bluuebunny/crossref_metadata_2025 huggingface dataset:
DOP133 Spatiotemporal analysis of Crohn’s disease reveals PECAM2 signaling at the basis of inflammation-to-fibrosis transition
Authors: Ungaro F , Massimino L , Parigi T L , Riva M , Nicolò S , Spanò S , Mino S , Bugatti M , Frontali A , Scarfò F , Vignali A , Municchi A , Villanacci V , Albarello L , Ponzoni M , Solitano V , Jairath V , Peyrin-Biroulet L , Sileri P , Danese S
Published: January 2025

Background

Crohn’s disease is a chronic inflammatory disease of the bowel often complicated by fibrotic strictures(1,2,3). Medical treatment is lacking, and surgery is commonly required(3). The mechanisms underlying the progression from chronic inflammation to fibrosis are not yet defined. We aim to unravel Crohn’s disease pathogenesis using cutting-edge computational and bioinformatics tools (Figure 1).

Methods

Spatial transcriptomics was performed on 13 surgical specimens of inflamed and fibrotic Crohn’s disease and healthy controls. Spatial transcriptomics results were integrated with single-cell data to track the cellular and molecular evolution from healthy intestine to fibrosis by employing CellChat(4) and pseudotime analysis. Computational data were confirmed by immunostaining of tissues from an independent cohort of Crohn’s patients.

Results

We demonstrated that intestinal cytoarchitecture was rearranged while chronic inflammation progressed. Crohn’s disease-associated fibrosis evolved within the mesenchymal compartment, driven by PECAM2 signaling through PECAM1-CD38 interaction. Notably, CD38 positivity was found in the mesenchymal compartment in an independent cohort of Crohn’s disease patients. In parallel, ApoA signaling, particularly APOA1-ABCA interaction, emerged as relevant for maintaining epithelial and stromal homeostasis, while its downregulation was associated with fibrosis development (Figure 2).

Conclusion

Our results provide insights into CD38-driven fibrosis and suggest that PECAM2 signaling blockade could reduce the development of strictures in patients with Crohn’s disease, potentially offering a new treatment target.

References

1.Torres J, Mehandru S, Colombel J-F, et al. Crohn’s disease. Lancet 2017;389:1741–1755.

2.Lichtenstein GR, Loftus EV, Isaacs KL, et al. ACG clinical guideline: management of crohn’s disease in adults. Am. J. Gastroenterol. 2018;113:481–517.

3.Rieder F, Kessler S, Sans M, et al. Animal models of intestinal fibrosis: new tools for the understanding of pathogenesis and therapy of human disease. Am. J. Physiol. Gastrointest. Liver Physiol. 2012;303:G786-801.

4.Jin S, Plikus MV, Nie Q. CellChat for systematic analysis of cell-cell communication from single-cell and spatially resolved transcriptomics. BioRxiv 2023.
P0010 Characterization of proliferating memory CD4⁺ T cells reveals a distinct immune signature in anti-α4β7 integrin therapy non-responders Inflammatory Bowel Disease patients
Authors: Cancino C , Horn V , Obermayer B , Fritz K , Nguyen A , Juhran K , Bösel D , Conrad T , Sonnenberg E , Weidinger C , Siegmund B , Thurley K , Hegazy A
Published: January 2025

Background

Failure to respond to the anti-α4β7 integrin therapy, vedolizumab, is observed in a subset of inflammatory bowel disease (IBD) patients, potentially linked to distinct T cell activation profiles. This study aims to characterize the transcriptional and phenotypic features of circulating memory CD4⁺ T cells in vedolizumab non-responders and their association with treatment resistance.

Methods

We performed multidimensional flow cytometry and single-cell RNA sequencing to analyze IBD patients' circulating memory CD4⁺ T cell compartment, comparing pre- and post-treatment peripheral blood samples from vedolizumab responders and non-responders. Proteogenomic analysis and TCR repertoire profiling of CD4⁺ T cells were conducted using matched peripheral blood samples. Flow cytometry characterized T cell subsets based on surface markers, migration receptors, transcription factors, and cytokine expression, focusing on differences in activation and migration profiles.

Results

Ki67⁺ cells were enriched in the memory CD4⁺ T cell population of vedolizumab non-responders in both UC and CD, confirmed by two independent cohorts. TCR repertoire analysis showed increased diversity in memory CD4⁺ T cells post-treatment. Single-cell sequencing and proteomics revealed these Ki67⁺ cells had a high activation and proliferation profile (HLA-DR, CD40LG, MKI67) with reduced IL7R and SELL expression. They displayed elevated integrins (α4, β1, β7) and gut-homing receptors (CXCR3, CCR6). Upregulated T-bet, EOMES, and RORγt and increased IL-17A, indicated a pro-inflammatory Th1/Th17 phenotype in non-responders.

Conclusion

Ki67⁺ memory CD4⁺ T cells are significantly enriched in the peripheral blood of vedolizumab non-responders, displaying a distinct transcriptional and phenotypic profile characterized by Th1/Th17 polarization, high integrin α4β1 expression, and enhanced gut-homing capacity. These findings indicate that proliferating effector memory CD4⁺ T cells could contribute to therapy resistance in IBD, emphasizing the need for alternative therapeutic strategies targeting these cells in anti-α4β7 integrin therapy non-responders.
P0014 Mesenteric inflammation-associated fibroblasts are critical in the pathogenesis of Crohn’s disease
Authors: Park J , Joo Y H , Song E M , Park Y , Noh G T , Choi E , Byun J , Byun J R , Kim H S , Ahn Y H , Jung S A
Published: January 2025

Background

The Mesentery, initially considered as a structure support of abdominal organs, has emerged as an active participant in the inflammatory processes of Crohn's disease (CD). It serves as a central hub for immune cell activity, including macrophages, T cells, and B cells, driving the propagation of inflammation. Additionally, mesenteric fibroblasts play a crucial role in CD-associated fibrosis. These fibroblasts are activated by pro-fibrotic cytokines such as TGF-β and IL-13, leading to excessive extracellular matrix (ECM) deposition, which contributes to intestinal wall thickening and the formation of strictures. In this study, we aim to explore the specific role of mesenteric fibroblasts in the pathogenesis of CD.

