Pathogenesis Of Inflammatory Bowel Disease Research Articles

P131 Therapeutic effects of KPG-818 in a trinitrobenzene sulfonic acid - induced mouse Crohn's/Ulcerative Colitis model

Abstract Background Immunomodulatory drugs have the potential therapeutic benefit not only in malignancy but also in diseases characterized by an inflammatory phenotype. In inflammatory bowel diseases (IBD), colonic inflammation results from dysregulation of the mucosal immune responses to intestinal flora together with genetic and environmental factors. Inflammatory cytokines are implicated in pathogenesis of IBD. Treatments for IBD that improve clinical outcomes and limit adverse effects are needed. KPG-818 is a novel generation of lenalidomide derivative and small molecule immunomodulatory drug. KPG-818 is currently under clinical development for therapy of multiple myeloma, lymphoma (phase 1), and systemic lupus erythematosus (SLE) (phase 2a). Remarkable immunomodulatory effects of KPG-818 are observed in SLE patients. In preclinical studies, KPG-818 significantly inhibits inflammatory cytokines interleukin-6 and TNF-α in human peripheral blood mononuclear cells, inhibits tumor growth in multiple myeloma and lymphoma xenograft tumor models. Here we report the significant therapeutic effects of KPG-818 in a chronic Crohn’s/ulcerative colitis mouse model. Methods Trinitrobenzene Sulfonic Acid (TNBS) was intra-colonially administered to induce Balb/c mouse Crohn’s/ulcerative colitis model at 50 mg/kg, once a week for 4 weeks. KPG-818 was orally administered at doses of 0.03, 0.12, 0.4, and 1.0 mg/kg, once a day for 6 consecutive weeks. Upadacitinib served as a positive control drug. Vehicle solution served as a negative control. To evaluate the therapeutic effects of the KPG-818, disease activity index (DAI) of ulcerative colitis, the degree of weight loss, fecal morphology, and rectal bleeding were scored once every other day for six consecutive weeks. At the end of the study, colonic samples were collected for gross observation and scoring of macroscopic damage, measurement of length and weight, and histopathological diagnosis and scoring. At the end of study, peripheral blood samples were also collected for analyses of immune cells and cytokines. Results Compared to the vehicle control, KPG-818 significantly improved the body weight loss, disease activity of ulcerative colitis, gross degree of colorectal injury, pathological lesions of colorectal tissue, inhibited the inflammatory cytokines in a dose-response manner. The effective dose was as low as 0.03 mg/kg. KPG-818 showed similar therapeutic effects to Upadacitinib. Conclusion In this mouse model of chronic Crohn’s/ulcerative colitis, KPG-818 showed remarkable therapeutic effects in a dose-dependent manner. These data encourage the clinical evaluation of KPG-818 in inflammatory bowel diseases.

P043 RIPK3 – a new marker for assessment of activity in Crohn’s disease

Abstract Background RIPK3 (Receptor Interacting Protein Kinase-3) is a key molecule involved in the process of necroptosis. Necroptosis is a regulated form of cell death that involves the activation of specific signaling pathways leading to cell lysis. The released endogenous molecules - DAMPs (Danger Associated Molecular Patterns) activate the immune system and lead to inflammation. This relationship suggests a role for necroptosis in the pathogenesis of IBD (Inflammatory Bowel Disease). Methods The aim of the study was to evaluate RIPK3 expression in patients with Crohn's disease (CD) according to disease activity and some clinical parameters. Tissue expression of RIPK3 in the inflamed areas of 85 patients with Crohn's disease was determined immunohistochemically. The CDAI (Crohn’s Disease Activity Index) was used to assess activity, and the Montreal Classification to determine disease location and bahavior. Results A correlation was found between RIPK3 expression and CDAI (p=0.038), with patients in remission having the lowest expression (154.67), while patients with severe activity had overexpression of the marker (180.0) (Fig.1). According to the localization of CD, a significant difference was found in the expression of RIPK3 (p<0.05), with the highest expression of the marker in the ileum (L1) and colon (L2) with involvement of the upper GIT (L4) – L1+L4 (225.50) and L2 + L4 (218.00). Isolated involvement of terminal ileum (L1) was characterized by the lowest expression of the marker, while patients with colonic localization (L2) had high expression of RIPK3 (184.87). Regarding disease behavior, it was found that the highest expression of the marker is in the patients with phenotype B2+B3 – both stricturing and penetrating disease, followed by inflammatory disease (B1) (Fig.2). Perianal disease further increased RIPK3 expression, with patients with concurrent perianal disease showing high expression of the marker (181.85 vs. 167.86 for those without perianal disease; p<0.05). Conclusion High RIPK3 expression in CD patients correlated with severe disease activity as measured by CDAI, involvement of upper GIT - L1+L4 and L2+L4, stricturing with penetrating disease - B2+B3 and concurrent perianal disease.

