described in a first in vivo study in IBD patients a significantly enhanced, but minor accumulation of non-functionalized poly(lactic-co-glycolic acid) (PLGA) microparticles in ulcerous lesions very recently. AimThe aim of this study was therefore the assessment of an increased targeting potential of different non-, chitosan- and polyethylene glycol (PEG)-functionalized PLGA micro- and nanoparticles to inflamed intestinal mucosa compared to healthy mucosa. Materials and methodsFor the quantification of nano- and microparticles, fluoresceinamine-labeled-PLGA was synthesized by carbodiimide reaction. Fluorescent chitosan-, PEG-, and non-functionalized PLGA micro- and nanoparticles with mean hydrodynamic diameters of 3000nm and 300nm were prepared by solvent evaporation technique. The targeting efficiencies in terms of particle translocation and deposition were investigated in Ussing chamber experiments. Healthy and inflamed macrobiopsies were received from routine endoscopic examinations of patients with IBD as well as control patients. ResultsOne-hundred and one Ussing chamber experiments of patients with IBD (Crohn’s disease: n=7 and ulcerative colitis: n=9) as well as healthy control patients (n=5) were performed. Histomorphological and electrophysiological investigations of inflamed mucosal tissues confirmed a significant alteration of mucosal barrier integrity in IBD patients (TER: healthy: 34.1Ωcm2; inflamed: 21.6Ωcm2; p=0.034). In summary, nanoparticles showed an increased translocation and deposition compared to microparticles in healthy and in inflamed mucosa. Chitosan-functionalized particles adhered onto the tissue surface and thus showed the lowest particle translocation and deposition in healthy and inflamed tissues. PEG-functionalized nanoparticles showed the highest translocation through healthy (2.31%) and inflamed mucosa (5.27%). Moreover, PEG-functionalized microparticles showed a significantly increased translocation through inflamed mucosa (3.33%) compared to healthy mucosa (0.55%; p=0.045). Notably, the particle deposition of PEG-functionalized microparticles was significantly increased in inflamed mucosa (10.8%) compared to healthy mucosa (4.1%; p=0.041). ConclusionsBased on the targeted translocation and deposition to inflamed intestinal mucosa, PEG-functionalized PLGA microparticles were qualified as an innovative drug delivery system. These particles may serve as a selective treatment strategy to inflamed mucosal areas in IBD with the potential to improve therapeutic efficacy and to reduce adverse events.

Identification of serum and tissue micro-RNA expression profiles in different stages of inflammatory bowel disease

The altered expression of micro-RNA (miRNA) has been associated with Crohn's disease (CD) and ulcerative colitis (UC). The aim of this study was to establish specific miRNA expression patterns in the serum and mucosa of inflammatory bowel disease (IBD) patients (UC and CD with colonic involvement) at different stages of the disease. Serum and biopsies from nine active CD (aCD), nine inactive CD (iCD), nine active UC (aUC) and nine inactive UC (iUC) and serum from 33 healthy subjects were collected. Up to 700 miRNAs were evaluated by the TaqMan human miRNA array. The ΔCt values were obtained using the mean expression values of all expressed miRNAs in a given sample as a normalization factor for miRNA real-time quantitative polymerase chain reaction data. The levels of serum miRNAs in CD and UC patients were different to healthy subjects. Thirteen serum miRNAs were expressed commonly in CD and UC patients. Two miRNAs were higher and four miRNAs were lower in the serum of aCD than iCD. No serum miRNA was regulated exclusively in aUC compared with iUC patients. Four miRNAs were higher and three miRNAs were lower in the mucosa of aCD than iCD. Two miRNAs were higher and three miRNAs were lower in the mucosa of aUC than iUC. No serum miRNAs coincided with tissue miRNAs in aCD and aUC patients. Our results suggest the existence of specific miRNA expression patterns associated with IBD and their different stages and support the utility of miRNA as possible biomarkers. This pilot study needs to be validated in a large prospective cohort.

