bluuebunny/crossref_metadata_2025_split huggingface dataset:
DOP83 Intestinal fibrosis in Crohn’s disease patients is marked by up-regulation of innate immune cells and mucosal B cells
Authors: Creyns B , Gabriele D , Jonathan C , Verstockt B , Bislenghi G , Marc F , Vermeire S , Van Assche G , Ceuppens J , D’Hoore A , De Hertogh G , Breynaert C 
Published: January 2020

Abstract

Background
Intestinal fibrosis represents a significant complication of inflammatory bowel disease (IBD). An urgent need is present for the identification of pathways and markers involved in fibrogenesis to prevent and intervene with intestinal fibrosis. As intestinal fibrosis mainly occurs in the intestinal deeper layers, mucosal biopsies for studies in intestinal fibrosis are of limited information. Therefore, characterisation of the relative contribution of innate and adaptive immune cells in the mucosa and deeper layers in fibrotic distal ileum of Crohn’s disease (CD) patients was performed.

Methods
Seventeen CD patients undergoing right hemicolectomy (RHC) for stricturing disease and 6 colorectal cancer (CRC) patients undergoing RHC were recruited. The resected ileum was divided in macroscopically inflamed and fibrotic tissue and single-cell suspensions were made from mucosal and deeper intestinal layers for immune cell characterisation with flow cytometry. For comparison, proximal unaffected CD tissue and control ileum from CRC patients was included. Fibrosis and inflammation were confirmed on HE stained histological sections. Wilcoxon matched-pairs signed-rank test between CD samples and Kruskal–Wallis testing with Dunn’s multiple comparisons test compared with CRC ileum were performed.

Results
From 12 CD patients, an additional macroscopically inflamed region could be identified next to the fibrotic area, with a decreased fibrosis score as compared with the fibrotic area (4.00 vs. 6.00, p = 0.016) (Figure 1). Both fibrotic and inflamed regions had increased inflammation as compared with proximal unaffected CD and control non-IBD ileum (score: 4.00 and 4.50 vs. 1.00 and 0.00, p &lt; 0.001 for all). In the inflamed ileum, no differences in immune populations were observed between mucosa and deeper layers, reflecting the transmural nature of CD. In contrast, CD19+ B cells were specifically enriched in the mucosa of fibrotic ileum, as compared with proximal CD mucosa (32.20 vs. 20.40% of CD45+ cells, p = 0.008) (Figure 2). In the deeper layers of fibrostenotic CD ileum FcεR+ Siglec 8+ eosinophils (1.13 vs. 1.17 % of CD45+ cells, p = 0.027), mature CD11c+ dendritic cells (3.95 vs. 2.96 % of CD45+ cells, p = 0.042) and M2 CD206+ macrophages (0.35 vs. 0.18% of CD45+ cells, p &lt; 0.001) were enriched as compared with the mucosa overlying the fibrotic tissue (Figure 3).

Conclusion
These results argue that inflammation in the deeper intestinal layers is different from the inflammatory signature seen in mucosal inflamed regions. We here report alternative innate immune cells expanded specifically in the deeper intestinal layers of fibrostenotic CD ileum that could identify targets for new anti-fibrotic therapies.
DOP84 Crohn’s disease modifies the DNA methylome of human adipose-stem cells, which is only partially re-established in remission
Authors: Serena C , Millan M , Ejarque M , Saera-Vila A , Monfort-Ferré D , Bautista M , Menacho M , Martí M , Espin E , Vendrell J , Fernández-Veledo S 
Published: January 2020

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.
P010 Measurement of colonic mucosal content of biologics with the use of magnetic resonance imaging (MRI): A pilot study in patients with ulcerative colitis
Authors: Filip R , Bar P , Bartusik-Aebisher D , Aebisher D 
Published: January 2020

Abstract

Background
Anti-tumour necrosis factor antagonists (infliximab) as well as other molecules with different modes of action, including anti-integrin agents (vedolizumab), are currently used in patients with ulcerative colitis (UC Numerous studies have demonstrated a positive correlation between serum biologic drug concentrations and favourable therapeutic outcomes, whereas low or undetectable drug concentrations can lead to treatment failure. However, despite immunological issues, lack of and or loss of response may also be attributed to drug pharmacokinetics, of which penetration to the target tissue (colon wall) may play a crucial role

Methods
We used MRI to perform biochemical analyses of infliximab, adalimumab and vedolizumab concentrations testing the hypothesis that MRI relaxation time can be used to track antibodies in both mucosal biopsy samples and serum. All MR scans were performed with an Optima MR360 from General Electric Healthcare. To determine spin–lattice (T1) and spin–spin (T2) relaxation times, the Fast Spin Echo (FSE) sequence was used.

