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Background: Intestinal epithelial cell (IEC) mitochondrial dysfunction involvement in inflammatory bowel diseases (IBD), including Crohn's disease affecting the small intestine, is emerging in recent studies. As the interface between the self and the gut microbiota, IECs serve as hubs of bidirectional cross-talk between host and luminal microbiota. However, the role of mitochondrial-microbiota interaction in the ileum is largely unexplored. Prohibitin 1 (PHB1), a chaperone protein of the inner mitochondrial membrane required for optimal electron transport chain function, is decreased during IBD. We previously demonstrated that mice deficient in PHB1 specifically in IECs (Phb1 i∆IEC ) exhibited mitochondrial impairment, Paneth cell defects, gut microbiota dysbiosis, and spontaneous inflammation in the ileum (ileitis). Mice deficient in PHB1 in Paneth cells (epithelial secretory cells of the small intestine; Phb1 ∆PC ) also exhibited mitochondrial impairment, Paneth cell defects, and spontaneous ileitis. Here, we determined whether this phenotype is driven by Phb1 deficiency-associated ileal microbiota alterations or direct effects of loss of PHB1 in host IECs., Results: Depletion of gut microbiota by broad-spectrum antibiotic treatment in Phb1 ∆PC or Phb1 i∆IEC mice revealed a necessary role of microbiota to cause ileitis. Using germ-free mice colonized with ileal microbiota from Phb1-deficient mice, we show that this microbiota could not independently induce ileitis without host mitochondrial dysfunction. The luminal microbiota phenotype of Phb1 i∆IEC mice included a loss of the short-chain fatty acid butyrate. Supplementation of butyrate in Phb1-deficient mice ameliorated Paneth cell abnormalities and ileitis. Phb1-deficient ileal enteroid models suggest deleterious epithelial-intrinsic responses to ileal microbiota that were protected by butyrate., Conclusions: These results suggest a mutual and essential reinforcing interplay of gut microbiota and host IEC, including Paneth cell, mitochondrial health in influencing ileitis. Restoration of butyrate is a potential therapeutic option in Crohn's disease patients harboring epithelial cell mitochondrial dysfunction. Video Abstract., (© 2023. The Author(s).)

Objectives: Gut and joint inflammation commonly co-occur in spondyloarthritis (SpA) which strongly restricts therapeutic modalities. The immunobiology underlying differences between gut and joint immune regulation, however, is poorly understood. We therefore assessed the immunoregulatory role of CD4 + FOXP3 + regulatory T (Treg) cells in a model of Crohn's-like ileitis and concomitant arthritis., Methods: RNA-sequencing and flow cytometry was performed on inflamed gut and joint samples and tissue-derived Tregs from tumour necrosis factor (TNF) ∆ARE mice. In situ hybridisation of TNF and its receptors (TNFR) was applied to human SpA gut biopsies. Soluble TNFR (sTNFR) levels were measured in serum of mice and patients with SpA and controls. Treg function was explored by in vitro cocultures and in vivo by conditional Treg depletion., Results: Chronic TNF exposure induced several TNF superfamily (TNFSF) members (4-1BBL, TWEAK and TRAIL) in synovium and ileum in a site-specific manner. Elevated TNFR2 messenger RNA levels were noted in TNF ∆ARE/+ mice leading to increased sTNFR2 release. Likewise, sTNFR2 levels were higher in patients with SpA with gut inflammation and distinct from inflammatory and healthy controls. Tregs accumulated at both gut and joints of TNF ∆ARE mice, yet their TNFR2 expression and suppressive function was significantly lower in synovium versus ileum. In line herewith, synovial and intestinal Tregs displayed a distinct transcriptional profile with tissue-restricted TNFSF receptor and p38MAPK gene expression., Conclusions: These data point to profound differences in immune-regulation between Crohn's ileitis and peripheral arthritis. Whereas Tregs control ileitis they fail to dampen joint inflammation. Synovial resident Tregs are particularly maladapted to chronic TNF exposure., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Nanoplastics (NPs), regarded as the emerging contaminants, can enter and be mostly accumulated in the digest tract, which pose the potential threat to intestinal health. In this study, mice were orally exposed to polystyrene (PS), PS-COOH and PS-NH 2 NPs with the size of ∼100 nm at a human equivalent dose for 28 consecutive days. All three kinds of PS-NPs triggered Crohn's ileitis-like features, such as ileum structure impairment, increased proinflammatory cytokines and intestinal epithelial cell (IEC) necroptosis, and PS-COOH/PS-NH 2 NPs exhibited higher adverse effects on ileum tissues. Furthermore, we found PS-NPs induced necroptosis rather than apoptosis via activating RIPK3/MLKL pathway in IECs. Mechanistically, we found that PS-NPs accumulated in the mitochondria and subsequently caused mitochondrial stress, which initiated PINK1/Parkin-mediated mitophagy. However, mitophagic flux was blocked due to lysosomal deacidification caused by PS-NPs, and thus led to IEC necroptosis. We further found that mitophagic flux recovery by rapamycin can alleviate NP-induced IEC necroptosis. Our findings revealed the underlying mechanisms concerning NP-triggered Crohn's ileitis-like features and might provide new insights for the further safety assessment of NPs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objective: Although immunoglobulin A (IgA) is abundantly expressed in the gut and known to be an important component of mucosal barriers against luminal pathogens, its precise function remains unclear. Therefore, we tried to elucidate the effect of IgA on gut homeostasis maintenance and its mechanism., Design: We generated various IgA mutant mouse lines using the CRISPR/Cas9 genome editing system. Then, we evaluated the effect on the small intestinal homeostasis, pathology, intestinal microbiota, cytokine production, and immune cell activation using intravital imaging., Results: We obtained two lines, with one that contained a <50 base pair deletion in the cytoplasmic region of the IgA allele (IgA tail-mutant; IgA tm/tm ) and the other that lacked the most constant region of the IgH α chain, which resulted in the deficiency of IgA production (IgA -/- ). IgA -/- exhibited spontaneous inflammation in the ileum but not the other parts of the gastrointestinal tract. Associated with this, there were significantly increased lamina propria CD4 + T cells, elevated productions of IFN-γ and IL-17, increased ileal segmented filamentous bacteria and skewed intestinal microflora composition. Intravital imaging using Ca 2+ biosensor showed that IgA -/- had elevated Ca 2+ signalling in Peyer's patch B cells. On the other hand, IgA tm/tm seemed to be normal, suggesting that the IgA cytoplasmic tail is dispensable for the prevention of the intestinal disorder., Conclusion: IgA plays an important role in the mucosal homeostasis associated with the regulation of intestinal microbiota and protection against mucosal inflammation especially in the ileum., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Metabolic changes associated with tissue inflammation result in significant extracellular acidosis (EA). Within mucosal tissues, intestinal epithelial cells (IEC) have evolved adaptive strategies to cope with EA through the up-regulation of SLC26A3 to promote pH homeostasis. We hypothesized that EA significantly alters IEC gene expression as an adaptive mechanism to counteract inflammation. Using an unbiased RNA sequencing approach, we defined the impact of EA on IEC gene expression to define molecular mechanisms by which IEC respond to EA. This approach identified a unique gene signature enriched in cyclic AMP response element-binding protein (CREB)-regulated gene targets. Utilizing loss- and gain-of-function approaches in cultured epithelia and murine colonoids, we demonstrate that EA elicits prominent CREB phosphorylation through cyclic AMP-independent mechanisms that requires elements of the mitogen-activated protein kinase signaling pathway. Further analysis revealed that EA signals through the G protein-coupled receptor GPR31 to promote induction of FosB, NR4A1, and DUSP1. These studies were extended to an in vivo murine model in conjunction with colonization of a pH reporter Escherichia coli strain that demonstrated significant mucosal acidification in the TNFΔARE model of murine ileitis. Herein, we observed a strong correlation between the expression of acidosis-associated genes with bacterial reporter sfGFP intensity in the distal ileum. Finally, the expression of this unique EA-associated gene signature was increased during active inflammation in patients with Crohn's disease but not in the patient control samples. These findings establish a mechanism for EA-induced signals during inflammation-associated acidosis in both murine and human ileitis., Competing Interests: The authors declare no competing interest.

Electrolytes (NaCl) and fluid malabsorption cause diarrhea in inflammatory bowel disease (IBD). Coupled NaCl absorption, mediated by Na + /H + and Cl - /HCO 3 exchanges on the intestinal villus cells brush border membrane (BBM), is inhibited in IBD. Arachidonic acid metabolites (AAMs) formed via cyclooxygenase (COX) or lipoxygenase (LOX) pathways are elevated in IBD. However, their effects on NaCl absorption are not known. We treated SAMP1/YitFc (SAMP1) mice, a model of spontaneous ileitis resembling human IBD, with Arachidonyl Trifluoro Methylketone (ATMK, AAM inhibitor), or with piroxicam or MK-886, to inhibit COX or LOX pathways, respectively. Cl - exchanges on the intestinal villus cells brush border membrane (BBM), is inhibited in IBD. Arachidonic acid metabolites (AAMs) formed via cyclooxygenase (COX) or lipoxygenase (LOX) pathways are elevated in IBD. However, their effects on NaCl absorption are not known. We treated SAMP1/YitFc (SAMP1) mice, a model of spontaneous ileitis resembling human IBD, with Arachidonyl Trifluoro Methylketone (ATMK, AAM inhibitor), or with piroxicam or MK-886, to inhibit COX or LOX pathways, respectively. Cl - /HCO 3 - exchange, measured as DIDS-sensitive 36 Cl uptake, was significantly inhibited in villus cells and BBM vesicles of SAMP1 mice compared to AKR/J controls, an effect reversed by ATMK. Piroxicam, but not MK-886, also reversed the inhibition. Kinetic studies showed that inhibition was secondary to altered K m with no effects on V max . Whole cell or BBM protein levels of Down-Regulated in Adenoma (SLC26A3) and putative anion transporter-1 (SLC26A6), the two key BBM Cl - /HCO 3 - exchangers, were unaltered. Thus, inhibition of villus cell Cl - /HCO 3 - exchange by COX pathway AAMs, such as prostaglandins, via reducing the affinity of the exchanger for Cl - , and thereby causing NaCl malabsorption, could significantly contribute to IBD-associated diarrhea.

