Pathogenesis Of Ulcerative Colitis Research Articles

Transcriptional landscape of intestinal environment in DSS-induced ulcerative colitis mouse model.

Ulcerative colitis (UC) is a chronic inflammatory disease that targets the colon and has seen an increasing prevalence worldwide. In our pursuit of new diagnostic and therapeutic approaches for UC, we undertook a sequencing of colons from UC mouse models. We focused on analyzing their differentially expressed genes (DEGs), enriching pathways, and constructing protein-protein interaction (PPI) and Competing Endogenous RNA (ceRNA) networks. Our analysis highlighted novel DEGs such as Tppp3, Saa3, Cemip, Pappa, and Nr1d1. These DEGs predominantly play roles in pathways like cytokine-mediated signaling, extracellular matrix organization, extracellular structure organization, and external encapsulating structure organization. This suggests that the UC pathogenesis is intricately linked to the interactions between immune and non-immune cells with the extracellular matrix (ECM). To corroborate our findings, we also verified certain DEGs through quantitative real-time PCR. Within the PPI network, nodes like Stat3, Il1b, Mmp3, and Lgals3 emerged as significant and were identified to be involved in the crucial cytokine-mediated signaling pathway, which is central to inflammation. Our ceRNA network analysis further brought to light the role of the Smad7 Long non-coding RNA (lncRNA). Key MicroRNA (miRNAs) in the ceRNA network were pinpointed as mmu-miR-17-5p, mmu-miR-93-5p, mmu-miR-20b-5p, mmu-miR-16-5p, and mmu-miR-106a-5p, while central mRNAs included Egln3, Plagl2, Sema7a, Arrdc3, and Stat3. These insights imply that ceRNA networks are influential in UC progression and could provide further clarity on its pathogenesis. In conclusion, this research deepens our understanding of UC pathogenesis and paves the way for potential new diagnostic and therapeutic methods. Nevertheless, to solidify our findings, additional experiments are essential to confirm the roles and molecular interplay of the identified DEGs in UC.

Growth Factors-Loaded Temperature-Sensitive Hydrogel as Biomimetic Mucus Attenuated Murine Ulcerative Colitis via Repairing the Mucosal Barriers.

The pathogenesis of ulcerative colitis (UC) is associated with the shedding of the gut mucus. Herein, inspired by the biological functions of mucus, growth factors-loaded in situ hydrogel (PHE-EK) was designed for UC treatment by integrating dihydrocaffeic acid-modified poloxamer as a thermosensitive material with hyaluronic acid (colitis-specific adhesive), epigallocatechin-3-gallate (antibacterial agent), and bioactive factors (KPV tripeptide and epidermal growth factor). PHE-EK presented good thermosensitive properties, as a flowable liquid at room temperature and gelled within 10 s when exposed to body temperature. PHE-EK hydrogel presented good mechanical strength with a strain of 77.8%. Moreover, PHE-EK hydrogel displayed antibacterial activity against Escherichia coli. Importantly, in vitro and in vivo adhesive tests showed that the PHE-EK hydrogel could specifically adhere to the inflamed colon via electrostatic interaction. When PHE-EK as a biomimetic mucus was rectally administrated to colitis rats, it effectively hindered the body weight loss, reduced the disease activity index and improved the colonic shorting. Moreover, the expression of pro-inflammatory cytokines (e.g., IL-1β, IL-6, and TNF-α) at the laminae propria or epitheliums of the colon for colitis rats was substantially inhibited by PHE-EK. Besides, the colonic epitheliums were well rearranged, and the tight junction proteins (Zonula-1 and Claudin-5) between them were greatly upregulated after PHE-EK treatment. Collectively, PHE-EK might be a promising therapy for UC.