P012 Intestinal epithelial cells as targets of thiopurines in Inflammatory Bowel Disease paediatric patients-derived organoids

Abstract Background Thiopurines, azathioprine (AZA) and mercaptopurine (MP), are immunomodulators used to maintain remission in patients with inflammatory bowel disease (IBD). Intestinal epithelial cells play a key role in the pathogenesis of IBD, however, their role as target cells for thiopurines has not been elucidated. The aim of the project is to establish an in vitro IBD model using organoids starting from intestinal biopsies of IBD paediatric patients to investigate thiopurine effects on intestinal epithelium. Methods Forty-four IBD patients (mean age 14.4 years, 21 males) were enrolled. Intestinal biopsies were used to isolate crypts and generate adult stem cell-derived organoids. The cytotoxicity of thiopurines (0.2-200 µM for 72 hours) was evaluated by CellTiter-Glo 3D assay. Gene expression was evaluated using TaqMan assays. Thiopurine metabolites were quantified by LC-MS/MS analysis. Proteomic profiles were analyzed by Q-Exactive Plus mass spectrometer, western blot and REACTOME pathway software. Statistical analysis was performed using GraphPad and R software. Results IBD patients-derived organoids were sensitive to thiopurine treatment in a dose-dependent manner with a high interpatient variability (Two-way ANOVA p-value<0.0001; IC50 2.24 µM for AZA (95% CI: 1.28–5.53 µM) and 2.65 µM for MP (95% CI: 1.43–8.27 µM)).A significant negative correlation was observed between the viability after thiopurine exposure and mRNA expression of candidate genes involved in thiopurine pharmacokinetics (ITPA, TPMT, PACSIN2 and NUDT15). The most abundant thiopurine metabolite measured on organoids treated with IC50 of MP was MeTIMP, with a concentration correlated with cell viability after MP exposure (p=0.0056, ρ=-0.99). Proteomic analysis on organoids treated with IC20 of thiopurines (0.2 μM) showed 45 differentially expressed proteins in common between AZA and MP (p<0.05). REACTOME enrichment analysis revealed as the most altered pathways: vesicular traffic, autophagy, protein ubiquitination and interferon signalling, while no proteins related to apoptosis were induced. Among these proteins, TRIM32, a ubiquitin ligase involved in STING activity, a key regulator of both interferon I expression and NF-kB activation, resulted downregulated by the treatment (t-test AZA: p=0.0006; MP: p=0.014) and its basal levels negatively correlated with viability rates after thiopurine exposure (AZA: p=0.006, ρ=-0.85; MP: p=0.002, ρ=-0.88). Organoid exposure to thiopurines also decreased p-STING/STING ratio, particularly after MP treatment (t-test p=0.025), and increased autophagy, assessed as LC3-II levels (t-test AZA: p=0.0006; MP: p=0.014). Conclusion Organoid cultures provide new insights on the mechanism of action of thiopurines in the intestinal epithelium.

P082 Downregulated apelin expression in T cells in the setting of Chronic Colitis

Abstract Background APJ is a G protein-coupled receptor and was originally found as an orphan receptor. Isolation of apelin (APL) from the alimentary tract was found to be the endogenous ligand for APJ. It has been reported that APL is upregulated in the colonic tissues of a murine model of acute colitis induced by dextran sodium sulfate (DSS), and thus it may be suggested to be associated with the pathogenesis of inflammatory bowel diseases (IBD) in humans. However, the mechanism and function of APL in the context of IBD are still not understood. Here, we analyzed APL expression in an animal model of chronic colitis to define its function in the pathogenesis of IBD. Methods Each cell type in the colonic tissue, including epithelial cells and lamina propria lymphocytes, were first isolated from wild type C57BL6 mice (WT) to assess APL expression. Next, naïve T cells isolated from WT were adoptively transferred into Rag deficient mice (Rag-/-) to induce chronic colitis, and then APL expression was measured either in the colonic tissue or in the isolated splenic and colonic CD4+ T cells from these T cell-reconstituted Rag-/- to compare with those of WT or Rag-/- without colitis. In addition, WT naïve T cells were differentiated into either Th1, Th2 or Th17 in vitro to analyze APL expression. Finally, the Rag-/- receiving naïve T cells were administered a synthetic APL peptide, APL-13, to assess the severity of colitis. Results Semi-quantitative PCR (qPCR) revealed that CD4+ T cells express relatively higher level of APL compared to other cell types including the epithelial cells in colonic tissue from WT. However, APL expression in the colonic tissues from the Rag-/- induced chronic colitis was unexpectedly downregulated compared to control groups without colitis; this is not consistent with the previous report using acute DSS colitis model. Subsequently, qPCR revealed significantly decreased APL expression in the splenic and colonic T cells from Rag-/- induced colitis compared to that of WT. APL expressions in the effecter T cells in vivo and all of the differentiated T cells in vitro were also significantly downregulated compared to naïve T cells and non-differentiated control T cells, respectively. Given these results, APL-13 was injected into the Rag-/- that underwent T cell reconstitution to antagonize the APL downregulation. This resulted in reduced severity of colitis compared to that of vehicle control-injected group. Conclusion These results suggest that T cells can be one of the major sources of APL in colonic tissues, and APL downregulation in effecter T cells may lead to the development of chronic colitis. In addition, this study also suggests that APL may be a novel therapeutic target for IBD.

P153 Artificial Intelligence to predict interleukin-23 signalling activity from H&E stained whole slide images of Inflammatory Bowel Disease

