Pathogenesis Of Inflammatory Bowel Disease Research Articles

P0001 Single-cell profiling reveals distinct γδ T cell subsets with protective and pathogenic roles in Ulcerative Colitis

Abstract 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.

P1308 The ApaI polymorphism in the vitamin D receptor (VDR) gene affects the phenotype and complications of Crohn’s disease

Abstract Background Vitamin D, through its receptor (VDR), exerts pleiotropic effects that support gut barrier homeostasis. Dysregulation of the vitamin D/VDR signaling is implicated in the pathogenesis of inflammatory bowel disease (IBD).1,2 Our study aims to elucidate how the ApaI, BsmI, TaqI, and FokI single-nucleotide polymorphisms (SNPs) influence disease phenotype and the development of related outcomes. Methods In this observational study, 76 patients with Crohn’s disease (CD) and 68 with ulcerative colitis (UC) were consecutively enrolled. Blood samples were collected, and genomic DNA was extracted. VDR SNPs were genotyped via predesigned TaqMan SNP assays on a Step One Plus PCR system. Fisher’s exact test was used for contingency table analysis. A multivariate logistic regression with backward selection evaluated adverse outcome risk, including variables with p<0.05 from univariate analysis. Results Genotype frequencies for all SNPs were in Hardy-Weinberg equilibrium, with no differences observed in distribution between CD and UC groups. The AA genotype of ApaI was accompanied by higher rates of penetrating (B3) disease in CD (36.7% vs. 8.7%; p=0.006) compared to the Aa/aa genotypes. Conversely, Aa/aa genotype carriers were more likely to develop stenotic (B2) CD (67.4% vs. 40%; p=0.032). The FokI SNP was associated with disease location, with the ff genotype being more common among patients with upper GI involvement (L4) (44.4% vs. 10.4%; p=0.022) and colonic disease (23.8% vs. 2.9%; p=0.018) compared to the FF/Ff genotypes. Additionally, patients harboring the f allele (Ff/ff) experienced higher rates of steroid-refractory or steroid-dependent CD (35.5% vs. 12.9%; p=0.035). In the overall cohort, the AA/Aa genotypes were also linked to failure to respond to steroids (37.6% vs. 11.1%; p=0.011). In CD, the risk of IBD-related hospitalization and surgery was significantly higher in patients homozygous for the dominant ApaI and TaqI SNPs than in those carrying the corresponding recessive alleles. Patients with the AA genotype demonstrated the highest surgery rates, followed by those with the Aa/aa genotype (56.7% vs. 23.9%; p=0.007) and then the aa genotype (56.7% vs. 7.1%; p=0.003), suggesting a possible additive effect of the ApaI SNP. In multivariate analysis, the aa genotype showed a protective effect against hospitalization (aOR=0.17; p=0.013), whereas the TT genotype was identified as an independent risk factor for hospitalization (aOR=4.79; p=0.044). Moreover, the aa genotype was independently associated with a decreased risk of IBD-related surgery (aOR=0.055; p= 0.014). Conclusion VDR SNPs, particularly ApaI, influence disease phenotype, progression, and treatment response in CD.

P1312 Clickable nanozyme enhances colonization of Saccharomyces boulardii by spatiotemporal guidance for ameliorating inflammatory bowel disease

Abstract Background Compelling evidence indicates that dysregulated in the gut microbiota significantly contribute to the progression and pathogenesis of inflammatory bowel disease (IBD). Probiotic therapeutic interventions aimed at modulating the microbiota offer potential alternative approaches for treating IBD, but currently available probiotics often suffer from low intestinal colonization and limited targeting capability. Methods Here, we developed azido (N3)-modified Prussian blue nanozyme (PB@N3) spatio-temporal guidance enhances the targeted colonization of probiotics to alleviate intestinal inflammation. First, IVIS and Photoacoustic imaging demonstrated clickable PB@N3 targets intestinal inflammation, simultaneously, it scavenges reactive oxygen species (ROS). Subsequently, utilizing click chemistry “N3-DBCO” to spatio-temporally guide targeted colonization of dibenzocyclooctyne (DBCO)-modified Saccharomyces boulardii (S.Br@DBCO). The therapeutic effects of PB@N3 + S.Br@DBCO were verified by using the dextran sulfate sodium (DSS)-induced colitis mice model. Weight loss, fecal conditions, colon length and histopathological changes were monitored. Results PB@N3 targets and anchors to inflamed region. The "click" reaction between PB@N3 and S.Br@DBCO has excellent specificity and efficacy both in vivo and in vitro. Despite the complex physiological environment of IBD, "click" reaction can prolong the retention time of probiotics in the intestine. Dextran sulfate sodium (DSS)-induced colitis mice model, demonstrates that the combination of PB@N3 and S.Br@DBCO effectively enhances the colonization of probiotics, restores intestinal barrier function, and alleviates intestinal inflammation. Conclusion This is the first time that “click” chemistry was employed to combine fungal probiotics and nanozyme. PB@N3 spatio-temporal guidance enhances targeted colonization of S.Br@DBCO provides a promising medical treatment strategy for IBD.