Results
The measured values of T1 relaxation times for infliximab, adalimumab, and vedolizumab were 2227 ± 35 ms, 2000 ± 22 ms and 1288 ± 15 ms, respectively. The obtained T2 relaxation times were 130 ± 11 ms, 90 ± 5, and 75 ± 10 ms, respectively. A decrease of both T1 and T2 values of 15 ± 3% are observed in serum from patients with ulcerative colitis. The values of infliximab and adalimumab were similar; the values of vedolizumab measurements in serum were about 50% lower. We find primary evidence that in T1 and T2 decreased in serum samples with ulcerative colitis and increase with the administration of infliximab, adalimumab and vedolizumab drugs. Samples of healthy tissue have T1 and T2 in the range of 2700 ± 5 ms and 150 ms ± 5 ms, respectively. A 30% decrease in T1 and T2 are observed for samples with ulcerative colitis. In this pilot study, we observed that values of T1 and T2 for tissues and serum that contain infliximab and adalimumab are similar, but vedolizumab shows a difference of about 30% when compared with infliximab and adalimumab.

Conclusion
MRI is an excellent method for quantitative and qualitative measurements of drug content in tissues and biological fluids. This is an innovative use of magnetic resonance imaging to develop a methodology for imaging of drugs that act as contrast agents via interaction with water in serum and tissue.
P038 A single-cell transcriptomics approach reveals high-resolution cellular signatures in Crohn’s disease
Authors: Garrido Trigo A , Corraliza A M , Veny M , Lozano J , Ricart E , Ordás I , Masamunt M C , Panés J , Hemberg M , Salas A 
Published: January 2020

Abstract

Background
Single-cell RNA sequencing (sc-RNAseq) provides high-resolution analysis of individual cells and cell clusters within complex tissues like the intestinal mucosa. No study to date has applied this novel technology to understand the complexity of intestinal inflammation in Crohn’s disease (CD) patients. Our aim is to identify the cellular subsets present in healthy and inflamed colon, and to describe how individual subsets are regulated in active CD.

Methods
Colon biopsies from a healthy control (HC) and two patients with endoscopically active colonic CD were collected and processed by enzymatic digestion and cell sorting to discard dead cells. Single-cell suspensions were processed by droplet-based protocol 10× chromium system followed by library preparation and sequencing. Sequencing data were aligned and quantified using Cell Ranger. Downstream analysis was performed using the R package Seurat.

Results
scRNA-seq analysis identified 14 transcriptionally different cell subsets in the healthy colon, including diverse types of T cells, plasma cells, mesenchymal cells, epithelial cells and myeloid populations, as well as a subset of MHCII+ CD20+ B cells. Overall, the number of transcriptionally differentiated cellular subsets was greater (16 and 17 subsets per sample) in the two CD colonic samples revealing higher cellular heterogeneity in active CD. In particular, in the HC we identified three T-cell subsets including a CD4+, CD8+ and a smaller subset of intraepithelial lymphocytes. In contrast, samples from CD patients presented four to six clearly differentiated T-cell clusters depending on the patient, with a marked enrichment of cytotoxicity genes (GZMA, GZMB, NKG7, CCL5 and AOAH) and T-regulatory genes (FOXP3, CTLA4, BATF and IL2RA). In addition, the HC sample presented three differentiated subsets of antibody-secreting plasma cells with predominant IgA and IgM production. In contrast, a clear expansion of IgG-producing plasma cells was observed in both CD patients. Moreover, the profiles of specific subsets significantly changed revealing transcriptional regulation within the individual subsets. Specifically, macrophages within the HC mucosa expressed the M2 markers CD163 and CD209 together with a whole signature of 163 additional genes, while in CD, they lost expression of these markers and upregulated CXCL8 and IL1B which were co-expressed with 268 additional genes common to macrophage populations in both CD patients.

