Inactive CD Research Articles

Genome-wide DNA Methylome and Transcriptome Profiling Reveals Key Genes Involved in the Dysregulation of Adipose Stem Cells in Crohn's Disease.

Crohn's disease [CD] is characterised by the expansion of mesenteric adipose tissue [MAT], named creeping fat [CF], which seems to be directly related to disease activity. Adipose-stem cells [ASCs] isolated from the CF of patients with CD are extremely pro-inflammatory, which persists during disease remission. We hypothesised that the dysfunctional ASCs in CD accumulate epigenetic modifications triggered by the inflammatory environment, that could serve as molecular markers. Genome-wide DNA methylome and transcriptome profiling were performed in ASCs isolated from MAT biopsies of patients with active and inactive disease and from non-Crohn's disease patients [non-CD]. A validation cohort was used to test the main candidate genes via quantitative polymerase chain reaction in other fat depots and immune cells. We found differences in DNA methylation and gene expression between ASCs isolated from patients with CD and from non-CD subjects, but we found no differences related to disease activity. Pathway enrichment analysis revealed that oxidative stress and immune response were significantly enriched in active CD, and integration analysis identified MAB21L2, a cell fate-determining gene, as the most affected gene in CD. Validation analysis confirmed the elevated gene expression of MAB21L2 in MAT and in adipose tissue macrophages in active CD. We also found a strong association between expression of the calcium channel subunit gene CACNA1H and disease remission, as CACNA1H expression was higher in ASCs and MAT from patients with inactive CD, and correlates negatively with C-reactive protein in peripheral blood mononuclear cells. We identified a potential gene signature of CD in ASCs obtained from MAT. Integration analysis highlighted two novel genes demonstrating a negative correlation between promoter DNA methylation and transcription: one linked to ASCs in CD [MAB21L2] and the other [CACNA1H] related to disease remission.

NON-INVASIVE DETERMINATION OF DISEASE ACTIVITY IN CROHN’S DISEASE BY SERUM LUMINEX PROFILING

Abstract BACKGROUND & AIMS Crohn’s disease (CD) is a chronic inflammatory disease with a complex pathogenesis; treatment and management largely depend on disease activity assessment, which can be challenging and invasive. The aim of this study was to assess the ability of multiplex Luminex technology to identify serum cytokine and/or chemokine markers of disease activity. METHODS Serum samples were prospectively obtained from 103 CD and 40 healthy controls undergoing colonoscopy. CD activity was determined by histologic assessment of colonoscopic biopsies. The Simple Endoscopic Score for Crohn’s Disease (SES) was available for 94 CD patients. We determined if the SES score was predictive of histologic activity (normal, quiescent, mild, moderate, or severe). Patient demographics and clinical history were obtained by chart review. Serum cytokines and chemokines were measured by a 47-plex Luminex assay. Analytes that had more than 70% of values below the lower limit of detection were excluded (GMCSF, IL3, IL17A, IL22, and TNFβ). Analyte profiles in controls v. CD patients were compared using the Mann-Whitney U test with Benjamini-Hochberg (BH) adjustment given multiple comparisons, and by disease activity (control v. inactive v. active) using the Kruskal-Wallis test, with post-hoc Dunn’s test with BH adjustment. RESULTS 34 CD patients were inactive (normal, quiescent) and 69 CD patients had active disease (mild, moderate, severe) on histology. Use of steroids, immunomodulators, and biologics did not differ significantly between the active and inactive CD groups. There was a strong positive correlation between SES score and histologic disease severity (Spearman’s r = 0.58, p<0.001). After BH adjustment, 16 analytes were significantly increased in CD v. controls: Fractalkine, GROα, IFNα2, IL1β, IL4, IL6, IL10, IL13, IL25, IL17F, IL18, MCP3, MCSF, PDGFAA, PDGFABBB, and TGFα. As shown in Table 1, when categorized by disease activity, 4 analytes were significantly increased and one analyte was significantly decreased in inactive disease v. control; 18 analytes were significantly increased in active v. control; and one analyte, CXCL9, which attracts effector T cells to sites of inflammation, was significantly increased in active v. inactive disease. CONCLUSIONS In CD patients, 16 of 42 serum cytokines and chemokines detectable by Luminex assay were increased v. healthy controls. When stratified by histologic disease activity, multiple analytes were found to be increased in inactive or active CD v. controls. Thus, CD is associated with alterations in serum cytokines and chemokines, which was much more frequent in CD patients with histologically active disease. Serum cytokine/chemokine profiling may be a non-invasive strategy to reduce the need for frequent colonoscopy in CD to assess disease activity. Table 1 Serum analyte profiles compared by disease activity (control v. inactive v. active). Data are shown in pg/mL as mean ± SD. p-values are calculated using Kruskal-Wallis test with post-hoc Dunn’s test with BH adjustment for multiple comparisons where 0.05 was considered significant. *p

P074 Inflammatory Environment-Induced Transcriptomic Alterations in Crohn's Disease Adipose Stem Cells

