bluuebunny/crossref_metadata_2025 huggingface dataset:
P050 Intraperitoneally injected 3-dimensional cultured tonsil-derived mesenchymal stem cells in murine colitis model form aggregates with mouse immune cells: A preliminary study
Authors: Byeon J R , Song E M , Joo Y H , Park Y , Shim K N , Choe A R , Tae C H , Kim S E , Moon C M , Jung H K , Hong J T , Jung S A
Published: January 2023

Background

3-dimensional (3D) cultured tonsil-derived mesenchymal stem cells (TMSCs) showed higher therapeutic efficacy in murine colitis model than conventional 2D-cultured TMSCs via increased anti-inflammatory cytokine expression. Here, we evaluated the in-vivo localization and formation of 3D-cultured TMSCs using electron microscopy.

Methods

Chronic mouse colitis was induced by oral administration of 1.5% dextran sulfate sodium for 30 days. 3D-cultured TMSCs and 2D-cultured TMSCs was injected intraperitoneally on day 6 and day 16 after inducing colitis in mice. TMSC was 3D-cultured as a spheroid for 24 hours before intraperitoneal injection. On day 31, mice were euthanized and the intraperitoneal localization of 3D-cultured TMSCs was evaluated using immunofluorescence analysis. Moreover, the in-vivo characterization of 3D-cultured TMSCs was evaluated using electron microscopy.

Results

In vitro 3D-cultured TMSC spheroids were measured to have an average size of 182.4 μm on day 1, 169.4 μm on day 2, and 154.1 μm on day 3; however, the size of spheroids gradually decreased after 2 days of culture. In mice treated with 2D-cultured TMSCs, there were no detectable TMSCs at the time of sacrifice. In mice treated with 3D-cultured TMSCs, white variable sized spherical aggregates were observed. These aggregates were attached to peritoneal cavity including omentum and mesentery. In fluorescence microscopy, these aggregates were stained with human nuclear antigen antibody, indicating that the transplanted 3D-cultured TMSCs formed a cluster in the peritoneum even 14 days after intraperitoneal injection. In electron microscopy, these aggregates were covered with mouse lymphocytes. In the inside of spheroids, suspected mesenchymal cell and mesothelial cell were distributed.

Conclusion

Intraperitoneally injected 3D-cultured TMSCs were found as aggregates in the peritoneal cavity of mouse even 14 days after injection. In electron microscopy, these aggregates were covered with mouse lymphocytes. Based on the results of current study, functional studies evaluating the role of TMSC as a cell therapy, including cell-to-cell communication between TMSC and immune cells, are needed.
P054 Secreted inflammatory protein profiles following treatment of ex-vivo human inflammatory bowel disease explants with licenced biologic therapies
Authors: Corcoran R , O'Connell F , O'Sullivan J , Kevans D
Published: January 2023

Background

Patient-derived inflammatory bowel disease (IBD) ex-plants have potential for biomarker and therapy discovery. Infliximab (IFX), ustekinumab (USTK) and vedolizumab (VDZ) are biologic therapies licenced for the induction and maintenance of remission in ulcerative colitis (UC) and Crohn’s disease (CD). All are efficacious in IBD despite differing mechanisms of action. Treatment of IBD explants ex-vivo with licensed biologic therapies may result in further insights into their mechanism of action. We aimed to evaluate the effect of IFX, USTK and VDZ on inflammatory protein secretion profiles in ex-vivo human IBD ex-plants.

Methods

Patients with IBD, undergoing endoscopy, were prospectively recruited. Biopsies were collected from the sigmoid and IBD ex-plants generated as per previously described methods. IBD explants were then co-cultured for 24 hours with an IgG control vehicle, IFX, USTK and VDZ. After 24 hours, tissue conditioned media (TCM) from IBD explants was collected. TCM secreted inflammatory protein profiles were quantified using 54 V-plex ELISA (Meso Scale Diagnostics, USA). Secreted inflammatory protein profiles were compared between IgG vehicle (control) and USTK, IFX, VDZ treated ex-plants. All continuous variables are presented as median [interquartile range (IQR)]. P values < 0.05 were considered significant in analyses.

Results

37 patients with IBD were included (51% CD, 49% UC); age (median, [IQR]) 41[33-53] years, 49% male; disease duration (median, [IQR]) 9 [5-14] years; 51% of patients were anti-TNF naïve. TNF-a secretion was significantly lower in IBD explants treated with IFX compared with IgG control, p=0.006. IL-23 secretion was significantly lower in IBD explants treated with USTK compared with IgG control, p=0.043. Biologic therapy ex-vivo resulted in significant differences in angiogenesis and vascular injury panel biomarkers compared to control; CRP, VEGF-A and SAA. Pro-inflammatory cytokines and chemokines were also significantly decreased with ex-vivo biologic therapy compared to control; IL-2, IL-10, IL-6, MCP-1, IL-1a, TNF-a and MDC. Three Th17 panel proteins were significantly decreased with ex-vivo biologic therapy compared to control; IL-22, IL-17A, IL17A Gen B.

Conclusion

The statistically significant decrease in both TNF-a and IL-23 secretion with ex vivo IFX and USTK treatment respectively demonstrates that the IBD explant model recapitulates expected IBD disease biology and therapy responses. These data demonstrate that treatment with licenced biologic therapies ex-vivo reduces multiple pro-inflammatory cytokines, chemokines and secreted proteins in IBD explants. Further study is required to completely understand the effects of licenced biologic therapies on the IBD tissue microenvironment.
P060 Restoration of occludin and claudin-1 expression in patients with Crohn’s disease receiving anti-TNF treatment.
Authors: Tsounis E , Geramoutsou C , Aggeletopoulou I , Lourida T , Pastras P , Theocharis G , Diamantopoulou G , Zolota V , Assimakopoulos S F , Bravou V , Thomopoulos K , Triantos C
Published: January 2023

Background

Occludin and claudin-1 are integral components of tight junctions (TJs) that contribute to intestinal barrier integrity. The aim of this study was to prospectively evaluate the expression of occludin and claudin-1 in the intestinal mucosa of patients with Crohn’s disease (CD) undergoing treatment with tumor necrosis factor (TNF) inhibitors.