ARGINASE 2 AMELIORATES EXPERIMENTAL COLITIS THROUGH ANTIOXIDANT EFFECTS VIA SPERMIDINE PRODUCTION

Abstract BACKGROUND Spermidine which is a type of polyamine, shows elevated levels in patients with ulcerative colitis and is implicated in the pathogenesis of ulcerative colitis (UC). Arginase 2 (ARG2) plays a critical role in spermidine production. This study aimed to determine the role and mechanism of endogenous spermidine by using ARG2-deficient mice. METHODS The protective role of spermidine against oxidative stress was examined by intracellular reactive oxygen species levels and cell viability. The physiological role of spermidine was investigated using Arg2 knockdown cells with reduced spermidine. The importance of ARG2 in inflammation was examined in the dextran sulfate sodium-induced colitis model. Protein expression levels of ARG2 and accumulation of spermidine in patients with UC were evaluated by immunohistochemistry of the rectum. RESULTS ARG2 protein expression and the accumulation of spermidine were upregulated in the rectum of mice with dextran sulfate sodium colitis. Spermidine upregulated antioxidant genes in the cells. Arg2 knockdown cells showed reduced antioxidant activity. Dextran sulfate sodium colitis was more severe in Arg2-deficient mice, and organoids derived from Arg2-deficient mice showed diminished tolerance to oxidative stress. Furthermore, in the rectum of patients with UC, ARG2 and spermidine expression levels were higher in the active phase than in the remission phase. CONCLUSIONS Endogenous spermidine in the intestinal epithelium induced by ARG2 has a protective role in colitis. Spermidine may be a promising new therapeutic target for inflammatory bowel disease.

ULCERATIVE COLITIS-DERIVED INTESTINAL EPITHELIAL CELLS ARE PROTECTED FROM CELL DEATH BY THE IMMUNE MEDIATORS RELEASED FROM RESTING AND ACTIVATED SYSTEMIC T CELLS

Abstract Immune dysregulation is a major driver in the pathogenesis of ulcerative colitis (UC), but the specific mechanisms of disease initiated by the adaptive immune system remain unknown. While many studies have focused on the contribution of individual T cell subsets, disrupted communication between the epithelium and T cells in the underlying lamina propria is not fully understood in UC. This study examines the impact of T cells isolated from healthy participants on primary cultures of epithelial cells derived from UC donors. Blood derived human CD4+ and CD8+ T cells were isolated by immunomagnetic negative selection and activated by anti-CD3/CD28 cross-linking. Activated and resting T cells were then co-cultured with UC-derived colonic epithelial spheroids, consisting predominantly of stem cells, suspended in Matrigel. Spheroid damage and epithelial cell death were measured by histological scoring of brightfield microscopic images and zombie violet positive staining, detected using flow cytometry, respectively. Our earlier studies showed that activated CD4+ T cells co-cultured with spheroids from healthy donors stimulated significant spheroid damage and epithelial cell death in a dose- and time-dependent manner. In addition, we reported that the addition of conditioned media (CM) from activated CD4+ T cells to healthy spheroids also induces spheroid damage and epithelial cell death. Neither resting CD4+ nor CD8+ T cells nor activated CD8+ T cells stimulate the same response (Panel A). Our preliminary results indicate that UC-derived epithelial cells, isolated from spheroids, exhibit a higher base line of spontaneous lethal sectoring (42%), when compared to spheroids isolated from healthy colons (10%). In stark contrast to healthy spheroids, addition of CM from resting and activated CD4+ or CD8+ T cells protect the ulcerative colitis-derived spheroids from damage and isolated UC epithelial cells from spontaneous death. We have not yet identified which T cell subset are protecting UC-derived epithelial cells from damage and death. Thus, our results distinguish the healthy epithelial spheroid response to T cell CM from UC spheroids in three distinct ways: [1] CM is protective in UC, [2] Both CD4 and CD8 cells mediate this response in UC, and [3] CM from resting and activated T cells secrete the responsible immune mediators. Overall, these results signify that the biological response of UC-derived epithelial cells to immune mediators is different from that seen with healthy epithelial cells, indicating a metabolic and genetic difference in the epithelium from the UC patient.

