bluuebunny/crossref_metadata_2025 huggingface dataset:
DOP131 Multi-ancestry genome-wide association study reveals new associated loci and nominates disease relevant cell types and novel effector genes for Crohn’s disease and ulcerative colitis
Authors: Fachal L , Zhang R ,   International IBD Genetics Consortium
Published: January 2025

Background

Genome-wide association studies (GWAS) have identified 370 signals associated with inflammatory bowel disease (IBD) and its two main subtypes, Crohn’s disease (CD) and ulcerative colitis (UC). Revealing new association signals and the genes dysregulated by those significantly improves our biological understanding of the disease. However, nominating these genes remains challenging since they are predominantly located within the non-coding genome.

Methods

We performed a multi-ancestry GWAS meta-analysis of 67 cohorts, from European, East and South Asian populations. The final dataset comprised 125,992 IBD patients (59,734 with CD; 57,565 with UC) and >1.2M population controls.

We ran fine-mapping (FM) analyses to define the most likely causal variants driving each signal.

To investigate whether the associated variants are preferentially located at cell-type specific regulatory regions, and thus infer cell-disease susceptibility relationships, we ran heritability enrichment analysis integrating ATAC-seq and single cell (sc) ATACseq data from five studies.

To nominate dysregulated genes, we combined cis expression QTL (eQTL) and sc-eQTL data for >30 relevant tissues and cell types via colocalization analyses; as well as two large protein QTL studies implementing mendelian randomization (MR).

Results

We identified 290 new independent disease signals. While most association signals are shared between CD and UC ~38% were subtype-specific.

IBD susceptibility variants were significantly enriched within regulatory elements of blood and tissue resident T cells, macrophages, and B cells. In contrast, we observed a larger enrichment of UC variants, when compared to CD variants, in regulatory elements of intestinal epithelial cells.

Combining FM, colocalization and MR, we prioritised 626 genes (Table). This set is enriched in well-established pairs of receptor-ligands, such as SERPINE1-ITGAV or VTN-PLAUR. Thirteen of the nominated genes have been previously linked to monogenic forms of IBD, such as CARD11, SHARPIN or IL10RB. Orthogonal lines of evidence provide further support for the effect (and the direction of effect) of the causal variant on disease susceptibility mediated through the molecular phenotype (Fig). For example, three novel risk signals (2 in CD, 1 in UC) mediate higher levels of expression of PDCD1, SELL, and MMP10, respectively. This was echoed by our MR analysis, which suggests that higher levels in serum of PDCD1 and SELL have a causal effect on CD-risk, and MMP10 on IBD-risk.

Conclusion

This study provides new insights into the cellular types by which genetic variants influence disease and nominates genes regulated by disease susceptibility variants, expanding the list of targets to prioritise for therapeutic interventions.
DOP134 Uncovering genetic archetypes to explain patient heterogeneity in IBD
Authors: Gaite-Reguero A , Sanchez-Mayor M , Lao O , Mars Z , Bujanda L , Marigorta U M
Published: January 2025

Background

Genome-wide association studies (GWAS) have identified 300 loci linked to inflammatory bowel disease (IBD) risk, underscoring the genetic complexity of the disease. But these findings do not explain the significant clinical heterogeneity observed across IBD patients. Without clear biomarkers to define subtypes, the clinical impact of these genetic variants remains limited. We hypothesized that distinct genetic archetypes underlie the variability in symptoms observed among patients. We developed a battery of polygenic risk scores to capture these genetic profiles and explore their potential for more precise patient stratification.

Methods

We applied a three-step strategy to develop partitioned polygenic risk scores (PRS) to characterize distinct genetic profiles. First, we used a phenome-wide association approach to classify genetic risk variants into non-overlapping clusters based on their association with traits related with IBD. Second, we used functional genomics datasets to identify partitioned PRSs based on similarity in gene enhancer landscapes, gene expression changes across IBD-relevant tissues, co-expression networks in patients, and publicly available gene ontologies and biological pathways. Finally, we used a genetic algorithm to integrate the abovementioned molecularly-based PRSs and assign patients into independent clusters. This algorithm utilized artificial intelligence principles based on genetic programming, focusing on optimizing the discovery of groups of patients with most genetic differences among them.

Results

Our analyses identified distinct genetic archetypes in IBD patients, revealing clusters associated with specific biological pathways. The phenome-wide approach classified genome-wide significant variants into seven non-overlapping clusters, some linked to inflammatory (e.g. multiple sclerosis) and behavioral traits (e.g. sedentary behaviour) that are relevant to the comorbidity patterns observed in each patient. Through functionally-informed partitioning of genetic risk, we proposed 18,470 novel pathway-specific risk scores adapted for IBD. Notably, 25% of them significantly differentiated patients from controls. Finally, the genetic algorithm integrating these pathway-specific PRSs uncovered patient clusters with unique genetic profiles, highlighting the diverse biological underpinnings of IBD heterogeneity.