Methods

Magnetic-activated cell sorting was utilized to isolate mesenteric inflammation-associated fibroblasts (IAFs) and normal fibroblasts (NFs) from surgical samples of patients with Crohn's disease (CD) and noncancerous colonic polyps (control group). Various cell-based assays, including cell viability, invasion, and collagen contraction assays, were conducted to investigate phenotypic differences between NFs and IAFs. To assess the impact of NFs and IAFs on colonic epithelium, Co-culture experiments were performed with the isolated fibroblasts and Caco-2 and HT-29 colorectal cancer cell lines using transwell and 3D Matrigel culture systems.

Results

IAFs exhibited a significantly faster growth rate than NFs (Figure 1B; fold change [FC] 1.60 ± 0.04, P < 0.001). Furthermore, IAFs showed markedly higher wound healing (Figure 1C; FC 1.44 ± 0.15, P = 0.004), invasion (Figure 1D; FC 1.45 ± 4.7, P = 0.035), and collagen contraction activities (Figure 1E; FC 1.26 ± 4.73, P = 0.007) compared to NFs. In a 3D Matrigel co-culture system with Caco-2 spheres, abnormal lumen formation with invasive structures of Caco-2 spheres was significantly higher in IAFs compared to NFs (Figure 2A; FC 1.3 ± 0.08, P < 0.001). Similarly, a fluorescein transport assay using a transwell co-culture system revealed that IAFs caused significantly greater epithelial disruption compared to NFs (Figure 2B; FC 1.25 ± 0.04, P = 0.04). Conditioned media from IAFs upregulated the expression of mesenchymal markers, such as ZEB1 (FC 2.67 ± 0.2, P < 0.01) and CDH2 (FC 2.11 ± 0.54, P < 0.05), in Caco-2 cells when compared with NFs (Figure 2C).

Conclusion

These findings suggest that mesenteric IAFs play an active role in the inflammatory and fibrotic processes in the pathogenesis of CD. Targeting mesenteric IAFs and their associated pathways may provide a promising therapeutic approach for managing CD.
P0015 The local intestinal interstitial cytokine profile provides an insight into inflamed bionaive patients with Ulcerative Colitis
Authors: Rogoll E , Seeger A , Zigra G , Wilhelm A , Kräker K , Müller D N , Siegmund B , Haag L M
Published: January 2025

Background

Proinflammatory cytokines identified in the mucosal microenvironment at the site of inflammation are involved in the pathophysiology of chronic inflammatory bowel diseases such as ulcerative colitis (UC). We hypothesize that the local cytokine production is significantly altered in inflamed regions of the intestine and cannot be predicted by plasma measurements. Our goal is to identify personalized cytokine signatures in UC patients' interstitial space depending on their disease progression. This can significantly advance our understanding of UC and support the development of more personalized treatment strategies as cytokine signaling is already a target of current therapies.

Methods

We isolated interstitial fluid (IF) from the intestinal mucosa using the tissue elution method in bionaive UC patients (mild activity (UCEIS 1-4) n = 6; inflamed (UCEIS 5-8) n = 6) and healthy controls (n = 25). We took biopsies from 4 different intestinal segments (rectum, descending colon, ascending colon and ileum) with an average weight of 13,4 mg in 400 mg NaCl for 2h elution in 4°C. Obtained IF was used for multiplex immunoassays of 18 proinflammatory cytokines in the same way as plasma cytokines.

Results

Cytokines such as VEGF, IL-16 and IL-12p40 are present in healthy controls and inflamed patients in the IF. Depending on the disease's severity, more cytokines can be detected. Cytokine concentrations decrease in the more aboral localizations of the colon with the rectum showing a unique cytokine pattern. Cytokines that can be detected in plasma show much lower concentrations compared to IF. Inflamed patients can be distinguished from mild activity in local interstitial profiles. In plasma, different cytokines appear to drive inflammation – those can be identified by linear discriminant analysis, and their composition differs from that in the rectum. Moreover, plasma and rectal interstitial mucosa cytokine patterns correlate well with endoscopic and clinical UC scores.

Conclusion

Isolation of IF is a valuable approach to assessing inflammation in UC patients, improving our understanding of mild residual activity and more severe conditions compared to traditional plasma measurements. We can identify cytokines that drive inflammation in UC and characterize the inflammatory cytokine profile in the intestinal interstitial space associated with a healthy gut. Future targeted therapies should also consider the unique cytokine profiles of individual patients being expressed in the rectum. Comparing bionaive cytokine profiles with profiles from patients treated with biologicals, will allow us to demonstrate how the network changes in response to immunosuppression.
P0016 Comprehensive Characterization of local Cytokine Signatures in Ulcerative Colitis in Deep Remission
Authors: Rogoll E , Seeger A , Gluschke H , Zigra G , Wilhelm A , Kräker K , Kühl A A ,   I.C. The TRR2415, Müller D N , Siegmund B , Haag L M
Published: January 2025

Background

Ulcerative colitis (UC) is a chronic relapsing-remitting disease characterized by distinct molecular patterns and proinflammatory pathways. Through advances in therapy and monitoring-strategies, long lasting remission can be achieved in an increasing number of patients. Our aim is to investigate cytokine signatures in the interstitial microenvironment at protein and RNA level in UC patients showing deep remission (DR) to better understand the response to existing therapies. We hypothesize that the DR forms a distinct group that differs in its local intestinal cytokine profile from mild activity and non-IBD controls.