Methods

Clinical assessment, laboratory testing, and colonoscopy were performed on all participants prior to receiving anti-TNF therapy. Colonic or ileal biopsies were obtained from the affected intestinal areas. A follow-up exam and colonoscopy were performed at least 6 months after the start of anti-TNF therapy, and paired biopsies were obtained from the initially affected segments. The expression of the TJ proteins was evaluated by immunohistochemistry (IHC).

Results

In this prospective observational study, 21 consecutive patients with active CD [median age 32.5 years (IQR: 18.5-51), 66.7% male] and 10 healthy controls [36 years (IQR: 29-58), 60% male] were enrolled. Patients with CD received treatment with TNF inhibitors (infliximab, adalimumab, and certolizumab: 61.9%, 33.3%, and 4.8%, respectively), according to current guidelines, and a follow-up examination was conducted after a median period of 25 months (IQR: 9.5-66). Before treatment administration, patients with active CD exhibited an increased expression of both occludin [2 (IQR: 1.75-2.75) vs. 1.5 (IQR: 0-1.75); p = 0.032] and claudin-1 [2.5 (IQR: 2-3) vs. 1 (IQR: 0.75-2); p = 0.01] in comparison to healthy controls. In control mucosa, IHC staining for occludin and claudin-1 was weak to moderate, cytoplasmic, and membranous, confined to the surface epithelium, while staining in intestinal crypts was negative. On the other hand, an enhanced expression of both proteins in the enteric crypts and surface epithelium was demonstrated in CD patients upon initial examination. Treatment with TNF inhibitors resulted in a significant reduction of claudin-1 expression (p = 0.018) as well as a notable decline of occludin expression (p = 0.08). Occludin and claudin-1 immunostaining was restricted to the surface epithelium in post-treatment biopsies. In addition, the activity of the disease, as estimated by the Crohn’s Disease Activity Index (r = 0.476; p = 0.014) and C-reactive protein (r = 0.411; p = 0.039), was positively correlated with the expression of claudin-1 in the mucosa.

Conclusion

Active CD is plagued with a disruption of the expression and distribution of claudin-1 and occludin throughout the affected intestinal mucosa. Successful treatment with biological agents induces the restoration of intestinal barrier function, which is associated with an improvement in the patients' clinical condition.
P067 Fractal analysis of extracellular matrix as a new histological method for observer-independent quantification of intestinal fibrosis in Crohn’s Disease
Authors: Weber M C , Schmidt K , Buck A , Kasajima A , Becker S , Wilhelm D , Friess H , Neumann P A
Published: January 2023

Background

The prevention of intestinal fibrosis formation is still an unsolved problem in the management of Crohn’s Disease (CD) as no specific antifibrotic therapies are available yet. Appropriate analysis of fibrosis severity is essential to assess the therapeutic efficacy of potential anti-fibrotic drugs. However, up to now, no standardized method exists, that allows for automated observer-independent histological evaluation of intestinal fibrosis severity. The aim of this study was thus to develop an objective and observer-independent histological method for the quantification of intestinal fibrosis in surgical specimens of Crohn’s patients using structural analysis of the extracellular matrix (ECM).

Methods

To quantify the structural complexity of the total ECM, fractal dimension analysis was performed using the FracLac plugin in FIJI/ImageJ in fibrotic and control sections of surgical specimens from patients with CD (n = 28) stained with Masson's trichrome. To selectively assess the structure of the collagen matrix, polarized light microscopy was used on Picrosirius red-stained sections. A score to quantify collagen fiber alignment and the color of the polarized light was established. As a reference, sections were scored by a pathologist using an established histological fibrosis score (0 - 4), the proportionate collagen fraction was measured and COL1A1 and COL3A1 mRNA expression was assessed.

Results

Fractal dimension as a measure for the structural complexity correlated significantly with the histological fibrosis score (r = .68, p < .0001), and differences in fractal dimension differed significantly between sections with different fibrosis scores, indicating that during fibrosis progression, there is not only an accumulation of ECM but also structural changes within the ECM network. Using Picrosirius polarized light microscopy, we found that sections with low fibrosis scores showed a meshwork-like alignment of the collagen fibers while sections with high fibrosis scores showed a parallelization of long collagen fibers. Fiber alignment (r =.71, p < .0001) positively correlated with the histological fibrosis score. All measurements were significantly higher in sections from the fibrotic focus compared to the resection margin.

Conclusion

Fractal dimension analysis was suitable for fibrosis quantification in histological samples from surgical specimen of CD patients. Picrosirius polarized light microscopy underlined our findings on structural changes of the ECM network during fibrosis progression in CD. Automated structural analysis of the ECM network using fractal analysis has the potential to serve as an observer-independent histological tool for fibrosis quantification in surgical specimens of patients with CD.
P070 Global cytokine—cytokine interaction framework to uncover communication channels between elements of the immune system
Authors: Olbei M , Modos D , Cozzetto D , Powell N , Korcsmaros T
Published: January 2023

Background

Cytokines are small peptides that signal between a variety of cell types and are one of the fundamental communication elements of the immune system. A variety of cytokine—cytokine interactions have previously been investigated in the literature, describing cytokines activating or inhibiting other cytokines in target cell types, and thus progressing the immune response to infection and illness. Disruption of cytokine communication is often an important goal in drug development as these compounds allow quelling and managing an overactive immune response in chronic diseases, such as Inflammatory Bowel Disease (IBD). In our previous work, we have introduced CytokineLink, a novel computational framework developed to establish cytokine—cytokine interactions from transcriptomics data (Olbei et al., Cells, 2021).