P0065 Circulating cell-free DNA and mitochondrial DNA differentiate disease activity in paediatric-onset inflammatory bowel disease: Interim analysis from Scotland-wide Mini-MUSIC study (2020-26)

Abstract Background Mitochondrial dysfunction has been implicated in the pathogenesis of inflammatory bowel disease (IBD) particularly paediatric-onset IBD (PIBD).1,2 Circulating cell-free DNA (cfDNA) and mitochondrial DNA (mtDNA) are damage-associated molecular patterns (DAMPS) released into the bloodstream during tissue damage and cell death and can be detected in peripheral blood samples.3 Recent studies have suggested that levels of cfDNA and mtDNA may correlate with disease activity in adult IBD4 but their utility in paediatric populations remains unexplored. This study aims to present initial data on the potential use of cfDNA and mtDNA as biomarkers in PIBD. Methods Mini-MUSIC and GI-DAMPS are ongoing longitudinal and cross-sectional multi-centre translational research studies in Scotland (2020 – 2026) with a primary aim of investigating the inflammatory mechanisms of IBD with a focus on multi-omics, immune- and microbiome-profiling aligned to prospective clinical follow-up. We measured circulating cfDNA (GAPDH) and mtDNA (ND2), using digital droplet PCR, in the peripheral blood of patients diagnosed under 17 years of age (A1 phenotype) and correlated this with phenotypic data and disease activity scores. Results Sixty-five A1 patients were included (39/65 (60%) male; 51 Crohn’s disease (CD), 6 ulcerative colitis (UC), 1 inflammatory bowel disease unclassified (IBDU) and 7 symptomatic non-IBD controls) with a median (IQR) age at diagnosis of 13 (10.0-15.0) years. mtDNA levels were significantly higher in those with highly active disease compared to those in remission (median [IQR] 145.1 (89.5-673.7 ng/μl) vs 64.4 (24.3-167.5 ng/μl), p=0.008) and compared to non-IBD symptomatic controls (median [IQR] 145.1 (89.5-673.7 ng/μl) vs 28.8 (12.6 – 138.7 ng/μl), p=0.036). Circulating cell-free DNA levels were significantly higher in those with highly active disease than non-IBD controls (4.94 (1.1-9.9 ng/ul) vs median [IQR] 0.53 (0.0-1.4 ng/ul), p=0.017). mtDNA levels were positively correlated with endoscopic disease activity (SES-CD) in Crohn’s disease (Pearson r=0.7619, p=0.004). Conclusion Our initial data from ongoing work, demonstrate that the levels of cfDNA and mtDNA in the blood are potentially valuable biomarkers for differentiating disease activity in PIBD and to identify PIBD-patients where mitochondrial dysfunction is dominant and thereby with the potential to stratify future human mechanistic studies and mitochondrial-targeted therapies.

P1318 Temporal Changes of Gut Microbiota and Micro-RNAs according to Disease Activity in Chronic Colitis

Abstract Background Both gut microbiota and micro-RNAs (miRNAs) have been implicated in the pathogenesis of inflammatory bowel disease. We aimed to investigate the temporal changes and their interactions between gut microbiota and miRNAs according to the disease activity using multi-omics analysis in a murine chronic colitis model. Methods Chronic colitis was induced in the C57BL/6 mice by three rounds of 2.5% DSS exposure. Mice were divided into three groups (n= 8 per group): control group, active group (sacrified at 3 days after the third DSS), and recovery group (sacrificed at 80 days after the third DSS). Clinical activities including weight change and histologic findings of colonic segments were examined. DNA was extracted from mouse feces, and 16S rRNA gene sequencing was performed to evaluate changes in gut microbiota. miRNA expression levels were measured by microarray analysis from mouse colonic tissues. Correlation and network analyses were performed to evaluate the interactions between gut microbiota and miRNAs. Results Alpha diversity indices were increased in the active group compared to control and recovery group. PCoA of metagenomic sequencing showed that the gut microbial profile of the active group was distinctly different from the control group, while the gut microbial profile of the recovery group was similar to the control group. On the other hand, PCoA of miRNA microarray showed that the active group had a significantly different miRNA expression pattern compared to the control group, while the recovery group had a similar miRNA expression pattern to the active group. In taxonomic and miRNA analyses, 147 differential abundant bacterial ASVs and 30 differential expressed miRNAs were identified in the active group compared to the recovery group. Spearman correlation and network analysis between gut microbiota and miRNAs, which showed significant differences between the active and the recovery groups, revealed significant associations between the genera Akkermansia and Phocaeicola and miR-34c-3p, miR-483-5p, miR-200b-3P, and miR-378a-3p. Conclusion In the active phase of chronic colitis, the gut microbial composition and miRNA expression were significantly altered, while in the recovery phase of chronic colitis, the gut microbial profile was similar to that of control, but a significantly different miRNA expression pattern was observed. Specific miRNAs were associated with specific bacterial ASVs, suggesting the interactions between miRNAs and gut microbiota in the development of chronic colitis. Our data suggested significant correlations between the genera Akkermansia and Phocaeicola and miR-34c-3p, miR-483-5p, miR-200b-3P, and miR-378a-3p in the active phase of chronic colitis.