Abstract Background Crohn’s disease (CD) is characterised by expansion of mesenteric adipose tissue, called creeping fat, which seems to be directly related to disease activity. Adipose stem cells (ASCs) isolated from the creeping fat of CD patients exhibit dysfunction, featuring impaired adipogenesis and an intensely pro-inflammatory phenotype. This study aims to explore the transcriptome of ASCs isolated from active and inactive CD subjects in comparison with healthy subjects, seeking key markers of this dysregulation. Methods Patients were recruited at Hospital Joan XXIII of Tarragona and Hospital Vall d’Hebron of Barcelona in accordance with the principles of the Helsinki Declaration. Transcriptome profiling was performed in ASCs isolated from adipose-tissue biopsies of visceral origin: CF and hMES in Crohn subjects (n=7, each) and hMES in inactive CD patients (n=7) and healthy control subjects (n=7). Groups were matched by age, gender, and BMI. Finally, we examined the pathways involving the differentially expressed gene (DEGs) in ASCs isolated from patients with CD with different clinical activity by gene set enrichment analysis (GSEA). Results Patients with CD across different clinical activity stages exhibited similar transcriptome patterns, indicating persistent ASC dysregulation even during remission state (Fig 1A). ASCs isolated from both creeping fat and mesenteric adipose tissue distant from intestinal damage displayed similar dysregulation, implying a global dysregulation of all mesenteric fat in CD. Specifically, transcriptomic analysis identified significant dysregulation of the NK2 Homeobox 3 (NKX2-3) gene in ASCs from active CD patients. Although higher NKX2-3 expression has been associated with B cells and intestinal tissues in CD1-3, our study is the first to link elevated expression of this gene also to creeping fat. Pathway enrichment analysis revealed significant enrichment in oxidative stress and immune response pathways in active CD (Fig 1B). Furthermore, our findings indicated also an elevated NKX2-3 expression in ASCs of inactive CD compared to controls, with oxidative stress pathway up-regulation (Fig 1C). Notably, no significant DEGs were found between ASCs from active and inactive CD groups (Fig 1D), suggesting a similar profile in both states. Conclusion Our study reveals profound ASC dysregulation in CD, in both active and inactive phases. Elevated NKX2-3 expression, previously associated with increased risk of complications in IBD, is now demonstrated in the mesenteric adipose tissue, persisting even in inactive CD patients. Ref: 1. Yamazaki K, et al Gut. 2009 Feb;58(2):228-32. 2. Yu W, et al. J Crohn’s Colitis. 2009 Sep;3(3):189-95. 3. Hong SN et al. Gut. 2016 May;65(5):788-96.

Altered resting-state brain functional activities and networks in Crohn’s disease: a systematic review

BackgroundCrohn’s disease (CD) is a non-specific chronic inflammatory disease of the gastrointestinal tract and is a phenotype of inflammatory bowel disease (IBD). The current study sought to compile the resting-state functional differences in the brain between CD patients and healthy controls.MethodsThe online databases PubMed, Web of Science Core, and EMBASE were used to find the published neuroimage studies. The search period was from the beginning through December 15, 2023. The predetermined inclusion and exclusion criteria allowed for the identification of the studies. The studies were assembled by two impartial reviewers, who also assessed their quality and bias.ResultsThis review comprised 16 resting-state fMRI studies in total. The included studies generally had modest levels of bias. According to the research, emotional processing and pain processing were largely linked to increased or decreased brain activity in patients with CD. The DMN, CEN, and limbic systems may have abnormalities in patients with CD, according to research on brain networks. Several brain regions showed functional changes in the active CD group compared to the inactive CD group and the healthy control group, respectively. The abnormalities in brain areas were linked to changes in mood fluctuations (anxiety, melancholy) in patients with CD.ConclusionFunctional neuroimaging helps provide a better understanding of the underlying neuropathological processes in patients with CD. In this review, we summarize as follows: First, these findings indicate alterations in brain function in patients with CD, specifically affecting brain regions associated with pain, emotion, cognition, and visceral sensation; second, disease activity may have an impact on brain functions in patients with CD; and third, psychological factors may be associated with altered brain functions in patients with CD.

A large and overlooked Cd source in karst areas: The migration and origin of Cd during soil formation and erosion