Conclusion
We show that sc-RNAseq can be applied to study at a high cellular resolution human intestinal biopsies. Understanding the transcriptomic profile of the CD-related subsets will help dissect disease pathophysiology and provide knowledge for therapeutic targets.
P043 Small intestinal inflammation but not colitis drives pro-inflammatory nutritional antigen-specific T-cell response
Authors: Rodríguez Sillke Y , Schumann M , Lissner D , Branchi F , Glauben R , Siegmund B 
Published: January 2020

Abstract

Background
Inflammatory bowel disease (IBD) represents a dysregulation of the mucosal immune system. The pathogenesis of Crohn’s disease (CD) and ulcerative colitis (UC) is linked to the loss of intestinal tolerance and barrier function. The healthy mucosal immune system has previously been shown to be inert against food antigens. Since the small intestine is the main contact surface for antigens and therefore the immunological response, the present study served to analyse food-antigen-specific T cells in the peripheral blood of IBD patients.

Methods
Peripheral blood mononuclear cells of CD, with an affected small intestine, and UC (colitis) patients, either active or in remission, were stimulated with the following food antigens: gluten, soybean, peanut and ovalbumin. Healthy controls and celiac disease patients were included as controls. Antigen-activated CD4+ T cells in the peripheral blood were analysed by a magnetic enrichment of CD154+ effector T cells and a cytometric antigen-reactive T-cell analysis (‘ARTE’ technology) followed by characterisation of the effector response.

Results
The effector T-cell response of antigen-specific T cells were compared between CD with small intestinal inflammation and UC where inflammation was restricted to the colon. Among all tested food antigens, the highest frequency of antigen-specific T cells (CD4+CD154+) was found for gluten. Celiac disease patients were included as control, since gluten has been identified as the disease-causing antigen. The highest frequency of gluten antigen-specific T cells was revealed in active CD when compared with UC, celiac disease on a gluten-free diet (GFD) and healthy controls. Ovalbumin-specific T cells were almost undetectable, whereas the reaction to soybean and peanut was slightly higher. But again, the strongest reaction was observed in CD with small intestinal involvement compared with UC. Remarkably, in celiac disease on a GFD only antigen-specific cells for gluten were detected. These gluten-specific T cells were characterised by up-regulation of the pro-inflammatory cytokines IFN-γ, IL-17A and TNF-α. IFN-g was exclusively elevated in CD patients with active disease. Gluten-specific T-cells expressing IL-17A were increased in all IBD patients. Furthermore, T cells of CD patients, independent of disease activity, revealed a high expression of the pro-inflammatory cytokine TNF-α.

Conclusion
The ‘ARTE’-technique allows to analyse and quantify food antigen specific T cells in the peripheral blood of IBD patients indicating a potential therapeutic insight. These data provide evidence that small intestinal inflammation in CD is key for the development of a systemic pro-inflammatory effector T-cell response driven by food antigens.
P046 Vitamin D decreases PDIA3 and prevents the enhanced migration of fibroblasts from stricturing Crohn’s disease
Authors: Gisbert-Ferrandiz L , Cosín-Roger J , Coll S , Bauset C , Ortiz-Masiá D , Navarro-Vicente F , Alos R , Calatayud S , Barrachina M D 
Published: January 2020

Abstract

Background
Fibrosis is a common complication in Crohn’s disease (CD) patients and fibroblasts play an important role in the fibrogenic process. Low vitamin D (VD) levels and a defective VD-signalling pathway have been reported in CD. VD signals through both vitamin D receptor (VDR) and protein disulfide-isomerase A3 (PDIA3) and we have previously demonstrated that VDR protein levels are reduced in fibroblasts isolated from CD patients and that VD increased VDR expression in these cells (A-2080; ECCO 2019). We aim to analyse here the effect of VD on both PDIA3 protein levels and migration in CD fibroblasts.