P0137 Primary bile acids promote epithelial necroptosis via Stat1 signaling

Abstract Background Gut barrier dysfunction and microbial dysbiosis play a crucial role in the pathogenesis of Inflammatory Bowel Disease (IBD), including Ulcerative colitis (UC) and Crohn´s disease (CD)1. Although IBD is marked by dynamic changes in the intestinal microbiome, little is known about the corresponding changes in the gut metabolism, the molecular interface between the host and its microbiota. In CD, changes in bile acid (BA) metabolism and composition have been observed, with a notable the enrichment of the primary BA chenodeoxycholic acid (CDCA) specifically in CD2, suggesting a potential involvement in disease pathogenesis. However, it is still poorly understood how BAs influence epithelial homeostasis. Methods Serum bile acid (BA) profiles of CD patients and healthy controls were analyzed using BA-targeted metabolomics. Data normalization and statistical analyses were performed to identify significant differences in BA composition between groups. Human ileal organoids were established from biopsy samples obtained from CD patients, while murine small intestinal organoids were derived from wildtype, Stat1-/-, and farnesoid X receptor (FXR) deficient mice. Bulk RNA sequencing was performed on organoids treated with CDCA to investigate transcriptional changes and identify molecular mechanisms underlying CDCA-induced toxicity. A cell death inhibitor screen was applied to delineate the pathways contributing to CDCA-induced IEC toxicity. Results Our findings reveal a significant difference in BA profiles between CD patients and healthy controls, with a notable enrichment of the primary BA, CDCA, in CD patients. CDCA exerts a pronounced toxic effect on both human and murine small intestinal organoids (SIO) in a dose-dependent manner, whereas the secondary BA, deoxycholic acid (DCA), does not. Importantly, the cytotoxicity of CDCA was markedly reduced when it was conjugated with glycine or taurine, suggesting that conjugation mitigates CDCA's impact on IEC viability. Mechanistically, CDCA disrupts BA signaling pathways, enhances the interferon (IFN) response, and activates cell death pathways within SIO models. CDCA specifically promotes epithelial necroptosis via STAT1 signaling, highlighting a pathway through which CDCA may exacerbate epithelial damage in CD. Therapeutically, both tofacitinib (Tofa) and glucocorticoids (GCs) demonstrate a protective effect, significantly suppressing the CDCA-induced IFN response. These findings suggest that modulation of BA signaling and inhibition of the IFN response may be promising strategies to protect intestinal epithelial integrity in CD patients. Conclusion These findings suggest that modulation of BA signaling may be promising strategies to protect intestinal epithelial integrity in CD patients.

P0171 IL-36 signaling as a drug target in Crohn’s disease patients with IL36RN mutations

Abstract Background The IL-36 signaling pathway has recently been identified as a key regulator of intestinal homeostasis and inflammation. However, the role of mutations in the IL-36R signaling pathway in the pathogenesis of inflammatory bowel disease remains unclear. Methods Mutations in the IL-36 receptor antagonist (IL-36RA, IL36RN) were identified in a cohort of 86 ulcerative colitis patients, 244 Crohn’s disease patients and 45 non-inflamed controls by whole exome sequencing followed by targeted Sanger sequencing. In-depth immune cell profiling of one IL36RN-mutated patient was performed using mass cytometry and multiplexed immunoassays. Overexpression experiments and functional assays characterized identified IL36RN mutations. Clinical and molecular responses of one IL36RN-mutated patient to a combined treatment with the IL-36R-blocking antibody spesolimab and the TNF-blocker certolizumab-pegol were assessed by fecal calprotectin, endoscopy, multiplexed immunoassays and mass cytometry. Results We identified four Crohn’s disease patients with heterozygous missense mutations in IL36RN. Experimental overexpression and functional assays demonstrated that two identified mutations resulted in a reduced protein expression of IL-36RA. In-depth immune profiling of one IL36RN-mutated patient revealed an increased response of PBMCs to IL-36 stimulation and elevated serum levels of IL-36-regulated cytokines. Administration of the IL-36R-blocking antibody spesolimab in combination with certolizumab-pegol over a period of 22 weeks to this patient resulted in a reduction of intestinal inflammation and alterations in immune cell composition and function. Conclusion Our findings indicate that pathogenic IL36RN mutations may contribute to the pathogenesis of Crohn’s disease in a subset of patients and that inhibiting IL-36 signaling in combination with TNF-blockade could offer a personalized therapeutic approach for these patients. Disclaimer Spesolimab was made available based on an out-of-scope request. Boehringer Ingelheim was given the opportunity to review this abstract as a courtesy. Boehringer Ingelheim had no role in the design, analysis or interpretation of the results in this study.

P1097 Phase 1/2 study to assess safety, tolerability, and efficacy of XmAb942 (anti-TL1A) in healthy participants and participants with ulcerative colitis

Abstract Background Tumour necrosis factor-like ligand 1A (TL1A) mediates a broad spectrum of pro-inflammatory and pro-fibrotic effects that have been linked to the pathogenesis of inflammatory bowel disease (IBD), as well as other autoimmune disorders. Recent clinical trials have demonstrated that antagonist antibodies that interfere with the interaction between TL1A and its cognate receptor, Death Receptor 3, can result in clinical remission in the two primary variants of IBD, ulcerative colitis and Crohn’s disease. XmAb942 is an anti-TL1A IgG1 antibody with ablated Fc-mediated effector function that incorporates the Xtend™ half-life extension technology. XmAb942 binds trimeric TL1A with high affinity and specificity and is highly potent across multiple preclinical assays. Allometric scaling of the non-human primate half-life of 23 days predicts a half-life of ≥ 70 days in humans. Quantitative systems pharmacology modelling supports the potential for superior drug exposure with less frequent dosing as compared to first-generation anti-TL1A antibodies. We now present details of the ongoing seamless Phase 1/2 study in healthy participants and participants with ulcerative colitis. Methods Phase 1 (Parts A and B) is a single ascending dose administered intravenously or subcutaneously followed by repeat dosing in healthy participants. Phase 2 (Part C) is in participants with ulcerative colitis with emerging Phase 1 data to determine dosing. Results The primary objectives of Parts A and B are to assess the safety and tolerability of single and repeat doses of XmAb942. Secondary objectives include assessment of pharmacokinetics (PK). Exploratory objectives include assessment of immunogenicity and pharmacodynamics. The primary objective of Part C is to evaluate the effect of repeat doses of XmAb942 on clinical remission in participants with moderately to severely active ulcerative colitis. Secondary objectives include evaluation of endoscopic improvement, endoscopic remission, clinical response, histologic-endoscopic improvement, histologic-endoscopic remission, as well as safety and PK assessments. Exploratory objectives include assessment of immunogenicity and pharmacodynamic effects. Conclusion The seamless Phase 1/2 study is designed to assess XmAb942 as a next-generation, potent, and long-acting anti-TL1A antibody therapeutic with potential best-in-class drug exposure and less frequent dosing. This study is currently enrolling in Australia, with Phase 2 to be expanded globally. Interim data are expected in the first half of 2025. References Clinical trials identification: NCT06619990