There is increasing concern regarding the substantial enrichment of Cd during the weathering of carbonate rocks and subsequent risks posed to the ecological environment and food security in karst areas. However, the incomplete understanding of Cd migration mechanisms and material sources restricts soil pollution control and land management. This study investigated the migration regulation of Cd during soil formation and erosion in karst areas. The results demonstrate that soil Cd concentration and bioavailability are both significantly higher in alluvium compared with those in eluvium. This increase is primarily attributed to the chemical migration of active Cd, rather than the mechanical migration of inactive Cd. Additionally, we analyzed the Cd isotopic characteristics of rock and soil samples. The isotopic composition of the alluvial soil was −0.18 ‰ ± 0.01 ‰, which is obviously heavier than the δ114/110Cd value of the eluvium (−0.78 ‰ ± 0.06 ‰). The Cd isotopic fingerprint revealed that the active Cd in the alluvium of the study profile was probably derived from the corrosion of carbonate rocks rather than by eluviation of the eluvium. Moreover, Cd tends to occur in soluble mineral components of carbonate rocks rather than in residues, which suggests that carbonate weathering has a great potential to release active Cd into the environment. It is estimated that the Cd release flux caused by carbonate weathering is 5.28 g Cd km−2 yr−1, accounting for 9.30 % of the anthropogenic Cd flux. Therefore, the corrosion of carbonate rocks is a substantial natural Cd source and poses significant potential risks to the ecological environment. It is suggested that the contribution of Cd from natural sources should be considered during ecological risk assessments and studies of the global Cd geochemical cycle.

Subcutaneous injection of infliximab CT-P13 results in stable drug levels within 14-day treatment cycle in Crohn's disease.

CD patients in clinico-biological remission under maintenance therapy with intravenous (iv) IFX/CT-P13 were switched to CT-P13 sc 8weeks (W) after the last infusion. They were treated with CT-P13 sc, 120mg every 2W. Assessments were performed from 8W after starting CT-P13 sc and patients had to attend 6 visits on 2 consecutive cycles of treatment (cycles A and B). Visits were scheduled on days 4-6 (visit 1), days 7-9 (visit 2) and day 14 (visit 3) of each cycle, where days 1 and 14 were the days of sc injection of CT-P13. At each visit, peripheral blood was collected to measure serum IFX levels and anti-drug antibodies. Twenty patients underwent 120 evaluations. Large intra-individual variations of serum drug levels of IFX were observed. When pooling the 120 evaluations, the mean drug level was 11.3±4.9μg/ml, and the median drug level was 10.9μg/ml (IQR 7.5-15.5). During each cycle, the median drug levels were similar between visits 1 and 2 as well as between visits 1 and 3 and between visits 2 and 3. In cycle A, median drug levels were 11.1μg/ml (7.8-14.5), 12.0μg/ml (7.2-16.1) and 11.0μg/ml (7.5-15.1) at V1, V2 and V3, respectively. Similar results were obtained in cycle B, where median drug levels were 11.6μg/ml (7.9-14.9), 11.4μg/ml (8.1-15.2) and 10.9μg/ml (7.9-15.6) at V1, V2 and V3, respectively. In univariate analysis, we failed to identify factors predictive of low drug levels. IFX drug levels are quite stable within 14-day treatment cycle, without trough levels in CD patients in remission during the maintenance therapy with CT-P13 sc. In patients with inactive CD under maintenance therapy with CT-P13 sc, therapeutic drug monitoring of IFX can be performed at any time between two CT-P13 sc injections.

P042 Succinate promotes the conversion from white to beige adipose tissue in Crohn’s Disease

Abstract Background Crohn’s disease (CD) is characterized by severe transmural inflammation with subsequent destruction of the intestinal barrier. Bacterial infiltration across this leaky gut facilitates access to the mesenteric fat and the development of a subsequent inflammatory reaction in the surrounding adipose tissue named creeping fat (CF). Dysbiosis in CD patients has been associated with an increase in succinate-producing bacteria and a decrease in succinate-consuming bacteria. In fact, elevated succinate levels have been found in the intestine and feces of CD patients. Furthermore, succinate is an inductor of beige adipogenesis in white adipose tissue progenitors. Methods Visceral and subcutaneous adipose tissue biopsies (VAT and SAT), serum, and plasma were obtained from a cohort of active CD (n=20), inactive CD (n=12), and control group (n=15). From adipose tissue biopsies, adipose-derived stem cells (ASCs) and adipose tissue macrophages (ATMs) were isolated. Different experiments were performed such as protein uncoupling protein 1 (UCP1)-immunohistochemistry and immunofluorescence, gene expression studies, adipose tissue explants, proteomic analysis of ASCs, succinate determinations, bio-impedancemetry, and infrared photographs. Results Succinate treatment increased markers of beige adipose tissue in ASCs from controls and inactive CD patients (Fig. A). Surprisingly, the markers of beige adipose tissue were already high in ASCs isolated from active CD, due to being exposed to an environment with high levels of succinate in vivo. Proteomic analysis of isolated CD-ASCs revealed that these cells secrete beige adipose tissue-related proteins compared to Control-ASCs (Fig. B). Furthermore, bacterial translocation increases the conversion of pre-adipocytes from white to beige adipose tissue in CD individuals (Fig. C). Immunohistochemical study of UCP-1, confirms increased staining in adipose samples from CD patients from both depots (SAT and VAT) (Fig. D). Altogether, adipose tissue depots from CD patients had a conversion from white to beige adipose tissue with a high protein UCP1-gene expression. Thermic images show an increase of temperature in the intestinal area of active CD patients that is not observed in inactive CD patients or the control group. Conclusion Understanding the role of beige adipose tissue in CD could help develop new therapeutic or diagnostic strategies in these patients.