THE FUNCTION OF THE CYSTATHIONINE-GAMMA-LYASE/HYDROGEN SULFIDE AXIS IN THE PATHOGENESIS OF ULCERATIVE COLITIS

Abstract INTRODUCTION Current combination therapies in ulcerative colitis (UC) target different inflammatory cascades, including tumor necrosis factor-alpha (TNF-alpha) signaling. However, most patients do not achieve long-term remission, suggesting that the molecular pathophysiology of UC is still poorly understood. Endogenously produced hydrogen sulfide (H2S) has emerged as a critical physiological mediator of gastrointestinal homeostasis, limiting mucosal inflammation and promoting tissue healing in response to epithelial injury. Inhibition of cystathionine-gamma-lyase (CSE)-dependent H2S production in animal models of UC has been shown to exacerbate colitis and delay tissue repair. Our research group has strong evidence revealing that the CSE/H2S axis regulates TNF-alpha signaling in colonic epithelial cells. Therefore, we hypothesize that decreased CSE/H2S pathway activity in colonic mucosa cells leads to dysregulation of TNF-alpha signaling and uncontrolled inflammatory/immune responses with aberrant wound repair mechanisms contributing to the pathogenesis of UC. RESULTS We found a significant decrease in CSE mRNA and protein expression in colonic biopsies from patients with UC. Using a preclinical animal model of epithelium injury-induced chronic colitis (DSS), we confirmed a sustained downregulation of CSE, as well as upregulation of TNF-alpha and tumor necrosis receptor 2 expressions in colonic mucosa samples. Treatment with the slow-releasing H2S-donor GYY4137 significantly reduced the severity of DSS-induced inflammation and improved mucosal integrity and restitution responses. In epithelial cell and colonoid cultures, the inhibition of CSE-dependent H2S synthesis decreased the activation of extracellular-signal-regulated-kinase-1/2 (ERK1/2), increased IL-6 and TNF-alpha mRNA levels and suppressed basic and TNF-alpha–mediated colonocyte migration. CONCLUSION These studies demonstrate that CSE regulates mucosal TNF-alpha signaling, pro- and anti-inflammatory responses as well as colonic epithelial repair. Adding the H2S donor GYY4137 reduces inflammation, suggesting the restoration of normal TNF-alpha signaling function. Acknowledgments: KM is supported by the DoD Career Development Award (W81XWH2010641) and ACS Research Scholar Grant (RSG–21–027–01–CSM).

P1229 The Role and Niche-Specific Adaptation of Malassezia in patients with Ulcerative colitis

Abstract Background Accumulating evidence has underscored the role of gut fungal microbiota (mycobiota) in the development of inflammatory bowel disease. We aimed to isolate a Malassezia strain directly from the human intestine mucosal surface from the patients with ulcerative colitis (UC) and investigated its genome and virulence in comparison with the same fungal species isolated from the human skin. Methods Mucosal lavage samples were collected separately from colonic areas with and without inflammation in patients with UC. Samples from healthy individuals (HT) were obtained in the same manner as from patients with UC at sigmoid or descending colon. Skin samples were taken from HT in our previous work. DNA was extracted from these lavage samples, and fungal isolation was conducted using PCR amplification with ITS4 and ITS5 primers. Comprehensive analysis and comparison of the genomes, transcriptomes, and virulence between M. globosa gut isolates and those of M. globosa strains isolated from the skin were performed. To determine the contribution of M. globosa gut isolates to exacerbating inflammation, 1107 fungal cells were orally gavaged to DSS-induced colitis mouse model for three days. Results Total 56 and 11 intestinal water-lavage samples from 29 UC patients and 11 HT were obtained respectively. The α- and b-diversities of mycobiota showed no significant differences between the groups, patients with UC vs. HT or the sites with inflammation vs. non-inflammation of the patient with UC. Malassezia was the fifth most frequently found fungal genus throughout the samples, and live fungal strains belong to 28 and 7 different species were isolated from the patients with UC and HT, respectively. The patients with UC tend to have higher frequency of M. globosa and M. restricta than HT in their gut mucosal surface with inflammation. Whole genome sequencing showed no specific genomic characteristics between gut-isolated M. globosa and skin-isolated M. globosa. However, gut-isolated M. globosa were suffered more from the higher oxygen levels than the skin isolates in different oxygen concentrations. In a mouse model, gut-isolated M. globosa exhibited a more pronounced exacerbation of DSS-induced colitis and elevated production of inflammatory cytokines, including TNF-a, IL-6, IL-12p40, IL-1b, and IL-18, while the skin isolates showed no difference compared to the negative control (Figure). Conclusion Our data shed new light on the pivotal role of M. globosa in the pathogenesis of UC, highlighting the potential influence of niche-specific adaptations on the virulence of this fungus. These findings provide critical insights into the complex interplay between the member of the gut mycobiota and host health.