Methods
We used intestinal fibroblasts isolated from surgical resections of the damaged mucosa of CD patients with stricturing behaviour (B2). Control fibroblasts were obtained from the non-damaged intestine of patients with colorectal cancer. Fibroblasts were treated with VD (100 nM) or its vehicle for 24 h and PDIA3 protein levels were measured by Western Blot. In the wound healing analysis, a single scraping was done in the centre of the fibroblasts monolayer and FBS-free medium was added to the cells, which allows us to determine the ability of fibroblasts to migrate and close the wound. Photos were taken at 0, 24 and 48 h. Results of wound healing were expressed as the percentage of the wound at each time point for the maximal wounded area (time 0, 100%). Statistical significance was measured by ANOVA or t-test.

Results
No significant differences in PDIA3 protein levels were detected between control and CD fibroblasts but VD significantly decreased PDIA3 expression in CD fibroblasts (Figure 1A). In the wound healing assay, we detected that CD-B2 fibroblasts migrate faster than control cells, resulting in a reduced wounding area, 48 h later (Figure 1B). Treatment of these CD-B2 cells with VD decreased their migration rate, and 48 h later cells exhibited a higher and significant wounding area than vehicle-treated cells (Figure 1C).

Conclusion
Vitamin D decreased PDIA3 expression and prevented the accelerated migration detected in intestinal fibroblasts from B2-CD patients which suggest an anti-fibrotic effect of VD mediated by a direct effect of this hormone on intestinal fibroblasts.
P054 The impact of intestinal stenosis on the expression of Crohn’s disease in young mice with genetic predisposition
Authors: Georgopoulos I , Mavrigiannaki E , Stasinopoulou S , Renieris G , NIkolakis G , Kollias G , Tiniakos D , Papaconstantinou I 
Published: January 2020

Abstract

Background
In the majority of patients, the areas of the intestine affected in Crohn’s disease (CD) are the terminal ileum and less commonly the rectum. The terminal ileum is affected in the majority of genetic animal models of CD as well. Additionally, CD recurs at sites of tight anastomosis or strictures postoperatively. These observations lead to the assumption that increased intraluminal pressure related to the presence of a valve, sphincter or stenosis is associated with CD pathogenesis. We investigated the hypothesis that the creation of a partial intestinal stenosis could have an impact on disease severity, in a genetic animal model of CD (TNFΔare/+). TNFΔare/+ mice overexpress TNFα leading to a Crohn-like colitis in the terminal ileum.

Methods
Twenty-nine TNFΔare/+ mice, 6 weeks old, were divided into three intervention groups: stenosis, sham and control. In the stenosis group (n = 11), a partial small bowel obstruction was created via a novel triple suture technique, approximately 3 cm from the ileocecal valve. In the control group (n = 9), a loose single suture was placed at the aforementioned site to test the direct effect of the foreign material on the intestinal wall. The sham group (n = 9) received a sham operation. The triple suture stenosis was also performed on wild-type (WT) C57BL/6 mice (group WT, n = 9). 6 weeks post-surgery all animals were sacrificed and samples from the ileum 3 cm proximal and 3 cm distal to the intervention site were collected for histopathological evaluation. The Crohn-like changes were assessed using a modified colitis histological scoring system (based on Katakura et al., JCI 2005; 115: 695–702).

Results
Proximal to the intervention, the mean colitis score of stenosis group (10.18 ± 0.87) was significantly higher compared with sham (6.33 ± 0.97, p: 0.009) and control group (5.00 ± 0.91, p: 0.001). There was no difference between sham and control group (p: 0.332). No significant differences between the groups were reported distal to the intervention. The triple suture technique led to Crohn-like inflammatory lesions only in the TNFΔare/+ mice, as shown from the significantly increased score compared with WT mice proximal and distal to the stenosis (10.18 ± 0.87 vs. 0.67 ± 0.37, p &lt; 0.001 and 9.20 ± 1.09 vs. 0.33 ± 0.24, p &lt; 0.001).