P083 An integrative analysis of DNA methylation and RNA-Seq data in human adipose-stem cells of Crohn’s Disease patients with different clinical activity

Abstract Background Crohn’s disease (CD) is characterized by the expansion of mesenteric fat attached to the inflamed segments of the intestine, also known as “creeping fat” which seems to be directly related to disease activity. Our group revealed that adipose-stem cells (hASCs) isolated from the creeping fat of CD subjects are dysfunctional (showing a high inflammatory profile, high invasive and phagocytic capacities, and worse immunosuppressive properties); and this dysfunction is maintained even in hASCs isolated from CD subjects in remission of the disease.1 Methods 1) Culture and characterization of hASCs isolated from adipose tissue biopsies of active CD, inactive CD, and healthy subjects (n=25). Visceral origin: creeping fat in active CD subjects and mesenteric fat in CD subjects in remission and healthy subjects. 2) Comparative DNA methylome analysis in hASCs isolated from the included population (Infinium Human-Methylation EPIC (850K) BeadChip; Illumina, Inc). 3) Transcriptomic study in hASCs isolated from the included population (Illumina HiSeq 2500 system). 4) Bioinformatic analysis & data integration. Results Methylation and transcriptomic studies revealed a multi-omic profile of hASCs isolated from CD subjects compared to control individuals (Heatmaps Fig 1A). Indeed, the integration of genome-wide methylation changes and gene expression differences revealed the main candidate’s genes (Figure 1B). The most significant upregulated gene in CD was Mab-21 like 2 (MAB21L2) (Fig 1B-C). This gene encodes a downstream target of transforming growth factor-beta signaling. MAB21L2 was validated in an independent cohort of hASC isolated from control, active, and inactive CD subjects (n=7/each group) using qPCR. In agreement, MAB21L2 gene expression was significantly higher in hASCs isolated from the active and inactive group than in the control group (Fig 1D). Another interesting candidate gene found was tyrosine kinase 2 (TYK2) which is significantly upregulated in active CD subjects. This gene promulgates cytokine signals by phosphorylating receptor subunits. It is also a component of both the type I and III interferon signaling pathways.2 Finally, another gene upregulated in remission of the disease is the calcium voltage-gated channel subunit alpha 1H (CACNA1H), which has been involved in low-grade inflammation and has been proposed as an important player in IBD during the remission phase.3 Conclusion Integration of data revealed genes involved in the dysregulation of hASCs associated with CD and genes related to disease remission. Reference 1. Serena C Stem Cell Rep 2017 9(4):1109–23; 2De Vries LCS JCC 2021 617-30; 3Picard E Br J Pharmacol 2019;176:950–63

Probing predilection to Crohn's disease and Crohn's disease flares: A crowd-sourced bioinformatics approach

BackgroundCrohn's Disease (CD) is an inflammatory disease of the gastrointestinal tract that affects millions of patients. While great strides have been made in treatment, namely in biologic therapy such as anti-TNF drugs, CD remains a significant health burden. MethodWe conducted two meta-analyses using our STARGEO platform to tag samples from Gene Expression Omnibus. One analysis compares inactive colonic biopsies from CD patients to colonic biopsies from healthy patients as a control and the other compares colonic biopsies from active CD lesions to inactive lesions. Separate tags were created to tag colonic samples from inflamed biopsies (total of 65 samples) and quiescent tissue in CD patients (total of 39 samples), and healthy tissue from non-CD patients (total of 30 samples). Results from the two meta-analyses were analyzed using Ingenuity Pathway Analysis. ResultsFor the inactive CD vs healthy tissue analysis, we noted FXR/RXR and LXR/RXR activation, superpathway of citrulline metabolism, and atherosclerosis signaling as top canonical pathways. The top upstream regulators include genes implicated in innate immunity, such as TLR3 and HNRNPA2B1, and sterol regulation through SREBF2. In addition, the sterol regulator SREBF2, lipid metabolism was the top disease network identified in IPA (Fig. 1). Top upregulated genes hold implications in innate immunity (DUOX2, REG1A/1B/3A) and cellular transport and absorption (ABCG5, NPC1L1, FOLH1, and SLC6A14). Top downregulated genes largely held roles in cell adhesion and integrity, including claudin 8, PAQR5, and PRKACB.For the active vs inactive CD analysis, we found immune cell adhesion and diapedesis, hepatic fibrosis/hepatic stellate cell activation, LPS/IL-1 inhibition of RXR function, and atherosclerosis as top canonical pathways. Top upstream regulators included inflammatory mediators LPS, TNF, IL1B, and TGFB1. Top upregulated genes function in the immune response such as IL6, CXCL1, CXCR2, MMP1/7/12, and PTGS2. Downregulated genes dealt with cellular metabolism and transport such as CPO, RBP2, G6PC, PCK1, GSTA1, and MEP1B. ConclusionOur results build off established and recently described research in the field of CD. We demonstrate the use of our user-friendly platform, STARGEO, in investigating disease and finding therapeutic avenues.