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

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

Sa1778 Genetically-Determined Cytokine Programs of Human Natural Killer (NK) Cells Uncover a Novel Mechanism of NK Cells and Kir Genes in Crohn's Disease Susceptibility

BACKGROUND AND AIMS. Intestinal macrophages play a central role in the pathogenesis of inflammatory bowel disease (IBD). The phenotype of mucosal macrophages changes dramatically at the site of inflammation, where high numbers of CD33+CD68+ macrophages overexpress toll-like receptors, CD14 and triggering receptor expressed on myeloid cells 1 (TREM-1). TREM-1 is expressed on the surface of neutrophils and macrophages and contributes to the amplification of inflammatory responses by enhancing degranulation and secretion of pro-inflammatory mediators. After comparing the percentage of TREM-1-expressing macrophages in IBD versus control mucosa, we explored the ex vivo effects of an activating anti-TREM-1 antibody on the intestinal immune response in IBD. METHODS. Lamina propria mononuclear cells (LPMCs) were isolated from the inflamed colon of 11 IBD patients (5 ulcerative colitis and 6 Crohn's disease), and from normal colon of 8 control subjects, and the expression of CD68, CD33 and TREM-1 was analysed by flow cytometry. TREM1 mRNA expression in CD33+ LPMCs was evaluated by qRT-PCR. IBD biopsies from inflamed mucosa were cultured ex vivo with an activating anti-TREM-1 antibody or its control isotype, and the production of pro-inflammatory cytokines, namely interleukin (IL)1beta, IL-6 and IL-8, was determined by ELISA. RESULTS. The percentage of mucosal CD33+CD68+ macrophages was significantly (p,0.05) higher both in Crohn's disease and ulcerative colitis (17%±1.4 and 15%±7.6, respectively) in comparison to control subjects (6.0%±1.0). TREM-1 expression by mucosal macrophages was significantly (p,0.05) higher both in ulcerative colitis and in Crohn's disease (6.4%±0.2 and 4.4%±0.6) than in control subjects (0.8%±0.1). TREM-1 transcripts were significantly (p,0.05) higher in CD33+ LPMCs from inflamed IBD compared to control subjects. TREM-1 activation significantly (p,0.01) increased IL-1beta, IL-6 and IL-8 production by IBD biopsies cultured ex vivo. CONCLUSIONS. TREM-1 is overexpressed on macrophages infiltrating inflamed IBD mucosa, and its activation amplifies the production of pro-inflammatory cytokines. Further studies using chromatin immunoprecipitation assays are underway in order to establish whether TREM-1 overexpression in IBD may derive from epigenetic changes.

Sa1777 Ex Vivo Activation of Triggering Receptor Expressed on Myeloid Cells 1 Enhances the Production of PRO-Inflammatory Cytokines by Inflammatory Bowel Disease Mucosa

Sa1777 Ex Vivo Activation of Triggering Receptor Expressed on Myeloid Cells 1 Enhances the Production of PRO-Inflammatory Cytokines by Inflammatory Bowel Disease Mucosa

Sa1750 Epithelial Notch-1 Signaling Modulates Regulatory/Effector T Cell Signature: A Role for the Epithelial Barrier

BACKGROUND/AIMS: We previously reported that inhibition of Bone Morphogenetic Protein (BMP) signaling in vivo, leads to enhancement of Helicobacter-induced gastric inflammation suggesting that the BMPs exert anti-inflammatory actions in the gut. The aim of this study was to examine the expression of BMP-4 and the localization of BMP-generated signals during gastrointestinal (GI) inflammation. METHODS: 3 month-old transgenic (TG) mice expressing the β-galactosidase (gal) gene under the control of a BMP responsive element (BRE) and C57BL/6-BMP-4β-gal/+ mice were either infected with H. felis (HF) or given 3% DSS-containing water for 4 days, to induce gastric and colonic inflammation, respectively. Animals were analyzed 2 months after HF infection and 3 days after DSS administration. Expression of BMP-4, BRE-β-gal and BMP Receptor 1A (BMPR1A) were assessed by X-Gal staining, IHC with anti-β-gal antibodies (abs) and QRT-PCR. Morphology of the GI mucosa was examined in sections stained with H&E. Phosphorylation of the BMP-signal transducers Smad1-5-8 was determined by IHC with anti-p-Smad1-5-8 abs. Identification of myofibroblasts, B and T lymphocytes, neutrophils and macrophages was achieved by IHC with antiα smooth muscle actin (SMA), -CD19, -CD3, -MPO, and -F4/80 abs, respectively. Biopsies were obtained from the colonic mucosa of normal (16 samples from 4 patients) and Inflammatory Bowel Disease (IBD) individuals (12 samples from 4 patients) with severe inflammation. RESULTS: BMP-4 is expressed in the mesenchymal layers of the GI mucosa while BMP signaling targets cells of the GI epithelium. BMP-4 staining was seen in α-SMA-positive cells of both the gastric and the colonic mucosa. HF-induced inflammation led to decreased BMP4 expression and signaling. No BMP-4 staining was seen in CD19-, CD3-, F4/80and MPOpositive cells of the gastric mucosa. Similarly, no p-Smad1-5-8 signals were seen in cells of the inflammatory infiltrates. 3% DSS caused inflammatory infiltrates, stromal expansion and loss of glands. The inflamed areas exhibited decreased BMP-4 expression and signaling and reduced BMPR1A mRNA expression. BMP-4 and BMPR1A mRNAs were also decreased in biopsies from patients with severe colonic IBD. CONCLUSIONS: BMP-4 is expressed in αSMA-positive cells of the GI mucosa but not in immune cells. BMP signals target epithelial cells but not cells of the inflammatory infiltrates. Inflammation reduces both the expression of BMP-4 and the intensity of BMP signaling in mouse models of gastric and colonic inflammation and in biopsies from IBD patients. Thus, BMP signaling, a pathway that exerts anti-inflammatory effects, is reduced during severe GI inflammation. Induction and reactivation of this mechanism might therefore represent a possible, novel modality for the treatment of inflammatory diseases of the gut.