Conclusion

By combining GWAS results, functional genomics, and AI algorithms, we uncovered distinct genetic archetypes in IBD patients that reflect diverse biological pathways, including inflammatory and behavioral traits. This strategy improves our understanding of the genetic underpinnings of IBD heterogeneity and may serve to lay the groundwork for more precise patient stratification.
P0001 Single-cell profiling reveals distinct γδ T cell subsets with protective and pathogenic roles in Ulcerative Colitis
Authors: Mayer L S , Arnold J , Reuter N , Pattekar A , Ohtani T , Bruder K , Ribeiro M , Siwicki R , Obwegs D , Stahl E , Kolter J , Mansoori Moghadam Z , Alaswad A , Li Y , Lewis J D , Wu G D , Bewtra M , Tomov V T , Bengsch B , Thimme R , Hasselblatt P , Picelli S , Hofmann M , Sagar S
Published: January 2025

Background

Comprising about 40% of mucosal lymphocytes, γδ T cells represent a significant lineage in the gut, and thus are key players in the intestinal immune homeostasis. In contrast to αβ TCRs, γδ TCRs are able to recognize antigen independent from MHC-restricted antigen presentation. While antigens recognized by γδ TCRs are largely undefined, activation of γδ T cells is initiated by phospho-antigens and members of the butyrophilin (BTN) family. Data from both murine and human studies suggest a protective role of γδ T cells in the pathogenesis of inflammatory bowel diseases (IBD). However, significant species-specific variations of γδ T cells demand more detailed investigation of human intestinal γδ T cells. To date, no single-cell study has comprehensively assessed subset-related roles of γδ T cells in ulcerative colitis (UC).

Methods

In this study, we performed multimodal profiling of intestinal γδ T cells in ulcerative colitis (UC) at single-cell resolution using FLASH-seq (healthy donors (HD) n=2, active UC n=5) and cytometry by time of flight (CyTOF, HD n=29, inactive UC n=26, active UC n=24). FLASH-seq analysis of additional samples is ongoing. Peripheral blood samples were analyzed in a subset of patients.

Results

Our analysis revealed that healthy gut epithelium is predominantly populated by CD103+ intraepithelial γδ lymphocytes (γδ IELs). However, in the inflamed intestinal environment, two distinct although clonally related γδ T cell subsets emerge: one TCF-1+ PD-1+ memory-like subset, resembling stem-like αβ T cells previously described in chronic infections and cancer, and the other exhibiting a cytotoxic phenotype, characterized by increased expression of GZMB, PRF1, and TBX21. The decline in CD103+ γδ T cells, coupled with the expansion of these two subsets, was associated with reduced expression of butyrophilin-like (BTNL) molecules, BTNL3 and BTNL8, and increased expression of BTN3A1 and BTN3A3, along with transcriptional and metabolic changes associated with enhanced γδ T cell-mediated killing in intestinal epithelial cells. Patients responding to therapies showed a return to a healthy γδ T cell subset composition, with the contraction of these subsets after remission, further supporting their pathogenic role. Additionally, significant opposing phenotypic changes were observed in peripheral blood γδ T cell subsets in UC.

Conclusion

Overall, our data provide a comprehensive single-cell landscape of γδ T cells in UC, identifying novel subsets that differentially carry out protective and pathogenic roles. These subsets may serve as valuable biomarkers, and may represent novel therapeutic targets.
P0006 Developmentally endothelial locus-1 promotes intestinal inflammation resolution through inhibition of the Cmpk2-cGAS-STING pathway and regulates reparatory macrophage transition
Authors: Tao M , Chen C , Fu Y
Published: January 2025

Background

Abnormalities in inflammation resolution function are closely associated with chronic inflammation, and proresolution therapies may provide new opportunities for the treatment of inflammatory bowel disease (IBD). Developmental endothelial locus 1 (DEL-1) is an important endogenous molecule that regulates tissue immune homeostasis and promotes resolution of inflammation, but its effect on IBD remains unknown. Here, we aimed to explore the expression and roles of DEL-1 in IBD.

Methods

DEL-1 levels were examined in patients with IBD, mice with colitis, and LPS-induced RAW264.7 cells. DEL-1 overexpression plasmids and/or DEL-1-Fc intervened in cells and mice to explore it effects in the acute and recovery phases of inflammation. Moreover, flow cytometry, RNA-Seq, chromatin immunoprecipitation (ChIP), dual-luciferase reporter assays and 16S rRNA were used to illustrate the potential mechanism of DEL-1 in colitis.

Results

DEL-1 levels were remarkably lower in IBD patients, colitis mice, and LPS-induced RAW264.7 cells, while the levels increased with inflammation to resolve. Transfection with DEL-1 overexpression plasmid and/or DEL-1-Fc intervention reduces levels of inflammatory cytokines in both phases and upregulates reparative gene levels in the recovery phase. Mechanistically, we demonstrated that DEL-1 inhibits Cytosine monophosphate kinase 2 (Cmpk2)-dependent mtDNA synthesis, thereby inhibiting the cGAS-STING pathway to ameliorate intestinal inflammation. Moreover, DEL-1 promoted reparative macrophage transition in the repair model of colitis. Spi1 was identified as a transcription factor that regulates Cmpk2 and the reparative gene Il10. Intervention with overexpression plasmid of Spi1 or Cmpk2 or the STING agonist DMXAA reverses the effects of DEL-1. DEL-1 also inhibits neutrophil recruitment, repairs the intestinal barrier, and improves intestinal microbiota dysbiosis.

Conclusion

This is the first study to show that DEL-1 effectively attenuates colonic inflammation in mice with DSS-induced colitis by inhibits the Cmpk2-cGAS-STING pathway and regulates reparatory macrophage transition, thus providing a novel therapeutic target for IBD.
P0009 Comparative analysis of ileal mucus in healthy individuals and Crohn’s Disease patients using scanning electron microscopy suggests changes in network structure
Authors: Mönch P W , Kramer C , Timm S , Kerkhoff Y , Ludwig K , Ochs M , Siegmund B
Published: January 2025

Background

Ileal mucus is vital for intestinal health, acting as a mesh-like barrier against pathogens while permitting nutrient absorption [1,2]. Structural changes in mucus are linked to diseases like Crohn’s disease (CD) [3,4]. This study used scanning electron microscopy (SEM) to qualitatively and quantitatively compare the ultrastructure of ileal mucus in healthy individuals and CD patients.