Endotrophin and C6Ma3, serological biomarkers of type VI collagen remodelling, reflect endoscopic and clinical disease activity in IBD

In inflammatory bowel disease (IBD), the chronic inflammation deeply affects the intestinal extracellular matrix. The aim of this study was to investigate if remodeling of the intestinal basement membrane type VI collagen was associated with pathophysiological changes in Crohn’s disease (CD) and ulcerative colitis (UC). Serum from IBD patients (CD: n = 65; UC: n = 107; irritable bowel syndrome: n = 18; healthy subjects: n = 20) was investigated in this study. The serological biomarkers C6Ma3 (a matrix metalloproteinase (MMP) generated fragment of the type VI collagen α3 chain) and PRO-C6, also called endotrophin (the C-terminus of the released C5 domain of the type VI collagen α3 chain) were measured by ELISAs. Serum C6Ma3 was increased in CD patients with moderate to severe and mild endoscopically active disease compared to endoscopic remission (p = 0.002, p = 0.0048), respectively, and could distinguish endoscopically active disease from remission with an AUC of 1.0 (sensitivity: 100%, specificity: 100%) (p < 0.0001), which was superior to CRP. C6Ma3 was increased in CD patients with moderate to severe clinical disease compared to mild and remission (p = 0.04; p = 0.009). Serum PRO-C6, endotrophin, was increased in CD patients in clinically remission compared to mild disease (p = 0.04) and moderate to severe disease (p = 0.065). In UC, fecal calprotectin was the only marker that alone could distinguish both clinical and endoscopic active and inactive disease. Type VI collagen degradation of the α3 chain mediated by MMPs was increased in CD patients with endoscopically active disease, measured by the serological biomarker C6Ma3, which was able to distinguish endoscopically active from inactive CD.

P001 Succinate, a gut microbiota-derived metabolite, modulates the inflammatory status of the creeping fat in Crohn’s disease

Abstract Background Crohn’s disease [CD] is characterized by severe transmural inflammation with subsequent destruction of the intestinal barrier. Recent works suggest that bacterial infiltration across this leaky gut facilitates access to the mesenteric fat and the development of a subsequent inflammatory reaction in the surrounding adipose tissue named creeping fat [CF]. Dysbiosis in CD patients has been associated with an increase in succinate-producing bacteria and a decrease in succinate-consuming bacteria. In fact, elevated succinate levels have been found in the intestinal and faeces of CD patients. Succinate has been classically considered as a marker of hypoxia and tissue damage, assisting as a pro-inflammatory signal that triggers immune activation. However, recent observations support potential additional functions of succinate. We and others have shown that succinate plays a key role in fine-tuning of the inflammatory response, acting both as an alarmin and resolving molecule. Our hypothesis is that succinate, a microbiota-derived metabolite, is a new determinant of adipose tissue dysfunction in CD. Methods A well-characterized cohort was used to obtained mesenteric adipose tissue biopsies including a) 10 subjects with active CD that require surgery for their underlying pathology b) 10 subjects with inactive CD and c) 10 healthy controls undergoing surgery for non-acute process (herniorrhaphy, cholecystectomy for lithiasis, etc.). The groups were comparable in age, sex, and body mass index. We studied the effect of exogenous succinate in adipose tissue explants, adipose-stem cells (ASC), and adipose tissue macrophages (ATM) isolated from adipose tissue biopsies of CD patients with different clinical activity. Circulating succinate levels and inflammatory variables including hs-CRP and faecal calprotectin were also measured. Results We observed an increased expression of SUCNR1 in CF, including ASCs and ATMs, mainly in patients suffering from an active disease (figure 1A). Furthermore, succinate appears to elicit a different response in adipose tissue from CD patients, when activity status is considered. Thus, our results indicate that in an inflammatory local and systemic environment, such as occurs in CD active patients, succinate triggers a pro-inflammatory response in VAT depot, while when subjects are in a remission period, the response of VAT explants to succinate is similar to than the observed in healthy subjects, promoting an anti-inflammatory expression profile (Figure 1B). Interestingly, we found elevated circulating succinate levels in active CD patients but those decrease drastically in patients in remission of the disease (Figure 1C). Conclusion Succinate has a relevant role in adipose tissue dysfunction in CD.