Epithelial expression of interleukin-37b in inflammatory bowel disease

Interleukin (IL)-37 is a member of the IL-1 cytokine family. We investigated IL-37b expression in the inflamed mucosa of inflammatory bowel disease (IBD) patients. Furthermore, we analysed IL-37b expression in human colonic epithelial cells. The human colonic epithelial cell line T84 and human colonic subepithelial myofibroblasts (SEMFs) were used. IL-37b expression in the IBD mucosa was evaluated by immunohistochemistry. IL-37b mRNA and protein expression were determined by real time-polymerase chain reaction (PCR) and Western blotting, respectively. IL-37b was not detected in the normal colonic mucosa. In the inflamed mucosa of IBD patients, epithelial IL-37b expression was increased markedly. In ulcerative colitis (UC) and Crohn's disease (CD) patients, IL-37b expression was enhanced in the affected mucosa. In the intestinal epithelial cell line T84, the expression of IL-37b mRNA and protein was enhanced by tumour necrosis factor (TNF)-α. This IL-37b induction by TNF-α was mediated by nuclear factor (NF)-κB and activator protein (AP)-1 activation. Furthermore, IL-37b inhibited TNF-α-induced interferon-γ-inducible protein (IP)-10 expression significantly in human colonic SEMFs. Epithelial IL-37b expression was increased in IBD patients, especially UC patients. IL-37b may be involved in the pathophysiology of IBD as an anti-inflammatory cytokine and an inhibitor of both innate and acquired immune responses.

MiR-200b inhibits TGF-β1-induced epithelial-mesenchymal transition and promotes growth of intestinal epithelial cells

Inflammatory bowel disease (IBD), which consists of Crohn's disease (CD) and ulcerative colitis (UC), is a chronic, inflammatory disorder of the gastro-intestinal tract with unknown etiology. Current evidence suggests that intestinal epithelial cells (IECs) is prominently linked to the pathogenesis of IBD. Therefore, maintaining the intact of epithelium has potential roles in improving pathophysiology and clinical outcomes of IBD. MicroRNAs (miRNAs) act as post-transcriptional gene regulators and regulate many biological processes, including embryonal development, cell differentiation, apoptosis and proliferation. In this study, we found that miR-200b decreased significantly in inflamed mucosa of IBD, especially for UC, when compared with their adjacent normal tissue. Simultaneously, we also found that the genes of E-cadherin and cyclin D1 were reduced significantly and correlated positively to the miR-200b. In addition, the upregulation of transforming growth factor-beta 1 (TGF-β1) was inversely correlated to the miR-200b in IBD. To investigate the possible roles of miR-200b in IECs maintaining, we used TGF-β1 to induce epithelial-mesenchymal transition (EMT) in IEC-6 initially. After sustained over-expressing miR-200b in IEC-6, the EMT was inhibited significantly that was characterized by downregulation of vimentin and upregulation of E-cadherin. Furthermore, we found that miR-200b enhanced E-cadherin expression through targeting of ZEB1, which encode transcriptional repressors of E-cadherin. SMAD2 was found to act as a target of miR-200b with direct evidence that miR-200b binding to the 3′ UTR of SAMD2 and the ability of miR-200b to repress SMAD2 protein expression. With SMAD2 depletion, the expression of vimentin decreased correspondingly, which suggested miR-200b might reduce vimentin through regulating the SMAD2. With endogenous over-expression of miR-200b, the proliferation of IEC-6 cells increased significantly by increasing S-phase entry and promoting expression of the protein cyclin D1. Summarily, our study suggested a potential role for mir-200b in maintaining intact of intestinal epithelium through inhibiting EMT and promoting proliferation of IECs.