Methods

Ileal mucus samples from five healthy donors and three CD patients were collected during colonoscopies and analyzed via SEM. Systematic random sampling ensured unbiased, representative image collection. Images were captured at predefined intervals using a meandering scan pattern. Morphological parameters, including pore diameter and filament properties, were measured manually with ImageJ using an unbiased counting frame. An automated tool was also employed for comparison. Rheological measurements estimated mucus mesh size, which was compared to SEM-derived pore diameters.

Results

Automated analysis of CD samples showed smaller median pore diameters (28.93 nm vs. 33.86 nm in healthy samples) and thicker filaments (71.58 nm vs. 36.73 nm). CD samples also had longer filaments (120.51 nm vs. 93.53 nm) and a mean pore diameter of 46.19 nm, slightly larger than healthy samples (43.73 nm). Structural differences, including altered cellular components like echinocytes, were observed in CD samples. The calculated mesh size from rheological measurements trends aligned with SEM pore diameters, validating the findings. Healthy samples were analyzed manually so far, while CD samples were analyzed using an automated tool under validation for airway mucus.

Conclusion

The study reveals significant structural differences in ileal mucus between healthy individuals and CD patients, offering a robust method for advancing mucosal biology research.

References

1.Johansson, M. E. V., Sjövall, H., & Hansson, G. C. (2013). The gastrointestinal mucus system in health and disease. Nature Reviews Gastroenterology & Hepatology, 10(6), 352-361. https://doi.org/10.1038/nrgastro.2013.35

2.Bansil, R., & Turner, B. S. (2018). The biology of mucus: Composition, synthesis and organization. Advanced Drug Delivery Reviews, 124, 3-15. https://doi.org/10.1016/j.addr.2017.09.019

3.Kramer, C., RulD, H., Ziegler, J. F., et al. (2024). Ileal mucus viscoelastic properties diDer in Crohn’s disease. Mucosal Immunology, 17(4), 713-722. https://doi.org/10.1038/s41385-023-00567-9

4.Atreya, R., Siegmund, B. (2021). Location is important: differentiation between ileal and colonic Crohn’s disease. Nature Reviews Gastroenterology & Hepatology, 18(8), 544-558. https://doi.org/10.1038/s41575-021-00452-3
P0020 Type 1 and CD103+ type 2 conventional dendritic cells are decreased in patients with active ulcerative colitis but not with Crohn’s disease.
Authors: Bernardo D , Arribas-Rodríguez E , G de Castro C , Fiz-López A , De Prado A , Martín-Muñoz A , Fernández-Salazar L , Barrio J , Izquierdo S , García-Alonso F J , de Andrés B , García-Abril J M , Romero A , Sánchez-González J , Arranz E , Chaparro M , Gisbert J P , Garrote J A
Published: January 2025

Background

Human intestinal conventional dendritic cells (cDC) maintain the mechanisms of immune tolerance in health. However, their specific contribution to the development of inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn´s disease (CD) remains elusive

Methods

Human intestinal cDC were identified by flow cytometry in the human intestinal mucosal, and divided into CD103- cDC2, CD103+ cDC2 and cDC1. Their phenotype was determined in health and IBD, together with their stimulatory capacity over naïve T-cells.

Results

cDC1 (minority) and cDC2 (majority) did not change their proportion throughout the healthy gut. However, CD103+ cDC2 were the main subset in the duodenum as opposed to CD103- cDC2 which were predominant in the colon and ileum. CD103+ cDC2 displayed higher levels of PD-L1 together with an enhanced production of IL-10. CD103+ cDC2 also increased their proportion following culture, referred to their CD103- cDC2 precursors, being this process abrogated in the presence of LPS. All human intestinal cDC subsets primed the generation of IL-10+ helper T-cells, being ileal cDC more stimulatory than their colonic counterparts. In IBD, cDC had a constitutive lower expression of SIRPα irrespectively of IBD type (CD/UC) or condition (inflamed/not-inflamed). Of note, the inflamed colon from UC patients, but not from CD, displayed a specific reduction of both cDC1 and CD103+ cDC2, while colonic cDC from CD patients were more stimulatory and primed IL-17+ T-cells in the inflamed tissue.

Conclusion

All human intestinal cDC subsets prime the generation of IL-10+ helper T-cells. The specific reduction of cDC1 and CD103+ cDC2 in the inflamed mucosa from UC, but not CD, suggests the presence of different pathogenic mechanisms occurring in IBD.
P0023 Methionine Restriction Promotes Intestinal Mucosal Repair by Regulating Selenbp1 and Modulating Macrophage Polarization
Authors: Wu L , Chen Y , Chen R , Tang W , Liu Y
Published: January 2025

Background

Intestinal epithelial homeostasis is critical for maintaining gut integrity and function. Methionine is an essential amino acid involved in various cellular processes, and its dietary regulation may impact intestinal injury and repair mechanisms. Selenbp1, an oxidase of the methionine metabolite methanethiol, plays an important role in this process. This study investigates the role of methionine intake and Selenbp1 in colonic injury and epithelial repair.

Methods

Animal models with radiation-induced intestinal injury were used to assess the effects of methionine restriction and supplementation. Intestinal epithelial-specific knockout of Selenbp1 and control mice were used for in vivo experiments. Colonic organoids, macrophage co-culture assays, and biochemical assays, including qPCR, immunoblotting, and flow cytometry, were employed to study epithelial proliferation, SUMOylation of Satb2, and macrophage polarization. Clinical samples from patients with Crohn's disease were also analyzed for macrophage markers, as well as Satb2 and SUMO2/3 expression levels.