Methods

In this project, we generated a global network of cytokine—cytokine interactions based on cytokine response transcriptomics data from over 2000 datasets deposited in the CytoSig database. Using CytoSig’s significance analysis protocol, we established statistically significant cytokine—cytokine interactions, and determined the likely intracellular pathways connecting upstream and target cytokines using the OmniPath interaction resource.

Results

The resulting network is a signed, directed network of cytokine communication, containing 488 cytokine—cytokine interactions of 91 cytokines. The interactions in the network are annotated with the intracellular pathways that the included cytokines are anticipated to utilise, as well as the stimulatory and inhibitory effects that the cytokines have on one another. The resource captures the cytokine—cytokine networks of cytokines crucial in the pathophysiology of IBD, such as TNF, IL2, IL21, and OSM, which may grant novel insights into the cytokine pathways important in the molecular mechanisms underlying chronic diseases like IBD. The resource can be used by the community as a knowledge base for hypothesis generation, and is freely available through the NDEx platform.

Conclusion

In our work, we generated a novel computational framework collating how cytokines differentially regulate the expression of one another based on cytokine response transcriptomics data. The resulting interactions are signed, highlighting the inhibitory or stimulatory nature of the associations, and the change in expression associated with each link. The resource could be used to identify previously unknown network pharmacology targets, and to better understand the cytokine dysregulation in chronic diseases such as IBD by illustrating the interplay of the most influential IBD associated cytokines with other cytokines.
P075 Characterisation and structural analysis of a new murine model to study postoperative fibrotic recurrence in Crohn’s disease combining ileocecal resection with chronic colitis
Authors: Schmidt K , Li C , Buck A , Wilhelm D , Friess H , Weber M C , Neumann P A
Published: January 2023

Background

The prevention of postoperative fibrotic recurrence in Crohn’s disease is still an unsolved problem in the management of patients undergoing bowel resection due to fibrotic intestinal strictures. Appropriate murine models are needed to examine the effect of surgery associated factors as well as mechanical and microbial alterations after ileocecal resection on the development of postoperative fibrotic recurrence in Crohn’s disease.

Methods

We developed a murine model combining chronic dextrane sodium sulfate(DSS)-colitis with ileocecal resection. 9 to 11-week-old C57BL/6 wild-type mice were exposed to 1% DSS via the drinking water for one week followed by ileocecal resection using a microsurgical technique. After surgery, mice recovered for one week after which two more cycles of DSS-administration were performed, now using 1.5% DSS. Between the two postoperative DSS-cycles one week of recovery was implemented. Histology was performed on the distal ileum, the anastomotic area, the proximal colon, and the distal colon.

Results

The inflammation (proximal colon: median score 5 vs. 0; p < 0.001, distal colon: median score 3 vs. 0; p = 0.003) and fibrosis scores (proximal colon: median score 2 vs. 0; p < 0.001, distal colon: median score 2 vs. 0; p = 0.008) were higher in the colon of mice with chronic DSS-colitis compared to mice without colitis. Furthermore, histomorphometric parameters such as the proportionate collagen area (proximal colon: mean area 6.6% vs. 2.6%; p < 0.001, distal colon: mean area 5.4% vs. 3.7%; p = 0.17) and the submucosal thickness (proximal colon: mean 127.4µm vs. 39.7µm; p < 0.001, distal colon: mean 71.5µm vs. 37.2µm; p = 0.049) were significantly higher in mice with chronic colitis. Interestingly, the most profound inflammatory and fibrotic changes were found in the proximal colon compared to the distal colon. No fibrotic changes were found in the small intestine.

Conclusion

The combination of ileocecal resection and chronic DSS colitis led to inflammatory and fibrotic changes within the colon. Histopathological features of fibrosis were more prominent in the proximal colon as opposed to the known results from the DSS-colitis model, where inflammatory and fibrotic changes of the intestinal wall worsen towards the distal colon. We thus hypothesize that mechanical and microbial changes due to the altered physiology of the lower gastrointestinal tract after ileocecal resection are responsible for these findings. Continuing research on this model might enable the examination of mechanisms of recurring fibrosis after ileocecal resection in Crohn’s disease.
P080 Mucosal clock and inflammation regulatory genes are disrupted in treatment naïve pediatric patients with ulcerative colitis
Authors: Labes S , Tabach Y , Matar M , Shamir R , Shouval D S , Froy O , Weintraub Y
Published: January 2023

Background

The human circadian clock is present in cells throughout the body. It consists of CLOCK and BMAL1 that heterodimerize and bind to E-box sequences to mediate transcription of a large number of genes, including Periods (PERs) and Cryptochromes (CRYs). PERs and CRYs constitute part of the negative feedback loop and inhibit CLOCK:BMAL1-mediated transcription. Recently, we have shown that patients with active ulcerative colitis (UC) display tissue-specific misalignment of the molecular clock that reverts to normal following effective treatment. Several circadian clock genes also function as anti-inflammatory regulators. RORa, PPARa and PPARg are positive regulators of the clock mechanism and induce the expression of IkB, a suppressor of NFkB. In the same manner, PCG1a induces transcription of IL10. Our aim was to characterize the expression of these regulators compared to that of core clock genes and determine their correlation in UC patients.