P073 Ex vivo activation of triggering receptor expressed on myeloid cells 1 enhances the production of pro-inflammatory cytokines by inflammatory bowel disease mucosa

BACKGROUND AND AIMS. Intestinal macrophages play a central role in the pathogenesis of inflammatory bowel disease (IBD). The phenotype of mucosal macrophages changes dramatically at the site of inflammation, where high numbers of CD33+CD68+ macrophages overexpress toll-like receptors, CD14 and triggering receptor expressed on myeloid cells 1 (TREM-1). TREM-1 is expressed on the surface of neutrophils and macrophages and contributes to the amplification of inflammatory responses by enhancing degranulation and secretion of pro-inflammatory mediators. After comparing the percentage of TREM-1-expressing macrophages in IBD versus control mucosa, we explored the ex vivo effects of an activating anti-TREM-1 antibody on the intestinal immune response in IBD. METHODS. Lamina propria mononuclear cells (LPMCs) were isolated from the inflamed colon of 11 IBD patients (5 ulcerative colitis and 6 Crohn's disease), and from normal colon of 8 control subjects, and the expression of CD68, CD33 and TREM-1 was analysed by flow cytometry. TREM1 mRNA expression in CD33+ LPMCs was evaluated by qRT-PCR. IBD biopsies from inflamed mucosa were cultured ex vivo with an activating anti-TREM-1 antibody or its control isotype, and the production of pro-inflammatory cytokines, namely interleukin (IL)1beta, IL-6 and IL-8, was determined by ELISA. RESULTS. The percentage of mucosal CD33+CD68+ macrophages was significantly (p,0.05) higher both in Crohn's disease and ulcerative colitis (17%±1.4 and 15%±7.6, respectively) in comparison to control subjects (6.0%±1.0). TREM-1 expression by mucosal macrophages was significantly (p,0.05) higher both in ulcerative colitis and in Crohn's disease (6.4%±0.2 and 4.4%±0.6) than in control subjects (0.8%±0.1). TREM-1 transcripts were significantly (p,0.05) higher in CD33+ LPMCs from inflamed IBD compared to control subjects. TREM-1 activation significantly (p,0.01) increased IL-1beta, IL-6 and IL-8 production by IBD biopsies cultured ex vivo. CONCLUSIONS. TREM-1 is overexpressed on macrophages infiltrating inflamed IBD mucosa, and its activation amplifies the production of pro-inflammatory cytokines. Further studies using chromatin immunoprecipitation assays are underway in order to establish whether TREM-1 overexpression in IBD may derive from epigenetic changes.

Gut-lung crosstalk in pulmonary involvement with inflammatory bowel diseases

Pulmonary abnormalities, dysfunction or hyper-reactivity occurs in association with inflammatory bowel disease (IBD) more frequently than previously recognized. Emerging evidence suggests that subtle inflammation exists in the airways among IBD patients even in the absence of any bronchopulmonary symptoms, and with normal pulmonary functions. The pulmonary impairment is more pronounced in IBD patients with active disease than in those in remission. A growing number of case reports show that the IBD patients develop rapidly progressive respiratory symptoms after colectomy, with failure to isolate bacterial pathogens on repeated sputum culture, and often request oral corticosteroid therapy. All the above evidence indicates that the inflammatory changes in both the intestine and lung during IBD. Clinical or subclinical pulmonary inflammation accompanies the main inflammation of the bowel. Although there are clinical and epidemiological reports of chronic inflammation of the pulmonary and intestinal mucosa in IBD, the detailed mechanisms of pulmonary-intestinal crosstalk remain unknown. The lung has no anatomical connection with the main inflammatory site of the bowel. Why does the inflammatory process shift from the gastrointestinal tract to the airways? The clinical and subclinical pulmonary abnormalities, dysfunction, or hyper-reactivity among IBD patients need further evaluation. Here, we give an overview of the concordance between chronic inflammatory reactions in the airways and the gastrointestinal tract. A better understanding of the possible mechanism of the crosstalk among the distant organs will be beneficial in identifying therapeutic strategies for mucosal inflammatory diseases such as IBD and allergy.