Results

Methionine restriction alleviated radiation-induced colonic injury in mice, as evidenced by improved histological scores and reduced inflammatory markers. In contrast, high methionine levels decreased Selenbp1 expression in the colon. Intestinal epithelial-specific Selenbp1 knockout exacerbated radiation-induced colonic mucosal injury, with significantly lower organoid survival rates and budding efficiency post-irradiation. However, this detrimental effect of Selenbp1 knockout was reversed upon methionine restriction, demonstrating the potential of dietary modulation. Furthermore, methionine supplementation led to a reduction in the SUMOylation of Satb2, a key regulator of intestinal epithelial homeostasis. The lower the level of Satb2 SUMOylation, the higher the clinical pathological features of Crohn's disease, such as disease activity. In co-culture assays, methionine supplementation enhanced M1 polarization of macrophages, further indicating a role of methionine in modulating immune responses. In clinical Crohn's disease (CD) specimens, we also found that a higher proportion of M1 macrophages correlated with lower expression levels of Selenbp1.

Conclusion

Methionine restriction promotes intestinal mucosal repair by regulating Selenbp1 and macrophage polarization, suggesting its potential as a therapeutic strategy for colonic repair.
P0025 The effect of very small superparamagnetic nanoparticles on human blood cells as a diagnostic tool in Inflammatory Bowel Disease
Authors: Hussein S , Kühl A A , Golusda L , Plattner C , Heinze N , Sturm G , Freise C , Traube H , Trajanoski Z , Taupitz M , Schnorr J , Siegmund B , Paclik D
Published: January 2025

Background

The field of application of organic or inorganic nanoparticles (1-100 nm size) is extensive and involves their use in medicine. This includes using nanoparticles in human beings or animals (life-stock) which holds advantages but also bears risks. Therefore, nanoparticles used in diagnostics and therapy should be inert not affecting tissues or immune cells. Additionally, nanoparticles should not aggregate to form clusters exceeding the size of 100 nm or accumulate in sensitive structures like bone marrow or brain for long-term deposition.

We developed in-house very small superparamagnetic iron oxide nanoparticles (VSOP) with a size of 7 nm that were successfully used in preclinical magnetic resonance imaging (MRI) for the detection of intestinal inflammation and sclerosis. This study elucidates the effect of nanoparticles on human blood cells, including monocytes as well as monocyte-derived macrophages, in vitro as a first step toward clinical application in inflammatory bowel disease (IBD).

Methods

Whole blood of healthy donors and patients suffering from IBD as well as monocytes and monocyte-derived macrophages were treated with VSOP in vitro and analysed for changes in their transcriptome, phenotype and function. For transcriptome analysis, RNA sequencing was performed. Cell abundance, activation and proliferation were measured by mass cytometry. Migratory behavior of monocytes was analyzed by live microscopy. Cell metabolism was detected by Seahorse assay.

Results

RNA sequencing of monocytes showed that the most significantly regulated gene is encoding for the transferrin receptor (Tcfr). As a response to VSOP treatment and VSOP uptake thereby increasing the levels of intracellular iron, Tcfr is probably downregulated to restrict further iron uptake. While RNA sequencing of whole blood showed only significant differences in non-coding genes (C3orf86P, ENSG00000278996, ENSG00000259002), CyTOF analyses revealed that VSOP-treated monocytes are neither activated nor showing increased proliferation. Also, the migratory behavior is not influenced by VSOP uptake. Additionally, VSOP uptake resulted in an enhanced oxygen consumption rate and extracellular acidification rate. In comparison to the uptake of Latex beads, these increased rates are rather attributed to phagocytosis.

Conclusion

Analyses of our data with PBMC, monocytes and monocyte-derived macrophages suggest, that treatment with VSOP has no major affect on phenotype and function of immune cells. Therefore, VSOP are a promising tool for the early detection of IBD through MRI.
P0028 Discovery of GLP-2R agonists using computational molecular design techniques
Authors: Scagnoli B , Righino B , Pirolli D , Lopetuso L , Chieppa M , Mocci G , Petito V , Scaldaferri F , Gasbarrini A , Papa A , De Rosa M C
Published: January 2025

Background

Computational drug discovery is a powerful, cost-effective approach for accelerating drug development, providing key insights into modulating the GLP-2 receptor (GLP-2R), an important target for gastrointestinal diseases like short bowel syndrome1.

Methods

This study uses diverse in silico molecular design methods, mainly virtual screening2-3, to discover nonpeptidic small-molecule GLP-2R agonists that overcome the drawbacks of peptide-based treatments. The first strategy identifies virtual high-affinity binders on the contact surface between GLP-2 and GLP-2R, which may stabilize the GLP-2R/GLP-2 complex forming hydrogen bonds with both partners to enhance receptor activation. A second strategy uses fragment-based virtual screening to find critical fragments that bind strongly to GLP-2R residues essential for activation. The goal of using these fragments to screen various hit-like and lead-like molecule libraries is to discover small molecules with strong activation potential. Given the difficulty of targeting the large, shallow orthosteric site of GLP-2R with small molecules, a third computational approach focuses on finding small moleculesthat bind to allosteric sites, identified through computational modeling.

Results

In silico studies have led to the identification of a number of potential hit compounds that will undergo experimental testing to evaluate their effectiveness in activating GLP-2R.

Conclusion

This study lays the foundation for further lead optimization, where computational approaches will help find new drug candidates, that may advance specialized therapies for gastrointestinal disorders with limited treatment options.