Abstract Background Interleukin23 (IL-23) is a cytokine that plays a crucial role in the pathogenesis of inflammatory bowel disease (IBD), making it a highly validated therapeutic target. Understanding the role of IL-23 in IBD at the histopathological level is crucial for determining effective treatment strategies, providing insights into IBD patients who fail to respond to targeted therapies, or predicting those who are likely to lose response. In this context, there is a surge in utilizing artificial intelligence (AI) for histopathological data in IBD and other disease indications. Here, we present an automated computer vision approach to predict IL-23 signalling activity directly from routinely stained Hematoxylin and Eosin (H&E) images Methods A total of 1502 samples with matched clinical data and H&E biopsy images were included from 991 Crohn’s disease (CD) and 511 ulcerative colitis (UC) samples. IL-23 signalling activity was calculated using gene set variation analysis on RNA-seq data collected from the same tissue biopsies. The data were obtained from the IBD Plexus program of the Crohn’s & Colitis Foundation. The proposed approach is based on vision transformers (ViTs) which is a type of deep learning model. ViTs divide the input image into fixed-size patches, transform it into linear embedding, and analyze it with the self-attention mechanism. This enables ViTs to incorporate relationships between different patches of the input image to identify regions predictive of IL-23. Our approach was trained in a weakly supervised manner to automatically identify tissue regions that correlate with IL-23 signaling activity. The model produces interpretable heatmaps to interrogate model predictions and allow clinicians to visualize and interpret the significance of different tissue regions predictive of IL-23 Results We performed 5-fold cross-validation on the splits obtained at the patient level, retaining the data distribution of IL-23 signaling activity, biopsy location, and diagnosis. We separately validated the performance of the proposed model on both disease categories, including CD and UC. The proposed approach achieved an area under the curve (AUC) of 0.82 ± 0.04 on unseen data from CD and an AUC of 0.80 ± 0.02 for UC. The 5-fold results for both disease categories are shown below Conclusion The presented results highlight the significance of computational pathology algorithms to identify IL-23 signalling activity from H&E images. Pathological interpretation from the heatmaps may help understand disease pathomechanism and optimize the treatment options for IBD patients by timely identification of IL-23 status. We are further validating the clinical utility of such heatmaps and expanding the use of H&E to predict other patient-centric endpoints

P145 Phagocyte responses to gut-derived C. albicans are modified in inflammatory bowel disease

Abstract Background Fungi are a key component of the human gut microbiota and have been strongly implicated in the pathogenesis of inflammatory bowel disease (IBD). Candida albicans typically exists as a commensal yeast within healthy hosts but can form tissue-invasive filaments that secrete Candidalysin (CLYS) toxin to disrupt epithelial barriers. Dynamics of this morphological switch are influenced by genotypic and phenotypic variation of gut-resident C. albicans strains, which may impact host response and extent of mucosal inflammation. Methods C. albicans isolates were derived from healthy control (HC) and IBD gut biopsies. Phenotypic, genomic, and proteomic analyses were conducted on these isolates. To further understand host interplay with intestinal C. albicans under physiologically relevant conditions, a human ‘gut-on-a-chip’ system was developed to assess tissue invasion potential in vitro. Results IBD-derived isolates were more readily able to form filaments, displayed altered cell wall composition, and modulated expression of adhesion-associated genes including IHD1. Subsequent innate leukocyte responses to these strain-dependent profiles were investigated to understand impact on host immunity. When exposed to IBD-derived C. albicans strains, blood neutrophils from healthy donors displayed increased swarming behaviour and NETosis induction, whereas monocyte-derived macrophages released greater amounts of pro-inflammatory IL-1b. These responses were substantially diminished in neutrophils and macrophages from IBD patients, suggesting altered reactivity to fungal stimuli in disease settings. The gut-on-a-chip model system confirmed that IBD-derived strains display increased adhesion and translocation in a dynamic human gut environment. Conclusion Gut-adapted C. albicans strains from HC and IBD donors exhibit distinct biological profiles that impact on host immune responses and shape interactions with the gut barrier in a novel organ chip model.

P165 Guselkumab binding to CD64+ IL-23–producing myeloid cells enhances potency for neutralizing IL-23 signaling

Abstract Background IL-23 is implicated in the pathogenesis of inflammatory bowel disease (IBD) and myeloid cells that express FcγRI (CD64) have been identified as the primary cellular source of IL-23 in inflamed IBD gut tissue. Guselkumab (GUS) and risankizumab (RZB) are monoclonal antibodies (mAbs) specifically directed against the IL-23p19 subunit. GUS is a fully human IgG1 mAb with a native Fc region while RZB is a humanized IgG1 mAb with a mutated Fc region. Here, we evaluated CD64 and IL-23 expression in IBD patient gut biopsies, binding of GUS and RZB to CD64, and the functional consequences of CD64 binding by IL-23p19 subunit mAbs, in in vitro assays. Methods IL23A and FCGR1A (CD64) expression was analyzed from bulk and single-cell RNAseq datasets. Binding of mAbs to IFNγ-primed human monocytes, as well as binding to IL-23–secreting inflammatory monocytes and capture of endogenously secreted IL-23, were assessed by flow cytometry. Internalization of IL-23, GUS, and RZB within CD64+ macrophages was evaluated using live cell confocal imaging. Potency of GUS and RZB for inhibiting IL-23 signaling was determined in a co-culture of THP-1 (a CD64+ monocyte cell line activated to produce IL-23) and an IL-23 reporter cell line (measuring biologically active IL-23). Results Analysis of RNAseq datasets showed that FCGR1A, IL23A, and IL12B were significantly increased in inflamed versus non-inflamed IBD gut biopsies and that IL23A was predominantly expressed by FCGR1A-expressing myeloid cells. In in vitro assays GUS, but not RZB, showed Fc-mediated binding to CD64 on IFNγ-primed monocytes. CD64-bound GUS simultaneously captured IL-23 secreted from the same cells. GUS, but not RZB, bound to the surface of CD64+ macrophages and mediated internalization of IL-23 to low pH intracellular compartments. GUS and RZB demonstrated similar potency for inhibiting signaling by IL-23 present in THP-1–conditioned media. However, in a co-culture of IL-23–producing THP-1 cells with an IL-23–responsive reporter cell line, GUS demonstrated enhanced potency compared to RZB for inhibition of IL-23 signaling. Conclusion Our transcriptomic analysis supported previous observations of CD64+ myeloid cells as a key source of IL-23 production in inflamed IBD gut tissue. GUS binding to CD64 on IL-23–producing cells likely contributed to the enhanced functional potency of GUS compared to RZB for inhibition of IL-23 signaling in the co-culture assay. These in vitro data support a hypothesis for optimal enrichment of GUS in inflamed tissues where CD64+ IL-23–producing myeloid cells are increased and in proximity to IL-23–responsive lymphoid cells, enabling GUS to more potently neutralize IL-23 by targeting IL-23 at its source of production.