Up-regulation and Pre-activation of TRAF3 and TRAF5 in Inflammatory Bowel Disease

Objective: TRAF3 and TRAF5 share a common ancestral gene, and interact as essential components of signaling pathways in immunity. TRAF3 and TRAF5 are overexpressed in the colon of rat/mouse models with colitis. However, the expressions of TRAF3 and TRAF5 in patients with inflammatory bowel disease have not been elucidated. The aim of the present study is to explore the potential roles of TRAF3 and TRAF5 in patients with inflammatory bowel disease.Methods: Plasma levels of TRAF3 and TRAF5 proteins were detected by Enzyme-linked Immunosorbent Assay (ELISA). Colonic expression of TRAF3 and TRAF5 proteins was detected by western blot analysis. Quantitative Real-time PCR (qRT-PCR) was applied for gene expression. Inflamed intestinal mucosa and non-inflamed intestinal mucosa in patients with inflammatory bowel disease and normal mucosa was analyzed from healthy controls.Results: The plasma levels of TRAF3 and TRAF5 were significantly higher both in patients with Crohn's disease and ulcerative colitis than in healthy controls. Only soluble TRAF5 showed a weak correlation with endoscopic disease activity index (Baron score) in patients with ulcerative colitis (spearman's r=0.358, P=0.022). Gene expressions of TRAF3 and TRAF5 in peripheral blood mononuclear cells were significantly higher both in patients with Crohn's disease and ulcerative colitis than in healthy controls (all P<0.0001). Gene and protein expressions of TRAF3 and TRAF5 were significantly higher in inflamed colonic mucosa of patients with Crohn's disease and ulcerative colitis than in non-inflamed colonic mucosa and normal mucosa of healthy controls (all P<0.0001). Furthermore, gene and protein expressions of TRAF3 and TRAF5 were also significantly higher in non-inflamed colonic mucosa of patients with Crohn's disease and ulcerative colitis than in normal mucosa of healthy controls.Conclusions: TRAF3 and TRAF5 are overexpressed in inflammatory bowel disease. Although the endoscopic appearance can be normal, TRAF3 and TRAF5 pre-activation can be detected in non-inflamed colonic segments.

Why is damage limited to the mucosa in ulcerative colitis but transmural in Crohn’s disease?

It has been a big puzzle as why the inflammation of ulcerative colitis (UC) is limited to the mucosa, while in Crohn's disease (CD) the inflammation is transmural and can be seen in all layers of the gut. Here, I give a tentative explanation extended from the unified hypothesis I proposed on the etiology of inflammatory bowel disease. This hypothesis suggested that both UC and CD are caused by weakening of the gut barrier due to damage of the protective mucus layer and the underlying tissue by the poorly inactivated digestive proteases resulting from a reduction of gut bacteria by dietary chemicals like saccharin and sucralose. However, the large amounts of bacteria in the colon make the recruitment of neutrophils and formation of crypt abscess the main manifestation of UC, while the infiltration of antigens and dietary particles in the small and large intestine mainly cause the recruitment of macrophages and formation of granulomas as the main manifestations in CD. The fast reacting and short life span of neutrophils make the fight and damage limited to the surface of the mucosa. In contrast, the long life span and constant movement of macrophages may bring the harmful agents deep into the tissue. Therefore, the pathogenesis of UC may be more like bacterial pneumonia, while CD may be more like pneumoconiosis or tuberculosis of the lung.

Neurotensin receptor 1 overexpression in inflammatory bowel diseases and colitis-associated neoplasia

To explore the association of neurotensin receptor 1 (NTSR1) with inflammatory bowel diseases (IBD) and colitis-associated neoplasia. NTSR1 was detected by immunohistochemistry in clinical samples of colonic mucosa with IBD colitis, colitis-associated raised low-grade dysplasia (LGD) including dysplasia-associated lesions or masses (DALMs, n = 18) and adenoma-like dysplastic polyps (ALDPs, n = 4), colitis-associated high-grade dysplasia (HGD, n = 11) and colitis-associated colorectal carcinoma (CACRC, n = 13), sporadic colorectal adenomatous polyp (SAP, n = 17), and sporadic colorectal carcinoma (SCRC, n = 12). The immunoreactivity of NTSR1 was semiquantitated (as negative, 1+, 2+, and 3+) and compared among different conditions. NTSR1 was not detected in normal mucosa but was expressed similarly in both active and inactive colitis. LGD showed a significantly stronger expression as compared with non-dysplastic colitic mucosa, with significantly more cases showing > 2+ intensity (68.75% in LGD vs 32.26% in nondysplastic mucosa, P = 0.001). However, no significant difference existed between DALMs and ALDPs. CACRC and HGD showed a further stronger expression, with significantly more cases showing 3+ intensity than that in LGD (61.54% vs 12.50% for CACRC vs LGD, P = 0.022; 58.33% vs 12.50% for CACRC/HGD vs LGD, P = 0.015). No significant difference existed between colitis-associated and non-colitic sporadic neoplasia. NTSR1 in colonic epithelial cells is overexpressed in IBD, in a stepwise fashion with sequential progress from inflammation to dysplasia and carcinoma.