P653 Impact of ozanimod on type III collagen turnover biomarkers and the association with ozanimod efficacy in patients with moderately to severely active ulcerative colitis: Results from the phase 3 True North study

Abstract Background Ozanimod (OZA), an oral sphingosine 1-phosphate 1 and 5 receptor modulator, was efficacious and well tolerated for up to 52 wk in adults with moderately to severely active ulcerative colitis (UC) in the phase 3 True North study (NCT02435992). Alterations in the degradation and formation of type III collagens, a major component of the extracellular matrix of the intestinal wall, may play a role in the pathogenesis of UC. The objective of this analysis was to assess the effect of OZA on type III collagen levels and turnover and the association of type III collagen levels with OZA efficacy in the True North study. Methods In a 10-wk induction period, patients (pts) in Cohort 1 were randomised to receive oral OZA 0.92 mg (n=429) or placebo (PBO) once daily (n=216) in a double-blind manner and pts in Cohort 2 received open-label OZA at the same daily dose (n=367). At Week (W) 10, pts with a clinical response to OZA in either cohort underwent rerandomisation to receive double-blind OZA (n=457) or PBO (n=69) for 52 wk (maintenance period). Type III collagen biomarker plasma levels were assessed at baseline (BL), W10, and W52. Associations between type III collagen levels and disease activity or efficacy outcomes were assessed using Spearman’s rho correlation and logistic regression, respectively. Results At W10, treatment with OZA resulted in significantly decreased collagen type III degradation (C3M) (Figure 1A), increased collagen formation (PRO-C3) (Figure 1B), and decreased collagen turnover (C3M/PRO-C3) (Figure 1C). Significant reductions in C3M and C3M/PRO-C3 were observed in pts with vs without a clinical response in all treatment groups (P<0.001), while significant increases in PRO-C3 were observed in responders vs nonresponders (P<0.001; Cohort 1 only). The significantly greater reductions in C3M/PRO-C3 were observed with OZA vs PBO regardless of prior exposure to biologics or tumour necrosis factor inhibitors. Changes in C3M, PRO-C3, and C3M/PRO-C3 levels from BL to W10 were significantly correlated with improvements in all disease activity scores from BL to W10 with OZA and PBO. These changes in C3M, PRO-C3, and C3M/PRO-C3 continued through W52 (Figure 1D–F) in pts who continued OZA. At W52, significantly greater reductions in C3M/PRO-C3 ratios were observed in responders vs non-responders in pts who continued OZA. Conclusion These data show that pts with UC who were treated with OZA had decreased plasma type III collagen degradation and turnover, with greater reductions in OZA responders than nonresponders. This further supports the evaluation of collagen type III fragments as potential markers of treatment response in pts with 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.

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.