P1317 Oral microbiota is linked to the propensity for mucosal healing: A prospective oral-gut-stool microbiome analysis in IBD

Abstract Background The oral cavity is the second most complex human microbial niche and is the conduit through which initial gut microbial colonisation occurs during early life. While the gut microbiome is implicated in the pathogenesis of inflammatory bowel disease (IBD), the role of the oral microbiome remains underexplored. We aimed to characterise the oral-gut microbiome in IBD and its association with disease activity and complete mucosal healing (CMH), a key prognostic marker for long-term remission. Methods All patients with Crohn’s disease (CD) and Ulcerative Colitis (UC) were prospectively recruited via www.musicstudy.uk. We conducted a 3-stage experimental approach. (1) We profiled the oral microbiota in IBD vs healthy controls (HC) (CD = 34, UC = 24 and HC = 25). (2) We carried out trans-anatomical oral-ileal-stool microbial source tracking1 within the same patients at a single time-point (CD = 23; inflamed vs. non-inflamed mucosal samples), alongside longitudinal oral-stool sampling (CD = 32, UC = 17). (3) Our machine learning (ML) modelling integrated stool microbiota taxonomic profiles (CD = 53, UC = 11; sampled at 0, 3, and 6 months) with ~80 clinical variables to classify CMH using random forest, AdaBoost, XGBoost, logistic regression, and neural networks. Results 16S rRNA sequencing revealed significant divergence between IBD and HC oral microbiota (PCoA, Bray-Curtis; PERMANOVA, p=0.003). Active disease also differed from remission (PCoA, Bray-Curtis; PERMANOVA, p=0.01). Higher oral Veillonella abundances were associated with IBD; bacteria commonly enriched in the IBD gut. We determined within patient oral microbiota abundances in gut samples and found no difference between inflamed ileal mucosa compared to non-inflamed (p=0.52). Patients who did not achieve CMH exhibited significantly higher oral microbiota abundances in stool compared to those with CMH (β = 0.046, p=0.022, linear mixed-effects model). Oral microbiota abundances in stool correlated with clinical markers of disease activity, calprotectin (r = 0.23, p=0.009) and albumin (r = –0.28, p=0.002; Benjamini-Hochberg adjusted). ML models integrating stool microbiota taxonomic profiles and clinical variables outperformed clinical markers alone in predicting CMH (AUC: 0.79 vs. 0.55), indicating that the inclusion of stool microbiota features could be valuable in predicting CMH in the clinical setting. Conclusion Our data show distinct oral microbiota taxonomic profiles in IBD and active disease, with higher oral microbiota abundances in stool associated with lack of CMH. These findings highlight the oral microbiome as a potential upstream regulator of microbial-immune crosstalk from the site of IBD-inflammation.

P0021 Long non-coding RNAs as predictors of response to anti-TNF therapy in Ulcerative Colitis patients

Abstract 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.

P0148 Studying the role of adipose tissue in intestinal inflammation using acquired generalized lipodystrophy

Abstract Background Crohn’s disease (CD) is associated with creeping fat, characterized by hyperplasia of mesenteric adipocytes and increased secretion of adipokines, including leptin (1). However, limited models currently exist to study the effect of adipose tissue on intestinal inflammation. Therefore, acquired generalized lipodystrophy and Crohn’s disease (AGLCD), a rare metabolic disorder characterized by the complete loss of fat tissue and intestinal inflammation, could provide a unique model to further elucidate the contribution of adipose tissue to the pathogenesis of inflammatory bowel disease. Methods To decipher the role of adipose tissue, we characterized the immune cell compartment in a patient with AGLCD using mass cytometry, whole exome sequencing and single cell sequencing. Samples collected for the analysis include lamina propria mononuclear cells (LPMCs) isolated from ileum biopsies and peripheral blood mononuclear cells (PBMCs) from blood obtained from 6 CD patients, one AGLCD patient, and 4 non-inflamed controls. Results Using mass cytometry, we were able to identify 17 intestinal immune cell clusters in the LPMCs. Namely, there was a higher frequency of CD8+ CD45RO+ T cells, CD11c+ CD11b+ myeloid cells and CD19+ CCR7+ B cells in the AGLCD sample compared to the fat-proficient CD controls. In addition, single cell sequencing of CD3+ T cells isolated from the blood of the AGLCD patient showed an expansion of CD8+ and CD4+ effector memory T cells compared to a healthy donor, suggesting that adipose tissue plays a role in T cell homeostasis in CD. To confirm that leptin plays a role in the regulation of T cells, we analyzed PBMCs from the AGLCD patient pre and post treatment with recombinant leptin. An increase in the expression of IL-17 producing CD4 T cells were present after treatment with leptin. To further investigate the expansion of CD8+ T cells, analysis of whole exome sequencing of the AGLCD showed a mutation in the NRAS gene, an essential regulator of hematopoietic development. The mutation was confirmed only in CD8 and CD4 T cells in the blood and intestinal biopsies of the AGLCD patient using Sanger sequencing. Conclusion These observations demonstrate that AGLCD is a unique model that can be used to further study the effects of adipose tissue in intestinal inflammation. References (1) Coffey JC, O'Leary DP. The mesentery: structure, function, and role in disease. Lancet Gastroenterol Hepatol 2016;1:238-247.