Methods

Two cohorts of UC patients along with non-IBD controls were characterized. We isolated interstitial fluid (IF) from the rectum and colonic mucosa of 14 UC patients in DR (UCEIS 0 + Mayo 0), 11 in mild activity (UCEIS 1-4 and/or Mayo 1-4) and 25 non-IBD controls as part of the InFlame study (DRKS00031203). Obtained IF was used for multiplex immunoassays of 18 proinflammatory cytokines in the same way as plasma cytokines of this cohort. RNA isolates of colonic biopsies of the other cohort (IBDome) (DR n=22; mild activity n=14, non-IBD controls n=18) underwent bulk RNA sequencing. After normalization, expression profiles of genes corresponding to measured cytokines were compared.

Results

RNA expression of genes corresponding to IL-15, IL-17 and VEGF were significantly upregulated in DR versus non-IBD samples in colonic samples. On the contrary, there were no significant differences in cytokine levels in the IF of the rectum and colon. 14 out of 18 (78 %) genes were differentially expressed between DR and mild activity. Cytokine levels of IL-8, IL-10, IL-15, IL-16, IL-17, IFN-gamma and TNF-a were also upregulated in the IF. VEGF and IL-12B were only significantly upregulated at the protein level. DR and non-IBD controls could not be satisfyingly distinguished in PCA clustering based on interstitial cytokines or the corresponding gene expression. However, PCA clustering could separate DR from mild activity in both datasets. Within the mild activity group, patients with a higher clinical score appeared further away from the DR group. Generally, the characterization of disease activity in the interstitial space was significantly more robust in the rectum than in the descending colon. At the plasma level, DR cannot be distinguished from mild activity, and neither can the healthy state.

Conclusion

Cytokines in the IF can reliably differentiate between UC in different activity stages. Furthermore, RNA expression of corresponding cytokines is correlating. Nevertheless, DR with very stringent criteria seems to resemble non-IBD controls more than UC with mild activity.
P0017 Single-cell transcriptomic analysis reveals insights into the mechanism of action and immune modulation by granulocyte-monocyte apheresis
Authors: Kioroglou D , Cabriada J L , Marigorta U M , Rodríguez-Lago I
Published: January 2025

Background

Granulocyte-monocyte apheresis (GMA) with Adacolumn® is a non-pharmacological therapy used for ulcerative colitis (UC). Despite its clinical efficacy, the precise immunological mechanisms of GMA have not been completely characterized. This study aimed to uncover the immune cell composition and signalling changes induced by GMA using single-cell RNA sequencing (scRNA-Seq) of peripheral blood mononuclear cells (PBMCs) in UC patients.

Methods

Patients with steroid-dependent UC receiving GMA were included. scRNA-Seq was performed on PBMCs obtained at a) immediately pre-treatment, b) post-first session (outflow catheter), and c) 1 month after induction GMA. We applied an in-house standard pipeline for quality control, including batch correction and data integration, followed by differential gene expression analyses to ascertain the alterations in immune cell profiles and regulatory pathways induced by GMA.

Results

Five patients were included and 12 samples were analysed, averaging 8,997 cells and 19,424 genes each. We observed pervasive modulations in immune cell composition, most notably reductions in classical monocytes and expansions in plasmacytoid dendritic cells (pDCs) and Natural Killer (NK) cells. The pathway analyses highlighted downregulation of NF-κB and IL6/JAK/STAT3 signalling across multiple immune cell types, implying widespread decrease in inflammatory activity. This downregulation was particularly pronounced in samples collected immediately after the first session (outflow catheter), suggesting an acute suppression of inflammatory signalling.

Classical monocytes showed reduced expression of key pro-inflammatory genes, including TNF, NFKBIZ, and NFKBIA, indicating suppressed pro-inflammatory potential. In turn, non-classical monocytes displayed upregulation of anti-inflammatory markers such as IL1R2, suggesting a reorientation toward balanced immune control.

By the second visit (one month after induction), we observed a marked return to homeostasis. This included increased expression of immunoglobulin-related genes, such as IGHA1 and IGKC, across various T cell subsets, such as regulatory T cells (Tregs) and tissue-resident memory T cells (Trm). These patterns suggest a heightened capacity for immune regulation and improved balance within the immune response over time.

Conclusion

This first single-cell transcriptomic analysis of GMA in UC patients reveals selective impacts on immune cell composition and signalling, with sustained reductions in inflammation and enhanced regulatory balance over time. Our work underscores the potential of GMA as a targeted therapeutic approach to managing immune dysregulation in UC, paving the way for future exploration into its application and optimization in immune-mediated diseases.
P0018 Fat rich dietary habits: Further increasing IBD risk in first degree relatives
Authors: Koentgen S , Cosier D , Tavakoli P , Leach S T , Dutt S , Ghaly S , Connor S J , Lemberg D A , Lambert K , Hold G L
Published: January 2025

Background

Western diets characterised by high intake of meat and processed food, are implicated in IBD development1. Dietary habits are often shared amongst family members however, where familial diets diverge a difference in IBD risk may arise, compounding in members adhering to western diets. In this study, we compared macronutrient intake and dietary patterns between unaffected first degree relatives (FDRs), their IBD relatives and healthy controls (HCs).

Methods

70 FDRs were recruited within the Australian IBD Microbiome (AIM) study. FDRs were matched ~1:3 to healthy participants (HC; n=200), IBD relatives (adult; n=25 and paediatric; n=25; no other groups contained paediatric participants) were also included for comparison. Macronutrient intake was assessed from 3-day food records. Dietary patterns were computed using principal component analysis (PCA) of food frequency questionnaires (FFQ).