In Inflammatory Bowel Disease (IBD), malabsorption of electrolytes (NaCl) results in diarrhea. Inhibition of coupled NaCl absorption, mediated by the dual operation of Na:H and Cl:HCO 3 exchangers on the brush border membrane (BBM) of the intestinal villus cells has been reported in IBD. In the SAMP1/YitFcs (SAMP1) mice model of spontaneous ileitis, representing Crohn's disease, DRA (Downregulated in Adenoma) mediated Cl:HCO 3 exchange was shown to be inhibited secondary to diminished affinity of the exchanger for Cl. However, NHE3 mediated Na:H exchange remained unaffected. Mast cells and their secreted mediators are known to be increased in the IBD mucosa and can affect intestinal electrolyte absorption. However, how mast cell mediators may regulate Cl:HCO 3 exchange in SAMP1 mice is unknown. Therefore, the aim of this study was to determine the effect of mast cell mediators on the downregulation of DRA in SAMP1 mice. Mast cell numbers and their degranulation marker enzyme (β-hexosaminidase) levels were significantly increased in SAMP1 mice compared to control AKR mice. However, treatment of SAMP1 mice with a mast cell stabilizer, ketotifen, restored the β-hexosaminidase enzyme levels to normal in the intestine, demonstrating stabilization of mast cells by ketotifen. Moreover, downregulation of Cl:HCO 3 exchange activity was restored in ketotifen treated SAMP1 mice. Kinetic studies showed that ketotifen restored the altered affinity of Cl:HCO 3 exchange in SAMP1 mice villus cells thus reinstating its activity to normal. Further, RT-qPCR, Western blot and immunofluorescence studies showed that the expression levels of DRA mRNA and BBM protein, respectively remained unaltered in all experimental conditions, supporting the kinetic data. Thus, inhibition of Cl:HCO 3 exchange resulting in chloride malabsorption leading to diarrhea in IBD is likely mediated by mast cell mediators.

Background and Aims: Nucleotide oligomerization domain 2 [NOD2] mutations are key risk factors for Crohn's disease [CD]. NOD2 contributes to intestinal homeostasis by regulating innate and adaptive immunity together with intestinal epithelial function. However, the exact roles of NOD2 in CD and other NOD2-associated disorders remain poorly known., Methods: We initially observed that NOD2 expression was increased in epithelial cells away from inflamed areas in CD patients. To explore this finding, Nod2 mRNA expression, inflammation, and cytokines expression were examined in the small bowel of wild-type [WT], Nod2 knockout and Nod2 mutant mice after rectal instillation of 2,4,6-trinitrobenzene sulphonic acid [TNBS]., Results: In WT mice, Nod2 upregulation upstream to rectal injury was associated with pro-inflammatory cytokine expression but no overt histological inflammatory lesions. Conversely, in Nod2-deficient mice the inflammation spread from colitis to ileum and duodenum., Conclusions: Nod2 protects the gut from colitis spreading to small intestine., (Copyright © 2019 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The serotonin transporter (SERT) functions to regulate the availability of serotonin (5-HT) in the brain and intestine. An intestine-specific mRNA variant arising from a unique transcription start site and alternative promoter in the SERT gene has been identified (iSERT; spanning exon 1C). A decrease in SERT is implicated in several gut disorders, including inflammatory bowel diseases (IBD). However, little is known about mechanisms regulating the iSERT variant, and a clearer understanding is warranted for targeting SERT for the treatment of gut disorders. The current studies examined the expression of iSERT across different human intestinal regions and investigated its regulation by HNF4α (hepatic nuclear factor-4α), a transcription factor important for diverse cellular functions. iSERT mRNA abundance was highest in the human ileum and Caco-2 cell line. iSERT mRNA expression was downregulated by loss of HNF4α (but not HNF1α, HNF1β, or FOXA1) in Caco-2 cells. Overexpression of HNF4α increased iSERT mRNA concomitant with an increase in SERT protein. Progressive promoter deletion and site-directed mutagenesis revealed that the HNF4α response element spans nucleotides -1,163 to -1150 relative to the translation start site. SERT mRNA levels in the intestine were drastically reduced in the intestine-specific HNF4α-knockout mice relative to HNF4α FL/FL mice. Both HNF4α and SERT mRNA levels were also downregulated in mouse model of ileitis (SAMP) compared with AKR control mice. These results establish the transcriptional regulation of iSERT at the gut-specific internal promoter (hSERTp2) and have identified HNF4α as a critical modulator of basal SERT expression in the intestine.

Crohn's disease (CD) is an intractable inflammatory bowel disease, and dysbiosis, disruption of the intestinal microbiota, is associated with CD pathophysiology. ER stress, disruption of ER homeostasis in Paneth cells of the small intestine, and α-defensin misfolding have been reported in CD patients. Because α-defensins regulate the composition of the intestinal microbiota, their misfolding may cause dysbiosis. However, whether ER stress, α-defensin misfolding, and dysbiosis contribute to the pathophysiology of CD remains unknown. Here, we show that abnormal Paneth cells with markers of ER stress appear in SAMP1/YitFc, a mouse model of CD, along with disease progression. Those mice secrete reduced-form α-defensins that lack disulfide bonds into the intestinal lumen, a condition not found in normal mice, and reduced-form α-defensins correlate with dysbiosis during disease progression. Moreover, administration of reduced-form α-defensins to wild-type mice induces the dysbiosis. These data provide novel insights into CD pathogenesis induced by dysbiosis resulting from Paneth cell α-defensin misfolding and they suggest further that Paneth cells may be potential therapeutic targets., (© 2020 Shimizu et al.)

Background: Lymphocytes recirculate from tissues to blood following the sphingosine-1-phosphate (S1P) gradient (low in tissues, high in blood), maintained by synthetic and degradative enzymes, among which the S1P lyase (SPL) irreversibly degrades S1P. The role of SPL in the intestine, both during homeostasis and IBD, is poorly understood. We hypothesized that modulation of tissue S1P levels might be advantageous over S1P receptor (S1PR) agonists (eg, fingolimod, ozanimod, etrasimod), as without S1PR engagement there might be less likelihood of potential off-target effects., Methods: First we examined SPL mRNA transcripts and SPL localization in tissues by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. The in vivo effects of the SPL inhibitors 4-deoxypyridoxine hydrochloride (30 mg/L) and 2-acetyl-4 (tetrahydroxybutyl)imidazole (50 mg/L) were assessed through their oral administration to adult TNF∆ARE mice, which spontaneously develop Crohn's-like chronic ileitis. The effect of SPL inhibition on circulating and tissue lymphocytes, transcriptional regulation of proinflammatory cytokines, and on the histological severity of ileitis was additionally examined. Tissue S1P levels were determined by liquid chromatography-mass spectrometry. Mechanistically, the potential effects of high S1P tissue levels on intestinal leukocyte apoptosis were assessed via terminal deoxynucleotidyl transferase dUTP nick end-labeling assay and annexin 5 staining. Finally, we examined the ability of T cells to home to the intestine, along with the effects of SPL inhibition on cellular subsets within immune compartments via flow and mass cytometry., Results: S1P lyase was ubiquitously expressed. In the gut, immunohistochemistry predominantly localized it to small intestinal epithelia, although the lamina propria leukocyte fraction had higher mRNA transcripts. Inhibition of SPL markedly increased local intestinal S1P levels, induced peripheral lymphopenia, downregulated proinflammatory cytokines, and attenuated chronic ileitis in mice. SPL inhibition reduced T and myeloid cells in secondary lymphoid tissues and the intestine and decreased naïve T-cell recruitment. The anti-inflammatory activity of SPL inhibition was not mediated by leukocyte apoptosis, nor by interference with the homing of lymphocytes to the intestine, and was independent of its peripheral lymphopenic effect. However, SPL inhibition promoted thymic atrophy and depleted late immature T cells (CD4+CD8+ double positive), with accumulation of mature CD4+CD8- and CD4-CD8+ single-positive cells., Conclusions: Inhibition of the S1P lyase alters the S1P gradient and attenuates chronic ileitis via central immunosuppression. SPL inhibition could represent a potential way to tame an overactive immune response during IBD and other T-cell-mediated chronic inflammatory diseases., (© 2019 Crohn’s & Colitis Foundation. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background and Aims: Magnetic resonance enterography [MRE] is the gold standard for assessing ileal inflammation in Crohn's disease [CD]. The aim of the present study was to correlate faecal calprotectin [FC] to MRE via a simple score in an exclusive ileal cohort with long-term follow-up for association with time to surgery or biologic therapy., Methods: In total, 150 MRE studies with matched FC [±30 days] were identified from the Edinburgh FC Register [2008-12; n = 18138]. Scans were re-read blinded to clinical data, independently, by two expert gastrointestinal radiologists, to generate a simple MRE score [range 0-10] from assessment of the worst intestinal segment plus total disease extent., Results: In total, 119 MRE scans were evaluated from 104 patients with ileal CD [L1 or L3 with panproctocolectomy]. Receiver operating characteristic analysis showed an area under the curve of 0.77 [0.67-0.87, p < 0.0001] for FC and MRE score >1, with an optimal cut-off of 145 μg/g for severe inflammation on MRE with 69.3% [57.6-79.5] sensitivity and 71.4% [53.7-85.4] specificity. Long-term follow-up over a median [interquartile range] of 2086 days [1786-2353] revealed FC ≥ 145 μg/g was associated with reduced biologic-free survival until 3 years following MRE, whereas MRE score [severe vs absent] was associated with reduced surgery- and biologic-free survival throughout follow-up. Backwards stepwise logistic regression revealed that length of ileal disease (odds ratio [OR] 3.8, 1.1-13.2, p = 0.034) and increased bowel wall thickness at MRE [OR 4.2, 1.6-10.7, p < 0.0001] or female sex [OR 5.2, 1.5-18.7, p = 0.011] increased the risk of biologic use or surgery, respectively., Conclusions: FC correlates well with MRE assessment of ileal CD with MRE parameters associated with long-term biologic- and surgery-free remission., (Copyright © 2018 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Epidemiological studies indicate that the use of artificial sweeteners doubles the risk for Crohn's disease (CD). Herein, we experimentally quantified the impact of 6-week supplementation with a commercial sweetener (Splenda; ingredients sucralose maltodextrin, 1:99, w/w) on both the severity of CD-like ileitis and the intestinal microbiome alterations using SAMP1/YitFc (SAMP) mice., Methods: Metagenomic shotgun DNA sequencing was first used to characterize the microbiome of ileitis-prone SAMP mice. Then, 16S rRNA microbiome sequencing, quantitative polymerase chain reaction, fluorescent in situ hybridization (FISH), bacterial culture, stereomicroscopy, histology, and myeloperoxidase (MPO) activity analyses were then implemented to compare the microbiome and ileitis phenotype in SAMP with that of control ileitis-free AKR/J mice after Splenda supplementation., Results: Metagenomics indicated that SAMP mice have a gut microbial phenotype rich in Bacteroidetes, and experiments showed that Helicobacteraceae did not have an exacerbating effect on ileitis. Splenda did not increase the severity of (stereomicroscopic/histological) ileitis; however, biochemically, ileal MPO activity was increased in SAMP treated with Splenda compared with nonsupplemented mice (P < 0.022) and healthy AKR mice. Splenda promoted dysbiosis with expansion of Proteobacteria in all mice, and E. coli overgrowth with increased bacterial infiltration into the ileal lamina propria of SAMP mice. FISH showed increase malX gene-carrying bacterial clusters in the ilea of supplemented SAMP (but not AKR) mice., Conclusions: Splenda promoted gut Proteobacteria, dysbiosis, and biochemical MPO reactivity in a spontaneous model of (Bacteroidetes-rich) ileal CD. Our results indicate that although Splenda may promote parallel microbiome alterations in CD-prone and healthy hosts, this did not result in elevated MPO levels in healthy mice, only CD-prone mice. The consumption of sucralose/maltodextrin-containing foods might exacerbate MPO intestinal reactivity only in individuals with a pro-inflammatory predisposition, such as CD.