P028 A pathogenic role of activated mucosal-associated invariant T cells in an animal model of Inflammatory Bowel Disease

Abstract Background It is known that the mucosal-associated invariant T (MAIT) cells are the innate-like T cells that are restricted by the major histocompatibility complex-related molecule 1 (MR1), and that these cells express a semi-invariant T cell receptor. We have previously reported that the activation status of the circulating MAIT cells in patients with Ulcerative Colitis (UC) is associated with disease activity, and these cells infiltrate the inflamed colonic mucosa. These findings imply that MAIT cells are involved in the pathogenesis of Inflammatory Bowel Disease (IBD). However, the role of MAIT cells in the setting of IBD has not been revealed. Therefore, we investigated the role of MAIT cells in an animal model of UC. Methods To this end, we utilized MR1 deficient mice (MR1-/-), which lack MAIT cells, and isobutyl 6-formyl pterin (i6-FP), which is a synthetic antagonistic MR1 ligand. MR1-/- on the C57BL/6 background, their littermate wild-type controls, and C57BL/6 mice were sensitized by rectal administration of oxazolone to induce colitis. These were then administered an oral i6-FP. Splenocytes (SPL) and colonic lamina propria lymphocytes (LPL) were isolated from mice receiving i6-FP to measure cytokine production. MR1-/-, i6-FP-treated mice and their controls were orally administered FITC-dextran to analyze intestinal permeability. The peripheral blood mononuclear cells (PBMC) from the patients with UC were also isolated to study the effect of i6-FP on cytokine production by MAIT cells. Results MR1 deficiency or i6-FP treatment resulted in reduced severity of oxazolone-induced colitis. Mice treated with i6-FP resulted in reduced MAIT cell production of pro-inflammatory cytokines such as IFN-g and TNF in both SPL and colonic LPL. Similar results were also observed in PBMC isolated from the patients with UC when incubated with i6-FP. Although MR1 deficiency resulted in increased intestinal permeability, i6-FP administration did not affect gut integrity in mice. Conclusion The current studies indicate that MAIT cells have a pathogenic role in colitis and suppressing activation of these cells may reduce the severity of colitis without affecting gut integrity. Thus, MAIT cells may be potential therapeutic targets for IBD including UC.

DOP26 Metagenomic and metabolomic profiles in IBD: understanding microbial and metabolic shifts from a large deeply phenotyped cohort

Abstract Background Alterations in gut microbiota composition and functions are involved in the pathogenesis of Inflammatory Bowel Disease (IBD) and the role of specific bacterial taxa has been particularly pointed out. The role of microbiota-derived metabolites, including those produced from tryptophan, are major actors in host-microbiota interactions in health and in IBD. Large studies analyzing both gut microbiota and metabolomics data are scarce. Methods In the current study, we analyzed a total of 764 individuals from Saint Antoine Hospital cohort, including 447 patients with Crohn's disease (CD), 262 patients with Ulcerative Colitis (UC) and 55 healthy subjects. We performed shotgun metagenomic sequencing on fecal samples and integrated the results with deep clinical phenotyping and targeted metabolomics data encompassing 294 different molecules. Results We observed strong changes in the taxonomic composition and functional capabilities of the microbiota in CD and UC patients compared to healthy subjects. Besides disease itself, the most important drivers of microbiota composition were the disease location (Montreal classification), recent antibiotic treatment, disease activity (flare vs remission) and history of ileocecal resection. Interestingly, IBD diagnosis explained much more the variations of microbiota functions than taxonomy. The decrease in microbiota diversity was stronger in CD than in UC. In parallel to a decreased amount of Faecalibacterium in IBD, we also observed a decrease in the diversity of Faecalibacterium strains, with a stronger decrease in CD. Our multifactorial analysis revealed specific microbial taxa and functions affected by disease-related factors. We particularly identified many correlations between tryptophan metabolites and microbial abundance. Targeted gene analysis of tryptophan-related enzymes in metagenomes further supported these findings. A network analysis considering bacterial taxa and metabolites revealed profound alterations in IBD with some specificities between CD and UC. Conclusion We pointed out new microbiome and metabolome alterations associated with IBD, with some phenotype specificities. Overall, our findings provide crucial information and a substantial resource for understanding the interactions between the host and microbiome in the context of IBD.