Corticosteroid enhances epithelial barrier function in intestinal organoids derived from patients with Crohn\u2019s disease

Corticosteroids (CS), first-line therapeutics for Crohn’s disease (CD) with moderate or severe disease activity, were found to restore intestinal permeability in CD patients, whereas the underlying molecular events are still largely unknown. This study aimed to investigate the effect and mechanisms of CS prednisolone on epithelial barrier using CD patient-derived intestinal organoids. 3D intestinal organoids were generated from colon biopsies of inactive CD patients. To mimic the inflammatory microenvironment, a mixture of cytokines containing TNF-α, IFN-γ, and IL-1β were added to the organoid culture with or without pre-incubation of prednisolone or mifepristone. Epithelial permeability of the organoids was assessed by FITC-D4 flux from the basal to luminal compartment using confocal microscopy. Expression of junctional components were analyzed by qRT-PCR, immunofluorescence staining, and western blot. Activity of signaling pathways were analyzed using western blot. Exposure of the cytokines significantly disrupted epithelial barrier of the intestinal organoids, which was partially restored by prednisolone. On the molecular level, the cytokine mixture resulted in a significant reduction in E-cadherin and ILDR-1, an increase in CLDN-2, MLCK, and STAT1 phosphorylation, whereas prednisolone ameliorated the abovementioned effects induced by the cytokine mixture. This study demonstrates that prednisolone confers a direct effect in tightening the epithelial barrier, identifies novel junctional targets regulated by prednisolone, and underscores intestinal barrier restoration as a potential mechanism that contributes to the clinical efficacy of prednisolone in CD patients.Key messagesPrednisolone confers a direct preventive effect against cytokine-induced barrier dysfunction.Prednisolone regulates the expression of CLDN-2, E-cadherin, and ILDR-1.The effect of prednisolone is GR-, MLCK-, and STAT1-dependent.

In search for interplay between stool microRNAs, microbiota and short chain fatty acids in Crohn\u2019s disease - a preliminary study

BackgroundInflammatory bowel diseases are classic polygenic disorders, with genetic loads that reflect immunopathological processes in response to the intestinal microbiota. Herein we performed the multiomics analysis by combining the large scale surveys of gut bacterial community, stool microRNA (miRNA) and short chain fatty acid (SCFA) signatures to correlate their association with the activity of Crohn’s disease (CD).MethodsDNA, miRNA, and metabolites were extracted from stool samples of 15 CD patients, eight with active disease and seven in remission, and nine healthy individuals. Microbial, miRNA and SCFA profiles were assessed using datasets from 16S rRNA sequencing, Nanostring miRNA and GC-MS targeted analysis, respectively.ResultsPairwise comparisons showed that 9 and 23 taxa differed between controls and CD patients with active and inactive disease, respectively. Six taxa were common to both comparisons, whereas four taxa differed in CD patients. α-Diversity was lower in both CD groups than in controls. The levels of 13 miRNAs differed (p-value < 0.05; FC > 1.5) in CD patients and controls before FDR correction and 4 after. Of six SCFAs, the levels of two differed significantly (p-value < 0.05, FC > 1.5) in CD patients and controls, and the levels of four differed in patients with active and inactive CD. PLS-DA revealed models with smallest error rate for controls in bacterial component and inactive disease in metabolites.ConclusionA complex interrelationship may exist between gut dysbiosis, miRNA profiling and SCFA level in response to intestinal inflammation.

Adipose stem cells from patients with Crohn\u2019s disease show a distinctive DNA methylation pattern

BackgroundCrohn’s disease (CD) is characterized by persistent inflammation and ulceration of the small or large bowel, and expansion of mesenteric adipose tissue, termed creeping fat (CF). We previously demonstrated that human adipose-derived stem cells (hASCs) from CF of patients with CD exhibit dysfunctional phenotypes, including a pro-inflammatory profile, high phagocytic capacity, and weak immunosuppressive properties. Importantly, these phenotypes persist in patients in remission and are found in all adipose depots explored including subcutaneous fat. We hypothesized that changes in hASCs are a consequence of epigenetic modifications.MethodsWe applied epigenome-wide profiling with a methylation array (Illumina EPIC/850k array) and gene expression analysis to explore the impact of CD on the methylation signature of hASCs isolated from the subcutaneous fat of patients with CD and healthy controls (n = 7 and 5, respectively; cohort I). Differentially methylated positions (p value cutoff < 1 × 10−4 and ten or more DMPs per gene) and regions (inclusion threshold 0.2, p value cutoff < 1 × 10−2 and more than 2 DMRs per gene) were identified using dmpfinder and Bumphunter (minfi), respectively. Changes in the expression of differentially methylated genes in hASCs were validated in a second cohort (n = 10/10 inactive and active CD and 10 controls; including patients from cohort I) and also in peripheral blood mononuclear cells (PBMCs) of patients with active/inactive CD and of healthy controls (cohort III; n = 30 independent subjects).ResultsWe found a distinct DNA methylation landscape in hASCs from patients with CD, leading to changes in the expression of differentially methylated genes involved in immune response, metabolic, cell differentiation, and development processes. Notably, the expression of several of these genes in hASCs and PBMCs such as tumor necrosis factor alpha (TNFA) and PR domain zinc finger protein 16 (PRDM16) were not restored to normal (healthy) levels after disease remission.ConclusionshASCs of patients with CD exhibit a unique DNA methylation and gene expression profile, but the expression of several genes are only partially restored in patients with inactive CD, both in hASCs and PBMCs. Understanding how CD shapes the functionality of hASCs is critical for investigating the complex pathophysiology of this disease, as well as for the success of cell-based therapies.Graphical abstractHuman adipose-stem cells isolated from subcutaneous fat of patients with Crohn’s disease exhibit an altered DNA methylation pattern and gene expression profile compared with those isolated from healthy individuals, with immune system, cell differentiation, metabolic and development processes identified as the main pathways affected. Interestingly, the gene expression of several genes involved in these pathways is only partially restored to control levels in patients with inactive Crohn’s disease, both in human adipose-stem cells and peripheral blood mononuclear cells. Understanding how Crohn’s disease shapes the functionality of human adipose-stem cells is critical for investigating the complex pathophysiology of this disease, as well as for the success of cell-based therapies.