Inflammatory bowel disease (IBD) has a complex pathophysiology with limited treatments. Structural and functional changes in the intestinal lymphatic system have been associated with the disease, with increased risk of IBD occurrence linked to a history of acute intestinal injury. To examine the potential role of the lymphatic system in inflammation recurrence, we evaluated morphological and functional changes in mouse mucosal and mesenteric lymphatic vessels, and within the mesenteric lymph nodes during acute ileitis caused by a 7-day treatment with dextran sodium sulfate (DSS). We monitored whether the changes persisted during a 14-day recovery period and determined their potential consequences on dendritic cell (DC) trafficking between the mucosa and lymphoid tissues. DSS administration was associated with marked lymphatic abnormalities and dysfunctions exemplified by lymphangiectasia and lymphangiogenesis in the ileal mucosa and mesentery, increased mesenteric lymphatic vessel leakage, and lymphadenopathy. Lymphangiogenesis and lymphadenopathy were still evident after recovery from intestinal inflammation and correlated with higher numbers of DCs in mucosal and lymphatic tissues. Specifically, a deficit in CD103 + DCs observed during acute DSS in the lamina propria was reversed and further enhanced during recovery. We concluded that an acute intestinal insult caused alterations of the mesenteric lymphatic system, including lymphangiogenesis, which persisted after resolution of inflammation. These morphological and functional changes could compromise DC function and movement, increasing susceptibility to further gastrointestinal disease. Elucidation of the changes in mesenteric and intestinal lymphatic function should offer key insights for new therapeutic strategies in gastrointestinal disorders such as IBD. NEW & NOTEWORTHY Lymphatic integrity plays a critical role in small intestinal homeostasis. Acute intestinal insult in a mouse model of acute ileitis causes morphological and functional changes in mesenteric and intestinal lymphatic vessels. While some of the changes significantly regressed during inflammation resolution, others persisted, including lymphangiogenesis and altered dendritic cell function and movement, potentially increasing susceptibility to the recurrence of gastrointestinal inflammation.

Purpose: In this study, we showed the beneficial effects of donkey milk (DM) on inflammatory damages, endogenous antimicrobial peptides levels and fecal microbiota profile in a mice model of Crohn's disease. Nowadays, new strategies of microbiome manipulations are on the light involving specific diets to induce and/or to maintain clinical remission. Interest of DM is explained by its high levels of antimicrobial peptides which confer it anti-inflammatory properties., Methods: C57BL/6 mice were orally administered with or without indomethacin for 5 days and co-treated with vehicle, DM or heated DM during 7 days. Intestinal length and macroscopic damage scores (MDSs) were determined; ileal samples were taken off for microscopic damage (MD), lysozyme immunostaining and mRNA α-defensin assessments. Ileal luminal content and fecal pellets were collected for lysozyme enzymatic activity and lipocalin-2 (LCN-2) evaluations. Fecal microbiota profiles were compared using a real-time quantitative PCR-based analysis., Results: Administration of indomethacin caused an ileitis in mice characterized by (1) a decrease in body weight and intestinal length, (2) a significant increase in MDS, MD and LCN-2, (3) a reduction in both α-defensin mRNA expression and lysozyme levels in Paneth's cells reflected by a decrease in lysozyme activity in feces, and (4) a global change in relative abundance of targeted microbial communities. DM treatment significantly reduced almost of all these ileitis damages, whereas heated DM has no impact on ileitis., Conclusions: DM consumption exerts anti-inflammatory properties in mice by restoring the endogenous levels of antimicrobial peptides which contribute in turn to reduce microbiota imbalance.

The aim of the study described here was to assess whether the analysis of time-intensity curves obtained after microbubble contrast agent injection could differentiate inflammatory from fibrotic ileal strictures among patients with Crohn's disease. Sixty-five consecutive patients (40 male and 25 female; mean age ± SD, 42.2 ± 12.22 y) with stricture of the terminal ileal loop from Crohn's disease were scanned after microbubble injection. Time-intensity curves were obtained from quantitative analysis, and peak enhancement, rise time, time to peak, area under the time-intensity curve (AUC), AUC during wash-in (AUCWI) and AUC during wash-out (AUCWO) were compared between patients with inflammatory strictures and patients with fibrotic strictures. Inflammatory (n = 40) and fibrotic (n = 25) strictures differed (p < 0.05) in peak enhancement, wash-in rate, wash-in perfusion index, AUC, AUCWI and AUCWO. The quantitative analysis of small bowel wall contrast enhancement after microbubble contrast agent injection may differentiate inflammatory from fibrotic ileal strictures in patients with Crohn's disease., (Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.)

Bile acids regulate the expression of intestinal bile acid transporters and are natural ligands for nuclear receptors controlling inflammation. Accumulating evidence suggests that signaling through these receptors is impaired in inflammatory bowel disease. We investigated whether tauroursodeoxycholic acid (TUDCA), a secondary bile acid with cytoprotective properties, regulates ileal nuclear receptor and bile acid transporter expression and assessed its therapeutic potential in an experimental model of Crohn's disease (CD). Gene expression of the nuclear receptors farnesoid X receptor, pregnane X receptor and vitamin D receptor and the bile acid transporters apical sodium-dependent bile acid transporter and organic solute transporter α and β was analyzed in Caco-2 cell monolayers exposed to tumor necrosis factor (TNF)α, in ileal tissue of TNF ΔARE/WT mice and in inflamed ileal biopsies from CD patients by quantitative real-time polymerase chain reaction. TNF ΔARE/WT mice and wild-type littermates were treated with TUDCA or placebo for 11 weeks and ileal histopathology and expression of the aforementioned genes were determined. Exposing Caco-2 cell monolayers to TNFα impaired the mRNA expression of nuclear receptors and bile acid transporters, whereas co-incubation with TUDCA antagonized their downregulation. TNF ΔARE/WT mice displayed altered ileal bile acid homeostasis that mimicked the situation in human CD ileitis. Administration of TUDCA attenuated ileitis and alleviated the downregulation of nuclear receptors and bile acid transporters in these mice. These results show that TUDCA protects bile acid homeostasis under inflammatory conditions and suppresses CD-like ileitis. Together with previous observations showing similar efficacy in experimental colitis, we conclude that TUDCA could be a promising therapeutic agent for inflammatory bowel disease, warranting a clinical trial.

Zinc transporter 3 (ZnT3) is a member of the solute-linked carrier 30 (SLC 30) zinc transporter family. It is closely linked to the nervous system, where it takes part in the transport of zinc ions from the cytoplasm to the synaptic vesicles. ZnT3 has also been observed in the enteric nervous system (ENS), but its reactions in response to pathological factors remain unknown. This study, based on the triple immunofluorescence technique, describes changes in ZnT3-like immunoreactive (ZnT3-LI) enteric neurons in the porcine ileum, caused by chemically-induced inflammation. The inflammatory process led to a clear increase in the percentage of neurons immunoreactive to ZnT3 in all "kinds" of intramural enteric plexuses, i.e., myenteric (MP), outer submucous (OSP) and inner submucous (ISP) plexuses. Moreover, a wide range of other active substances was noted in ZnT3-LI neurons under physiological and pathological conditions, and changes in neurochemical characterisation of ZnT3⁺ cells in response to inflammation depended on the "kind" of enteric plexus. The obtained results show that ZnT3 is present in the ENS in a relatively numerous and diversified neuronal population, not only in physiological conditions, but also during inflammation. The reasons for the observed changes are not clear; they may be connected with the functions of zinc ions and their homeostasis disturbances in pathological processes. On the other hand, they may be due to adaptive and/or neuroprotective processes within the pathologically altered gastrointestinal tract.

Pathogen infections, autoimmune diseases, and chronic inflammatory disorders are associated with systemic antibody responses from the host immune system. Disease-specific antibodies can be important serum biomarkers, but the identification of antigens associated with specific immune reactions is challenging, in particular if complex communities of microorganisms are involved in the disease progression. Despite promising new diagnostic opportunities, the discovery of these serological markers becomes more difficult with increasing complexity of microbial communities. In the present work, we used a metagenomic M13 phage display approach to select immunogenic oligopeptides from the gut microbiome of transgenic mice suffering from chronic ileitis. We constructed three individual metaproteome phage display libraries with a library size of approximately 10 7 clones each. Using serum antibodies, we selected and validated three oligopeptides that induced specific antibody responses in the mouse model. This proof-of-concept study provides the first successful application of functional metaproteome display for the study of protein-protein interactions and the discovery of potential disease biomarkers.