P1196 Genomic structural variation and polygenic risk score for Inflammatory Bowel Disease

Abstract Background Genome-wide association studies (GWAS) of patients with inflammatory bowel disease (IBD) have identified multiple single-nucleotide polymorphisms (SNPs) and genetic variants involving structural changes in the DNA sequence. SNPs and structural variants (SVs) can play a crucial role in IBD pathogenesis. In this study, we evaluated large deletions induced by SVs in patients with IBD. Additionally, we evaluated the ethnic contribution to a polygenic risk score (PRS) for IBD. Methods Seventy-five Patients with IBD of Korean ancestry from Seoul National University Bundang Hospital (SNUBH) were subjected to whole-genome sequencing (49 patients with UC and 26 patients with CD). SVs involving large deletions were detected and analyzed by using the Genome Aggregation Database (gnomAD) and AnnotSV. The SVs were prioritized based on a list of 241 IBD-associated SNPs identified in the previous GWAS. The prediction performance of European- and Korean-derived PRSs for IBD was also evaluated. For calculating PRS, we applied previously published models derived from the European and Korean populations, respectively, and used Korean Genome and Epidemiology Study (KoGES) database as the Korean controls. Results We found a total of 146 SVs involving large deletions in the IBD cohort. Among them, 10 SVs were annotated as likely pathogenic, according to AnnotSV and American College of Medical Genetics and Genomics guidelines; 24 SVs had the probability of being loss-of-function intolerant (pLI ≥ 0.9). We found that the frequency of large deletions that overlapped with the regions coding NOTCH1, HGF, PTPN2, and SENP7 were rare in the IBD cohort compared to the gnomAD population (Table). The large deletions that overlapped with the regions coding IL2RA, ITGA4, BANK1, and STAT3 were identified in the IBD cohort but not in the gnomAD population. The PRS for IBD in the IBD cohort was significantly higher than that in the Korean controls under the Korean- or European-derived model (Figure A). However, the PRSs for CD or UC were not able to distinguish patients with CD and UC in the IBD cohort (Figure B and C). Conclusion Our finding suggests that SVs involving large deletion may be an important genomic variability in predicting the development of IBD. The PRS for IBD can predict IBD development without ethnic specificity.

D-ECCO Grant Personalizing the dietetic strategy in children with IBD: exploring an antioxidant approach

Abstract Background and Aims Diet is a key element in the pathogenesis and treatment of Inflammatory Bowel Disease (IBD). Also, oxidative stress (OS) is involved in IBD onset and evolution. Our group is conducting a multicenter trial (the OxIBDiet study, NCT0451301) to explore the OS imbalance in both children and adults with IBD, compared to matched controls, the genetic variants of genes implicated in OS and the effects of an Antioxidant diet (AoD) on these patients. Preliminary results have shown a great imbalance in the total antioxidant capacity in IBD compared to healthy matched controls, particularly in IBD children, and a clear improvement of the antioxidant capacity and oxidant parameters after 3 months of AoD. Aims of this study are: 1. To identify IBD subjects’ responders to a specific dietetic treatment (on the basis of clinical, biochemical and genetic characteristics); 2. To evaluate the effect of antioxidant compounds on stem cell derived 3D models of the intestinal epithelium by organ-on-a-chip technology; 3. To characterize the effect of AoD and Mediterranean diet in terms of inflammatory pattern. Methods A machine-learning algorithm that integrates clinical parameters, anthropometrics, blood parameters and genetic data measured in OxIBDiet cohort will be generated and validated in a new cohort of 30 IBD children (15 CD and 15 UC). Human intestinal organoids will be generated from 10 patients, in which an endoscopy is planned for disease reassessment, and levels of interleukins and TNF-alfa will be measured, after stimulation with antioxidant compounds. Thirdly, cytokine analysis in serum from OxIBDiet cohort who performed dietetic sub-study will be evaluated. Anticipated Impact Results of this study will provide useful indications on factors that influence redox imbalance in IBD and will permit to identify patients who may benefit from a personalized dietetic approach, especially an AoD.

P1105 Short-term exposure to environmental air pollution and inflammatory bowel disease flares: a time-stratified case cross-over study

Abstract Background Some epidemiological studies suggest that long-term exposure to environmental air pollutants, such as particulate matter (PM), could have a role in inflammatory bowel disease (IBD) incidence and pathogenesis. However, less is known about the potential effect of short-term air pollution exposure. The aim of this study is to investigate whether short-term exposure to environmental air pollution is associated with the occurrence of IBD flares. Methods Anonymized hospital discharge records (HDR) with ICD-9-CM codes 555 (Crohn’s disease) and 556 (ulcerative colitis) for years 2002-2009 from all hospitals of the Lombardy region were collected. All HDR of adult patients (18-75 years) resident in Milan were included, except for recurrent hospitalizations within 6 months. Each record was assigned daily PM ≤10 µm (PM10) concentrations at municipality level by averaging PM10 levels of the day of admission with the levels of the day before (lag01) and of each preceding day up to six days before (lag06). To evaluate the association between PM10 and IBD-related hospitalizations, a time-stratified case-crossover study design was applied. Conditional logistic regression models, calculating Odds Ratios (OR) and 95% Confidence Intervals (95%CI) per increments of 10 µg/m3 of PM10, were performed. Results Overall, 2,176 hospital admissions (males=1,137, Crohn’s disease=1,264, ulcerative colitis=912) were included. In the entire population no clear association between PM10 and IBD-related hospitalization was identified, notwithstanding a small risk increase for males (OR: 1.03; 95%CI: 0.99-1.08 at lag04). When further stratifying for ICD code, an increased risk of hospitalization for "left-sided ulcerative colitis" (ICD code: 556.5) was found in males only, for exposures in all days preceding hospitalization up to lag04 (1.28; 1.05-1.28; 59 cases, 207 controls) (Figure 1). In females, the association was weaker, although positive (1.14; 0.91-1.42; 49 cases, 164 controls). No relevant findings emerged when looking at other ICD codes. Conclusion We observed an increased risk of IBD-related hospitalization in male patients with left-sided ulcerative colitis per increasing levels of air pollution in the days preceding hospital admission. Although preliminary, our findings suggest the possible role of sex as an effect modifier of the influence that air pollution might exert on IBD flares. Further studies are needed to better comprehend the complex framework characterizing the interplay between environmental and individual factors contributing to IBD.