Results

There was no difference in mean daily energy (KJ), total fat or protein intake (g/day) between groups. Paediatric IBD patients matched FDRs in energy intake (8031KJ; □□□□=2788 and 8046KJ; □□□□= 2741 respectively, p>0.9999). Carbohydrate intake (g/day) was significantly higher in the paediatric IBD group than FDRs (p=0.0408), however variance in daily intake was high (paediatric IBD; 210g, □□□□=108g). FDRs had the lowest percentage of energy from carbohydrate intake compared to all other groups (36.4%, p<0.0004). Energy contribution from total fat was highest in FDRs (38.2%); significantly higher than HCs (35.1%, p=0.0063). Energy from protein consumption was consistent among groups (~20%). PCA of FFQs (all participants) produced 5 distinct dietary patterns from 27 food groups, explaining 47.6% of the total variance. These patterns were described as ‘Ultra-Processed’, ‘Balanced Whole Foods’, ‘Processed High Fat’, ‘Meat Centric’, and ‘Sweetened Beverages & Low Fibre’. Adherence to the ‘Processed High Fat’ dietary pattern was significantly higher in paediatric IBD patients and FDRs than HCs (p<0.0362). This dietary pattern was characterised by high consumption of butters/creams, processed meats, pizzas, savoury pastries, sauces/condiments and red meat.

Conclusion

FDRs have unique macronutrient intake and habitual dietary pattern adherence, diverging significantly from HCs. Similarity in adherence to the ‘Processed High Fat’ dietary pattern between paediatric IBD and FDR cohorts may reflect food decision-making by parental FDRs. Macronutrient and dietary pattern analysis indicate fat-rich dietary habits in FDRs; Increased IBD risk may in part be due to fat rich dietary habits.

References

1.Kaplan GG, Ng SC. Understanding and Preventing the Global Increase of Inflammatory Bowel Disease. Gastroenterology. 2017;152(2):313-321. doi:10.1053/j.gastro.2016.10.020
P0021 Long non-coding RNAs as predictors of response to anti-TNF therapy in Ulcerative Colitis patients
Authors: Heydari R , Tavassolifar M J , Derakhshan Nazari M H , Shahrokh S , Meyfour A
Published: January 2025

Background

Long non-coding RNAs (lncRNAs) are transcripts with more than 200 nucleotides that modulate molecular mechanisms in several ways, including epigenetic modification, transcription, post-transcription, translation, and post-translation. We have recently demonstrated the involvement of lncRNAs in the pathogenesis of inflammatory bowel disease (IBD) and their role in the accurate diagnosis of IBD patients 1,2. In this study, we aim to determine the potential of lncRNAs in predicting the response of ulcerative colitis (UC) patients to anti-TNF-α therapy.

Methods

Twenty-two UC patients who were naïve to Adalimumab biosimilar, CinnoRa enrolled in this prospective cohort study. Colonic tissue samples were collected at baseline and week 14 after treatment. Based on our previous reports, a list of important lncRNAs in IBD pathogenesis was prepared and their expression was evaluated by qRT-PCR. Machine learning algorithms were applied to evaluate the predictive potentiality of lncRNAs in differentiating UC non-responders (UCN) from UC Responders (UCR) at baseline.

Results

Among analyzed lncRNAs, significant dysregulation of H19 and TUG1 was observed in UCNs compared to UCRs before receiving anti-TNF therapy. Interestingly, this difference between the two patient groups was still detected in the 14th week after treatment. Machine learning algorithm results and receiver operating characteristic curve analysis demonstrated that these two lncRNAs could appropriately discriminate UCN patients from UCR with more than 90% accuracy and AUC > 0.8.

Conclusion

We concluded that expression profiles of lncRNAs are different in pretreatment lesions of UC patients before receiving anti-TNF therapy. Importantly, lncRNAs are involved in the distinctive molecular response of UC patients to anti-TNF monoclonal antibodies. Furthermore, H19 and TUG1 can serve as biomarkers in predicting the response of UC patients to anti-TNF therapy at baseline.

References

1.

2. Heydari R, Karimi P, Meyfour A. Long non-coding RNAs as pathophysiological regulators, therapeutic targets and novel extracellular vesicle biomarkers for the diagnosis of inflammatory bowel disease. Biomed Pharmacother. Jul 2024;176:116868. doi:10.1016/j.biopha.2024.116868
P0022 Development of primary human fibroblast cell cultures for fibrosis modelling in Inflammatory Bowel Diseases
Authors: Kajári L , Szabó V , Jójárt B , Bacsur P , Molnár T , Farkas K , Maléth J ,   Epithelial Cell Signaling and Secretion Research Group
Published: January 2025

Background

Inflammatory Bowel Diseases (IBD) are chronic immune-mediated diseases of the gastrointestinal tract. Fibrosis is frequent in IBD driven by inflammatory cytokines. During fibrosis, extracellular matrix proteins accumulate in the tissue, causing loss of epithelial function and stenosis, leading to malabsorption and intestinal motility problems. This can ultimately lead to serious complications, strictures, fistulas, or ileus. Anti-inflammatory drugs cannot target fibrosis, only surgical intervention can be used. No relevant primary in vitro cell culture model is available for intestinal fibrosis modelling. Therefore, we aimed to establish and optimize human primary fibroblast cell culture from intestinal biopsies of control and IBD patients. We characterized and compared the fibrosis phenotype and gene expression as well.