P108 Response to vedolizumab in ulcerative colitis is associated with reduced neutrophil extracellular traps formation and IL-1β production: insights from NEUTROVED study

Abstract Background Vedolizumab (VDZ) is a humanized mAb against integrin α4β7. VDZ inhibits gut inflammation by impeding intestinal T-cell homing, however, recent data suggest that effects of α4β7 blocking extend beyond T-cells to alterations in innate immunity. Neutrophils are key components of innate immune response and inflammation in ulcerative colitis (UC). Studies have provided evidence linking neutrophils with UC pathogenesis, through autophagy induction and production of IL-1β-enriched neutrophil extracellular traps (NETs). NEUTROVED aimed to investigate the effects of VDZ treatment on neutrophils that may be associated with clinical and endoscopic response. Methods Patients with moderate-to-severe UC who received VDZ in standard medical care were evaluated in two timepoints, 24 hours before treatment initiation (T1) and at week 14 of treatment (T2). Clinical/endoscopic scores and common laboratory indexes were evaluated. In parallel, RNA-sequencing in peripheral neutrophils was performed, and markers of NETs in plasma and intestinal biopsies were assessed. Results In total 5 patients completed the study until today (Table 1). In T2, 4 patients demonstrated complete remission, and one patient had mild disease. No adverse events were recorded. RNA-sequencing analysis of paired samples revealed 248 significantly downregulated and 109 upregulated genes in peripheral neutrophils after VDZ treatment. Bioinformatics analysis using the GeneCodis4 web-based tool revealed that downregulated DEGs clustered mainly in immune-related pathways, including neutrophil degranulation, Toll-like receptor cascades, signaling by interleukins, NETs formation, and HIF-1 signaling pathway, while upregulated DEGs were involved in RNA metabolism-related processes, and the ribosome biogenesis pathway. A comparative analysis between these differentially expressed genes (DEGs) and our previous RNA-sequencing data of peripheral neutrophils isolated from active UC patients (n=24) highlighted a set of 168 DEGs as restored targets after VDZ therapy. Of note, several unique genes implicated in neutrophil activation, including IL-1 signaling components, were found significantly downregulated after VDZ therapy. In line with the abovementioned transcriptome alterations, markers of NETs and IL1β-bearing NETs in plasma and colonic tissue were significantly reduced, recapitalizing clinical and endoscopic response to VDZ. Conclusion Response to VDZ treatment in UC is related to anti-inflammatory actions on neutrophils characterized by reduced NETosis and IL-1β production through NETs. Further mechanistic studies investigating the possible effects of VDZ on innate immunity and neutrophil-mediated responses are warranted.

P1238 Exploring the Role of Klebsiella variicola and Klebsiella pneumoniae in Pediatric Ulcerative Colitis Pathogenesis

Abstract Background Recent studies indicated that Klebsiella (K) pneumoniae (isolated from the stool of IBD patients) and K. variicola (isolated from the mesenteric tissue of Crohn disease patients) have the potential to induce inflammation in epithelial and preadipocyte cells, exacerbating colitis in murine models. We isolated these strains from pediatric ulcerative colitis (UC) patients' appendix (relevant to UC pathogenesis given the protective effects of appendectomy) and other non-inflamed colon sections. We hypothesized that these isolates are invasive and induce inflammation in UC. These strains have not been previously studied in UC. Methods K. variicola and K. pneumoniae were isolated from two pediatric UC patients' appendices and other non-inflamed colon sections and identified using 16S DNA Sanger sequencing. Virulence of the two strains was determined by infecting Caco2 cell lines and performing adhesion and invasion assays, quantifying biofilm formation, and assessing barrier functions using transepithelial electrical resistance (TEER) measurement. Importantly, we monitored the production of pro-inflammatory (e.g., IL-8, TNF-α) and anti-inflammatory (IL-10) cytokines using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Results K. variicola and K. pneumoniae exhibited high levels of biofilm formation compared to adherent-invasive Escherichia coli (AIEC), which are commonly isolated from IBD patients. Furthermore, the Klebsiella strains adhered to epithelial cells within 2-3 hours post infection. TEER experiments showed compromised barrier integrity after 6 hours and overnight infection. Except for K. pneumoniae isolated from the ascending colon, the tested strains exhibited a significant increase in IL-8 expression. Similarly, K. variicola from the peri-appendicular region demonstrated elevated TNF-α gene expression. Conclusion K. variicola and K. pneumoniae isolated from UC patients have the potential to form biofilms, disrupt barrier integrity, and trigger inflammatory responses. These findings unravel a potential role for pathogenic Klebsiella strains in driving UC pathogenesis. Importantly, these data shed light on the role of appendix-associated bacteria in the development of UC. Future work includes using comparative genomics to map the virulence determinants of these bacteria.