Human basophils interact with memory T cells to augment Th17 responses

AbstractBasophils are a rare population of granulocytes that have long been associated with IgE-mediated and Th2-associated allergic diseases. However, the role of basophils in Th17 and/or Th1 diseases has not been reported. In the present study, we report that basophils can be detected in the mucosa of Th17-associated lung and inflammatory bowel disease and accumulate in inflamed colons containing large quantities of IL-33. We also demonstrate that circulating basophils increased memory Th17 responses. Accordingly, IL-3– or IL-33–activated basophils amplified IL-17 release in effector memory T cells (TEM), central memory T cells (TCM), and CCR6+ CD4 T cells. More specifically, basophils promoted the emergence of IL-17+IFN-γ− and IL-17+IFN-γ+, but not IL-17−IFN-γ+ CD4 T cells in TEM and TCM. Mechanistic analysis revealed that the enhancing effect of IL-17 production by basophils in TEM involved the ERK1/2 signaling pathway, occurred in a contact-independent manner, and was partially mediated by histamine via H2 and H4 histamine receptors. The results of the present study reveal a previously unknown function for basophils in augmenting Th17 and Th17/Th1 cytokine expression in memory CD4 T cells. Because basophils accumulated in inflamed inflammatory bowel disease tissues, we propose that these cells are key players in chronic inflammatory disorders beyond Th2.

Induction of Lipocalin-2 in Colonic Epithelial Cells in Inflammatory Bowel Disease

In our gene expression analysis on colonic biopsies from an IBD cohort we found Lipocalin-2 (LCN2) to be among the most upregulated genes in inflamed mucosa compared to un-inflamed mucosa of both ulcerative colitis (UC) and Crohn’s Disease (CD). The protein LCN2 acts as an acute phase protein and has antimicrobial properties, making it a part of the innate immune system. We sought to identify localization and regulation of LCN2 in the colonic mucosa and also quantify LCN2 in serum. Microarray of colonic biopsies from an IBD population (n = 133) was done using Illumina Bead Chip technology. Immunohistochemical staining and in situ hybridization of colonic biopsies was done on a subset of biopsies to identify cellular sources of LCN2. We studied release of LCN2 from colonic epithelial cell lines HT-29 and SW620, upon pattern recognition receptor stimulation (PRR). LCN2 was quantified in serum using ELISA and hsCRP quantified using immunoturbidometric method. LCN2 mRNA was among the 10 most upregulated gene in inflamed vs uninflamed mucosa of UC and CD. LCN2 is located to the epithelial cells and infiltrating neutrophils in inflamed mucosa. The LCN2 mRNA synthesis is found solely in epithelial cells indicating the de novo synthesis to take place in the epithelium. LCN2 is abundantly and constitutively released from the colonic epithelial cell line HT-29 and its release is enhanced by both IL1b and the TLR3 ligand polyinosinic: polycytidylic acid (poly(I:C)). Serum LCN2 correlates with hsCRP. LCN2 is among the most upregulated genes in active IBD. The epithelial cells of colonic mucosa are an important source of LCN2. LCN2 seems to be released into the intestinal lumen and has a potential role in shaping the intestinal bacterial flora. The synthetic dsRNA analog poly(I:C) increased synthesis and release of LCN2 for colonic epithelial cell line HT-29 and the TLR3 mediates this release, supporting a role of TLR3 in intestinal homeostasis and mucosal inflammation.