Fundings

2.1 "Rafforzamento e potenziamento della ricerca biomedica del SSN", finanziato dall’Unione europea – NextGenerationEU, CUP C53C22001140007. 2022 PNRR Project"Changing the future of intestinal failure in intestinal chronic inflammation: towards innovative predictive factors and therapeutic targets" code: PNRR-MAD-2022-12376791.

References

1.Johnson TO, Akinsanmi AO, Ejembi SA, et al. Modern drug discovery for inflammatory bowel disease: The role of computational methods. World J Gastroenterol. 2023;29(2):310-331. doi:10.3748/wjg.v29.i2.310

2.Tripathi NM, Bandyopadhyay A. High throughput virtual screening (HTVS) of peptide library: Technological advancement in ligand discovery. Eur J Med Chem. 2022;243:114766. doi:10.1016/j.ejmech.2022.114766

3.Pirolli D, Righino B, Camponeschi C, Ria F, Di Sante G, De Rosa MC. Virtual screening and molecular dynamics simulations provide insight into repurposing drugs against SARS-CoV-2 variants Spike protein/ACE2 interface [published correction appears in Sci Rep. 2023 Feb 15;13(1):2707. doi: 10.1038/s41598-023-29766-8]. Sci Rep. 2023;13(1):1494. Published 2023 Jan 27. doi:10.1038/s41598-023-28716-8
P0032 Human Extrachromosomal Circular DNA (EccDNA) is a Novel Biomarker in Inflammatory Bowel Disease
Authors: Di Vincenzo F , Petito V , Gerovska D , Piazzesi A , Russo A , Turchini L , Masi L , Lopetuso L R , Regenberg B , Gasbarrini A , Putignani L , Aruzo-Bravo M J , Scaldaferri F
Published: January 2025

Background

Inflammatory Bowel Diseases (IBD), comprising Ulcerative Colitis (UC) and Crohn’s Disease (CD), are chronic, relapsing-remitting disorders of the gastrointestinal tract with unclear etiology. Identifying reliable biomarkers for predicting disease progression and treatment response remains a priority. Extrachromosomal circular DNA (eccDNA) has shown promise as a diagnostic and prognostic biomarker in oncology, but its potential in IBD patients, including the gene fragments involved, has not been extensively studied.

Methods

This monocentric, observational case-control study enrolled IBD patients and healthy controls (HC) undergoing colonoscopy for colorectal cancer screening at our center. IBD disease activity was assessed using the Partial Mayo Score and Mayo Endoscopic Subscore (MES) for UC, and the Harvey-Bradshaw Index and Simple Endoscopic Score (SES-CD) for CD. Colonic biopsies were collected from both inflamed and healthy mucosa of IBD patients, and from HCs. eccDNA was purified using the Circle-Seq method; detection was performed with the Circle Finder pipeline. The study analyzed differences in eccDNA abundances between IBD patients and HCs, focusing on Differentially Produced per Gene eccDNAs (DPpGCs).

Results

In total, 93 IBD patients (38 CD, 39 UC) and 16 HC were included. IBD patients showed a significantly higher abundance of eccDNAs compared to HC, with this increase observed across all chromosomes, suggesting a global upregulation of eccDNA production throughout the human genome. There was also a statistically significant trend towards larger eccDNA fragments in IBD biopsies. Notably, even when considering only eccDNA derived from genic elements ("genic eccDNAs") IBD patients exhibited a significantly higher abundance of eccDNA compared to HCs. Analysis of DPpGCs (fold change >1 log2, p < 0.01) identified 152 genes enriched in IBD, including GRID2 and NRG1. In CD patients, 70 genes, such as FGF14, SOX5, and ZBTB20, were enriched, while UC patients had 123 enriched genes, including NRG1, SPOCK3, and ATP13A4.

Patients in endoscopic remission (SES-CD< 3, MES 0-1) exhibited fewer eccDNAs, though this reduction was not statistically significant. Among identified DPpGCs, key genes included FARS2, HERC3, and PNK2. When analyzing UC and CD seperately, we found a statistically significant increase of eccDNA production in active UC, but not in CD, with NRG1 being notably overrepresented, suggesting its potential as a UC-specific biomarker.

Conclusion

Our study unveils a unique pattern of eccDNA production in IBD. We showed that circles harboring specific genes can distinguish UC and CD from HCs with statistical significance (p<0.01), indicating that eccDNA profiling may represent a novel diagnostic tool for IBD patients.
P0042 Hydrogel-integrated millifluidic system enhances function in primary small intestinal epithelial cells
Authors: Alzain N , Almalla A , Gerbeth L , Ziegler J , Kramer C , Glauben R , Weinhart M , Siegmund B
Published: January 2025

Background

The intestinal epithelium is protected by a vital mucus layer which is essential for maintaining gut homeostasis and regulating host-microbial interactions. However, studying this mucus layer in vitro has been challenging due to the lack of physiologically relevant models.

Methods

We hypothesized that culturing human adult intestinal stem cells (ISCs) under flow conditions on a biomimetic scaffold could better recapitulate in vivo epithelial differentiation and mucus production. To test this, we developed a hydrogel-integrated millifluidic chamber, where ISCs were cultured on decellularized and methacrylated porcine small intestinal submucosa (dSIS-MA) under both static and dynamic (laminar flow) conditions. RNA bulk sequencing was used later to explore gene expression profiles across early and late time points to assess the impact of dSIS-MA and fluid flow on ISC differentiation and function over time.

Results

Our results reveal significant changes in pathways related to epithelial lineage commitment and intestinal barrier function on dSIS-MA compared to tissue culture plastic and in dynamic compared to static conditions, highlighting the critical influence of matrix structure and fluid flow.