P101 Investigating the role of gut eukaryotic virome in contributing to colorectal cancer carcinogenesis

Abstract Background Eukaryotic-targeting viruses have recently attracted interest in gastrointestinal diseases such as ulcerative colitis (UC) and colorectal cancer (CRC). UC and CRC share several factors influencing their etiogenesis, including intestinal dysbiosis. Our group has recently published a work pinpointing a gut virome-associated Orthohepadnaviridae protein, the Hepatitis B protein X (HBx) to correlate with UC pathogenesis and promote intestinal inflammation in mice by disrupting the epithelial barrier and shaping the gut mucosal immune environment, independently of the microbiota. Moreover, we showed that HBx induces DNA damage-related biological processes and active processes related to the Wnt pathway known to be involved in CRC carcinogenesis. Based on this evidence we hypothesize that HBx may fill the gap existing between intestinal inflammation and CRC onset by stimulating pro-inflammatory and/or pro-carcinogenic effects. Methods To verify this hypothesis, we evaluated the presence of HBx-transcript in CRC-derived mucosal samples. HBx positivity of CRC patient-derived mucosa was assessed by RT-PCR. Caco-2 cells, an in vitro model of the gut barrier, were transduced with HBx protein-encoding lentiviral vectors or GFP as control and were characterized for their carcinogenic and pro-inflammatory phenotypes by FACS analysis. C57BL/6 mice underwent intramucosal injections of liposome-conjugated HBx- or the control. FACS analysis and transcriptomic analysis were performed on mice colonic samples. Results Metatranscriptomic analysis revealed that HBx was highly enriched in CRC-derived mucosal samples as compared to healthy individuals, strengthening the link between a virome protein-induced inflammation and tumor development in patients. Moreover, HBx overexpression in Caco-2 cells directly provokes DNA damage, as assessed by the significant increase of γH2AX+ cells. Furthermore, since HBx was recently shown by our group as capable of inducing colitis-like symptoms in mice, we investigated whether this viral protein may induce colitis-associated cancer over time. Interestingly, experiments conducted at longer time points (from 40 to 60 days long observation) revealed that mice developed polypoid structures in the colonic mucosa, characterized by a decreased abundance of T cells, including effector T and NKT cells. Of note, in vivo HBx modulated the expression of genes involved in the activation of the proto-oncogene PIM3, further supporting the role of this viral protein on CRC onset. Conclusion This study introduces unconventional and innovative insights for giving CRC studies a new direction for scientific and clinical investigations, looking at the gut virome as an active component and not as a mere bystander entity of the microbiota.