Salinity influences Cd accumulation and distribution characteristics in two contrasting halophytes, Suaeda glauca and Limonium aureum.

The potential for the phytoremediation of halophytes has been widely recognized. However, the effects of salt on Cd accumulation characteristics in different halophytic species, which may also be related to their salt tolerance, are still unclear. This study investigated the effects of salinity on Cd accumulation and distribution in two distinct halophytes, Suaeda glauca (euhalophyte) and Limonium aureum (recretohalophyte). Seedlings of the two species were treated with 0, 3, and 6mgkg-1 soil Cd in combination with or without 0.3% NaCl in a pot experiment. The amount of Cd within the rhizosphere and plant tissues, plant biomass, and the subcellular distribution and chemical forms of Cd were examined. Results showed that the addition of NaCl significantly increased Cd bioavailability at high Cd levels due to the rhizosphere acidification effect. Meanwhile, salinity differently impacted plant biomass allocation, and enhanced Cd uptake and translocation in both studied halophytes. Excess Cd was excreted from the leaf surface, possibly by salt glands of L. aureum, with the salinity facilitating this process. Majority of the Cd was found within the cell walls and vacuolar compartments of two species. However, S. glauca plants had higher proportions of inactive Cd (extracted by 2% HAc and 0.6M HCl) and lower proportions of active Cd (extracted by 80% ethanol and water), as opposed to L. aureum, which would better inform S. glauca's higher Cd accumulation. Based on these results, S. glauca seems more applicable for phytomanagement of Cd-contaminated saline soils due to its higher capacity for Cd enrichment and tolerance amplified by NaCl.

DOP84 Crohn’s disease modifies the DNA methylome of human adipose-stem cells, which is only partially re-established in remission

Abstract Background Crohn’s disease (CD) is characterised by the expansion of mesenteric adipose tissue, termed creeping fat (CF). We previously demonstrated that human adipose-tissue stem cells (hASCs) from CF exhibit a dysfunctional phenotype, including a pro-inflammatory profile, high phagocytic capacity and weak immunosuppressive properties. Importantly, these phenotypes persist in patients in remission, and are found in all adipose depots explored including subcutaneous fat. We hypothesised that this is a consequence of epigenetic modifications. Methods Epigenome-wide association studies using the 850k EPIC Illumina array were used to explore the impact of CD on the methylation signature of hASCs isolated from the subcutaneous fat (Figure 1). Changes in the expression of differentially methylated candidate genes were validated in hASCs of patients with active/inactive CD and healthy controls (Figure 1). Conclusion hASCs of patients with CD exhibit an alter DNA-methylation and gene expression profile. Remarkably, the expression of several genes is only partially restored in patients with inactive CD. Understanding how CD shapes the functionality of hASCs is critical for investigating the complex pathophysiology of this disease, as well as for the success of cell-based therapies.

Faecal Micro-RNAs in Inflammatory Bowel Diseases.

Faecal biomarkers are used as indicators of disease activity in inflammatory bowel diseases [IBD], which include Crohn's disease [CD] and ulcerative colitis [UC]. Micro-RNAs [miRNAs] are small non-coding RNAs detectable in extracellular fluids and can be used as clinical biomarkers. The aim of this study was to determine if faecal miRNA composition is altered in IBD. More than 800 different human faecal miRNAs were measured in stool samples from control individuals and patients with active CD by using NanoString technology. Selected miRNAs were quantified by qRT-PCR in faeces, serum and intestinal tissue of controls [n = 23] and patients with inactive or active CD [n = 22, n = 22] or UC [n = 11, n = 24] as well as patients with Clostridium difficile infection [CDI, n = 8]. In total, 150 miRNAs were significantly detected in faeces from controls and patients, and multivariate analyses showed that CD patients with high disease activities had a distinct miRNA profile and that miR-223 and miR-1246 were distinct from other faecal miRNAs. In a larger cohort, active UC patients displayed significantly higher levels of miR-223 and miR-1246 than controls while patients with CDI had higher levels of faecal miR-1246 but not miR-223. No differences were noted in serum samples. To our knowledge, this is the first comprehensive screen of faecal miRNAs performed in IBD. Further investigation will aim to confirm these findings in a larger cohort and to understand the biological function and cellular sources of faecal miRNAs.