Methods

Clock gene expression patterns using whole transcriptome RNA sequencing were retrieved from the IBD Transcriptome and Metatranscriptome Meta-Analysis platform (TaMMA IBD). We compared rectal biopsy gene expression of 12 clock genes and their isoforms (AMPK, BMAL1, CLOCK, CRY1, CRY2, PER1, PER2, PCG1a, PPARa, PPARg, REV-ERB, RORa, RORg and SIRT1) in 206 treatment naïve pediatric patients with UC (age 12.9±3.2; 54% male) and 20 healthy controls (age 13.9±3.3; 45% male). Spearman correlations were calculated between all 12 clock genes within each of the UC and control cohorts.

Results

Core clock genes: BMAL (p<0.0001), CLOCK (p<0.01) and CRY1 (p<0.0001), and the anti-inflammatory regulator gene RORa (p<0.0001) were upregulated in UC patients compared to controls. In contrast, expression of other anti-inflammatory regulatory genes: PCG1a (p<0.0001), PPARa (p<0.01), PPARg (p<0.0001) and RORg (p<0.0001) was decreased. Clock gene expression levels of healthy controls show discordant correlations compared to UC patients. For example, correlations between BMAL/REV-ERBa as well as PPARg/REV-ERBa were inverted between controls and UC patients (r=-0.44 vs. r=0.13 and r =-0.4 vs. r=0.3, respectively).

Figure 1. Expression of clock and inflammation regulatory genes in UC patients vs. healthy controls.

Conclusion

Core clock gene expression was disrupted, and anti-inflammatory regulator gene expression was decreased in patients with active UC vs. controls. The demonstration of discordant correlations of some positive vs. negative feedback loop genes in UC highlights the clock disruption in active inflammation. Since these clock regulators also ameliorate inflammation, their reduced expression may explain not only clock dysregulation but also increased tissue inflammation.
P081 The application of multiomics to investigate the evolution of inflammatory bowel disease in pediatric and adult patients
Authors: Maccalli C , Gupta I , Toufiq M , Habib T , Mathew R , Shobha Manjunath H , Missous G N , Mifsud W , Charles A , Ammar A A , Tomei S , van Panhuys N , Al-Kaabi S , Akobeng A , Al-Mohannadi M J , Elawad M
Published: January 2023

Background

The aim of this study is to monitor the evolution of Inflammatory Bowel Disease (IBD), including Crohn’s Disease (CD) and Ulcerative Colitis through the integration of genomic and immunological investigations to assess the mechanisms of IBD severity and the risk to develop malignancy

Methods

The study included pediatric (N=20) and adult (N=18) IBD patients at different stages of the disease (remission, mild, moderate and severe) and CRC patients with IBD history (N=13). Retrospectively collected tissues of IBD, inflamed non-IBD and normal gut tissues (N=10) were also utilized. Gut tissue biopsies (from both left and right side) and peripheral blood were collected from patients. DNA and RNA were extracted from fresh small size (2 mm in diameter) of gut tissues using the BioMasher II (Kimble) and All Prep DNA/RNA kits (Qiagen). Multiomics approach including MicroRNA (miRNA; N=700) and gene expression (N=800 genes) profiling were performed (cCounter platform; Nanostring) as well as the methylation profiling microarray (Infinium Methylation Epic Bead Chip kit, Illumina) to interrogate up to 850,000 methylation sites across the genome. These analyses were coped with immunological investigations. See Figure 1.

Results

Differential miRNA (such as miRNA92a-3p, 135-5p, 152-3p, 612; p<0.05) and gene signatures were found by the comparison of tissues from pediatric and adult patients with different stages of the disease. The differentially expressed genes belong to molecular pathways of inflammation, stemness, fibrosis, cell proliferation and migration (representative genes are: STAT3, CXCL2, MPM1, BIRC3, ISG15, ISG20, PECAM1). Pathways associated with the development of malignancy, such as PI3K/Akt/STAT and Wnt/GSK-3b were also detected as significantly differentially expressed. Differential profiles were also detected when comparing biopsies collected from right and left sides of gut in both pediatric and adult patients. Methylation sites at single nucleotide resolution have been also analyzed and correlated with miRNA and gene expression profiles

Conclusion

Although the results are currently under further investigation, the ongoing integration of data from multiple platforms provide insights of the overall molecular determinants in IBD patients along with the evolution of the disease and the clinical outcome
P082 The CCR9/CCL25 axis is specific to the ileum and is correlated with stricturing disease
Authors: Gehrmann U , Csomor E , Monkley S , Georgi B , Tian S , Cairns J , Angermann B , Khan E , Nys J , Marks D
Published: January 2023

Background

Crohn’s Disease (CD) is a chronic disease in which inflammation is sustained by continuous influx of inflammatory leukocytes into gut mucosa and may affect all segments of the gastrointestinal tract. Immune cell trafficking is regulated via chemokine gradients and adhesion molecules such as integrins. Here we explored the expression profile of gut-specific integrin and chemokine axes in different regions of the gut to identify possible targets for treating CD patients with different disease location or phenotype.

Methods

This study used transcriptomic data from gut tissue biopsies of the large and small intestine of 611 CD and 315 UC patients enrolled in a multicentred longitudinal Study of a Prospective Adult Research Cohort with IBD (SPARC IBD) obtained from the IBD Plexus program of the Crohn’s and Colitis Foundation. We mapped the expression of selected chemokine receptors and their ligands as well as gut-specific integrins onto the different segments of the intestine. Expression data were then linked to available clinical disease phenotypes, as well as genotype information.