Methods

Fibroblast cell cultures were generated from colonic biopsy samples by enzymatic digestion. In a suitable medium, the cells attach to the bottom of the vessel and proliferate, at 90% confluence the cultures were passaged. The phenotype was confirmed by immunofluorescent staining. Target proteins: COL1A1 (Collagen type 1 alpha 1), TGF-ß1 (Transforming growth factor beta), PAI-1 (Plasminogen activator inhibitor type 1), α-SMA (Alpha smooth muscle actin), and PHH3 (Phosphohistone H3). For gene expression analysis RNA was isolated and qRT-PCR technique was used for the following target genes: COL1A1, ACTA2 (Actin alpha 2), TGF-ß1, FN1 (Fibronectin 1), PAI-1, H3C4 (H3 clustered histone 4). We evaluated the collected data and performed statistical analysis.

Results

We can efficiently create and maintain human colon primary fibroblast cell cultures from colonic biopsies of healthy and CD origins. Cell morphology differences were observed from control and diseased patient samples. At the staining, the fluorescent intensity of fibrotic markers (COL1A1, TGF-ß1, PAI1) and the mitosis marker PHH3 were significantly higher in the CD samples, indicating that the cells maintained fibrotic phenotype, and the diseased fibroblasts had a higher proliferation rate. The myofibroblast marker, α-SMA didn’t show a significant difference. Interestingly, significant differences were not noted between the two groups in the qRT-PCR measurements.

Conclusion

In conclusion, we can generate primary human intestinal fibroblast cell cultures and maintain them to passage number 3. Fibrotic protein markers and morphology distinguish the diseased samples from the healthy subjects, therefore it could be useable for in vitro fibrosis modelling. However, the gene expression analysis doesn’t discriminate between the two groups at passage number 3. In the future, we would like to observe the fibrotic properties in earlier passage numbers.
P0024 Circadian regulator-mediated molecular subtypes depict the features of elderly ulcerative colitis
Authors: Zhang Y , Xu J , Cao S , Wang J , Lin Y , Liu Y
Published: January 2025

Background

The prevalence of UC in older individuals (i.e., aged > 60 years) is increasing globally because of an ageing population and a subset of population with elderly-onset (i.e., onset aged ≥ 60 years) UC, which causes high burden on healthcare. Circadian rhythms are 24-h oscillations that control various biological processes in the living system and have been recognized to be associated with the development of autoimmune diseases and aging. However, the implication of circadian rhythm in elderly UC remains largely unknown.

Methods

We downloaded datasets from the Gene Expression Omnibus (GEO) database. The differential expression of circadian regulators between early adult (15-25 years old) and elderly (≥ 60 years old) UC patients was identified. We constructed and compared random forest (RF) and support vector machine (SVM) models. Feature genes were selected to develop a nomogram model with the superior model. The receiver operator characteristic (ROC) curves were used to validate the performances of predictive models and signature genes. The infiltration of 22 kinds of immune cells in elderly UC was analyzed by ssGSEA, and the relationship between feature circadian genes and the proportion of immune infiltration was studied. We identified circadian subtypes based on significantly differentially expressed circadian regulatory genes. Comprehensive evaluation of the two circadian patterns was performed, including immune characteristics and potential functions.

Results

The expression levels of circadian regulatory genes were extracted and a total of 44 differentially expressed circadian regulators were identified between early adult and elderly UC patients. Five feature circadian regulatory genes, AGT, EGF, NR3C2, PPY, and PYY were determined (p < 0.05) to establish a nomogram model that can predict the incidence of elderly UC with the RF model. The signature circadian regulators were found to be correlated with activated memory CD4+ T cells, gamma delta T cells, and macrophages. We identified two circadian subtypes based on the five significant circadian regulatory genes. Cluster A had a higher immune infiltration of activated CD4+T cells, CD8+ T cells and B cells than cluster B (p < 0.01). The signature circadian genes can also be used to predict the risk of elderly UC and have good diagnostic efficacy.

Conclusion

The circadian regulatory genes play non-negligible roles in elderly UC. A nomogram model developed by five feature circadian regulatory genes could be used to predict the incidence of elderly UC. Two circadian patterns were identified and evaluated comprehensively, which may provide insights into the classification of elderly UC patients and guide treatment.
P0033 GPR84, which is increased in Ulcerative Colitis patients, regulates the cytokine secretion of macrophages
Authors: Cejudo-Garcés A , Patel J , Jarén A , Macias-Ceja D , Navarro-Vicente F , Calatayud S , Barrachina M D , Ortiz-Masiá D , Cosín-Roger J
Published: January 2025

Background

Inflammatory Bowel Disease (IBD) is a gastrointestinal disorder comprising Ulcerative Colitis (UC) and Crohn´s Disease (CD) characterized by an exacerbated and chronic immune response. Although GPR84 has emerged as a proinflammatory receptor in some pathologies such as atherosclerosis or reflux esophagitis, its role in IBD remains unclear. We aim to analyze the protein expression of GPR84 in IBD patients and its role in the cytokine secretion in macrophages.

Methods

Surgical resections from UC (n=5) and CD patients (n=5) were obtained. Healthy colon and ileum from colon-carcinoma patients (n=10) were used as controls. Protein expression of GPR84 and CD68 was analyzed by WesternBlot and immunohistochemistry. U937 monocytes were differentiated into macrophages with PMA (0,01μM) during 48 hours and treated with the GPR84 agonist decanoic acid (50-250μM) and a proinflammatory cocktail (0,1μM LPS + 20ng/mL IFNγ) during 24 hours. Some of them were treated with the GPR84 antagonist (10μM) or transiently silenced with a specific siRNA-GPR84. RNA was isolated and gene expression of pro- and anti-inflammatory cytokines was quantified by qPCR. Cytokine levels in supernatant were also quantified by Luminex. Data were expressed as fold induction vs control (mean±SEM) and compared by t-test. A p-value<0.05 was considered statistically significant.