Usefulness of fecal calprotectin by monoclonal antibody testing in adult Japanese with inflammatory bowel diseases: a prospective multicenter study.

Background/AimsNoninvasive objective monitoring is advantageous for optimizing treatment strategies in patients inflammatory bowel disease (IBD). Fecal calprotectin (FCP) is superior to traditional biomarkers in terms of assessing the activity in patients with IBD. However, there are the differences among several FCP assays in the dynamics of FCP. In this prospective multicenter trial, we investigated the usefulness of FCP measurements in adult Japanese patients with IBD by reliable enzyme immunoassay using a monoclonal antibody.MethodsWe assessed the relationship between FCP levels and disease or endoscopic activity in patients with ulcerative colitis (UC, n=64) or Crohn’s disease (CD, n=46) compared with healthy controls (HCs, n=64).ResultsFCP levels in UC patients strongly correlated with the Disease Activity Index (rs=0.676, P<0.0001) and Mayo endoscopic subscore (MES; rs=0.677, P<0.0001). FCP levels were significantly higher even in patients with inactive UC or CD compared with HCs (P=0.0068, P<0.0001). The optimal cutoff value between MES 1 and 2 exhibited higher sensitivity (94.1%). FCP levels were significantly higher in active UC patients than in inactive patients (P<0.001), except those with proctitis. The Crohn’s Disease Activity Index tended to correlate with the FCP level (rs=0.283, P=0.0565).ConclusionsOur testing method using a monoclonal antibody for FCP was well-validated and differentiated IBD patients from HCs. FCP may be a useful biomarker for objective assessment of disease activity in adult Japanese IBD patients, especially those with UC.

Expression of neutrophil gelatinase-associated lipocalin (NGAL) in the gut in Crohn\u2019s disease

The antimicrobial glycoprotein neutrophil gelatinase-associated lipocalin (NGAL) is strongly expressed in several infectious, inflammatory and malignant disorders, among these inflammatory bowel disease (IBD). Fecal and serum NGAL is elevated during active IBD and we have recently shown that fecal NGAL is a novel biomarker for IBD with a test performance comparable to the established fecal biomarker calprotectin. This study examines expression of NGAL in the healthy gut and in Crohn’s disease (CD), with emphasis on the previously unexplored small intestine. Pinch biopsies were taken from active and inactive CD in jejunum, ileum and colon and from the same sites in healthy controls. Microarray gene expression showed that the NGAL gene, LCN2, was the second most upregulated among 1820 differentially expressed genes in terminal ileum comparing active CD and controls (FC 5.86, p = 0.027). Based on immunohistochemistry and in situ hybridization findings, this upregulation most likely represented increased expression in epithelial cells. Double immunofluorescence showed NGAL expression in 49% (range 19–70) of Paneth cells (PCs) in control ileum with no change during inflammation. In healthy jejunum, the NGAL expression in PCs was weak to none but markedly increased during active CD. We further found NGAL also in metaplastic PCs in colon. Finally, we show for the first time that NGAL is expressed in enteroendocrine cells in small intestine as well as in colon.

Evaluation of interleukin-6 and its soluble receptor components sIL-6R and sgp130 as markers of inflammation in inflammatory bowel diseases

PurposeInterleukin-6 (IL-6) production and signalling are increased in the inflamed mucosa in inflammatory bowel diseases (IBD). As published serum levels of IL-6 and its soluble receptors sIL-6R and sgp130 in IBD are from small cohorts and partly contradictory, we systematically evaluated IL-6, sIL-6R and sgp130 levels as markers of disease activity in Crohn’s disease (CD) and ulcerative colitis (UC).MethodsConsecutive adult outpatients with confirmed CD or UC were included, and their disease activity and medication were monitored. Serum from 212 CD patients (815 measurements) and 166 UC patients (514 measurements) was analysed, and 100 age-matched healthy blood donors were used as controls.ResultsIL-6 serum levels were significantly elevated in active versus inactive CD and UC, also compared with healthy controls. However, only a fraction of IBD patients showed increased serum IL-6. IL-6 levels ranged up to 32.7 ng/mL in active CD (> 5000-fold higher than in controls), but also up to 6.9 ng/mL in inactive CD. Increases in active UC (up to 195 pg/mL) and inactive UC (up to 27 pg/mL) were less pronounced. Associations between IL-6 serum levels and C-reactive protein concentrations as well as leukocyte and thrombocyte counts were observed. Median sIL-6R and sgp130 levels were only increased by up to 15%, which was considered of no diagnostic significance.ConclusionsOnly a minority of IBD patients shows elevated IL-6 serum levels. However, in these patients, IL-6 is strongly associated with disease activity. Its soluble receptors sIL-6R and sgp130 do not appear useful as biomarkers in IBD.