Results

In both CD and UC patients, ITGA4 and ITGB7, the targets for Vedolizumab, and their endothelial ligand MADCAM1 were ubiquitously present in all regions of the gut. GPR15 and its ligand C10orf99 were predominantly expressed in colonic biopsies. On the contrary, expression of CCR9 and its ligand CCL25 were specific to the terminal ileum with only minimal transcriptional activity in the colon. Expression of CCL25 was significantly increased in patients with penetrating and/or stricturing CD, a disease feature associated with ileal CD that has high impact for patients and is currently an unmet for IBD patients. Further, we identified an expression quantitative trait loci (eQTL) upstream of the CCL25 promoter linked to small bowel expression of CCL25.

Conclusion

Our data highlight the potential for targeting the CCR9/CCL25 axis as a treatment for small bowel Crohn’s Disease which may have been incompletely targeted to date by small molecule inhibitors. Further investigations are warranted to identify a mechanistic link between this pathway and penetrating and stricturing disease.
P083 JAK/STAT pathway and IL-6 activity in moderate to severe Ulcerative Colitis
Authors: Calviño Suárez C , Martinez-Rodriguez A L , Baston-Rey I , Moreira-Alvarez D , Ferreiro-Iglesias R , Ardao-Palacios I , de la Iglesia-Garcia D , Porto-Silva S , Nieto-Garcia L , Varela-Liste M J , Brea-Floriani J M , Dominguez-Munoz J E , Loza-Garcia M , Barreiro-de Acosta M
Published: January 2023

Background

Ulcerative colitis (UC) is a chronic inflammatory bowel disease with a high social and health care system burden. Despite recent advances in UC treatment with available drugs acting in different signalling pathways related to the disease, there is still a significant proportion of non-responders who end up being overtreated and exposed to treatment's secondary effects. Therefore, it would be helpful to know the activity of each inflammatory pathway involved in UC pathogenesis in order to tailor the best treatment for each patient. The aim of our study was to measure the activation of JAK/STAT pathway and IL-6 levels in colonic biopsies from patients with UC.

Methods

A prospective, observational single-centre study was designed and it is currently on going. Adult UC patients with any endoscopic activity (Mayo Endoscopic Score (MES) > 0) in a routine colonoscopy were included. Biopsies from inflamed (endoscopically active) and non-inflamed (endoscopically inactive) colon of each patient were homogenised and processed by using RIPA buffer and ultrasounds to obtain cell lysates. Determination of the activation of JAK/STAT pathway was performed by detecting phosphorylated forms of JAK1, JAK2, JAK3, TYK2, STAT1, STAT3 and STAT4 by Western blot. Human Luminex discovery assay was used for IL-6 quantification. Data are shown as percentage, median, interquartile range (IQR) and mean ± standard deviation as appropriate.

Results

So far, 19 patients were consecutively included (median age 60.9 years (IQR 40.8-66.6), 58% female). About 47% had left-sided UC, 32% extensive UC and 21% had proctitis. Regarding endoscopic activity, 52.6% of patients showed MES-3, 42.1% MES-2 and 5.2% MES-1. IL-6 was increased in biopsies from inflamed colon, while no IL-6 was found in biopsies from non-inflamed colon (p=0.00). Median IL-6 in inflamed colonic biopsies from patients with MES-2 was 41.5pg/ml (IQR 7.4-106.7) and 51.9pg/ml (IQR 17.5-220.5) in patients with MES-3 (p=0.4) (Figure 1). Every patient showed, at least, one phosphorylated JAK protein and one phosphorylated STAT protein from JAK/STAT pathway in inflamed colonic biopsies. Nevertheless, the pattern of activation of JAK/STAT pathway was different in all patients as shown in Figure 2.

Conclusion

Different activated forms of JAK and STAT kinases are detected in inflamed colonic biopsies from UC patients, even though the activation pattern of JAK/STAT pathway differs among them. IL-6 levels are increased in inflamed colonic biopsies from UC patients while it is not detected in non-inflamed mucosa.
P085 Gut microbiota profile changes in patients with Ulcerative Colitis
Authors: Inčiūraitė R , Gedgaudas R , Lukosevicius R , Plingyte K , Muskieta T , Juzenas S , Kupcinskas J , Skieceviciene J
Published: January 2023

Background

Ulcerative Colitis (UC) is a chronic, relapsing inflammatory disease of the lower gastrointestinal tract. The frequency of UC is increasing worldwide, however, existing methods for both diagnostics and treatment of this disease are not efficient enough. It is known that besides comprised immune response, environmental and genetic factors, gut microbiota play a major role in the onset and course of UC. Therefore, efforts are currently being made to find and develop new gut microbiome-based tools to improve the management of UC. The aim of this study was to identify changes in the gut microbiome during active and quiescent UC.

Methods

Study included 72 subjects, who were divided into three age- and sex-matched groups: control (n=25), active UC (n=27) and quiescent UC (n=20). Total DNA was extracted from faeces, which was further subjected to the next generation sequencing of 16S rRNA-coding gene V1-V2 hypervariable region on MiSeq (Illumina) platform. Further, bioinformatics and statistical analysis were performed.