P1222 Gut microbiome differences regarding lifestyle and the history of COVID-19 infection in ulcerative colitis patients

Abstract Background The microbiome contributes to the pathogenesis of ulcerative colitis (UC) but the contribution of different lifestyle and environmental factors to the compositional variability of the gut microbiota is unclear. Also, it is known that microbiota plays an important role in infectious diseases like coronavirus disease 2019 (COVID-19). Methods In 6-month time (June 2021 to December 2021) 49 UC outpatients from Riga East Clinical University Hospital were included in a Cross-sectional study. All patients were divided into groups according to COVID-19 status (COVID-19posvs COVID-19neg) within 6 months. The effect of the lifestyle (smoking cigarettes, alcohol intake, sports activities, and high psychological stress level) and COVID-19 to the gut microbiota in patients with UC was investigated. For taxonomical classifying of the gut microbiome metagenome data, MetaPhlAn v.2.6.0 tool was used. In further analysis gut microbiome (mostly bacteria phyla) and related data was analysed with SPSS 20.0. Results Out of 49 patients, 31(63%) were male and 18(37%) were female, the mean age was Md=38 [IQR:34-51]. Fourteen patients (28.6%) have been COVID-19 pos and 35(71.4%) COVID-19 neg. There were no statistically significant differences between COVID-19 pos and COVID-19 neg patients and their microbiota, nor between COVID-19 pos symptomatic 12(24.5%) and asymptomatic 37(75.5%) patients and their gut microbiome. Out of 49, 24(49%) were doing sports, of those 18(51.4%) were COVD-19neg vs. 6 (42.9%) COVID-19pos, p=0.82. Evaluating differences in the gut microbiome regarding physical activities there was no statistical significance. Of 49 patients, 30(61.2%) have smoked before, while 19(38.8%) haven’t smoked. Medium smoking time was 8 years Md=8 [IQR:4.50-20.0]. Analysing gut microbiome in smokers/non-smokers, and the length of smoking, there were found no statistically significant differences. Thirty-five (71.4%) were using alcohol, and 14(28.6%) were not. Those who were using alcohol had statistically significantly more Firmicutes in their gut microbiome Md= 64.8[IQR:53.7-70.4] than no users Md=48[IQR:37.5-66.3], p=0.041. Regarding high stress levels, those who had less stress 18(36.7%), had more Actinobacteria in their gut microbiome Md=11.7[IQR:6.92-14.8] vs stressful patients 31(63.3%) Md=6.03[IQR:2.23-13.8], p=0.03. Conclusion There were no changes in gut microbiome in COVID-19 positive and COVID-19 negative patients, nor in smokers and no smokers. Alcohol users had more bacteria Firmicutes in their gut microbiome. There were no differences in gut microbiome in UC patients doing or lacking sports. UC patients with less stress had more Actinobacteria in their gut microbiome.

P173 Characterization of the gut microbiota and the colonic immune response in acute severe ulcerative colitis : the multi-omics ITAC project

Abstract Background Limited understanding exists regarding the pathogenesis of acute severe ulcerative colitis (UC). Microorganisms are proposed as potential triggers due to the resemblances to infectious colitis and the essential role played by gut microbiota in UC-related inflammation. Our aim was to identify microbiome elements and host factors associated with acute severe UC. Methods This was a prospective study across three referral centers comparing two UC patient groups: acute severe hospitalized UC (as per Truelove and Witts criteria) and non-severe UC. We analyzed gut microbiota using 16S rRNA gene sequencing and conducted single-cell RNA-Seq on rectal biopsies in a subgroup of patients to uncover cellular subtypes and pathways involved in mucosal inflammation. We utilized whole blood RNA-Seq to investigate the host pathways involved in mediating systemic inflammation. Results Forty-one patients (23 (56%) female, median (interquartile range IQR) age 42 (34 – 57) years were included: 19 with acute severe UC and 22 with non-severe UC. Compared to patients with non-severe UC, those with acute severe UC displayed distinct gut microbiota with reduced diversity, increased Proteobacteria (Escherichia/Shigella genus), and decreased Lachnospiraceae and Ruminococcaceae. In severe cases (n=4 - 13,047 cells), single-cell RNA-Seq analysis of rectal biopsies revealed distinctive patterns compared to non-severe cases (n=5 - 18,433 cells): plasmablasts showed an altered transcriptomic profile with heightened IgG expression, and there was an expanded cluster of interleukin (IL) 26 expressing T cell population. Innate immune cells exhibited a pro-inflammatory profile marked by increased expression of the IL1B gene. In blood samples, we observed no distinct differentiation of transcriptomic profiles between severe and non-severe cases. Conclusion Our multi-omics study reveals key cellular and bacterial components in acute severe UC pathogenesis. We identified Proteobacteria, especially Escherichia coli as a potential pathobiont in acute severe UC. At the colonic level, we observed an increased IgG/IgA ratio, while both T cells and innate immune cells indicated a pro-Th17 mucosal environment. Enhanced systemic inflammation in acute severe UC was not reflected by specific changes in immune circulating cells. These insights may pave the way for future research focusing on microbiome modulation, interventions targeting plasmablasts, or nuanced inhibition of the Th17/IL-23 axis in acute severe UC.