Conclusion

In summary, we have successfully established a model that mimics in vivo conditions and reveals that surface and shear stress strongly impact intestinal epithelial cell development and function. This advanced in vitro approach enhances our understanding of molecular processes underlying epithelial cell behavior in conditions closely resembling the gut environment, with applications in gut health and disease research.
P0045 Serologic levels of fatty acids as promising biomarkers for the severity of Inflammatory Bowel Disease
Authors: Cejudo-Garcés A , Patel J , Jarén A , Macias-Ceja D , Boscá-Watts M M , Boronat L , Calatayud S , Barrachina M D , Ortiz-Masiá D , Cosín-Roger J
Published: January 2025

Background

Inflammatory Bowel Disease (IBD) is a chronic and autoimmune disease which affects the gastrointestinal tract. Several changes in the metabolomic profile have been described in urine, blood and feces samples of IBD patients. However, little is known regarding the relationship between these metabolic changes and the severity of IBD. Therefore, we aim to characterize the metabolomic profile, specifically the fatty acid profile, in serum from both active and in remission IBD patients.

Methods

Blood samples from UC active patients (n=20), UC patients in remission (n=20), CD active patients (n=21), CD patients in remission (n=21) and non-IBD controls (n=21) were obtained. UC and CD patients were classified in active or remission according to the Mayo-score and Harvey-Bradshaw Index, respectively. Serum was obtained after centrifugation of the fresh blood. Metabolomic analysis was performed using Liquid Chromatography High-Resolution Mass Spectrometry. Data were expressed as mean±SEM (μg/mL) and compared by a t-test or ANOVA. A p-value<0.05 was considered statistically significant.

Results

A total of 42 fatty acids were quantified and results are shown in Table 1. 23 of them showed significant differences, either Control vs UC, Control vs CD, or CD vs UC. In addition, 9 fatty acids showed significant differences between active CD vs remission CD patients (FA 15:0, FA 16:1n10, FA 17:0, FA 18:0, FA 18:1n12 + FA 18:1n13 + FA 18:1n9trans, FA 20:0, FA 20:3n9, FA 20:4n6, FA 22:4n6). In case of UC, 24 fatty acids showed significant differences in active vs remission patients, especially those with large and very large chain (FA 14:0, FA 15:0, FA 16:1n10, FA 16:1n9, FA 17:0, FA 18:1n12 + FA 18:1n13 + FA 18:1n9trans, FA 18:1n7trans, FA 18:1n9, FA 18:2n6trans, FA 18:3n6, FA 19:0, FA 20:0, FA 20:1n9, FA 20:2n6, FA 20:3n3 + FA 20:3n6, FA 20:3n9, FA 20:4n6, FA 22:0, FA 22:1n9, FA 22:2n6, FA 22:3n3 + FA 22:3n6 + FA 22:3n9, FA 22:4n6, FA 22:5n6 and FA 24:1n9). Moreover, 4 fatty acids, specifically FA 20:0, FA 22:0, FA 22:1n9 and FA 22:4n6, showed areas under the curve higher than 0.8 in the ROC curves (0.87, 0.84, 0.89 and 0.81 respectively).

Conclusion

Differences in serum levels of fatty acids were detected between active and remission patients of both CD and UC. The ROC curves of some of them point these fatty acids as potential biomarkers indicating the severity of IBD in these patients.

Table 1. Levels of the fatty acids in serum of both non-IBD and IBD patients.
P0048 Alterations in circulating T-cell subpopulations reflect disease activity in Ulcerative Colitis
Authors: Profeta F , Rumi G , Notarnicola D , Capobianco I , Masi L , Emoli V , Troisi S , Varuolo M M , Turchini L , Lopetuso L R , Laterza L , Pugliese D , Papa A , Gasbarrini A , Petito V , Scaldaferri F
Published: January 2025

Background

Ulcerative colitis (UC) is a chronic condition characterised by dysregulation of the immune response and consequent uncontrolled inflammation. Circulating T cells play a crucial role in maintaining immune balance and their dysfunction contributes to disease progression1. This study aims to evaluate the expression of circulating T-cell subpopulations in UC patients and explore their association with disease activity.

Methods

Blood samples were collected from UC patients with active disease (partial Mayo score (PMS) > 1), UC patients in clinical remission (PMS 0-1) and healthy controls (HC). A cytofluorimetric analysis using the BD FACSLyric Flow Cytometry system was performed for T-cell subpopulations analysis [table 1]. ANOVA test was used to compare the expression levels of subpopulations across the three groups, while t-tests were conducted for pairwise analysis.

Results

21 UC patients (11 in clinical remission and 10 with active disease) and 10 HC were enrolled. Central memory CD4 T cells resulted significantly reduced in active UC compared to UC in remission and HC (p< 0.05), while effector memory and effector CD4 T cells were markedly elevated in active UC compared to UC in remission and HC. Similarly, central memory CD8 T cells were reduced in active UC, while effector memory CD8 T cells and effector CD8 T cells showed significant increase in active patients compared to patients in remission and HC (p< 0.01). Notably, regulatory T cells were significantly higher in remission UC compared to active disease (p< 0.05) [figure 1]. No significant differences were observed among other subpopulations analysed.

Conclusion

This study demonstrates significant alterations in circulating T-cell subpopulations in UC compared to controls, with distinct differences based on disease activity status. The progression of T cells toward effector phenotypes parallels increased inflammatory activity, linking T-cell differentiation and immune aging to disease progression. These findings underscore the potential of circulating T-cell subpopulations as non-invasive biomarkers for disease activity and as promising therapeutic targets.