A dysfunction of the Na(+)/H(+) exchanger isoform 3 (NHE3) significantly contributes to the reduced salt absorptive capacity of the inflamed intestine. We previously reported a strong decrease in the NHERF family member PDZK1 (NHERF3), which binds to NHE3 and regulates its function in a mouse model of colitis. The present study investigates whether a causal relationship exists between the decreased PDZK1 expression and the NHE3 dysfunction in human and murine intestinal inflammation. Biopsies from the colon of patients with ulcerative colitis, murine inflamed ileal and colonic mucosa, NHE3-transfected Caco-2BBe colonic cells with short hairpin RNA (shRNA) knockdown of PDZK1, and Pdzk1-gene-deleted mice were studied. PDZK1 mRNA and protein expression was strongly decreased in inflamed human and murine intestinal tissue as compared to inactive disease or control tissue, whereas that of NHE3 or NHERF1 was not. Inflamed human and murine intestinal tissues displayed correct brush border localization of NHE3 but reduced acid-activated NHE3 transport activity. A similar NHE3 transport defect was observed when PDZK1 protein content was decreased by shRNA knockdown in Caco-2BBe cells or when enterocyte PDZK1 protein content was decreased to similar levels as found in inflamed mucosa by heterozygote breeding of Pdzk1-gene-deleted and WT mice. We conclude that a decrease in PDZK1 expression, whether induced by inflammation, shRNA-mediated knockdown, or heterozygous breeding, is associated with a decreased NHE3 transport rate in human and murine enterocytes. We therefore hypothesize that inflammation-induced loss of PDZK1 expression may contribute to the NHE3 dysfunction observed in the inflamed intestine.

Background: Oral infection of C57BL/6J mice with the protozoan parasite Toxoplasma gondii leads to a lethal inflammatory ileitis., Principal Findings: Mice lacking the purinergic receptor P2X7R are acutely susceptible to toxoplasmic ileitis, losing significantly more weight than C57BL/6J mice and exhibiting much greater intestinal inflammatory pathology in response to infection with only 10 cysts of T. gondii. This susceptibility is not dependent on the ability of P2X7R-deficient mice to control the parasite, which they accomplish just as efficiently as C57BL/6J mice. Rather, susceptibility is associated with elevated ileal concentrations of pro-inflammatory cytokines, reactive nitrogen intermediates and altered regulation of elements of NFκB activation in P2X7R-deficient mice., Conclusions: Our data support the thesis that P2X7R, a well-documented activator of pro-inflammatory cytokine production, also plays an important role in the regulation of intestinal inflammation.

Objective: The purpose of this article was to assess the feasibility of golden-angle radial acquisition with compress sensing reconstruction (Golden-angle RAdial Sparse Parallel [GRASP]) for acquiring high temporal resolution data for pharmacokinetic modeling while maintaining high image quality in patients with Crohn disease terminal ileitis., Materials and Methods: Fourteen patients with biopsy-proven Crohn terminal ileitis were scanned using both contrast-enhanced GRASP and Cartesian breath-hold (volume-interpolated breath-hold examination [VIBE]) acquisitions. GRASP data were reconstructed with 2.4-second temporal resolution and fitted to the generalized kinetic model using an individualized arterial input function to derive the volume transfer coefficient (K(trans)) and interstitial volume (v(e)). Reconstructions, including data from the entire GRASP acquisition and Cartesian VIBE acquisitions, were rated for image quality, artifact, and detection of typical Crohn ileitis features., Results: Inflamed loops of ileum had significantly higher K(trans) (3.36 ± 2.49 vs 0.86 ± 0.49 min(-1), p < 0.005) and v(e) (0.53 ± 0.15 vs 0.20 ± 0.11, p < 0.005) compared with normal bowel loops. There were no significant differences between GRASP and Cartesian VIBE for overall image quality (p = 0.180) or detection of Crohn ileitis features, although streak artifact was worse with the GRASP acquisition (p = 0.001)., Conclusion: High temporal resolution data for pharmacokinetic modeling and high spatial resolution data for morphologic image analysis can be achieved in the same acquisition using GRASP.

Two experiments were conducted to determine the effects of increasing the dietary Zn content on growth performance, carcass characteristics, plasma Zn, and ileal mucosal inflammation mRNA expression of finishing pigs fed diets containing ractopamine HCl (RAC; Elanco Animal Health, Greenfield, IN). In Exp. 1, 312 pigs (327 × 1050; PIC, Hendersonville, TN; 94 kg BW) were used in a 27-d study. There were 2 pigs per pen and 26 pens per treatment. Treatments included a corn-soybean meal diet (control; 0.66% standardized ileal digestible [SID] Lys); a diet (0.92% SID Lys) with 10 mg/kg RAC; and the RAC diet plus 50, 100, or 150 mg Zn/kg from ZnO or 50 mg Zn/kg from a Zn AA complex (ZnAA; Availa-Zn; Zinpro, Eden Prairie, MN). All diets also contained 83 mg Zn/kg from ZnSO4 in the trace mineral premix. Pigs fed the RAC diet without added Zn had increased (P < 0.05) ADG, G:F, HCW, carcass yield, and loin weight compared with pigs fed the control diet. Increasing Zn from ZnO in diets containing RAC tended to increase (linear, P = 0.067) G:F and loin weight (quadratic, P = 0.064). Pigs fed diets with 50 mg Zn/kg from ZnAA tended to have increased (P = 0.057) ADG compared with pigs fed the RAC diet. In Exp. 2, 320 pigs (327 × 1050; PIC; 98 kg BW) were used in a 35-d study. There were 2 pigs per pen and 20 pens per treatment. Treatments included a control diet (0.66% SID Lys); a diet (0.92% SID Lys) with 10 mg/kg RAC; or the RAC diet plus 75, 150, and 225 mg Zn/kg from ZnO or ZnAA. All diets also contained 55 mg Zn/kg from ZnSO4 from the trace mineral premix. Pigs fed the RAC diet had increased (P < 0.05) ADG, G:F, HCW, loin depth, percentage lean, and liver weight compared with pigs fed the control diet. No Zn level or source effects or level × source interactions were observed for growth performance. A Zn level × source interaction (quadratic, P = 0.007) was observed in liver Zn concentrations. This resulted from liver Zn concentrations plateauing at 150 mg Zn/kg when ZnO was supplemented, while there was a linear increase when using ZnAA. Increasing Zn in diets containing RAC increased (linear, P < 0.05) plasma Zn on d 18 and 32. The expression of IL-1β was increased (P = 0.014) in mucosa of pigs fed the RAC diet compared with those fed the control diet. Expression of IL-1β decreased (linear, P = 0.026) in the mucosa of pigs fed increasing added Zn. In conclusion, adding Zn to diets containing RAC resulted in a trend for improved growth performance of pigs in 1 of 2 experiments. Also, additional Zn increased plasma Zn and reduced IL-1β.

Prostaglandins are important mediators responsible for many changes that occur during the inflammatory response. Specifically, in inflammatory bowel disease (IBD), prostaglandins are key players in maintenance of blood flow and mucosal defense. In blood vessels, prostaglandins modulate and inhibit transmigration. In lymphatic vessels, on the other hand, prostaglandin E2 (PGE2) and prostacyclin (PGI2) have been shown to potently inhibit lymphatic contractility. Inhibition of lymphatic contractility could impair proper tissue fluid drainage during inflammation, consequently leading to the submucosal oedema observed in IBD. Alterations in production of PGE2 and PGI2 during inflammation could have severe implications on lymphatic and vascular functions within the small intestine. Using the 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced ileitis guinea pig and rat models, we assessed by quantitative PCR changes in mRNA transcript of enzymes and receptors involved in the production and actions of prostaglandins in mesenteric lymphatic and blood vessels as well as in the affected ileum. Furthermore, we also assessed lymphatic tissue levels of PGE2 and PGI2 during inflammation. We observed significant changes in lymphatic mRNA expression of COX-1, COX-2, MPGES-1, PGIS, EP4 and IP and increases in PGE2 and PGI2 in tissues of TNBS-treated animals. Changes in mRNA in blood vessels from TNBS-treated animals included differences in COX-1, COX-2, MPGES-1, PGIS, EP1, EP2 and IP expression. Prostaglandin metabolites are differentially regulated in both lymphatic and blood vessels during intestinal inflammation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: The spondyloarthritides share genetic susceptibility, interleukin-23 (IL-23) dependence, and the involvement of microbiota. The aim of the current study was to elucidate how host genetics influence gut microbiota and the relationship between microbiota and organ inflammation in spondyloarthritides., Methods: BALB/c ZAP-70(W163C) -mutant (SKG) mice, Toll-like receptor 4 (TLR-4)-deficient SKG mice, and wild-type BALB/c mice were housed under specific pathogen-free conditions. SKG and wild-type BALB/c mice were maintained under germ-free conditions, and some of these mice were recolonized with altered Schaedler flora. All of the mice were injected intraperitoneally with microbial β-1,3-glucan (curdlan). Arthritis, spondylitis, and ileitis were assessed histologically. Microbiome composition was analyzed in serial fecal samples obtained from mice that were co-housed beginning at the time of weaning, using 454 pyrosequencing. Infiltrating cells and cytokines in the peritoneal cavity were measured by flow cytometry and enzyme-linked immunosorbent assay. Cytokine, endoplasmic reticulum (ER) stress marker, and tight junction protein transcription was measured by quantitative real-time polymerase chain reaction., Results: Microbiota content and response to curdlan varied according to whether T cell receptor signal strength was normal or was impaired due to the ZAP-70(W163C) mutation. Curdlan triggered acute inflammation regardless of the presence of the SKG allele or microbiota. However, no or limited microbiota content attenuated the severity of arthritis. In contrast, ileal IL-23 expression, ER stress, lymph node IL-17A production, goblet cell loss, and ileitis development were microbiota-dependent. Ileitis but not arthritis was suppressed by microbiota transfer upon co-housing SKG mice with wild-type BALB/c mice, as well as by TLR-4 deficiency., Conclusion: The interaction between immunogenetic background and host microbiota leads to an IL-23-dependent loss of mucosal function, triggering ileitis in response to curdlan., (Copyright © 2014 by the American College of Rheumatology.)

Impairment of gut epithelial barrier function is a key predisposing factor for inflammatory bowel disease, type 1 diabetes (T1D) and related autoimmune diseases. We hypothesized that maternal obesity induces gut inflammation and impairs epithelial barrier function in the offspring of nonobese diabetic (NOD) mice. Four-week-old female NOD/ShiLtJ mice were fed with a control diet (CON; 10% energy from fat) or a high-fat diet (HFD; 60% energy from fat) for 8 weeks to induce obesity and then mated. During pregnancy and lactation, mice were maintained in their respective diets. After weaning, all offspring were fed the CON diet. At 16 weeks of age, female offspring were subjected to in vivo intestinal permeability test, and then ileum was sampled for biochemical analyses. Inflammasome mediators, activated caspase-1 and mature forms of interleukin (IL)-1β and IL-18 were enhanced in offspring of obese mothers, which was associated with elevated serum tumor necrosis factor α level and inflammatory mediators. Consistently, abundance of oxidative stress markers including catalase, peroxiredoxin-4 and superoxide dismutase 1 was heightened in offspring ileum (P<.05). Furthermore, offspring from obese mothers had a higher intestinal permeability. Morphologically, maternal obesity reduced villi/crypt ratio in the ileum of offspring gut. In conclusion, maternal obesity induced inflammation and impaired gut barrier function in offspring of NOD mice. The enhanced gut permeability in HFD offspring might predispose them to the development of T1D and other gut permeability-associated diseases., (Published by Elsevier Inc.)