P033 Unrevealing new potential key mechanisms implicated in inflammatory bowel disease by multiomic approach

Abstract Background Inflammatory bowel diseases (IBD), comprising Crohn´s disease (CD) and ulcerative colitis (UC), are characterised by chronic, relapsing and remitting inflammation of the gastrointestinal tract. Despite significant efforts to understand the pathogenetic mechanisms of IBD, the elucidation of its etiopathology and progression remains incomplete. This study aimed to clarify IBD pathogenesis using a multiomic approach in serum, urine, intestinal tissue and stool samples. Methods We performed a multiomic analysis (transcriptomic, proteomic, metabolomic and metagenomic) in a discovery cohort of 53 patients with active CD, 50 patients with active UC, and 33 healthy controls (HC). Proteomic analysis of intestinal tissue was performed by liquid chromatography-mass spectrometry, serum and urine samples were used for metabolome study using nuclear magnetic resonance spectroscopy, and metagenomic analysis of stool samples was performed by 16rRNA gene sequencing. Results Gene expression profiles showed a detailed transcriptome landscape of intestinal tissue in patients with CD and UC. A total of 4,105 proteins were identified in mucosal biopsies, of which 2,128 were differentially expressed (DE) between CD and HC, 2,715 proteins DE in UC compared with HC, and 1,653 proteins DE between CD and UC. Metabolomic analyses revealed metabolites and pathways related to ketone, amino sugar and methyl histidine metabolism, Warburg effect, and ammonia recycling deregulated in IBD patients. Moreover, metagenomic results indicated that CD and UC differ in their microbial populations according to beta diversity. In comparison to HC, the microbial population in CD was different, and its alpha diversity was significantly lower (Figure 1). Conclusion The results showed that some analytes identified could play essential roles in the pathogenesis of CD and UC. New potential key mechanisms that could help to understand the information of omics layers that underlies the pathogenesis of IBD have been outlined.

P172 Spatial Transcriptomics Characterization of S1P Pathway Genes in Ulcerative Colitis and Crohn’s Disease Colon Tissues

Abstract Background Inflammatory bowel diseases (IBD), such as ulcerative colitis (UC) and Crohn’s disease (CD) are chronic inflammatory diseases of the gastrointestinal tract, related to the confluence of environmental and immunological factors in genetically predisposed individuals. Activated T and B cells migrate from the lymphoid tissue into the circulation through the interaction of Sphingosine-1-phosphate (S1P) and the S1P1 receptor (S1P1), which can perpetuate inflammation and lead to tissue damage. Targeting of cellular migratory pathways has emerged as a novel therapeutic paradigm in IBD. The aim of this study is to characterize the S1P pathway-related genes and signaling pathways driving the pathogenesis of UC and CD. Methods Spatial transcriptomics analysis was performed in health volunteer (HV, n=2), UC (n=4) and CD (n=2) FFPE colon tissues using the GeoMX platform and NanoString whole transcriptome atlas (WTA) assay. The WTA panel was run on 8 samples stained with antibodies to S1P1, pan Cytokeratin, and CD45. Six regions of interests (ROIs) were selected in each sample based on the staining and each ROI were split into two areas (CD45+ or CD45-). Differential gene expression and enriched pathways analysis including S1P pathway genes were performed in CD45+ and CD45- segments in HV, UC and CD colon tissues. Results A total of 6085 and 6054 differentially expressed genes were identified from the CD45+ segments in UC and CD respectively, compared to HV colon (P=0.05), including the genes that are involved in T cell activation (e.g., HLA-DPA1, CCDC13), immune cell migration (e.g., ADAM15, CCL5) and tissue damage (e.g., FABP6, PDK4). Interestingly, significant differences were identified in UC and CD in S1P pathway gene expression. Specifically, SMPD1, a gene involved in generating ceramide and regulating inflammatory responses, was the most significantly up-regulated gene from both CD45+ and CD45- compartments in UC compared to HV colon. While CERS2 and CERS6, genes related to S1P metabolism, were significantly down-regulated genes in CD45+ and CD45- compartment in UC respectively, as compared to HV colon. Additionally, RTN4 (participates in the intestinal epithelial barrier function) and PLPP3 (dephosphorylates extracellular S1P to sphingosine) were most significantly down-regulated genes from CD45+ segments in both UC and CD compared to HV colon. Conclusion In summary, our study successfully validated the GeoMx transcriptome analysis approach in FFPE colon tissue and assessed the S1P pathway gene signature difference in HV, UC and CD colon tissues supporting the pathway’s role in IBD. Results could also facilitate the identification of potential S1P pathway UC and CD disease biomarkers.