Results

Protein levels of GPR84 were significantly increased in UC patients (150.3±11.75 vs 100.0±11.85), while no differences were observed in CD patients vs healthy ileums. Immunohistochemistry revealed a slight staining of this receptor in intestinal epithelial cells, whereas several cells of the lamina propria were strongly stained. Double immunohistochemistry confirmed that intestinal CD68 macrophages expressed GPR84 in both UC and CD patients. U937-derived macrophages treated with decanoic acid showed a significant increased expression of IL1b (2.68±0.86) and IL8 (2.44±0.48) vs vehicle-treated macrophages (0.998±0.23 and 1.05±0.17, respectively). In addition, IL1b levels in supernatant from macrophages treated with decanoic acid were significantly increased compared with the supernatant from vehicle-treated macrophages (4.50±1.05 vs 1.00±0.19). Moreover, the cotreatment with decanoic acid and GPR84 antagonist significantly reduced the expression of IL1b (0.99±0.29) vs vehicle-treated macrophages (1.94±0.26) and the transiently silencing of this receptor significantly reduced the expression of IL8 (0.89±0.25) vs siCtrl macrophages (4.02±1.59).

Conclusion

GPR84 is increased in UC patients and expressed in intestinal CD68+ macrophages. It regulates the expression of several cytokines pointing GPR84 as a promising pharmacological target for UC patients.
P0034 Glycosylation as new molecular mechanism underlying Pediatric Inflammatory Bowel Disease
Authors: Amorim R , Santos-Pereira B , Dias A M , Sousa Pinto B , Lefeber D J , Espinheira C , Trindade E , Carneiro F , Gaifem J , Pinho S
Published: January 2025

Background

Pediatric Inflammatory Bowel Disease (IBD) can be a highly disabling chronic condition. Genetic, immune and microbial factors have been associated with the development of IBD. Recent studies have shown that changes in glycosylation increase the susceptibility to immune-mediated diseases in adults, including IBD. This study aims to investigate, for the first time in pediatric population, if changes in glycosylation play a role in the immunopathogenesis of IBD, envisioning the identification of novel biomarkers and therapeutic targets.

Methods

We designed a cross-sectional study in which blood samples and fresh colonic biopsies from pediatric patients, either with Crohn’s Disease (CD) or Ulcerative Colitis (UC) or without IBD, were collected at the time of colonoscopy or outpatient visit in a tertiary hospital. T lymphocytes were isolated from colonic tissue and blood, and their glycosylation profile was analyzed by fluorescence-activated cell sorting using specific lectins that recognize different glycans structures and surface antibodies as markers of immune cells (CD45, CD4, CD8).

Results

A total of 80 participants were included, 24 without IBD and 56 with IBD. Among the IBD patients, 23 were diagnosed with Ulcerative Colitis (UC) and 33 with Crohn's Disease (CD). We observed that T lymphocytes from patients with CD exhibit a glycosylation profile alteration distinct from that seen in patients with UC and children without IBD, both at the level of the colon mucosa and in circulation (blood). The levels of a specific free glycan in peripheral blood circulation were higher in patients with Crohn's Disease compared to those with UC and non-IBD (p<0.05).

Conclusion

Our data suggest that CD patients may have a distinctive glycosignature that can be detected in circulation. This may represent a promising tool to discriminate CD patients from UC and non IBD, thus reducing the need for invasive exams in these patients. Overall, our findings may contribute to the identification of a novel diagnostic biomarker with clinical impact in decision-making algorithms, as well as therapeutic targets to improve disease control.
P0036 NRG1, a gene significantly detected from intestinal mucosa eccDNA, may predict therapeutic response in Inflammatory Bowel Disease
Authors: Emoli V , Foscarini E , Di Vincenzo F , Masi L , Troisi S , Pane C , Migliore G , Piazzesi A , Turchini L , Pugliese D , Laterza L , Lopetuso L R , Papa A , Cammarota G , Putignani L , Gasbarrini A , Scaldaferri F , Petito V
Published: January 2025

Background

Many patients with Inflammatory Bowel Diseases do not respond to biological therapies. Extrachromosomal circular DNA(eccDNA) has been showed to play a role in the pathophysiology of IBD, as there is a higher presence of eccDNA in the intestinal mucosa of these patients. By characterizing the gene content of eccDNA, up-regulation of certain genes is observed; one of these is NRG1 (Neuregulin 1), an EGF ligand that guides tissue repair after injury and is associated with tissue plasticity. This gene is located on chromosome 8p12 and it is expressed in macrophages, Paneth cells, and stromal mesenchymal cells of the stem cell niche. In the context of IBD, where chronic inflammation is a hallmark, NRG1 could potentially modulate the immune response to reduce inflammation and promote healing. It is important to understand whether its expression within eccDNA may correlate with a particular course of the disease.

Methods

eccDNA was extracted from intestinal mucosa samples derived from patients diagnosed with IBD and amplified using the Circle-Seq analysis. Following sequencing, bioinformatic interpretation of the data was performed, through which a list of gene fragments present on the circles of individual patients and their localization was obtained. Finally, the presence of the gene found up-regulated by the bioinformatic analysis was confirmed using Real Time PCR.

Results

Starting from 99 intestinal mucosa samples from IBD patients, whose eccDNA was sequenced, circles containing fragments of NRG1 were found in 22 of them; its presence was compared to samples of diseased mucosa biopsies versus healthy mucosa from the same IBD patient. The number of circles from NRG1 is greater in diseased mucosa samples compared to healthy ones, in both UC and CD patients. Furthermore stratifying patients by response to therapy, about 70% of responders to IBD-therapy were NRG1- , with Odds Ratio <1.

Conclusion

Preliminary results show that NRG1, a gene significantly detected from intestinal mucosa eccDNA, may predict therapeutic response in Inflammatory Bowel Disease. Further analysis are needed to confirm these data.