Objective: Spondyloarthritides (SpA) occur in 1% of the population and include ankylosing spondylitis (AS) and arthropathy of inflammatory bowel disease (IBD), with characteristic spondylitis, arthritis, enthesitis, and IBD. Genetic studies implicate interleukin-23 (IL-23) receptor signaling in the development of SpA and IBD, and IL-23 overexpression in mice is sufficient for enthesitis, driven by entheseal-resident T cells. However, in genetically prone individuals, it is not clear where IL-23 is produced and how it drives the SpA syndrome, including IBD or subclinical gut inflammation of AS. Moreover, it is unclear why specific tissue involvement varies between patients with SpA. We undertook this study to determine the location of IL-23 production and its role in SpA pathogenesis in BALB/c ZAP-70(W163C)-mutant (SKG) mice injected intraperitoneally with β-1,3-glucan (curdlan)., Methods: Eight weeks after curdlan injection in wild-type or IL-17A(-/-) SKG or BALB/c mice, pathology was scored in tissue sections. Mice were treated with anti-IL-23 or anti-IL-22. Cytokine production and endoplasmic reticulum (ER) stress were determined in affected organs., Results: In curdlan-treated SKG mice, arthritis, enthesitis, and ileitis were IL-23 dependent. Enthesitis was specifically dependent on IL-17A and IL-22. IL-23 was induced in the ileum, where it amplified ER stress, goblet cell dysfunction, and proinflammatory cytokine production. IL-17A was pathogenic, while IL-22 was protective against ileitis. IL-22+CD3- innate-like cells were increased in lamina propria mononuclear cells of ileitis-resistant BALB/c mice, which developed ileitis after curdlan injection and anti-IL-22., Conclusion: In response to systemic β-1,3-glucan, intestinal IL-23 provokes local mucosal dysregulation and cytokines driving the SpA syndrome, including IL-17/IL-22-dependent enthesitis. Innate IL-22 production promotes ileal tolerance., (Copyright © 2014 by the American College of Rheumatology.)

Necrotizing enterocolitis (NEC) is associated with a high morbidity and mortality in very low birth weight infants. Several hypotheses regarding the pathogenesis of NEC have been proposed but to date no effective treatment is available. Previous studies suggest that probiotic supplementation is protective. We recently reported that probiotic (Bifidobacterium infantis) conditioned medium (PCM) has an anti-inflammatory effect in cultured fetal human intestinal cells (H4) and fetal intestine explants. In this study, we tested in vivo whether PCM protects neonatal mice from developing intestinal inflammation induced by exposure to Cronobacter sakazakii (C. sakazakii), an opportunistic pathogen associated with NEC. We found that infected neonatal mice had a significantly lower body weight than control groups. Infection led to ileal tissue damage including villous rupture, disruption of epithelial cell alignment, intestinal inflammation, apoptotic cell loss, and decreased mucus production. Pretreatment with PCM prevented infection caused decrease in body weight, attenuated enterocyte apoptotic cell death, mitigated reduced mucin production, and maintained ileal structure. Infected ileum expressed reduced levels of IκBα, which could be restored upon pretreatment with PCM. We also observed a nuclear translocation of NF-κB p65 in H4 cells exposed to C. sakazakii, which was prevented in PCM-pretreated cells. Finally, treatment of neonatal mice with PCM prior to infection sustained the capacity of ileal epithelial proliferation. This study suggests that an active component(s) released into the culture medium by B. infantis may prevent ileal damage by a pathogen linked to NEC.

Autism spectrum disorder (ASD) is a cluster of neurodevelopmental disorders characterized by impairments in communication, social interest and stereotypical behaviour. Dysfunction of the intestinal tract is reported in patients with ASD and implicated in the development and severity of ASD symptoms. However, more research is required to investigate the association of intestinal problems with ASD and the potential underlying mechanisms. The purpose of this study was to investigate comorbid symptoms of intestinal inflammation in a murine model of ASD induced by prenatal exposure to valproic acid (VPA). Pregnant BALB/c females were treated subcutaneously with 600 mg/kg VPA or phosphate buffered saline on gestational day 11. Offspring were housed with their mother until weaning on postnatal day 21 (P21). All pups were exposed to a social behaviour test on P28. Inflammatory correlates and activity of the serotonergic system were measured in brain and intestinal tissue. Here we demonstrate, in addition to reduced social behaviour and increased expression of neuroinflammatory markers in the brain, that VPA in utero- exposed male offspring showed epithelial cell loss and neutrophil infiltration in the intestinal tract. Furthermore, reduced levels of serotonin were not only observed the prefrontal cortex and amygdala of VPA in utero- exposed males, but also in the small intestine. Overall, we demonstrate that gender-specific inflammatory conditions are present in the small intestines of VPA in utero- exposed mice and are accompanied by a disturbed serotonergic system in the brain as well as in the intestinal tract., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background & Aims: Levels of microRNAs are altered in intestinal tissues of patients with Crohn's disease (CD). The adherent-invasive Escherichia coli (AIEC), which colonize the ileal mucosa of patients with CD, adhere to and invade intestinal epithelial cells. We investigated the mechanism by which AIEC infection alters the expression of microRNAs and the host immune response., Methods: Levels of microRNAs in human intestinal epithelial T84 cells and in mouse enterocytes were measured using quantitative reverse-transcription polymerase chain reaction. Luciferase assays were used to measure binding of microRNAs to the 3'-untranslated region of messenger RNA targets. Binding of nuclear factor-κB to promoters of genes encoding microRNAs was assessed by chromatin immunoprecipitation assays. Autophagy was measured by immunoblot analyses and immunofluorescent labeling of LC3. Anti-microRNAs were transferred to mice using ileal loops. Biopsy specimens from the terminal ileum of patients with ulcerative colitis (n = 20), CD (n = 20), or individuals without inflammatory bowel disease undergoing surveillance colonoscopies (controls, n = 13) were collected during endoscopic examination., Results: AIEC infection up-regulated levels of microRNA (MIR) 30C and MIR130A in T84 cells and in mouse enterocytes by activating nuclear factor-κB. Up-regulation of these microRNAs reduced the levels of ATG5 and ATG16L1 and inhibited autophagy, leading to increased numbers of intracellular AIEC and an increased inflammatory response. In ileal biopsy samples of patients with CD, there was an inverse correlation between levels of MIR30C and MIR130A and those of ATG5 and ATG16L1, supporting in vitro findings. Inhibition of MIR30C and MIR130A in cultured intestinal epithelial cells and in mouse enterocytes blocked AIEC-induced inhibition of ATG5 and ATG16L1 expression and restored functional autophagy. This resulted in more effective clearance of intracellular AIEC and reduced AIEC-induced inflammation., Conclusions: Infection with AIEC up-regulates microRNAs to reduce expression of proteins required for autophagy and autophagy response in intestinal epithelial cells. Ileal samples from patients with CD have increased levels of these same microRNAs and reduced levels of ATG5 and ATG16L1., (Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Rifaximin is used to treat patients with functional gastrointestinal disorders, but little is known about its therapeutic mechanism. We propose that rifaximin modulates the ileal bacterial community, reduces subclinical inflammation of the intestinal mucosa, and improves gut barrier function to reduce visceral hypersensitivity., Methods: We induced visceral hyperalgesia in rats, via chronic water avoidance or repeat restraint stressors, and investigated whether rifaximin altered the gut microbiota, prevented intestinal inflammation, and improved gut barrier function. Quantitative polymerase chain reaction (PCR) and 454 pyrosequencing were used to analyze bacterial 16S ribosomal RNA in ileal contents from the rats. Reverse transcription, immunoblot, and histologic analyses were used to evaluate levels of cytokines, the tight junction protein occludin, and mucosal inflammation, respectively. Intestinal permeability and rectal sensitivity were measured., Results: Water avoidance and repeat restraint stress each led to visceral hyperalgesia, accompanied by mucosal inflammation and impaired mucosal barrier function. Oral rifaximin altered the composition of bacterial communities in the ileum (Lactobacillus species became the most abundant) and prevented mucosal inflammation, impairment to intestinal barrier function, and visceral hyperalgesia in response to chronic stress. Neomycin also changed the composition of the ileal bacterial community (Proteobacteria became the most abundant species). Neomycin did not prevent intestinal inflammation or induction of visceral hyperalgesia induced by water avoidance stress., Conclusions: Rifaximin alters the bacterial population in the ileum of rats, leading to a relative abundance of Lactobacillus. These changes prevent intestinal abnormalities and visceral hyperalgesia in response to chronic psychological stress., (Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Objective: Long-term evolution of subclinical gut inflammation to overt Crohn's disease (CD) has been described in AS patients. The aim of this study was to evaluate macrophage polarization occurring in the inflamed gut of patients with AS., Methods: Twenty-seven HLA-B27(+) AS patients, 20 CD patients and 17 normal controls were consecutively enrolled. Classic M1 (iNOS(+)IL-10(-)), resolution phase (iNOS(+)IL-10(+)), M2 and CD14(+) macrophages were characterized by immunohistochemistry and flow cytometry. Quantitative gene expression analysis of IFN-γ, IL-4, IL-5, IL-33 and STAT6 was performed by real time PCR., Results: Classic M1 macrophages were expanded in CD and AS, where resolution phase macrophages predominate. A large increase in CD163(+) (M2) macrophages was observed in AS strictly correlated with the expression of IL-33, a Th2 cytokine involved in M2 polarization. Unlike in CD, CD14(+) macrophages were virtually absent in the gut of AS patients and controls., Conclusion: The absence of CD14(+) macrophages together with the expansion of resolution phase and M2 macrophages is the immunological signature of subclinical ileal inflammation in AS.