References

Casalegno Garduño R, Däbritz J. New Insights on CD8+ T Cells in Inflammatory Bowel Disease and Therapeutic Approaches. Front Immunol. 2021 Oct 11;12:738762. doi: 10.3389/fimmu.2021.738762. PMID: 34707610; PMCID: PMC8542854.
P0049 Bile acid profiling of ileal fluid in Crohn’s disease using metabolomics
Authors: De Greef I , Loopmans S , Bislenghi G , D'Hoore A , Terrasson I , Verstockt B , Sabino J , Ferrante M , Ghesquière B , Vermeire S , Verstockt S
Published: January 2025

Background

An ileal resection is known to cause bile acid malabsorption in Crohn’s disease (CD) 1, but its impact on the bile acid landscape remains understudied. Therefore, we studied bile acid composition in ileal fluid of CD and control patients using targeted metabolomics, and assessed the impact of sample preparation.

Methods

Ileal fluid was collected from CD patients (n=10) and non-IBD controls (n=10) having an ileostomy (patient characteristics in Table 1). Targeted metabolomics (LC-MS/MS) was applied on full ileal fluid samples as well as on their supernatants (ie. after centrifugation of a full fluid sample aliquot). Bile acid raw abundances were normalised to sample weight (g), and only bile acids detected in at least 40% of all samples (per sample type) were included for further analysis. A comparative analysis between full ileal fluid and its supernatants across all samples was assessed by Spearman correlations, while the bile acid abundances between CD and controls was compared using Mann-Whitney U tests. A nominal p value < 0.05 was considered significant.

Results

For the CD group, the median [min-max] length of resected ileum was 15.0 [0.0-33.5] cm and 0.0 [0.0-15.0] cm for the control group (Table 1). Ten primary, 6 secondary and 2 modified bile acids were present in full ileal fluid as well as in the corresponding supernatants. While bile acid abundances were 1.2 to 6.0 times higher in full ileal fluid compared to paired supernatants (Figure 1A), the overall bile acid profiles remained highly consistent. In particular, 13 out of 18 bile acids showed a strong correlation between both sample types (r>0.8, p<0.05), and the remaining 5 showed moderate but significant correlations (r=0.5-0.7, p<0.05). Lastly, while the abundances between the full CD and control groups were similar, a subanalysis comparing CD patients with an ileal resection (n=5) to controls with a total mesorectal excision (n=6), revealed an increase in primary bile acids glycocholic acid (GCA) (p=0.052) and glycochenodeoxycholic acid (GCDCA) (p=0.056) in the CD subgroup (Figure 1B).

Conclusion

In this pilot metabolomics study, we detected both primary and secondary bile acids in the ileal fluid of CD patients and controls. Bile acid composition in full ileal fluid aligns well with its derived supernatant, suggesting supernatant as a viable alternative for measurements, simplifying sample preparation. GCA and GCDCA, both primary bile acids typically reabsorbed in the ileum, tended to be increased after ileal resection in CD patients. A broader screening of bile acid and microbiota metabolites in ileal fluid on a larger scale may unravel novel targets contributing to disease recurrence after resection.

References

(1)Vitek L. Bile acid malabsorption in inflammatory bowel disease. Inflamm Bowel Dis. 2015;21(2):476-83
P0052 Enhancing gut epithelial integrity and reducing epithelial inflammation through supplementation of fecal metabolites, guided by novel ex vivo functional prioritization and testing
Authors: Elwahidi L , Abbas-Egbariya H , Braun T , Amir A , Haberman Y
Published: January 2025

Background

Inflammatory bowel disease (IBD) pathogenesis involves the gut epithelia, showing intensified inflammatory signals and a "leaky gut". However, there are no interventions targeting the epithelia. We aimed to prioritize and test whether fecal content from IBD patients, supplemented with metabolites directed by our functional assays, can reduce epithelial inflammation and improve integrity.

Methods

We established a model system utilizing whole human fecal samples from Crohn's disease (CD), Ulcerative colitis (UC), and healthy control subjects that were co-cultured with Caco-2 epithelial cells. Transepithelial electrical resistance (TEER) measured epithelial barrier integrity, and ELISA measured CXCL1 secretion. 16Sseq characterized fecal bacteria and metabolites were characterized using a semi-targeted predefined library (n=550).

Results

Fecal samples were processed by separating the supernatant, heat-killing the bacteria, and rejoining them. (Fig. 1A). We generated three pools per group (CD p#1-3, UC p#1-3, Controls p#1-3). All p#1 pools included samples from ten subjects, and p#2-3 each included samples from additional five independent subjects. Microbial characterization indicated that the fecal content pools captured the overall variations with separation between IBD and control samples (Fig. 1B). Co-culturing with p#1 revealed a significant increase in CXCL1 proinflammatory chemokine secretion to the media (Fig. 1C), and reduction in TEER (Fig. 1D) which was lower with pools obtained from CD and UC patients compared to the healthy pools. Independent validation with fecal pools p#2 and p#3 showed largely similar results (Fig. 1E-F). Characterizing metabolite levels in the nine pools and correlating them with disease state (IBD vs. controls) and the measured TEER outcomes highlighted several metabolites higher in controls and with improved TEER outcomes (Fig. 2A-B). Supplementing these prioritized metabolites with fecal contents pools, UC p#3 and CD p#2, which were linked with poorer epithelial outcomes, improved epithelial integrity, showing higher TEER when compared with original CD and UC pools (Fig. 2C). Similarly, supplementing UC p#3 and CD p#2 with those same metabolites mix significantly reduced CXCL1 secretion from epithelia in the model (Fig. 2D).