P052 Mucosal IgG-like naive B-cell expansion and maturation contribute to ulcerative colitis pathogenesis

Abstract Background A higher abundance of colonic naive B-cells and IgG plasma cells is implicated in the pathogenesis of ulcerative colitis (UC) during active inflammatory episodes. However, B-cell inhibition by targeting CD20 was found to be ineffective in achieving remission in UC patients indicating that the causal role of the B-cell expansion in UC is not well understood. In this study, we performed single cell RNA-sequencing (scRNA-seq) on mucosal tissue where we interrogated the B-cells in inflamed and non-inflamed UC colon biopsies. We subsequently explored the pathogenic role of B-cells in colitis mouse models. Methods We collected biopsies from the colon of 5 UC patients with active inflammation and 5 UC patients showing no inflamed mucosa. Three mucosal biopsies were collected from the same colon location for each patient and processed for scRNA-seq. We acquired usable transcriptomes of 21.475 cells. In a T-cell transfer colitis mice model, we evaluated the effects of the co-transfer of naive B-cells. Both CD4+ T and naive B-cells were transferred into SCID mice through intraperitoneal injection in ratios of 2:1, 1:1 and 1:2 (T:B ratio). The colitis course was followed for 35 days. Results Differential abundance analysis relative to all immune cells confirmed a higher abundance of naive B-cells (p= 0.03) and IgG plasma B-cells (p= 0.002) in inflamed relative to non-inflamed UC patient tissue, whereas no differences in abundance of transitional B-cells and memory B-cells were found. Pseudo-time analysis indicated a higher rate of maturation in the naive B-cells in inflamed tissue. This was evidenced by higher expression of IGHG1 (p= 0.025) and IGHG3 (p< 0.001) in naive B-cells in inflamed tissue. In a T-cell transfer model, we co-transferred naive B-cells to the CD4+ T-cell pool into SCID mice in various ratios (2:1, 1:1 and 1:2 T to B-cells respectively) and T-cell only control. Significant increase in disease severity was observed in a 1:2 T:B ratio based on body weight loss, increase of colon density, mouse colitis histology index and histological changes by disrupted mucosa. This observation was associated to significant elevated IgG concentration in serum and colon and B-cell presence in colon through immunohistochemistry. Conclusion We observed significantly higher abundance of naive B-cells and IgG plasma cells in inflamed relative to non-inflamed UC colon tissue, where naive B-cells were shown to be more mature and IgG-like in inflamed condition. Notably, transferring increased ratio of naive B-cells resulted in elevated disease severity in a colitis model, demonstrating its pathogenic role. Our findings suggest that specific targeting of the IgG-like naive B-cell may be beneficial for UC patients.

P424 Analysis of the intestinal microbiome using an Endoscopic Brush in Ulcerative colitis

Abstract Background The precise pathogenesis of the Ulcerative colitis is still yet unknown, but one of its cause is known to be microbial dysbiosis. The mucosa-associated microbiota are more deeply involved in the pathogenesis of UC. However, the optimal sampling of mucosa-associated microbiome has yet to be investigated. In this study, we investigated the mucosa-associated microbiome in patients with ulcerative colitis, using endoscopic brush samples. We hypothesized that endoscopic brushing is precise and noninvasive method to get sample of mucosa-associated microbiome. Methods Patients with UC who visited the gastroenterology department of Korea University Anam hospital were screened for this study. Clinical data such as medical records, colonoscopy and fecal samples were reviewed. Using a stool and saliva sample collector kit respectively, the subjects provided stool and saliva samples. Brushing samples were collected during the sigmoidoscopy procedure with 3-4 brush strokes on the colon mucosa using the cytology brush. The samples were analyzed for microbiome in the Korea University Medical Center. Results From July 2022 to January 2023, we prospectively enrolled 19 patients with UC. Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria were the most common species in microbiota of brush, stool and saliva.(Fig.1-1) The microbiome between stool and brush was no significant difference in alpha and beta diversities.(Fig.1-2) However, Oral microbiome was different from stool and brush in beta diversities.(Fig.1-3) Patients were categorized into to analyze the oral microbiome. A trend was observed where increased disease severity was associated with an increase in Firmicutes.(Fig.1-4) Conclusion The microbiome of stool and brush was no significantly different. However, the novel sampling of mucosa-associated microbiome, endoscopic brush, is not inferior compared to currently used sampling of stool. Also the analysis of the oral microbiome suggested that Firmicutes could be considered a useful biomarker for assessing disease severity. Therefore, it is necessary to conduct followup research by increasing the number of subjects.

P699 Identification of A Novel Activated NK-Associated Gene Score Associated with Diagnosis and Biological Therapy Response in Ulcerative Colitis

Abstract Background Natural killer (NK) cells are associated with the pathogenesis of ulcerative colitis (UC), while their precise contributions remain unclear. The present study sought to investigate the diagnostic value of activated NK-associated gene score (ANAG score) in UC and evaluate its predictive value in biological therapy response. Methods Bulk RNA-seq datasets were obtained from the Gene Expression Omnibus (GEO). GSE11223, GSE87466, and GSE92415 were integrated for diagnosis value analysis. GSE206285 and GSE92415 were used to analyze the prediction of response to ustekinumab (UST) and golimumab (GLM), respectively. The immune infiltration landscape was estimated by single-sample gene set enrichment analysis (ssGSEA) and CIBERSORT. Differentially expressed genes (DEGs) correlated with activated NK cells were identified as activated NK-associated genes (ANAGs). Key ANAGs were screened by protein-protein interaction (PPI) analysis and least absolute shrinkage and selection operator (LASSO) regression. Results The immune infiltration analysis revealed a higher abundance of activated NK cells in non-inflamed UC tissues. 54 DEGs correlated with activated NK cells were identified as ANAGs. PPI analysis and LASSO regression were utilized to screen out 4 key ANAGs (SELP, TIMP1, MMP7, and ABCG2). ANAG score was established with the following formula: ANAG score = 0.628 * SELP + 1.574 * TIMP1 + 0.740 * MMP7 - 0.361 * ABCG2 – 18.940. The ANAG scores were significantly higher in inflamed tissues, as well as in biological therapy non-responders (NR) tissues before treatment. The ANAG score demonstrated excellent diagnostic value (AUC = 0.979). Furthermore, according to quartile stratification, patients with a higher ANAG score before treatment were more likely to experience lack of response to ustekinumab and golimumab. Conclusion This study established a novel ANAG score with the ability to precisely diagnose UC and distinguish the efficacy of biological treatment.