Results

Bacterial α-diversity, as assessed by Richness, Shannon and Simpson diversity indexes, revealed that control patients had highest α-diversity compared to patients with active UC or quiescent UC (p<0.05), but there were no differences between UC disease states (p>0.05). Significant microbial community clusters (β-diversity), as assessed by the Bray-Curtis dissimilarity index, were identified between control subjects and patients with active or quiescent UC (p=0.02, p<0.01, respectively). However, no significant clusters were found between different disease states (p=0.22). In-between samples dissimilarity assessed by Bray-Curtis dissimilarity index showed that samples from control subjects had higher in-between sample similarity (mean 0.542 ± 0.117) than patients with active (mean 0.638 ± 0.161) and quiescent (0.6 ± 0.145) UC. In addition, 16, 13 and 27 core taxa were identified in active, quiescent UC and control group, respectively. Differential abundance of Cuneatibacter, Faecalibacterium and Prevotellamassilia genera was detected when comparing control vs UC (both active and quiescent), Paraprevotella and Cuneatibacter genera – control vs active UC, Faecalibacterium, Prevotellamassilia, Mediterraneibacter and Cuneatibacter genera – control vs quiescent UC.

Conclusion

In conclusion, this study revealed both qualitative and quantitative gut microbiota changes in active and quiescent UC.

Study was funded by the Research Council of Lithuania (Grant No. S-MIP-20-56).
P089 Colonic explant lactate concentration and inflammatory protein secretion in ulcerative colitis
Authors: McShane C , Corcoran R , O'Connell F , McDonagh P , O'Sullivan J , Kevans D
Published: January 2023

Background

Lactate, previously thought of as a waste product of cellular metabolism, is now known to act as an immunoregulator with pleiotropic effects. In murine models lactate has been shown to suppress colonic inflammation & restore colonic homeostasis. We aimed to investigate if lactate concentration in tissue cultured media (TCM) from ulcerative colitis (UC) patient-derived explants is associated with TCM concentration of inflammatory proteins known to be involved in UC pathogenesis.

Methods

UC patients were prospectively recruited in 2 cohorts: discovery (2018 – 2019) and validation (2020 - 2021). Endoscopic biopsies were collected from the sigmoid colon and TCM generated as per previously described methods. Total protein content of biopsies was quantified to allow normalisation of secretions. Patients demographics, baseline characteristics and disease behaviour were characterised. TCM secreted lactate was quantified using a colorimetric L-Lactate assay (Abcam, UK). Secreted inflammatory protein profiles were analysed via 54 V-plex ELISA (Meso Scale Diagnostics, USA).

Results

52 patients were recruited (discovery cohort n=28, replication cohort n=24), baseline characteristics are described in Figure 1. Median follow up time was significantly longer in discovery compared with validation cohort p=0.02. In the discovery cohort TCM lactate concentration was inversely correlated with CRP and CRP/Albumin ratio, however, this correlation was not replicated in the validation cohort (Figure 2). Baseline explant TCM lactate concentration did not correlate with TCM CRP concentration. Baseline explant TCM lactate concentration was correlated with IL-2 and IL-31 concentration in both discovery and validation cohorts (Figures 3 & 4). Multivariate analysis, including prior biologic exposure, clinical and endoscopic Mayo scores demonstrated TCM lactate concentration to be independently associated with IL-2 concentration in discovery and validation cohorts, p<0.001 respectively. A similar multivariate analysis demonstrated TCM lactate concentration to be independently associated with IL-31 concentration in the discovery (p<0.001), however, not validation cohort.

Conclusion

TCM lactate concentration is directly correlated with IL-2 & IL-31 in UC explants. The proposed immunoregulatory properties of lactate are in keeping with its positive correlation with TCM IL-2 concentration given reduced IL-2 secretion has previously been associated with UC disease progression. Further studies are required to define the effects of lactate in the UC tissue microenvironment.
P091 Gender focused investigation of sexual dysfunction, sexual satisfaction and body schema disorders in male and female patients with Inflammatory Bowel Disease
Authors: Kranzeder A , Lindenthal D , Hirning C , von Wietersheim J , Klaus J
Published: January 2023

Background

Gender-specific differences in Inflammatory Bowel Disease (IBD) have been reported for epidemiology, pathogenesis, clinical course and disease outcomes. However, when it comes to sexual health deviations regarding both biological sexes, research studies are scarce. In this context, gender could be an important modulating factor. The aim of this study was to investigate gender-specific sexual dysfunction, sexual satisfaction and body schema disorders in male and female patients with IBD.

Methods

A cross-sectional unicentric study was performed. 123 patients with IBD were recruited for 12 months. Of these, 50 patients were male and 73 female. Inclusion criteria included a diagnosed IBD (Crohn's disease or ulcerative colitis), age between 18 - 65 years and sufficient language skills to complete the questionnaires. Data was collected using standardised questionnaries (IBDQ, IBD-F, pMS, HBI, IIEF, BISF-W, FKKS, PHQ15, PHQ9, HADS-D). Sexual dysfunction, sexual satisfaction and body schema disorders were mainly assessed by the „Frankfurter Körperkonzeptskalen“ (FKKS), „The Brief Index of Sexual Functioning for Women“ (BISF-W) and „International Index of Erectile Function“ (IIEF).

Results

Significant differences (p < 0.05) were found for the pMS, FKKS, PHQ15 and PHQ9. Male patients with IBD scored higher in the FKKS, especially in the subscale „Health and physical condition scale (SGKB)“ but also on sexuality- and body-related questions, meaning that they had fewer problems with their sexuality and biological functionality. Due to the small number of only 13 male participants, the IIEF could only be assessed to a limited extent. No significant differences were found here. Female patients with IBD had higher scores in the pMS, PHQ15 and PHQ9, meaning that they suffered more from severe disease activity, somatic impairment and depression. In addition, they scored lower on the BISF-W dimension scores "Arousal" (D2) and "Pleasure/Orgasm" (D5).