Conclusion

Fecal content from CD and UC patients was associated with more pathologic epithelial signals, which was improved in part with the addition of specific prioritized metabolites. This pipeline and model system highlights the potential future use of metabolite supplementations to improve epithelial function and outcome in IBD.
P0054 Human colonoid-derived monolayers and Caco-2: comparative analysis of inflammatory responses
Authors: Chiara M L , Parente I A , Flammini L , Martina V , Vacondio F , Federica G , de’Angelis G L , Elisabetta B , Bertoni S
Published: January 2025

Background

Given the increasing incidence of inflammatory bowel diseases, there is a growing need for reliable preclinical models that replicate the inflamed intestinal epithelium. Immortalized Caco-2 cells are widely considered the standard in vitro model for intestinal studies, but human-derived colonoid monolayers (CDMs) could be proposed as a powerful alternative to more accurately mimic the pathophysiological features of intestinal inflammation. The present study aims to compare the functional and morphological responses induced by inflammatory cytokines TNFα and IFNγ on CDMs and Caco-2.

Methods

Human colonic crypts from biopsies of 5 healthy volunteers were embedded in Matrigel® and cultured for 5-7 days. IntestiCult™ OGM was used to expand cystic-like colonoids, and single cells were seeded onto coated 24-well inserts. Upon reaching confluence, the medium was switched to IntestiCult™ ODM. Caco-2 cells were cultured for 21 days on 24-well inserts. Differentiated CDMs and Caco-2 monolayers were exposed to 25 ng/mL TNFα-IFNγ for 24 hours. Cell differentiation was assessed by immunocytochemistry (ICC), and barrier integrity was evaluated by transepithelial electrical resistance (TEER) measurement. Cytotoxicity was assessed through the LDH assay. IL-8 and CCL20 release were quantified.

Results

Both models showed increasing TEER over the growth period, indicating the gradual development of epithelial barriers, up to 484±58 Ω*cm2 for CDMs and 700±184 Ω*cm2 for Caco-2. ICC staining for epithelial markers villin and MUC2 confirmed polarization and differentiation under basal conditions in both models, cytokines reduced the expression of epithelial markers. Inflammation reduced the barrier integrity of the monolayers (approximately 18%), although more uniformly in Caco-2 than in CDMs. IL-8 and CCL20 release was augmented by cytokines in both models, (for IL-8 of about 20 times in CDM and 2 times in Caco-2). As regards cell viability, CDMs (40% cytotoxicity) showed higher sensitivity to cytokines than Caco-2 (22% cytotoxicity).

Conclusion

Exposure to an inflammatory environment altered cell morphology and impaired epithelial barrier integrity in both CDMs and Caco-2 monolayers. These findings highlight the potential of CDMs as an advanced model for studying personalized responses in intestinal inflammation.
P0059 Mechanistic understanding of MB310: a consortium of gut commensal bacteria for the treatment of Ulcerative Colitis
Authors: Schreiber F , Villemin C , Sharpington T , Lawley T D , Robinson M J , Bakdash G
Published: January 2025

Background

Microbiotica is developing MB310, a live biotherapeutic product consisting of 8 bacterial strains for the treatment of ulcerative colitis (UC). These were selected based on association with clinical responses in UC patients in a fecal microbiota transplantation (FMT) clinical study. To understand how MB310 drives clinical benefit, we tested their impact on epithelial barrier repair, innate immune cells, and CD4+ T lymphocytes, which are key to the pathology of UC. We also investigated the effects of MB310 bacterial metabolites on the induction of regulatory T (Treg) cells that can counteract UC-associated inflammation.

Methods

Effects on barrier function were assessed by measuring electric impedance of Caco-2 cells in the presence or absence of lipopolysaccharide (LPS). Effects on human dendritic cells (DCs) and M1 macrophages were assessed by determining their cytokine production profile. The effects of MB310 or its metabolites on CD4+ T cells were assessed by measuring their expression of the Treg markers Foxp3 and interleukin (IL)-10 following polarization with MB310-treated DCs. Finally, the induced Treg cells were functionally validated in a T cell suppression assay.

Results

Investigating barrier effects, we observed that five MB310 strains individually increased the integrity of a gut epithelial cell monolayer, and 2 strains repaired the monolayer following damage by LPS challenge. Furthermore, primary human monocyte-derived M1 macrophages and LPS-activated DCs showed an increase in the release of the anti-inflammatory cytokine, IL-10 in comparison to the pro-inflammatory cytokine tumor necrosis factor (TNF), following exposure to all MB310 strains. The MB310-treated DCs were able to enhance IL-10 production by T cells. Investigating the mechanisms of Treg induction, we revealed that the bacterial metabolites from two MB310 strains induced DCs to polarize two distinct subsets of actively suppressive Treg cells: Foxp3+ Tregs and IL-10-producing Tregs. Another MB310 species induced Foxp3+ suppressive Treg cells through direct effect on CD4+ T cells.

Conclusion

The accumulated data suggest that the strains of MB310 interact with the host to oppose key UC disease pathologies by improving gut epithelial barrier integrity; promoting an anti-inflammatory profile of innate immune cells; and inducing Treg cells. Moreover, the metabolites of some MB310 strains lead to induction of different types of functionally suppressive Treg cells. This highlights that multifaceted effects of MB310 that can circumvent the challenge of UC disease heterogeneity, proposing it as a potential therapeutic for a wide spectrum of UC patients. COMPOSER-1 is an ongoing phase 1b trial testing MB310 in UC patients.