Purinergic signalling is remarkably plastic during gastrointestinal inflammation. Thus, selective drugs targeting the "purinome" may be helpful for inflammatory gastrointestinal diseases. The myenteric neuromuscular transmission of healthy individuals is fine-tuned and controlled by adenosine acting on A(2A) excitatory receptors. Here, we investigated the neuromodulatory role of adenosine in TNBS-inflamed longitudinal muscle-myenteric plexus of the rat ileum. Seven-day postinflammation ileitis lacks adenosine neuromodulation, which may contribute to acceleration of gastrointestinal transit. The loss of adenosine neuromodulation results from deficient accumulation of the nucleoside at the myenteric synapse despite the fact that the increases in ATP release were observed. Disparity between ATP outflow and adenosine deficit in postinflammatory ileitis is ascribed to feed-forward inhibition of ecto-5'-nucleotidase/CD73 by high extracellular ATP and/or ADP. Redistribution of NTPDase2, but not of NTPDase3, from ganglion cell bodies to myenteric nerve terminals leads to preferential ADP accumulation from released ATP, thus contributing to the prolonged inhibition of muscle-bound ecto-5'-nucleotidase/CD73 and to the delay of adenosine formation at the inflamed neuromuscular synapse. On the other hand, depression of endogenous adenosine accumulation may also occur due to enhancement of adenosine deaminase activity. Both membrane-bound and soluble forms of ecto-5'-nucleotidase/CD73 and adenosine deaminase were detected in the inflamed myenteric plexus. These findings provide novel therapeutic targets for inflammatory gut motility disorders.

Background and Aim: Human and animal studies have clearly established tumor necrosis factor (TNF)α as an important mediator of Crohn's disease pathogenesis. However, whether systemic or only local TNFα overproduction is required for the development of chronic intestinal inflammation and Crohn's disease remains unclear. The aim of this study was to assess the contribution of intestinal epithelial-derived TNFα to the development of murine Crohn's-like ileitis., Methods: We adapted the well-established TNF(∆ARE/+) mouse model of Crohn's disease (which systemically overexpresses TNFα) to generate a homozygous mutant strain that overexpress TNFα only within the intestinal epithelium. Intestinal-specific TNF(i∆ARE/i∆ARE) mice were examined for histopathological signs of gut inflammation and extraintestinal manifestations of Crohn's disease. The mucosal immune phenotype was characterized, and the contribution of specific lymphocyte populations to the pathogenesis of TNF(i∆ARE/i∆ARE) ileitis was assessed., Results: TNF(i∆ARE/i∆ARE) mice had increased mucosal and systemic TNFα levels compared to wild-type controls (P<0.001), as well as severe chronic ileitis with increased neutrophil infiltration and villous distortion, but no extraintestinal manifestations (P<0.001 vs. wild-type controls). The gut mucosal lymphocytic compartment was also expanded in TNF(i∆ARE/i∆ARE) mice (P<0.05), consisting of activated CD69(+) and CD4(+)CD62L(-) lymphocytes (P<0.05). FasL expression was significantly elevated in the mesenteric lymph nodes of TNF(i∆ARE/i∆ARE) mice (P<0.05). Adoptive transfer of mucosal TNF(i∆ARE/i∆ARE) lymphocytes resulted in ileitis in immunologically naïve severe combined immunodeficiency recipients (P<0.05 vs. wild-type controls), indicating an effector phenotype that was associated with increased production of both Th1 (IFNγ) and Th2 (IL-5, IL-13) cytokines., Conclusion: Intestinal epithelial-derived TNFα is sufficient for the induction of Crohn's-like ileitis, but not for the occurrence of extraintestinal manifestations, in TNF(i∆ARE/i∆ARE) mice. These effects were associated with generation of effector lymphocytes within the intestinal mucosa and dysregulated apoptosis. Thus, targeted intestinal blockade of TNFα may provide an effective means to neutralize gut-derived TNFα with reduced side effects.

Background: Antimicrobial peptides (AMPs) maintain a sterile environment in intestinal crypts, limiting microbial colonization and invasion. Decreased AMP expression is proposed to increase the risk for inflammatory bowel disease. Expression and function of inducible AMPs, human β-defensin 2 and 3 (hBD-2 and hBD-3), remain poorly characterized in healthy and chronically inflamed intestine., Methods: Peptide concentrations of hBD-2 and hBD-3 in serum and intestinal biopsies of subjects with ulcerative colitis and Crohn's disease (CD), and those of healthy subjects were measured by ELISA. Messenger RNA of hBD-2 and hBD-3 was quantified by quantitative PCR in biopsies from the terminal ileum (TI) of patients with CD and healthy controls. Peptide localization of hBD-3 in the TI was visualized by confocal microscopy., Results: Immunoreactive hBD-3 peptide is present in the TI and colon in healthy subjects. In the TI of patients with CD, hBD-3, but not hBD-2 peptide, is increased 4-fold, whereas hBD-2 peptide is elevated in the serum. Messenger RNA of hBD-3 in the CD TI remains unchanged and does not correlate with hBD-3 peptide expression. However, hBD-3 is localized to Paneth cell granules and the apical surface of the healthy columnar epithelium. In CD, hBD-3 peptide location switches to the basolateral surface of the columnar epithelium and is diffusely distributed within the lamina propria., Conclusion: The peptide hBD-3 throughout the healthy gastrointestinal tract suggests a role in maintaining balance between host defenses and commensal microbiota. Increased and relocalized secretion of hBD-3 toward the lamina propria in the CD TI indicates possible local immunomodulation during chronic inflammation, whereas increased serum hBD-2 in CD implicates its systemic antimicrobial and immunomodulatory role.

Mesenteric lymphatic vessels actively transport lymph, immune cells, fat, and other macromolecules from the intestine via a rhythmical contraction-relaxation process called lymphatic pumping. We have previously demonstrated that mesenteric lymphatic pumping was compromised in the guinea pig model of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced ileitis, corroborating clinical and experimental observations of a dilated and/or obstructed phenotype of these vessels in inflammatory bowel disease. Many mediators released during the inflammatory process have been shown to alter lymphatic contractile activity. Among them, nitric oxide (NO), an inflammatory mediator abundantly released during intestinal inflammation, decreases the frequency of lymphatic contractions through activation of ATP-sensitive potassium (K(ATP)) channels. The objective of this study was to investigate the role of NO and K(ATP) channels in the lymphatic dysfunction observed in the guinea pig model of TNBS-induced ileitis. Using quantitative real-time PCR, we demonstrated that expression of Kir6.1, SUR2B, and inducible NO synthase (iNOS) mRNAs was significantly upregulated in TNBS-treated animals. Pharmacological studies performed on isolated, luminally perfused mesenteric lymphatic vessels showed that the K(ATP) channels blocker glibenclamide, the selective iNOS inhibitor 1400W, and the guanylyl cyclase inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) significantly improved lymphatic pumping in quiescent lymphatic vessels from TNBS-treated animals. Membrane potential measurement with intracellular microelectrodes revealed that vessels from TNBS-treated animals were hyperpolarized compared with their sham counterpart and that the hyperpolarization was significantly attenuated in the presence of glibenclamide and ODQ. Our findings suggest that NO and K(ATP) play a major role in the lymphatic contractile dysfunction that occurred as a consequence of the intestinal inflammation caused by TNBS.

Aim: To determine whether the carbon monoxide (CO)-releasing molecules (CORM)-liberated CO suppress inflammatory responses in the small intestine of septic mice., Methods: The C57BL/6 mice (male, n = 36; weight 20 ± 2 g) were assigned to four groups in three respective experiments. Sepsis in mice was induced by cecal ligation and puncture (CLP) (24 h). Tricarbonyldichlororuthenium (II) dimer (CORM-2) (8 mg/kg, i.v.) was administrated immediately after induction of CLP. The levels of inflammatory cytokines [interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α)] in tissue homogenates were measured with enzyme-linked immunosorbent assay. The levels of malondialdehyde (MDA) in the tissues were determined. The levels of nitric oxide (NO) in tissue homogenate were measured and the expression levels of intercellular adhesion molecule 1 (ICAM-1) and inducible nitric oxide synthase (iNOS) in the small intestine were also assessed. NO and IL-8 levels in the supernatants were determined after the human adenocarcinoma cell line Caco-2 was stimulated by lipopolysaccharide (LPS) (10 g/mL) for 4 h in vitro., Results: At 24 h after CLP, histological analysis showed that the ileum and jejunum from CLP mice induced severe edema and sloughing of the villous tips, as well as infiltration of inflammatory cells into the mucosa. Semi-quantitative analysis of histological samples of ileum and jejunum showed that granulocyte infiltration in the septic mice was significantly increased compared to that in the sham group. Administration of CORM-2 significantly decreased granulocyte infiltration. At 24 h after CLP, the tissue MDA levels in the mid-ileum and mid-jejunum significantly increased compared to the sham animals (103.68 ± 23.88 nmol/mL vs 39.66 ± 8.23 nmol/mL, 89.66 ± 9.98 nmol/mL vs 32.32 ± 7.43 nmol/mL, P < 0.01). In vitro administration of CORM-2, tissue MDA levels were significantly decreased (50.65 ± 11.46 nmol/mL, 59.32 ± 6.62 nmol/mL, P < 0.05). Meanwhile, the tissue IL-1β and TNF-α levels in the mid-ileum significantly increased compared to the sham animals (6.66 ± 1.09 pg/mL vs 1.67 ± 0.45 pg/mL, 19.34 ± 3.99 pg/mL vs 3.98 ± 0.87 pg/mL, P < 0.01). In vitro administration of CORM-2, tissue IL-1β and TNF-α levels were significantly decreased (3.87 ± 1.08 pg/mL, 10.45 ± 2.48 pg/mL, P < 0.05). The levels of NO in mid-ileum and mid-jejunum tissue homogenate were also decreased (14.69 ± 2.45 nmol/mL vs 24.36 ± 2.97 nmol/mL, 18.47 ± 2.47 nmol/mL vs 27.33 ± 3.87 nmol/mL, P < 0.05). The expression of iNOS and ICAM-1 in the mid-ileum of septic mice at 24 h after CLP induction significantly increased compared to the sham animals. In vitro administration of CORM-2, expression of iNOS and ICAM-1 were significantly decreased. In parallel, the levels of NO and IL-8 in the supernatants of Caco-2 stimulated by LPS was markedly decreased in CORM-2-treated Caco-2 cells (2.22 ± 0.12 nmol/mL vs 6.25 ± 1.69 nmol/mL, 24.97 ± 3.01 pg/mL vs 49.45 ± 5.11 pg/mL, P < 0.05)., Conclusion: CORM-released CO attenuates the inflammatory cytokine production (IL-1β and TNF-α), and suppress the oxidative stress in the small intestine during sepsis by interfering with protein expression of ICAM-1 and iNOS.