Conclusion
The creation of a stenotic segment in the intestine of TNFΔare/+ mice, led to higher colitis score than expected. The probable mechanism is the increased intraluminal pressure proximal to the stenosis. This suggests that mechanical forces contribute as important co-factors in the pathophysiology of CD, in genetically predisposed populations.
P060 The pharmacologic inhibition of store-operated calcium entry pathway in inflammatory bowel disease
Authors: Letizia M , Yerinde C , Sand A , Schlickeiser S , Kaufmann U , Siegmund B , Feske S , Weidinger C 
Published: January 2020

Abstract

Background
Store-operated calcium entry (SOCE) represents the major calcium influx pathway in T cells which not only controls the activation and function of lymphocytes, but which also has been implicated in the metabolic homeostasis and survival of murine CD4+ and CD8+ T cells. Conditional knockout mice, in which SOCE signalling components are deleted in T cells, revealed that SOCE is required for the induction of intestinal inflammation in mouse models of colitis. However, the effects of SOCE inhibition have not been studied in inflammatory bowel disease (IBD) and it remains elusive, which immune cell subset is affected by the pharmaceutical blockade of SOCE. We therefore aim to investigate the effects of SOCE inhibitor BTP-2 on functions and metabolic homeostasis of human lymphocytes isolated from IBD patients.

Methods
PBMC and/or lamina propria lymphocytes were isolated from colitis patients undergoing colon resection and mass cytometry served in order to evaluate the cytokine production and activation of B, T, NK as well as myeloid cells. Additionally, Ca2+ influx measurement and Seahorse analyses were performed in order to assess the metabolic status of immune cell subsets after SOCE inhibition.

Results
Data on B, T, NK, myeloid cells and neutrophils isolated from peripheral blood or colon lamina propria revealed that each immune cell subset harbours a distinctive SOCE-dependent Ca2+ influx rate, suggesting that SOCE might differentially regulate the activation and function of each cell subtype. In particular, CD4+ and CD8+ T cells, B and NK cells as well as monocytes were highly susceptible to extracellular Ca2+ influx, followed by granulocytes. Furthermore, inhibition of SOCE in lymphocytes resulted in impaired metabolic fitness, reduced glycolytic capacity and impaired fatty acid oxidation. Finally, BTP-2 was able to decrease the production of key pro-inflammatory cytokines involved in IBD, including TNFα and IL-17 in lamina propria resident T cells.

Conclusion
Our data revealed for the first time that the cytokine production and the activation of several immune cell subtypes can be modulated by SOCE blockade in human intestinal inflammation, identifying SOCE as a novel therapeutic target in colitis. Moreover, we hope that a wide phenotypical characterisation of immune cells via mass cytometry will provide a better insight into positive as well as negative effects of SOCE inhibitors that might interfere with the clinical applicability of SOCE inhibitors for treating IBD.
P064 Exosomes from mesenchymal stromal cells reduce murine colonic inflammation via a macrophage-dependent mechanism
Authors: Wu X R , Lan P , Wu X J , Gao X 
Published: January 2020

Abstract

Background
Conventional treatments for inflammatory bowel disease (IBD) have multiple potential side effects. Therefore, alternative treatments are desperately needed. Exosomes from mesenchymal stromal cells (MSC-Exos) show potent immunomodulatory activities and protective effects in several diseases.

Methods
MSC-Exos were isolated from human bone marrow-derived mesenchymal stromal cells. Experimental colitis was induced by administration of dextran sulphate sodium (DSS) in C57BL/6 mice (male, 6–8 weeks), or 2, 4, 6-trinitrobenzenesulfonic acid (TNBS) in Balb/c mice (male, 7–9 weeks). Macrophage depletion was performed via intraperitoneal injection of clodronate liposomes. Colitic mice were treated intravenously with MSC-Exos or saline, and mortality and diverse disease signs were tracked.

Results
Systemic administration of MSC-Exos significantly mitigated colitis in mice. MSC-Exos treatment downregulated inflammatory responses, maintained intestinal barrier integrity and polarised M2 macrophages. Infused MSC-Exos mainly acted on colonic macrophages and their beneficial effect was blocked by macrophage depletion. Particularly, MSC-Exos were enriched in proteins involved in regulating multiple biological processes associated with the anti-colitic benefit of MSC-Exos.

Conclusion
MSC-Exos are critical regulators of immune/inflammatory responses and may be promising candidates for IBD treatment