OP20 Expanding the genetic spectrum of inflammatory bowel disease through large-scale exome sequencing

Abstract Background Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is highly heritable, with more than 50% of the variation in disease risk explained by genetics 1. Genome-wide association studies (GWASs) have identified over 300 IBD-associated loci 2, primarily underpinned by non-coding variants. Initial sequencing efforts have identified rare coding variants associated with CD 3, offering novel insights into disease mechanisms. However, the impact of low-frequency and rare coding variants in IBD remains underexplored. Methods We conducted single variant and meta-analysis across six different whole-exome/genome sequencing datasets. After quality control, 167,172 non-synonymous variants with minor allele frequency (MAF) between 0.0001 and 0.1 in gnomAD v4.1 (Non-Finnish European, NFE) were tested for association across 86,213 IBD cases (44,131 CD, 32,748 UC, and 9,334 IBD-uncertain) and 478,363 controls. Variants with P < 3×10-7 were considered significant. Additionally, very rare (MAF < 0.001) non-synonymous (NSYN) and protein-truncating variants (PTVs) were analysed for gene-level burden differences in each dataset and meta-analysed, with P < 2.5×10⁻⁶ as the threshold for significance. Results In the single variant analysis, we found 55 CD, 17 UC, and 20 IBD associated variants. After checking their linkage disequilibrium (LD) with known signals, 27 (CD), 14 (UC), and 19 (IBD) novel associations were defined (Figure 1). For example, a frameshift variant in TNFRSF6B (E45fs, MAF = 0.00055), missense variants in FAM120B (S431P, MAF = 0.0018) and IFNL2 (H32R, MAF = 0.0045) were significantly associated with CD, UC and IBD, respectively. Our results expand the list of TNF receptors involved in IBD susceptibility, and provide additional support to the role of IFNλ signalling on IBD pathogenesis. The burden test identified 18 genes (Figure 2) with a significantly different burden of very rare NSYNs or PTVs in CD (N = 9), UC (N = 4), or IBD (N = 12) cases versus controls. Among them, NOD2, ATG4C, ADCY7, and ITGAV had orthogonal support from GWASs, either from independent common coding variants or non-coding variants driving gene-expression differences, suggesting the joint contribution of common and rare-coding variants in these genes to IBD susceptibility. In addition, IBD patients showed an increased burden of very rare variants in IL10RA, a known monogenic IBD gene, expanding the phenotypic spectrum to include a more complex form of IBD. Conclusion In conclusion, we report novel rare variants and genes associated with IBD based on the meta-analysis of 86,213 IBD cases and 478,363 controls. This study provides novel insights into the biology of IBD and may prompt further investigation into new therapeutics.

P0189 Microbial and Molecular Profiling of the appendix of pediatric patients with inflammatory bowel diseases

Abstract Background Inflammatory bowel diseases (IBD), including Crohn disease (CD) and ulcerative colitis (UC), are chronic inflammatory conditions influenced by genetic, environmental, and microbial factors.1,2 Emerging evidence suggests a role for the appendix in IBD with immune and microbe impacts on the colon. Peri-appendicular patch (PAP) inflammation is observed in some UC patients; appendectomy has shown protective effects, reducing relapse and colectomy rates in UC.3,4 However, the mechanisms underlying these associations remain unclear. This study aims to explore these mechanisms by characterizing microbial species, molecular pathways, and host-microbiota interactions in the appendix of IBD patients. Methods Mucus from the appendix and non-inflamed colon of 10 pediatric IBD and 5 non-IBD patients undergoing elective surgical resection was collected for DNA and whole-genome sequencing. RNA and DNA from appendix tissues were used for RNA-seq and 16S rRNA sequencing, respectively. Metagenomic analysis was performed across all platforms. Fluorescence in-situ hybridization (FISH) and immunofluorescence defined localization. Results Proteobacteria were significantly enriched in IBD (mean = 27.23%±32%) compared to non-IBD mucus samples (mean = 1.6%±0.23%, p = 0.0052), with reduced bacterial diversity in the appendix of IBD patients. Differential abundance analysis revealed site-specific changes in MetaCyc pathways, notably in the IBD-appendix; for example, ABH and Lewis epitopes biosynthesis pathways were highly enriched in this organ. 38 of 50 MetaCyc pathways were linked to specific species, with E. coli contributing to 32 pathways. Immune and inflammatory pathways were significantly upregulated in the IBD-appendix. Strong correlations were observed between host tissue and microbial pathways in IBD mucus; for instance, Chondroitin Sulfate and Dermatan Sulfate Biosynthesis bacterial pathways were positively correlated with the upregulated host pathway Chemokine Receptors Bind Chemokines (Rho = 0.8851 and 0.88549, respectively), reflecting host-bacteria interaction in the appendix. Bacteria were observed in the mucus layer of IBD-appendix. Conclusion The appendix in IBD exhibits unique molecular and microbial features, offering key insights into its role in IBD pathogenesis and potential therapeutic avenues, reinforcing the efficacy of interventions such as appendectomy in UC.