A Rare Finding of Dysplasia Associated Mass or Lesion 6 Months after Diagnosis of Ulcerative Colitis

Purpose: A 19-year-old female was evaluated for loose stools of several years duration. Lab work revealed a normal hematocrit, c-reactive protein, albumin, and erythrocyte sedimentation rate. Stool studies were negative for infection. A colonoscopy found severe pancolitis consistent with ulcerative colitis (UC). Systemic steroids and a 5-aminosalicylic acid derivative were initiated. Repeat colonoscopy 6 months later was significant for a carpet like lesion at 25 cm from the anal verge in an otherwise normal appearing colon. Biopsies contained low grade dysplasia within the lesion and the surrounding mucosa, consistent with a dysplasia associated non-adenoma like lesion or mass (DALM). The patient underwent a total proctocolectomy with J pouch formation and ileoanal anastomosis. Upon follow up she was doing well. In inflammatory bowel disease (IBD), dysplasia is defined as unequivocal neoplastic epithelium that is confined to the basement membrane without invasion into the lamina propria. The rate at which colitic mucosa progresses to dysplasia, and ultimately to colorectal cancer (CRC) is unknown. The molecular biology of cancer in IBD is unique in that the accumulation of molecular and genetic alterations may occur more rapidly or in an unconventional sequence when compared to sporadic CRC. The duration and extent of colitis, early age of onset of colitis, family history of CRC, coexistent primary sclerosing cholangitis, and severity of microscopic inflammation are risk factors for the development of cancer in UC. Elevated lesions that are endoscopically visible, but not amenable to endoscopic resection are referred to as dysplasia associated lesion or mass (DALM). In a series of 12 patients with a DALM who underwent colectomy, 7 were found to have invasive carcinoma. One in 8 patients with UC will have dysplasia or cancer found on their initial screening colonoscopy. Patients with greater than 10 years of disease and those with extensive disease (pancolitis) are at highest risk for cancer. The incidence rate increases with each successive decade of disease activity, with cumulative probabilities of 2% at 10 years, 8% at 20 years, and 18% at 30 years. More recent data suggests that the risk may be on the decline with 0.6% after 10 years, 5.4% after 20 years, and 7.5% after 30 years of chronic UC. We present a case of UC with findings of a DALM that led to proctocolectomy only 6 months after the diagnosis of pancolitis in a 19-year-old patient. This presentation of a DALM is unusual because of the short duration of known UC and the patient's young age.

Effects of alanyl-glutamine dipeptide on the expression of colon-inflammatory mediators during the recovery phase of colitis induced by dextran sulfate sodium

Glutamine (Gln) is a nutrient with immunomodulatory effects in metabolic stressed conditions. This study investigated the effects of Gln on colonic-inflammatory-mediator expression and mucosal repair in mice with dextran sulfate sodium (DSS)-induced colitis. C57BL/6 mice received distilled water containing 3 % DSS for 5 d to induce colitis. One of the DSS-treated groups was intraperitoneally injected with an alanyl (Ala)-Gln solution 3 days before (G-DSS) while the other group was administered Ala-Gln 3 days after colitis (DSS-G) was induced. The Ala-Gln solution provided 0.5 g Gln/kg/d. The saline-DSS group (S-DSS) received an identical amount of saline before and after colitis was induced to serve as a positive control. The S-DSS group had a shorter colon length, higher plasma haptoglobin level, and more-severe colon inflammation. Also, the toll-like receptor (TLR)4 level, nuclear factor (NF)-κB activation, and inflammatory cytokine gene expression in the colon were higher than those of the normal control group. Gln administration either before or after colitis suppressed TLR4 protein levels, decreased plasma haptoglobin, and reduced colon inflammation. Histological inflammatory scores were also lowered. Compared to the post-colitis Gln group, preventive use of Gln had higher colon length, expressions of mucin 2, trefoil factor 3, and heat shock protein 72 genes were also upregulated in the colon. These results suggest that Gln administered either before or after the colitis mitigated inflammation of colitis that was not observed in group without Gln injection. Prophylactic treatment with Gln had more-beneficial effects on reducing inflammatory markers and enhancing the recovery of mucosa in DSS-induced colitis.

Study on protective mechanism of kushenin injection on colonic mucosa of experimental colitis rats

To study the effect of NOD2 on colitis pathogenesis in experimental rats, and discuss therapeutical effect and mechanism of kushenin injection (OMT) on colitis in experimental rats. Fourty Sprague-Dawley (SD) rats were randomly divided into four groups: the normal control group, the model group, the SASP group, and the OMT group, with 10 rats in each group. Except the normal control group, models were established in the remaining three groups with TNBS. The OMT group was injected with kushenin injection, the SASP group was orally administered with mesalazine suspension, the model group and the normal group were orally administered with distilled water for 15 days. Colon lesion score and histological score of experimental rats were observed. Expression of NOD2, NF-kappaB p65 protein in rats colonic mucous was detected by immunohistochemistry. Expression of IL-6 in rat colon mucous was detected by ELISA. Compared with normal control group, the expression of NOD2, NF-kappaB p65 and IL-6 in colonic mucosa of the model group were significantly increased (P < 0.01). The SASP group and the OMT group showed lower expressions of NOD2, NF-kappaB p65 and IL-6 in colonic mucosa than the model group (P < 0.01, P < 0.05). The over expression of colonic mucosa proteins NOD2 and NF-kappaB p65 and increasing secretion of IL-6 take part in the appearance and development of ulcerative colitis. OMT can attenuate ulcerative colitis and protect colonic mucosa by inhibiting expression of NOD2, NF-kappaB p65 and decreasing IL-6.