Exploring the role of IL-1β in inflammatory bowel disease pathogenesis.

Interleukin 1β (IL-1β) is a significant mediator of inflammation and tissue damage in IBD. The balance between IL-1β and its endogenous inhibitor-IL-1Ra-, plays a critical role in both initiation and regulation of inflammation. However, the precise role of IL-1β as a causative factor in IBD or simply a consequence of inflammation remains unclear. This review summarizes current knowledge on the molecular and cellular characteristics of IL-1β, describes the existing evidence on the role of this cytokine as a modulator of intestinal homeostasis and an activator of inflammatory responses, and also discusses the role of microRNAs in the regulation of IL-1β-related inflammatory responses in IBD. Current evidence indicates that IL-1β is involved in several aspects during IBD as it greatly contributes to the induction of pro-inflammatory responses through the recruitment and activation of immune cells to the gut mucosa. In parallel, IL-1β is involved in the intestinal barrier disruption and modulates the differentiation and function of T helper (Th) cells by activating the Th17 cell differentiation, known to be involved in the pathogenesis of IBD. Dysbiosis in the gut can also stimulate immune cells to release IL-1β, which, in turn, promotes inflammation. Lastly, increasing evidence pinpoints the central role of miRNAs involvement in IL-1β-related signaling during IBD, particularly in the maintenance of homeostasis within the intestinal epithelium. In conclusion, given the crucial role of IL-1β in the promotion of inflammation and immune responses in IBD, the targeting of this cytokine or its receptors represents a promising therapeutic approach. Further research into the IL-1β-associated post-transcriptional modifications may elucidate the intricate role of this cytokine in immunomodulation.

YAP represses intestinal inflammation through epigenetic silencing of JMJD3

BackgroundEpigenetics plays an important role in the pathogenesis of inflammatory bowel disease (IBD). Some studies have reported that YAP is involved in inflammatory response and can regulate target genes through epigenetic modifications. JMJD3, a histone H3K27me3 demethylase, is associated with some inflammatory diseases. In this study, we investigated the role of YAP in the development of IBD and the underlying epigenetic mechanisms.ResultsYAP expression was significantly increased in both in vitro and in vivo colitis models as well as in patients with IBD. Epithelial-specific knockout of YAP aggravates disease progression in dextran sodium sulfate (DSS)-induced murine colitis. In the TNF-α-activated cellular inflammation model, YAP knockdown significantly increased JMJD3 expression. Coimmunoprecipitation experiments showed that YAP and EZH2 bind to each other, and chromatin immunoprecipitation-PCR (ChIP-PCR) assay indicated that silencing of YAP or EZH2 decreases H3K27me3 enrichment on the promoter of JMJD3. Finally, administration of the JMJD3 pharmacological inhibitor GSK-J4 alleviated the progression of DSS-induced murine colitis.ConclusionOur findings elucidate an epigenetic mechanism by which YAP inhibits the inflammatory response in colitis through epigenetic silencing of JMJD3 by recruiting EZH2.

Dietary Emulsifier Exposure in People With Inflammatory Bowel Disease Compared With Healthy Controls: Is There a Cause for Concern?

Emulsifiers are implicated in the pathogenesis of inflammatory bowel disease (IBD). Few studies have examined emulsifier intake in people with existing IBD. We aimed to describe the frequency of exposure to 6 selected emulsifiers in a contemporary cohort of people with IBD and compare intake with healthy controls (HCs). Baseline food records from participants in an Australian prospective cohort study examining the microbiome of IBD patients and HCs were analyzed. Exposure to inflammatory emulsifiers polysorbate-80 (P80); carboxymethylcellulose (CMC); carrageenan; xanthan gum (XG); lecithin (soy and sunflower) and mono- and diglycerides of fatty acids (MDGs) were determined by examining ingredient lists. Frequency of emulsifier exposure between groups (IBD vs HC, Crohn's disease [CD] vs ulcerative colitis [UC], IBD children vs adults, active disease vs remission) was examined after controlling for confounders. Records from 367 participants were analyzed (n = 176 IBD, of which there were 101 CD, 75 UC, and 191 HC patients). In total, 5022 unique food items were examined, with 18% containing 1 or more emulsifier of interest. Inflammatory bowel disease participants had significantly higher total daily emulsifier exposure compared with HCs (2.7 ± 1.8 vs 2.3 ± 1.6, P = .02). In IBD participants, emulsifiers with the highest daily exposure were MDGs (1.2 ± 0.93), lecithin (0.85 ± 0.93), and XG (0.38 ± 0.42). There were no recorded exposures to P80. Inflammatory bowel disease participants were exposed to more emulsifiers than HCs. Intake of inflammatory emulsifiers were low or nonexistent, suggesting their presence in the food supply are not as common as frequently stated.