P0768 Ultra Processed Food in Inflammatory Bowel Disease – Friend or Foe? An examination into the contribution of UPFs to nutrient intake in an IBD remission cohort

Abstract Background Ultra-processed foods (UPFs) and their effect on the gut microbiome and their role in the pathogenesis and activity of inflammatory bowel disease (IBD) are attracting increasing interest. Dietary restrictions in Crohn’s Disease (CD) are common, whether self-imposed or result from adherence to advice from health professionals1. Increasing attention in UPFs may lead to further food restrictions and impact on nutrient intake. This study investigated the intake of UPFs of adults with CD during disease remission and explored their contribution to overall macro and micronutrient intake. Methods 195 CD outpatients in clinical remission (HBI <5 and faecal calprotectin <250µg) were recruited from a single UK IBD centre as part of the INTICO 2 observational study. Seven-day food diaries were electronically recorded (Nutritics® Libro) and used to determine dietary adequacy (number of nutrients <LRNI). UPF intake was determined using NOVA Scoring System2 to determine UPF (NOVA 4) intake by two registered dietitians. Product ingredient lists were examined to assign a NOVA score. Consensus on NOVA Scoring was achieved, and a data dictionary was prospectively updated to justify the assigned score. Results Mean Nova 4 score was 39.8 (SD 15.7) per 7 day food diary. Inadequacies of micronutrient intake were common and often multiple, with a mean of 6(SD 4) micronutrients below the LRNI per subject. UPFs contributed significantly to overall macro and micronutrient intake from food with 54% energy, 60% carbohydrates, 55% fat and 50% fibre intake derived from foods categorised as NOVA 4. There was no association between NOVA 4 intake and the degree of nutrient inadequacy as the number of nutrients below RNI in this cohort (Pearson’s Correlation P=0.629). Figure 1 and 2 describe the contribution of each NOVA group to micronutrients and fibre respectively. Conclusion In a CD remission cohort, UPF intake as captured by NOVA score was comparable with what was seen in other reports of healthy adults and other studies of patients with IBD. Eating more UPFs was not associated with a lower intake of micronutrients. Importantly, food categorised as NOVA-4 contributed to a significant proportion of energy, fibre, and micronutrient intake. NOVA scoring does not differentiate between UPFs high in fat, sugar, and salt and those that are not. Advice to "avoid UPFs" must be balanced against the potential consequences of reducing micronutrient intake, especially in those with low or marginal nutrient intakes. A targeted approach to reduce the intake of UPFs that provide little nutritional value is required to maintain nutrient intake. References 1)Holt D.Q., Strauss B.J. & Moore G.T. (2017) Patients with inflammatory bowel disease and their treating clinicians have different views regarding diet. J Hum Nutr Diet. 30, 66–72 doi: 10.1111/jhn.12400 2)SACN statement on processed foods and health. GOV.UK. https://www.gov.uk/government/publications/sacn-statement-on-processed-foods-and-healthOffice. SACN statement on processed foods and health - summary report. GOV.UK. Published July 11, 2023. https://www.gov.uk/government/publications/sacn-statement-on-processed-foods-and-health/sacn-statement-on-processed-foods-and-health-summary-report#results Figure 1: Micronutrient intake characterised by NOVA score Figure 2: Fibre intake characterised by NOVA Score

P0052 Enhancing gut epithelial integrity and reducing epithelial inflammation through supplementation of fecal metabolites, guided by novel ex vivo functional prioritization and testing

Abstract 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.

DOP124 Fecal content from IBD patients causes more severe epithelial damage than fecal content from healthy donors, which can be moderated by supplementation of metabolites cocktail