Conclusion

Sexual health seems to be impaired in male and female patients with IBD. However, it appears that men are less affected by their IBD than women in terms of sexual well-being, mental health, aesthetics and biological functionality. The reasons behind these results are diverse and cannot be clearly determined. It could be that men are overall more resilient because of their phsyical body structure or because they tend to hide supposed weaknesses.
P093 Creeping fat-derived long chain free fatty acids drive intestinal muscularis propria muscle cell proliferation via carnitine palmitoyltransferase 1 (CPT-1) – a relevant mechanism for stricturing Crohn’s disease
Authors: Le T H N , Mao R , Liu W , Czarnecki D , Chandra J , Gordon I O , Plesec T , Wang J , Lin S , Zhao S , Dejanovic D , Mukherjee P , West G , Fiocchi C , Brown J M , Rieder F
Published: January 2023

Background

Mesenteric fat wrapping around the intestinal wall, so called ‘creeping fat’ (CF), is spatially linked with stricture formation in Crohn’s disease (CD). Intestinal muscularis propria (MP) smooth muscle cell (HIMC) hyperplasia is a major contributor to luminal narrowing in stricturing CD. We investigated CF derived factors and their effect on HIMC hyperplasia in vitro and in vivo using human tissues, primary human cells and a colitis model.

Methods

Secretion of free fatty acids (FFA) by mesenteric fat (MF) or CF organ cultures was determined via lipidomic mass spectrometry. Effects of different length and types of FFA as well as CF and MF conditioned medium on proliferation of primary HIMF was assessed. Next generation sequencing (NGS) and lipidomics on HIMF was performed and relevant pathways inhibited with small molecules or siRNA knockdown. In the dextrane sodium sulfate (DSS) induced colitis model CPT-1 blockade was achieved via the small molecule etomoxir.

Results

Histopathology analysis of intestinal resection tissues revealed CD CF being located in the subserosa and its presence was linked with dramatic thickening of the MP. CF conditioned medium markedly upregulated HIMC proliferation compared to mesenteric fat from CD, UC and normal controls. CF released higher amounts of total, saturated and poly-unsaturated FFA with elevated levels of five long-chain (LC-)FFA, including palmitate. LC, but not medium or short chain FFA selectively increased proliferation in HIMC and fibroblasts, but not other intestinal cell types. NGS revealed gene regulation suggesting LC-FFA transport as a putative mechanism. Lipidomic analysis indicated the majority of palmitate being converted into phospholipids, predominantly phosphatidylcholine. Inhibition of LC-FFA uptake into cells via CD36, metabolism through inhibition of acyl-CoA synthetase, choline-phosphate cytidylyltransferase & choline kinase or blockade of LC-FFA uptake into mitochondria through CPT-1A reduced palmitate and CF conditioned medium induced HIMC proliferation. MP thickness increased in acute DSS colitis. Prophylactic inhibition of CPT-1 with etomoxir in acute DSS colitis did not reduce histopathologic inflammation or inflammatory cytokine gene expression, but reduced MP thickness and gene expression of the smooth muscle cell genes desmin and Sm22.

Conclusion

Subserosal CF releases LC-FFA inducing a selective proliferative response by HIMC. This effect was dependent on CD36, acyl-CoA synthetase and CPT-1. LC-FFA in HIMC are converted into phospholipids. Inhibtion of CPT-1 in DSS colitis reduced the increased MP thickness. These results point to CF as a novel contributor to stricture formation in CD.
P094 Impacts of 5-aminosalicylic acid on the intestinal microbiota leading to the anti-inflammatory effect
Authors: Wada H , Miyoshi J , Kuronuma S , Hibi N , Nishinarita Y , Oguri N , Matsuura M , Takeuchi O , Kobayashi T , Akimoto Y , Hibi T , Hisamatsu T
Published: January 2023

Background

5-aminosalicylic acid (5-ASA) is widely used for the treatment of inflammatory bowel disease (IBD), while its anti-inflammatory mechanisms remain controversial. The intestinal microbiota plays a certain role in the pathogenesis of IBD. We hypothesised that 5-ASA exerts anti-inflammatory effects by altering the gut microbiota. Elucidating the effects of 5-ASA on gut microbiota and its contribution to anti-inflammatory effects can lead to the development of effective and economical IBD medications.

Methods

Specific-pathogen-free (SPF) mice were orally administered 5-ASA for 4 weeks. The fecal bacterial compositions were examined with 16S rRNA gene amplicon sequencing analysis in mice of the 5-ASA-treatment group (5-ASA group) and no-treatment group (NT group). The immune profiles of the colonic mucosa were assessed. The feces from female mice in both groups were gavaged into germ-free (GF) female mice in separated isolators. In each isolator, these female mice (5-ASA dams and NT dams) were mated with GF male mice. The populations of CD4+ T cells in mesenteric lymph nodes (MLN) and colonic mucosal immune profiles were analysed in their pups. The pups were used for the dextran sulfate sodium (DSS)-induced colitis model to evaluate the anti-inflammatory effects of the gut microbiota altered by 5-ASA.

Results

The fecal bacterial compositions changed with 5-ASA treatment over time in SPF mice. At the genus level, the relative abundance of Allobaculum increased significantly in the females of the 5-ASA group compared to the NT group (corrected p < 0.05). The same tendency was observed in male counterparts. The mRNA expression of IL-10 and IL-22 in the colonic mucosa increased significantly in mice of the 5-ASA group compared to their counterparts (p < 0.05 in both sexes). In each isolator, the fecal bacterial compositions were similar between the dams and pups, suggesting that the gut microbiota was vertically transmitted from the dams to the pups in each group. CD4+RORgt+ T cells increased in MLN in the pups of 5-ASA dams compared to those of NT dams (p < 0.01). The mRNA expression of TGF-b, claudin-2 and claudin-3 in the colonic mucosa increased significantly (p < 0.001). The colonic inflammation induced by DSS was milder in 5-ASA pups compared to NT pups (Figure 1).