References

1.Di Vincenzo F, Yadid Y, Petito V. et al. Circular and Circulating DNA in Inflammatory Bowel Disease: From Pathogenesis to Potential Molecular Therapies. Cells. 2023 Jul 27;12(15):1953.

2.Møller H.D., Bojsen RK., Tachibana C. et al. Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells. J Vis Exp. 2016 Apr 4;(110):e54239

3.Møller H.D., Mohiyuddin M., Prada-Luengo I. et al. Circular DNA elements of chromosomal origin are common in healthy human somatic tissue. Nat Commun 9, 1069 (2018).
P0041 Assessment of real-world effectiveness of risankizumab in patients with moderate-severe Crohn’s disease in a global setting
Authors: Dignass A , Joshi N , Lukanova R , Dawod F , Jones W , Kligys K , Zambrano J A , Song A , Griffith J , Jairath V , Bossuyt P
Published: January 2025

Background

The primary treatment goals in Crohn’s disease (CD) are to control symptoms, such as abdominal pain and diarrhea, and induce long-term endoscopic remission. The biologic risankizumab (RZB) was approved in 2022 for the treatment of adult patients with moderate-severe CD. As data on RZB utilization and effectiveness are limited, this study aimed to assess the impact of RZB on disease remission and symptom control for optimizing treatment outcomes in patients with CD.

Methods

Data were drawn from the Adelphi Real World CD Disease Specific Programme, a cross-sectional survey with retrospective data collection of gastroenterologists (GIs) in France, Germany, Italy, Spain, the UK, and the USA from Feb 2023–Oct 2024. GIs provided data on their consulting patients with subjectively classified at RZB initiation moderate-severe CD reported demographics, clinical characteristics and symptoms at RZB initiation and at most recent consultation. Analyses were descriptive, Wilcoxon signed rank and McNemar tests were used to compare severity and symptoms at RZB initiation and at most recent consultation, respectively.

Results

Overall, 206 GIs reported data on 333 patients with moderate-severe CD receiving RZB. Mean (standard deviation; SD) patient age was 40.8 (13.0), disease duration was 4.7 (5.8) years; 54% were advanced therapy (AT, comprising biologics and small molecule therapies) naïve and 46% AT experienced (Table 1). Among all patients, 85% received RZB as a monotherapy; 6% received RZB with corticosteroids and 9% with 5-aminosalicylates and/or immunomodulators for; mean (SD) time on RZB among those with a known treatment duration (n=299) was 7.5 (7.5) months.

Among patients receiving RZB for >6 months (n=140), 55% were in GI-reported clinical remission at most recent consultation. Among all patients, GI-reported moderate-severe disease severity significantly reduced from RZB initiation (100% of patients) to most recent consultation (55%; p<0.0001; Fig 1A). Key symptoms experienced at RZB initiation (abdominal pain, 87%; bloody diarrhoea, 29%; non-bloody diarrhoea, 59%; fatigue, 54%; bowel urgency, 41%) significantly reduced at most recent consultation with continued RZB treatment (48%, 9%, 35%, 38%, 20%, respectively; p<0.0001; Fig 1B).

Conclusion

In a real-world global setting, patients with moderate-severe CD treated with RZB experienced a significant reduction in GI-reported disease severity and improvement in key symptoms, and over half of patients were in GI-reported clinical remission. The improvement in symptoms and severity were greater among patients who were treated for a longer duration with RZB. These results highlight the real-world effectiveness of RZB in managing disease activity and symptom burden among patients over time.
P0043 Fecal extracellular vesicles from healthy individuals: A novel therapeutic approach for patients with Inflammatory Bowel Disease
Authors: Ghandian A , Heydari R , Shahrokh S , Meyfour A
Published: January 2025

Background

Inflammatory bowel disease (IBD) is a chronic, relapsing inflammatory disease that consists of two main types: Crohn's disease and ulcerative colitis. One of the main causes of this disease is the disruption of the diversity of the gut microbiome and dysregulation of the interactions between microbes and the human immune system. So far, no definitive cure has been identified for the disease, and current treatments are only for temporary suppression of severe inflammatory responses. One treatment option currently being investigated in clinical trials is fecal transplants from healthy donors to patients with IBD. Extracellular vesicles (EVs), released by various cell types and microbes residingt in the gastrointestinal tract, can play an important role in the disease progression and treatment of IBD. In this study, we investigate the therapeutic potential of extracellular vesicles extracted from the feces of healthy individuals in the in vitro model of inflammatory bowel disease.

Methods

An in vitro model of IBD was established using a transwell co-culture system of Caco-2 intestinal epithelial cells and RAW264.7 macrophages. Inflammation was induced in macrophages using lipopolysaccharide (LPS). Stool samples were obtained from healthy donors and IBD patients. Stools were suspended in Tris-EDTA-based buffer in a 1:5 ratio and centrifuged. The supernatant was filtered and centrifuged at 100,000 x g to isolate EVs. Dynamic light scattering (DLS) and Transmission electron microscopy (TEM) were performed to characterize EVs in size and shape. The effects of EVs isolated from healthy individuals and IBD patients on the intestinal cells and expression of epithelial barrier markers were investigated in the in vitro IBD model.

Results

DLS results revealed that the size of isolated EVs was mainly distributed around 100-200 nm. TEM results showed that the EVs were round- or oval-shaped and their structures did not alter by ultracentrifugation. Inflammation induction using LPS led to changes in the morphology of intestinal cells. However, the shape of cells after treatment with EVs derived from feces of healthy donors was similar to the control group. Intriguingly, the cell shapes shifted toward the inflammatory phase when they were treated with stool-derived EVs of IBD patients. Molecular findings showed that stool-derived EVs of healthy individuals could restore the expression of epithelial cell markers in the inflamed intestinal cells, preserving epithelial barrier function, similar to what was observed in the non-inflamed cells.