Abstract Background Inflammatory bowel disease (IBD) pathogenesis involves the gut epithelia. However, no specific epithelial interventions exist, and models that capture epithelial and human gut variations for pre-clinical screening of therapies are limited Methods Monolayered organoides, derived from human rectal mucosal biopsies, were incubated with fecal samples from Crohn's disease (CD), ulcerative colitis (UC), and healthy control. Organoid viability was quantified by ATP levels and validated by live microscopy imaging. Fecal metabolites were measured using a semi-targeted library of 550 predefined metabolites. Gut bacteria were characterized using 16Sseq. Results Fecal samples were processed by separating the supernatant, heat-killing (HK) the bacteria, and rejoining the samples (Fig. 1A). We generated three pools per group (CD p#1-3, UC p#1-3, controls p#1-3). Each fecal p#1 (CD, UC, and controls) was a mix of ten subjects, and fecal pools p#2-3 were each a mix of five other independent subjects. An additional saliva/oral pool was processed similarly as an independent bacterial/metabolite source. Microbial characterization shows the overall variations of the original and pooled samples, with separation between IBD and control samples (Fig. 1B). We then inculcated the oral or fecal contents pools with differentiated colonoid and compared epithelial viability to untreated organoids and to those exposed to inflammatory triggers. Fecal content from UC and CD patients significantly reduced colonoid viability and ATP metabolism compared to healthy controls (p<0.001, Fig. 1C-D). This effect was mediated mainly by the fecal supernatant that also includes the metabolites, rather than the HK fraction (Fig. 1E). Live microscopy confirmed cell clumping already after 3 hours with the IBD fecal material (Fig. 1F). Characterizing metabolite levels in the joined and supernatant pools and correlating them with disease state (IBD vs. controls) and the colonoids viability outcomes highlighted several metabolites higher in controls and linked with improved viability (Fig. 2A-B). Notably, supplementing these identified metabolites with the fecal content pools, UC p#3 and CD p#2 improved epithelial viability (Fig. 2C-D). Lower metabolite supplementation mediated the improvement of the CD pool, but only higher concentrations resulted in improvement of the UC pools. Similarly, microscopy showed improvement in cell clumping when the IBD pools were supplemented with the prioritized metabolites (Fig. 2E). Conclusion Fecal content from IBD patients was associated with significantly reduced organoid viability and ATP metabolism compared to fecal content from controls, which improved, in part, with supplementation with prioritized metabolites.

P0828 The impact of etrasimod on neutrophils in the ELEVATE UC clinical programme

Abstract Background Neutrophils are thought to play a role in the pathogenesis of ulcerative colitis (UC); they are believed to be the principal source of faecal calprotectin and are a histological marker of disease activity in mucosal biopsies. Etrasimod is an oral, once-daily, selective sphingosine 1-phosphate (S1P)1,4,5 receptor modulator for the treatment of moderately to severely active UC. This post hoc analysis evaluated neutrophils in patients (pts) receiving etrasimod or placebo (PBO) in the ELEVATE UC clinical programme. Methods We assessed percentage changes from baseline (BL) in absolute neutrophil counts (ANC) throughout ELEVATE UC 52 (NCT03945188) and ELEVATE UC 12 (NCT03996369)1 for all pts and those who did or did not achieve clinical remission (clinical remitters) or endoscopic improvement-histologic remission (EIH remitters) at Week (Wk) 12. Transcriptional characterisation followed by cell type deconvolution analysis from bulk RNA-seq was performed on colonic biopsies to assess for neutrophils in clinical remitters vs nonremitters (at Wk 12). Incidence of neutropenia and potential associated infections were assessed. Results BL blood and colonic biopsy samples, respectively, were available for 432 and 340 pts (ELEVATE UC 52) and 353 and 249 pts (ELEVATE UC 12). Pts receiving etrasimod vs PBO had significant percent decreases from BL in ANC from Wk 4 (-14.9% vs -4.9%, p<0.05; ELEVATE UC 52) and Wk 2 (-12.0% vs 0.3%, p<0.05; ELEVATE UC 12); the greatest decrease was seen for pts receiving etrasimod at Wk 12 in ELEVATE UC 12 (-16.8%) and nadir was reached at Wk 20 in ELEVATE UC 52 (-24.5%) and maintained through Wk 52. Wk 12 clinical remitters vs nonremitters receiving etrasimod had significantly greater percent decrease in ANC from BL at most time points (p<0.05; Figure); a similar trend was seen for EIH remitters vs nonremitters. Colonic neutrophils were also significantly decreased at Wk 12 vs BL for pts receiving etrasimod (false discovery rate [FDR] <0.05) but not PBO, and for clinical remitters vs nonremitters receiving etrasimod (FDR <0.05). Across both trials, nine (etrasimod) and four (PBO) pts had neutrophil counts <1 × 109/L at any post-BL assessment. Of these pts, none reported subsequent serious, severe or opportunistic infections; one pt receiving etrasimod experienced a nonserious infection ≤60 days after reported neutropenia. Conclusion Our data support a relationship between decreased circulating and mucosal neutrophil levels and clinical and endoscopic improvement with etrasimod treatment in pts with UC, while showing a low likelihood of neutropenia.

P0816 Dietary habits of ulcerative colitis patients under follow-up in a Pediatric Inflammatory Bowel Disease Unit

Abstract Background A Western dietary pattern characteristics was suggested to have a role in the pathogenesis of ulcerative colitis (UC). Poor dietary pattern can exacerbate the patients’ symptoms and lead to significant nutrient deficiencies. However, data regarding dietary habits and nutritional needs are scarce. Our study aimed to prospectively evaluate dietary intake and habits in peadiatric patients with UC at a Spanish tertiary hospital. Methods In this prospective observational study, peadiatric UC patients were followed up, during which their dietary habits were collected. Patients were asked to complete the KIDMED questionnaire for adherence to the Mediterranean diet (MD) and a 3-day food diary. The degree of food processing was assessed using the NOVA classification system, which classifies all food products into four main groups. Results A total of 47 patients were included, with a mean age of 11.9±3.3 years (57% female), and a median follow-up of 2.8±5.4 years. The mean caloric intake was 1919.07 ± 292.8 kcal, and 55.31% of patients did not cover their energy requeriments. Protein and saturated fat intake exceeded the recommended daily intake (RDI): mean protein intake was 2.13±0.8g/kg (239.06% of RDI), and saturated fatty acids was 31.65±7.9% of total fat. In contrast, fiber, calcium and iron intake were below recommendations: mean fiber was 13.44±2.9g (75.1±24.7% of RDI), calcium was 457.7±239.3mg (41.6±26.2% of RDI) and iron 8.3±3.4mg (75.3±37.2% of RDI). The average KIDMED score was 6.2 points, with only 15 patients (31.9%) presenting high adherence to the MD. Of the remaining patients, 26 (55.3%) needed to improve their diet quality, and 6 (12.7%) had very low diet quality. Regarding the NOVA classification, 7 patients (14.9%) consumed more than 40% of their total energy from ultra-processed foods (NOVA 4), while 23 (48.9%) had less than 40% of their energy intake coming from unprocessed or minimally processed foods (NOVA 1). Conclusion Our results highlight that a substantial proportion of pediatric UC patients have suboptimal dietary patterns, characterized by high intake of UPF and saturated fats, alongside low intake of fiber, calcium, and iron. These findings suggest a need for tailored follow-up with a specialized dietitian within the multidisciplinary care team. Further research is essential to clarify the impact of diet on UC.