Conclusion

Orally-administered 5-ASA altered the intestinal microbiota affecting the host immune system and leading to less susceptibility to DSS-induced colitis. Alteration of gut microbiota could be a part of the anti-inflammatory mechanisms of 5-ASA.
P096 Characterization of suppressive immune cell subsets in mouse models of colitis-associated colorectal cancer
Authors: Frigerio S , Saris J , Franco Fernandez R , Koelink P , van Roest M , Wildenberg M , D’Haens G , Grootjans J
Published: January 2023

Background

Chronic colonic inflammation in inflammatory bowel disease (IBD) patients increases the risk of colitis-associated cancer (CAC). Cancer development in CAC is different from that observed in sporadic colorectal cancer (CRC). Most studies have focused on the role of pro-inflammatory immune cell subsets in CAC, yet it is becoming increasingly clear that immunosuppression may also drive cancer progression. We hypothesize that emergence of immunosuppressive cell subsets as a result of chronic intestinal inflammation, dampens anti-tumor immune responses and accelerates the development of CAC.

Methods

To simulate cancer formation in the background of chronic inflammation, mice were treated with azoxymethane (AOM) followed by three cycles of dextran sodium sulphate (DSS). Apc Min/+ mice treated with AOM were used to model sporadic CRC. After 10 weeks, immune cells from the proximal and distal colonic lamina propria, mesenteric lymph nodes (MLNs) and tumors were obtained for microscopy analyses and processed to obtain single cell suspensions for flow cytometry. Immune cell populations were studied by conventional flow cytometry and immunofluorescence.

Results

We observed a significant increase in absolute numbers of PD1+CTLA4+TIM3+ ‘exhausted’ CD4+ T cells in the lamina propria from proximal colons in CAC mice as compared to sporadic CRC mice. Interestingly, these numbers showed a positive correlation with the inflammation score in AOM/DSS, demonstrating that chronic inflammation is associated with the emergence of ‘exhausted’ T cells. In addition, we observed a trend to higher numbers of M2-like (MHCII-CD206+) suppressive macrophages and CD4+FoxP3+ regulatory T cells (Tregs) in tumors from CAC mice compared to sporadic CRC. When analysing T cell immune responses in the MLNs, we found a significant decrease in the ratio IFNγ/IL10-producing CD8+ T cells in CAC mice as compared to CRC mice, suggesting a shift towards an anti-inflammatory T cell response in CAC.

Conclusion

In summary, suppressive immune cell subsets were increased in colonic mucosa and tumors from CAC mice as a result of chronic inflammation, which may be associated with the development of dysplasia. Further functional studies are necessary to prove the role of suppressive immune subsets in IBD-associated dysplasia- and carcinoma progression.
P097 Various regulatory single nucleotide polymorphisms regulate similar biological pathways in Crohn’s disease
Authors: Modos D , Poletti M , Brooks-Warburton J , Bohar B , Madgwick M , Verstockt B , Vermeire S , Carding S , Korcsmaros T
Published: January 2023

Background

Single nucleotide polymorphisms (SNP) associated with Crohn’s disease (CD) are in both protein-coding and non-coding regions of the genome. Interpreting the effect of non-coding regulatory SNPs is challenging. We previously developed the iSNP pipeline, which determines how SNPs in transcription factor (TF) binding sites and miRNA target sites affect cellular regulatory networks and contribute to disease pathogenesis. We successfully used this method in Ulcerative Colitis, and now we developed this method further to study CD.

Methods

We applied the iSNP pipeline to identify CD-associated SNP affected genes in 1695 genotyped CD patients. The original iSNP approach can only analyse the direct interactors of SNP affected genes. Here, we implemented a heat propagation algorithm to uncover further downstream pathways, TFs and regulatory processes involved in the pathogenesis of CD. The heat was propagated from the proteins encoded by the SNP-affected proteins through a signalling network (using OmniPath) to TFs, and from them to their target genes (using DoRothEA).

Results

From the disease causing SNPs on the immunochip data of the patients, we identified 47 regulatory SNPs out of which 39 SNPs were in TF binding sites, 5 were affecting miRNA-target sites and 3 were affecting both. The pipeline predicted these SNPs regulate the expression of 83 proteins; 47 of them were present in the OmniPath signalling network. The network propagation analysis identified 518 proteins that were affected significantly in CD by these 47 proteins. The affected proteins were involved in antigen presentation through MHC-I, WNT signalling, C-type lectin signalling and NF-kB signalling pathways based on Gene Ontology analysis. GNAI1, RIPK2, ATG16L1, CLTC, and CARD9 were affected in 63% of the patients. TF-target gene analysis identified a downstream regulatory network containing 2067 potentially affected genes. These genes were involved in T cell activation, other immune functions, DNA repair and autophagy regulation. Genes encoding MMP9, RARA and SSB2 were affected in 70% of patients.

Conclusion

The affected signalling network showed the known importance of the WNT pathway, autophagy and type I antigen presentation in CD. We extended the list of affected processes with the network propagation modelling approach, and pointed out key functions that were commonly affected in the majority of patients with CD. Thus, patient-specific, various SNP sets converge on the same, few key functions, such as fibrosis and tissue remodelling (through MMP9 or RARA), and DNA repair (through SSB2). Overall, the new network propagation model in the iSNP pipeline pointed out genes affected in the majority of CD patients, highlighting their potential role in CD pathogenesis.