Downregulation of the Ubiquitin-Proteasome System in Normal Colonic Macrophages and Reinduction in Inflammatory Bowel Disease

Background: In normal mucosa, intestinal lamina propria macrophages (IMACs) maintain tolerance against food antigens and the commensal bacterial flora. Several mechanisms have been identified that mediate tolerance. The ubiquitin-proteasome system (UPS) is a large multiprotein complex that degrades cellular proteins. As the UPS may modulate immune functions of IMACs, we performed a detailed investigation of UPS expression and function under normal conditions and in cells derived from patients suffering from inflammatory bowel disease (IBD). Methods: IMACs were isolated from intestinal mucosa. mRNA expression of macrophages differentiated in vitro (i.v. MACs) and IMACs was compared by Affymetrix® oligonucleotide arrays. Quantitative Taqman-PCR was performed on five exemplary proteasomal and five ubiquitinylation genes each. Proteins were analyzed by immunohistochemistry and Western blotting. Proteasome function was assessed by a fluorimetric test. Results: Affymetrix analysis showed downregulation of mRNA expression of almost all represented proteasomal and of 22 ubiquitination-associated genes in IMACs as compared to i.v. MACs and monocytes. By quantitative PCR, up to tenfold higher mRNA expression of 10 exemplary genes of the UPS (UBE2A, UBE2D2, UBE2L6, USP14, UBB and ATPase2, β2, β5, β2i/MECL-1, β5i/LMP7) was demonstrated in i.v. MACs as compared to IMACs. Immunohistochemistry and Western blots confirmed these findings in intestinal mucosa of controls and patients suffering from diverticulitis. In contrast, a significant increase in protein amounts was found in mucosa of patients with IBD. Conclusion: Reduced expression of subunits of the UPS in IMACs of normal mucosa supports the concept of the presence of a nonreactive, anergic macrophage phenotype in the gut under normal conditions. Reinduction in IMACs of IBD mucosa reflects activated IMACs which can present antigenic peptides and thus support inflammation.

Ulcerative Colitis Impairs the Acylethanolamide-Based Anti-Inflammatory System Reversal by 5-Aminosalicylic Acid and Glucocorticoids

Studies in animal models and humans suggest anti-inflammatory roles on the N-acylethanolamide (NAE)-peroxisome proliferators activated receptor alpha (PPARα) system in inflammatory bowel diseases. However, the presence and function of NAE-PPARα signaling system in the ulcerative colitis (UC) of humans remain unknown as well as its response to active anti-inflammatory therapies such as 5-aminosalicylic acid (5-ASA) and glucocorticoids. Expression of PPARα receptor and PPARα ligands-biosynthetic (NAPE-PLD) and -degrading (FAAH and NAAA) enzymes were analyzed in untreated active and 5-ASA/glucocorticoids/immunomodulators-treated quiescent UC patients compared to healthy human colonic tissue by RT-PCR and immunohistochemical analyses. PPARα, NAAA, NAPE-PLD and FAAH showed differential distributions in the colonic epithelium, lamina propria, smooth muscle and enteric plexus. Gene expression analysis indicated a decrease of PPARα, PPARγ and NAAA, and an increase of FAAH and iNOS in the active colitis mucosa. Immunohistochemical expression in active colitis epithelium confirmed a PPARα decrease, but showed a sharp NAAA increase and a NAPE-PLD decrease, which were partially restored to control levels after treatment. We also characterized the immune cells of the UC mucosa infiltrate. We detected a decreased number of NAAA-positive and an increased number of FAAH-positive immune cells in active UC, which were partially restored to control levels after treatment. NAE-PPARα signaling system is impaired during active UC and 5-ASA/glucocorticoids treatment restored its normal expression. Since 5-ASA actions may work through PPARα and glucocorticoids through NAE-producing/degrading enzymes, the use of PPARα agonists or FAAH/NAAA blockers that increases endogenous PPARα ligands may yield similar therapeutics advantages.