P0139 Dynamics of the species structure of the gut microbial community in patients with ulcerative colitis

Abstract Background The potential role of microbiome in the pathogenesis, progression and treatment of ulcerative colitis (UC) is becoming a subject of considerable interest and research. The study of the microbial ecosystem of the colon in patients with UC in different stages of the disease provides an opportunity to identify key taxon-biomarkers for non-invasive monitoring of the severity of UC and the effectiveness of treatment. Methods The study included 21 patients (ages from 20 to 41, 13 men and 8 women) in the relapse stage of UC - left-sided UC with mild to moderate activity (Truelov-Witts scale, Mayo index); and 20 patients (ages from 26 to 52, 10 men and 10 women) in the stage of clinical endoscopic remission. The study of the coprofiltrate microbiota was carried out by NGS sequencing of the structure of the bacterial 16S ribosomal RNA gene using the Illumina MiSeq platform. Results Non-zero values of representation were found in 273 species in patients of the studied groups, however, differences in the presence of microorganisms were statistically significant only for 10 species presented in Table 1. A higher level of Pelomonas_aquatica, Granulicatella_adiacens / para-adiacens, Gemella_morbillorum, Escherichia/Shigella_coli, Enterococcus_azikeevi / casseliflavus / durans / faecalis / faecium / hirae / lactis / mundtii / raffinosus / ratti / rivorum / thailandicus / villorum, Solobacterium_moorei, Streptococcus_australis / parasanguinis and lower levels of Roseburia_inulinivorans, Ruminococcus_bicirculans, Ruminococcus_callidus were found in the relapse group. Conclusion The differences identified in the taxonomic structure at the species level among patients in the relapse and clinical-endoscopic remission stages of UC determine the potential of the colon microbiota in non-invasive assessment of the severity of the disease.

P0086 The role of enteric glial cells in patients with ulcerative colitis.

Abstract Background Enteric glial cells (EGCs) have traditionally been regarded as supportive cells within the enteric nervous system. However, recent studies indicate that, beyond their involvement in neural circuit formation, EGCs possess antigen-presenting capabilities and actively contribute to mucosal defense. In the mouse myenteric plexus, inflammation-inducing stimuli have been shown to trigger the expression of MHC class II on glial cells, which subsequently influences the differentiation of helper T-cell and regulatory T-cell subtypes, thereby maintaining immune homeostasis under inflammatory conditions. Nevertheless, research on EGCs in humans remains limited. Thus, this study aimed to elucidate the function and involvement of EGCs in the pathogenesis of ulcerative colitis (UC). Methods Normal colonic mucosa was obtained from histologically and macroscopically intact areas of patients with colorectal cancer (n=6). Colonic mucosa from UC patients, diagnosed based on established clinical, bacteriological, radiologic, and endoscopic criteria, was also obtained from surgically resected specimens (n=7). Lamina propria mononuclear cells were isolated through enzymatic digestion, and mesenchymal stromal fractions (CD31-CD45-EPCAM-CD90+), containing glial cells, were further isolated using flow cytometry for single-cell RNA sequencing (scRNA-seq) analysis and T cell differentiation assay. Results We identified a specific EGC population (Cluster 3: PDPN+ HLA-DR high) with high antigen-presenting capacity, which was selectively elevated at inflamed sites in UC. scRNA-seq analysis, transcription factor activity analysis, and gene-enriched pathway analysis indicated that IFNγ-STAT1 signaling activation within Cluster 3 enhances MHC class II expression. To assess T-cell effects, naive CD4+ T-cells were co-cultured with PDPN+ HLA-DR high cells isolated from UC samples. This co-culture induced differentiation into IL-10+ IL-17A+ CD4+ T-cells, with a positive correlation observed between the proportions of IL-10+ IL-17A+ CD4+ T-cells and PDPN+ HLA-DR high cells. Bulk RNA sequencing and qPCR analysis further demonstrated that PDPN+ HLA-DR high cells highly express the IL-27 subunit EBI3 and the TGF-β activator integrin αvβ8. To evaluate clinical relevance, we also examined the correlation between PDPN+ HLA-DR high cell numbers and clinical factors in 45 UC patients. Immunofluorescence staining for the glial marker CDH2 and HLA-DR revealed a negative correlation between CDH2+ HLA-DR+ cell numbers and preoperative Mayo endoscopic scores. Conclusion PDPN+ HLA-DR high glial cells are increased in inflamed areas of UC patients, suggesting a role in anti-inflammatory processes and the maintenance of immune homeostasis.