Conclusion

Stool-derived EVs of healthy individuals can be introduced as a new solution for the treatment of IBD patients, although further studies on animal models are needed to enter the clinic.
P0057 METTL3 potentiates aged CD4+T cell response via programming effector differentiation in elderly ulcerative colitis
Authors: Zhang Y , Xu J , Cao S , Wang J , Lin Y , Liu Y
Published: January 2025

Background

The burden of IBD in the elderly is increasing globally. However, the mechanisms underlying the age-related IBD susceptibility remain unclear. Aging leads to a progressive functional decline of the immune system, rendering the elderly increasingly susceptible to inflammatory and autoimmune disease. As a fundamental mRNA modification, N6-methyladenosine (m6A) participates in various pathological processes. However, the role of METTL3-mediated m6A RNA modification in elderly IBD remains unclear. Herein, we sought to determine the role of immunosenescence in age-dependent colitis and to explore the underlying mechanisms.

Methods

Young (2-3 months), middle-aged (10-12 months), and aged (20-24 months) mice were established models of colitis by 2% dextran sulfate sodium salt (DSS) treatment. We detected the senescence-associated-β-galactosidase (SA-β-Gal) activity in lymphocytes isolated from colon of mice. Single-cell and bulk RNA-seq of colonic immune cells in mice with different ages were performed to investigate the precise molecular mechanisms. CD4+CD25-CD45RBhi T cell adoptive transfer model was used to further analyze the role of senescent CD4+T cells in colitis. Mettl3fl/lfCd4Cre mice were obtained to explicit role of METTL3 in regulating CD4+T cell senescence.

Results

Ageing increased the severity of DSS-induced colitis. CD4+T cell senescence was implicated in the progression of aging-related experimental colitis, exhibiting an increase of SA-β-Gal and accumulation of CD4+ effector memory T (TEM) cells in colon. Aged colonic CD4+ T cells generate higher levels of IFN-γ and IL-17 comparing to young colonic CD4+ T cells. Single-cell analysis revealed that aging increased colonic CD4+ TEM cells, which were associated with T cell-mediated cytotoxicity and cytokine-mediated signaling pathway. Adoptive transfer of aged CD4+CD25-CD45RBhi T cells to Rag-/- mice induced more severe colitis compared with young CD4+CD25-CD45RBhi T cells transfer. Aged CD4+T potentiates Th1/Th17/Tfh differentiation and exacerbates the progression of colitis. METTL3 was increased in aged colonic CD4+T cells. Inhibiting METTL3 in CD4+T cell protects from cellular senescence, effector differentiation and colitis.

Conclusion

These results provide a significant insight into the contribution of senescent CD4+ T cells to age-dependent colitis and provide an attractive possibility that targeting T cell specifically might be a potential strategy to treat elderly IBD patients.
P0058 Modulation of early innate and adaptive immune responses in primary human intestinal tissue ex vivo
Authors: Beneke V , Grieger K , Dehmel S , Neuhaus V , Linge H , Kulik U , Aselmann H , Gundert B , Braun A , Hesse C , Sewald K
Published: January 2025

Background

Intestinal immune cells are organized within specialized tissue niches shaped by the local environment, including dietary components and microbiota. Dysregulation of these interactions can lead to chronic inflammation, a key feature of inflammatory bowel disease. Precision-cut intestinal slices (PCIS) preserve the intestinal microanatomy, including immune cells. However, functional immune responses in PCIS have not been analysed in detail. Here, we aimed to characterise early innate and adaptive immune responses and evaluate the modulatory effects of the bacterial metabolite butyrate in primary human intestinal slices ex vivo.

Methods

PCIS were prepared from ileum or colon tissue of Crohn's disease (CD) patients or patients with other background diseases (non-CD) and stimulated with lipopolysaccharides (LPS), anti-CD3/CD28 antibodies or Concanavalin A (ConA) ± butyrate (10 mM) for 24h. Secretion of 32 immune mediators was analysed in the supernatant by multiplex ELISA. Tissue viability was measured by LDH- and ATP-assay. PCIS were stained with haematoxylin-eosin for histological imaging.

Results

PCIS maintained typical morphology and viability during culture. Of the 32 immune mediators analyzed, 28 were detected in the supernatant and grouped according to their overall secretion levels. Low secretion (≤ 0.1 ng/mL) was observed e.g. for Th2-associated cytokines IL-4, IL-5 and IL-13 as well as epithelium-associated cytokines IL-18 and IL-33. Moderate secretion (≤ 1 ng/mL) was observed for IL-2, TRAIL, IL-17F, GM-CSF, TNF-α and CCL20. High secretion (≤100 ng/mL) was observed for IFN-γ, granzyme A/B, IL-17A, IL-22, CXCL5 and MCP-1. LPS, ConA and anti-CD3/CD28 upregulated the secretion of mediators in tissue from both non-CD and CD patients. Compared to ConA-stimulated tissue from non-CD patients, ConA-stimulated tissue from CD patients showed increased absolute secretion of IL-2 (log2FC: 1.03), TNF-α (log2FC: 3.32), IFN-γ (log2FC: 1.08), IL-17F (log2FC: 3.29), IL-22 (log2FC: 1.36), IL-21 (log2FC: 1.32) and IL-23 (log2FC: 1.50), though relative upregulation was higher in non-CD tissues. Butyrate reduced the ConA-induced release of proinflammatory mediators (e.g. IL-2, IL-17A: 2-3-fold) independent of the disease background.

Conclusion

Our data show robust induction of early innate and adaptive immune responses in primary human intestinal tissue ex vivo, highlighting PCIS as a promising translational model to study pathomechanisms and treatment responses in a patient-specific manner. Further research into immune cell regulation by butyrate and other microbial metabolites directly in patient tissues could support the development of novel therapeutic strategies for IBD.