Endoplasmic reticulum (ER) unfolded protein responses (UPR) are implicated in the pathogenesis of inflammatory bowel disease. Cytotoxic CD8αβ(+) intraepithelial lymphocytes (IEL) contribute to the development of Crohn's disease-like ileitis in TNF(ΔARE/+) mice. In this study, we characterized the role of ER-UPR mechanisms in contributing to the disease-associated phenotype of cytotoxic IEL under conditions of chronic inflammation. Inflamed TNF(ΔARE/+) mice exhibited increased expression of Grp78, ATF6, ATF4, and spliced XBP1 in CD8αβ(+) IEL but not in CD8αα(+) IEL or in lamina propria lymphocytes. Chromatin immunoprecipitation analysis in CD8αβ(+) T cells showed selective recruitment of ER-UPR transducers to the granzyme B gene promoter. Heterozygous Grp78(-/+) mice exhibited an attenuated granzyme B-dependent cytotoxicity of CD8αβ(+) T cells against intestinal epithelial cells, suggesting a critical activity of this ER-associated chaperone in maintaining a cytotoxic T cell phenotype. Granzyme B-deficient CD8αβ(+) T cells showed a defect in IL-2-mediated proliferation in Grp78(-/+) mice. Adoptively transferred Grp78(-/+) CD8αβ(+) T cells had a decreased frequency of accumulation in the intestine of RAG2(-/-) recipient mice. The tissue pathology in TNF(ΔARE/+) × Grp78(-/+) mice was similar to TNF(ΔARE/+) mice, even though the cytotoxic effector functions of CD8αβ(+) T cells were significantly reduced. In conclusion, ER stress-associated UPR mechanisms promote the development and maintenance of the pathogenic cytotoxic CD8αβ(+) IEL phenotype in the mouse model of Crohn's disease-like ileitis.

Oral infection of certain inbred mouse strains with the protozoan Toxoplasma gondii triggers inflammatory pathology resembling lesions seen during human inflammatory bowel disease, in particular Crohn's disease (CD). Damage triggered by the parasite is largely localized to the distal portion of the small intestine, and as such is one of only a few models for ileal inflammation. This is important because ileal involvement is a characteristic of CD in over two-thirds of patients. The disease induced by Toxoplasma is mediated by Th1 cells and the cytokines tumor necrosis factor-α and interferon-γ. Inflammation is dependent upon IL-23, also identified by genome-wide association studies as a risk factor in CD. Development of lesions is concomitant with emergence of E. coli that display enhanced adhesion to the intestinal epithelium and subepithelial translocation. Furthermore, depletion of gut flora renders mice resistant to Toxoplasma-triggered ileitis. Recent findings suggest complex CCR2-dependent interactions between lamina propria T cells and intraepithelial lymphocytes in fueling proinflammatory pathology in the intestine. The advantage of the Toxoplasma model is that disease develops rapidly (within 7-10 days of infection) and can be induced in immunodeficient mice by adoptive transfer of mucosal T cells from infected donors. We propose that Toxoplasma acts as a trigger setting into motion a series of events culminating in loss of tolerance in the intestine and emergence of pathogenic T cell effectors. The Toxoplasma trigger model is providing new leaps in our understanding of immunity in the intestine.

Background and Purpose: Cannabichromene (CBC) is a major non-psychotropic phytocannabinoid that inhibits endocannabinoid inactivation and activates the transient receptor potential ankyrin-1 (TRPA1). Both endocannabinoids and TRPA1 may modulate gastrointestinal motility. Here, we investigated the effect of CBC on mouse intestinal motility in physiological and pathological states., Experimental Approach: Inflammation was induced in the mouse small intestine by croton oil. Endocannabinoid (anandamide and 2-arachidonoyl glycerol), palmitoylethanolamide and oleoylethanolamide levels were measured by liquid chromatography-mass spectrometry; TRPA1 and cannabinoid receptors were analysed by quantitative RT-PCR; upper gastrointestinal transit, colonic propulsion and whole gut transit were evaluated in vivo; contractility was evaluated in vitro by stimulating the isolated ileum, in an organ bath, with ACh or electrical field stimulation (EFS)., Key Results: Croton oil administration was associated with decreased levels of anandamide (but not 2-arachidonoyl glycerol) and palmitoylethanolamide, up-regulation of TRPA1 and CB₁ receptors and down-regulation of CB₂ receptors. Ex vivo CBC did not change endocannabinoid levels, but it altered the mRNA expression of TRPA1 and cannabinoid receptors. In vivo, CBC did not affect motility in control mice, but normalized croton oil-induced hypermotility. In vitro, CBC reduced preferentially EFS- versus ACh-induced contractions. Both in vitro and in vivo, the inhibitory effect of CBC was not modified by cannabinoid or TRPA1 receptor antagonists., Conclusion and Implications: CBC selectively reduces inflammation-induced hypermotility in vivo in a manner that is not dependent on cannabinoid receptors or TRPA1., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

Background & Aims: Tumors that develop in patients with Crohn's disease tend be multifocal, so field cancerization (the replacement of normal cells with nondysplastic but tumorigenic clones) might contribute to intestinal carcinogenesis. We investigated patterns of tumor development from pretumor intestinal cell clones., Methods: We performed genetic analyses of multiple areas of intestine from 10 patients with Crohn's disease and intestinal neoplasia. Two patients had multifocal neoplasia; longitudinal sections were collected from 3 patients. Individual crypts were microdissected and genotyped; clonal dependency analysis was used to determine the order and timing of mutations that led to tumor development., Results: The same mutations in KRAS, CDKN2A(p16), and TP53 that were observed in neoplasias were also present in nontumor, nondysplastic, and dysplastic epithelium. In 2 patients, carcinogenic mutations were detected in nontumor epithelium 4 years before tumors developed. The same mutation (TP53 p.R248W) was detected at multiple sites along the entire length of the colon from 1 patient; it was the apparent founder mutation for synchronous tumors and multiple dysplastic areas. Disruption of TP53, CDKN2A, and KRAS were all seen as possible initial events in tumorigenesis; the sequence of mutations (the tumor development pathway) differed among lesions., Conclusions: Pretumor clones can grow extensively in the intestinal epithelium of patients with Crohn's disease. Segmental resections for neoplasia in patients with Crohn's disease might therefore leave residual pretumor disease, and dysplasia might be an unreliable biomarker for cancer risk. Characterization of the behavior of pretumor clones might be used to predict the development of intestinal neoplasia., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: We previously showed that the probiotic mixture, VSL#3, prevents the onset of ileitis in SAMP/YitFc (SAMP) mice, and this effect was associated with stimulation of epithelial-derived TNF. The aim of this study was to determine the mechanism(s) of VSL#3-mediated protection on epithelial barrier function and to further investigate the "paradoxical" effects of TNF in preventing SAMP ileitis., Methods: Permeability was evaluated in SAMP mice prior to the onset of inflammation and during established disease by measuring transepithelial electrical resistance (TEER) on ex vivo-cultured ilea following exposure to VSL#3 conditioned media (CM), TNF or VSL#3-CM + anti-TNF. Tight junction (TJ) proteins were assessed by qRT-PCR, Western blot, and confocal microscopy, and TNFRI/TNFRII expression measured in freshly isolated intestinal epithelial cells (IEC) from SAMP and control AKR mice., Results: Culture with either VSL#3-CM or TNF resulted in decreased ileal paracellular permeability in pre-inflamed SAMP, but not SAMP with established disease, while addition of anti-TNF abrogated these effects. Modulation of the TJ proteins, claudin-2 and occludin, occurred with a significant decrease in claudin-2 and increase in occludin following stimulation with VSL#3-CM or TNF. TNF protein levels increased in supernatants of SAMP ilea incubated with VSL#3-CM compared to vehicle, while IEC-derived TNFR mRNA expression decreased in young, and was elevated in inflamed, SAMP versus AKR mice., Conclusions: Our data demonstrate that the previously established efficacy of VSL#3 in preventing SAMP ileitis is due to direct innate and homeostatic effects of TNF on the gut epithelium, modulation of the TJ proteins, claudin-2 and occludin, and overall improvement of intestinal permeability.

Background and Aims: Understanding the interplay between genetic susceptibility, the microbiome, the environment and the immune system in Crohn's Disease (CD) is essential for developing optimal therapeutic strategies. We sought to examine the dynamics of the relationship between inflammation, the ileal microbiome, and host genetics in murine models of ileitis., Methods: We induced ileal inflammation of graded severity in C57BL6 mice by gavage with Toxoplasma gondii, Giardia muris, low dose indomethacin (LDI; 0.1 mg/mouse), or high dose indomethacin (HDI; 1 mg/mouse). The composition and spatial distribution of the mucosal microbiome was evaluated by 16S rDNA pyrosequencing and fluorescence in situ hybridization. Mucosal E. coli were enumerated by quantitative PCR, and characterized by phylogroup, genotype and pathotype., Results: Moderate to severe ileitis induced by T. gondii (day 8) and HDI caused a consistent shift from >95% gram + Firmicutes to >95% gram - Proteobacteria. This was accompanied by reduced microbial diversity and mucosal invasion by adherent and invasive E. coli, mirroring the dysbiosis of ileal CD. In contrast, dysbiosis and bacterial invasion did not develop in mice with mild ileitis induced by Giardia muris. Superimposition of genetic susceptibility and T. Gondii infection revealed greatest dysbiosis and bacterial invasion in the CD-susceptible genotype, NOD2(-/-), and reduced dysbiosis in ileitis-resistant CCR2(-/-) mice. Abrogating inflammation with the CD therapeutic anti-TNF-α-mAb tempered dysbiosis and bacterial invasion., Conclusions: Acute ileitis induces dysbiosis and proliferation of mucosally invasive E. coli, irrespective of trigger and genotype. The identification of CCR2 as a target for therapeutic intervention, and discovery that host genotype and therapeutic blockade of inflammation impact the threshold and extent of ileal dysbiosis are of high relevance to developing effective therapies for CD.

Background: Ileal involvement in Crohn's disease (CD) is associated with NOD2 mutations and granulocyte-macrophage colony stimulating factor autoantibodies (GM-CSF Ab), and GM-CSF blockade promotes ileitis in Nod2/Card15-deficient (C15KO) mice. RALDH2-expressing dendritic cells (DC) and IL-4 promote CCR9 imprinting and small bowel homing of T lymphocytes, in conjunction with CCL25 expression by ileal epithelial cells (IEC). We hypothesized that GM-CSF neutralization promotes ileal disease by modulating expression of CCL25 by IEC and CCR9 by T lymphocytes via Nod2-dependent and independent pathways., Methods: CCL25 and CCR9 expression were determined in pediatric CD patients stratified by GM-CSF Ab. Ileitis was induced in C15KO mice via GM-CSF Ab administration followed by nonsteroidal antiinflammatory drug (NSAID) exposure, and expression of CCL25, CCR9, FOXP3, intracellular cytokines, and RALDH2 was determined in IEC and immune cell populations., Results: The frequency of CCL25(+) IEC and CCR9(+) T lymphocytes was increased in CD patients with elevated GM-CSF Ab. In the murine model, GM-CSF blockade alone induced IEC CCL25 expression, and reduced the frequency of mesenteric lymph node (MLN) CD4(+) FOXP3(+) cells, while Card15 deficiency alone enhanced MLN DC RALDH2 expression. Both GM-CSF neutralization and Card15 deficiency were required for downregulation of MLN DC IL-10 expression; under these conditions NSAID exposure led to an expansion of IL-4(+) and IL-17(+) CCR9(+) lymphocytes in the ileum., Conclusions: GM-CSF prevents ileal expansion of CCR9(+) lymphocytes via Nod2-dependent and independent pathways. CCR9 blockade may be beneficial in CD patients with elevated GM-CSF Ab., (Copyright © 2011 Crohn's & Crohn's & Colitis Foundation of America, Inc.)

Background: Vascular endothelial growth factor (VEGF-A) and placental growth factor (PlGF) are major regulators of pathological angiogenesis, which is a prominent feature of both Crohn's disease (CD) and peripheral synovitis in spondyloarthritis., Objective: To investigate the presence of VEGF-A and PlGF in the gut of spondyloarthritis patients and to link this finding with subclinical gut inflammation in these patients., Methods: Intestinal biopsies from healthy controls, CD patients, spondyloarthritis patients with or without subclinical gut inflammation and rheumatoid arthritis (RA) patients were stained for VEGF-A, PlGF, CD31 and vascular cell adhesion molecule 1 (VCAM-1) and digitally analysed., Results: Spondyloarthritis patients with subclinical gut inflammation had markedly increased intestinal VEGF-A expression (p<0.001), mucosal vascularisation (p<0.001) and VCAM-1 expression (p<0.01) compared with healthy controls and RA patients, which, unlike in CD patients, was also seen when the gut inflammation was in a quiescent state. PlGF expression was highly increased in the subclinically inflamed gut of spondyloarthritis (p<0.01 compared with healthy controls), but not at all in CD., Conclusion: A pro-angiogenic intestinal phenotype is observed in spondyloarthritis patients with quiescent chronic gut inflammation. This favours an environment for enhanced trafficking of immune cells in this subpopulation.

The MAS-related gene (Mrg) receptor MrgE has been suggested to be expressed at all tissue levels involved in pain sensation and to influence the expression of another Mrg receptor, MrgF. Given the knowledge on the role of the enteric nervous system (ENS) in sensation, and the plasticity of enteric neurons during intestinal inflammation, it can be hypothesized that MrgE is expressed in enteric neurons, and that MrgE and MrgF change expression in intestinal inflammatory conditions. Therefore, we aimed to reveal the expression details of MrgE and MrgF in the murine ileum in normal and inflamed conditions. Using reverse transcriptase-PCR, quantitative-PCR and immunohistochemistry, we compared the ileum of non-inflamed control mice with that of two models of intestinal inflammation, i.e. intestinal schistosomiasis and chemically induced ileitis. MrgE and MrgF mRNAs were detected in control and inflamed conditions. MrgE and MrgF mRNAs showed a trend towards downregulation during intestinal schistosomiasis and a significant reduction during ileitis. MrgE and MrgF receptors were expressed in distinct enteric neuronal subpopulations, such as the sensory, secretomotor and vasodilator neurons, and in nerve fibres in the tunica muscularis and lamina propria of control and inflamed ileum. Only a minor proportion of enteric neurons co-expressed MrgE and MrgF. The number of enteric neurons expressing MrgE and MrgF receptors was significantly reduced during intestinal schistosomiasis and ileitis. This is the first report on the expression of MrgE and MrgF in the ENS in (patho)physiological conditions. The expression of MrgE and MrgF in enteric neurons was negatively affected by inflammation.

Collagenous ileitis (CI), characterized by subepithelial collagen deposition in the terminal ileum, is an uncommon condition. The few cases reported to date have been associated with collagenous colitis (CC) or lymphocytic colitis. Thirteen cases of CI retrieved over a 9-year period were retrospectively studied. There were 7 female and 6 male patients, with an age range of 39 to 72 years (mean, 64 y). Two groups were identified: (1) CI associated with collagenous or lymphocytic disease elsewhere in the gastrointestinal tract and (2) CI as an isolated process. Diarrhea was the presenting symptom in 11 cases. Most patients had no regular medication use. Subepithelial collagen thickness ranged from 15 to 100 μm (mean, 32 μm) and involved 5% to 80% of the subepithelial region of the submitted biopsies. Six cases had >25 intraepithelial lymphocytes (IELs)/100 epithelial cells, and villous blunting was observed in 11 cases. Chronic inflammation of the lamina propria was present in 9 cases, and focal neutrophil infiltration was identified in 3 cases. In biopsies taken from other sites, 7 of 13 colonic biopsies showed CC, 4 of 9 gastric biopsies showed collagenous gastritis, and 2 of 10 duodenal biopsies were abnormal with collagenous sprue (n=1) and partial villous atrophy and increased IELs (n=1) (both celiac disease related). Resolution of the subepithelial collagen deposition was found in the 1 case in which follow-up of terminal ileal biopsies were taken. There was partial or complete resolution of symptoms in 6 of 9 patients for whom follow-up information was available.

Background: Some literature data suggest that there is a regulatory neuronal circuit between the small and the large bowel. To verify this hypothesis the present study investigated: (i) the distribution, chemical coding and routing of caudal mesenteric ganglion (CaMG) neurons participating in an intestinointestinal reflex pathway involving ileal descending neurons and viscerofugal colonic neurons and (ii) possible changes in the neuroarchitecture of this pathway evoked by chemically induced ileitis in juvenile pigs (n=16)., Methods: Combined retrograde tract tracing and transections of the intermesenteric or caudal colonic nerves were applied. In addition, double immunostainings was used to investigate the chemical coding of retrogradely labeled CaMG neurons and intraganglionic nerve terminals apposed to them, under normal and inflammatory conditions., Key Results: The majority of the ileum-projecting neurons were found in the caudal part of CaMG. Disruption of particular nerve pathways resulted in diminished number of retrogradely labeled neurons, ipsilateral to the side of manipulation. In normal pigs, ileum-projecting CaMG neurons stained for tyrosine hydroxylase, dopamine-β-hydroxylase, neuropeptide Y (NPY), somatostatin and galanin (GAL). The number and chemical coding of the neurons in the inflamed animals were similar to those observed in the normal pigs. However, in the inflamed pigs, the number of NPY-, GAL- or substance P-positive nerve terminals supplying retrogradely labeled neurons was increased., Conclusions & Inferences: The present results suggest that inflammatory processes of the porcine ileum are able to induce changes in the intraganglionic architecture of a sympathetic ganglion located at discrete distance from the affected bowel segment., (© 2011 Blackwell Publishing Ltd.)

Background & Aims: A balance between effector and regulatory T-cell (Treg) responses is required to maintain intestinal homeostasis. To regulate immunity, T cells migrate to the intestine using a combination of adhesion molecules and chemokine receptors. However, it is not known whether the migration pathways of effector cells and Tregs are distinct or shared. We sought to determine whether interaction between the chemokine receptor 9 (CCR9) and its ligand, chemokine ligand 25 (CCL25), allows effectors or Tregs to localize to chronically inflamed small intestine., Methods: By using a mouse model that develops Crohn's-like ileitis (tumor necrosis factor Δadenosine uracyl-rich element [TNFΔARE] mice) we examined the role of CCL25-CCR9 interactions for effector and Treg traffic using flow cytometry, quantitative reverse-transcription polymerase chain reaction, immunohistochemistry, immunoneutralization, and proliferation analyses., Results: In TNFΔARE mice, expression of CCL25 and the frequency of CCR9-expressing lymphocytes increased during late-stage disease. In the absence of CCR9, TNFΔARE mice developed exacerbated disease, compared with their CCR9-sufficient counterparts, which coincided with a deficiency of CD4(+)/CD25(+)/forkhead box P3(+) and CD8(+)/CD103(+) Tregs within the intestinal lamina propria and mesenteric lymph nodes. Furthermore, the CD8(+)/CCR9(+) subset decreased the proliferation of CD4(+) T cells in vitro. Administration of a monoclonal antibody against CCR9 to TNFΔARE mice exacerbated ileitis in vivo, confirming the regulatory role of CD8(+)/CCR9(+) cells., Conclusions: Signaling of the chemokine CCL25 through its receptor CCR9 induces Tregs to migrate to the intestine. These findings raise concerns about the development of reagents to disrupt this pathway for the treatment of patients with Crohn's disease., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: Inflammatory bowel disease (IBD) of pediatric and adult onset differs in several aspects although little knowledge exists about pathogenic disparity. Regulatory T cells (Tregs) characterized as CD4+CD25+Foxp3+ are modulators of gut homeostasis, but their role in human IBD remains unclear., Objective: To evaluate the mucosal distribution of Foxp3+ and CD25+ cells in untreated pediatric IBD patients at the time of diagnosis., Material and Methods: Untreated pediatric (n = 14) and adult (n = 12) Crohn's disease (CD) patients were prospectively included together with age-matched symptomatic controls. Colonic and ileal mucosal biopsies collected at diagnosis were studied by immunohistochemistry for enumeration of T cells and for mucosal expression of Foxp3 and CD25. Multicolor immunofluorescence staining was performed in situ to phenotype Foxp3+ cells as Tregs and characterize the CD25+ cells., Results: The density of mucosal T cells displayed only small variations, while that of Foxp3+ cells and CD25+ cells was increased in CD patients. Multicolor immunofluorescence showed that most CD25+ cells were macrophages. Interestingly, in the ileum of pediatric CD patients the density of Foxp3+ cells was significantly higher than in adult CD patients. Co-expression of Foxp3 and CD25, as well as Foxp3 and CTLA-4, indicated that the Foxp3+ cells were Tregs., Conclusion: Mucosal numbers of Foxp3(+) Tregs and activated (CD25+) macrophages are elevated in both pediatric and adult ileal CD. The greater increase of ileal Foxp3+ Tregs in pediatric CD than in adult CD might contribute to the relatively less frequent phenotype of isolated ileal enteritis in CD children.