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.

Multi-targeting inulin-based nanoparticles with cannabidiol for effective prevention of ulcerative colitis

The pathogenesis of ulcerative colitis (UC) is closely related to severe inflammation, damaged colonic mucosal barrier, increased oxidative stress and intestinal ecological imbalance. However, due to the nonspecific distribution and poor bioavailability of drugs, UC treatment is still a serious challenge. Here, a mitochondria/colon dual targeted nanoparticles based on redox response was developed to effectively alleviate UC. Cannabidiol nanoparticles (CBD NPs) with a particle size of 143.2 ± 3.11 nm were prepared by self-assembly using polymers (TPP–IN–LA) obtained by modifying inulin with (5-carboxypentyl) triphenyl phosphonium bromide (TPP) and α-lipoic acid (α-LA). Excitingly, the constructed CBD NPs showed excellent mitochondrial targeting, with a Pearson correlation coefficient of 0.76 at 12 h. The results of animal imaging in vivo showed that CBD NPs could be effectively accumulated in colon tissue. Not only that, CBD showed significant glutathione stimulated release in the presence of 10 mM glutathione at pH 7.4. The results of in vivo animal experiments showed that CBD NPs significantly ameliorated DSS-induced colonic inflammation by modulating the TLR4-NF-κB signaling pathway. Moreover, CBD NPs significantly improved the histological damage of colon in UC mice, increased the expression level of tight junction protein ZO-1, and effectively restored the intestinal mucosal barrier function and intestinal mucosal permeability. More importantly, CBD NPs significantly improved the species composition, abundance and amount of short chain fatty acids of intestinal flora in UC mice, thus effectively maintaining the balance of intestinal flora. The dual-targeted and glutathione-responsive nanoparticles prepared in this study provide a promising idea for achieving targeted delivery of CBD for effective treatment of UC.

S100a10 deficiency in neutrophils aggravates ulcerative colitis in mice

Background and aimsS100a10 is a member of the S100 family of proteins, which plays a key role in the depression and tumor metastasis. However, the role of S100a10 is unclear in ulcerative colitis. MethodsThe effect of S100a10 was assessed using a murine ulcerative colitis model which was accompanied by parameters including body weight loss, disease activity index, histological score, colon weight and length. The quantity and role of immune cells was determined by flow cytometry and bone marrow chimeric mice. Neutrophils depletion, adoptive cell transfer and conditional knockout mice were used to ascertain which cells played the key role in ulcerative colitis. The function of neutrophils was evaluated by migration assay, phagocytosis assay, multiplex immunoassay and real-time PCR. ResultsIn this study, our data showed that S100a10-/- mice were prone to ulcerative colitis induced by dextran sodium sulfate. Neutrophils number increased in colon of S100a10-/- mice after dextran sodium sulfate treatment significantly. Meanwhile, adoptive transfer of neutrophils from wild type mice partially decreased the susceptibility of S100a10-/- mice to dextran sodium sulfate. There was no difference in ulcerative colitis between the groups of S100a10-/- mice without neutrophils and wild type mice. Finally, we found that S100a10-/- neutrophils had stronger function in secretion and synthesis of inflammatory factor. ConclusionsIn one word, these results suggest that S100a10 has a role in inhibiting the pathogenesis of ulcerative colitis through regulation of neutrophils function.

Ustekinumab Decreases Circulating Th17 Cells in Ulcerative Colitis.

Objective T helper (Th) cells play a central role in the pathogenesis of ulcerative colitis (UC). The present study analyzed the changes in circulating T cells by administration of ustekinumab (UST), an interleukin-12/23p40 antibody. Methods CD4 T cells were isolated from peripheral blood at 0 and 8 weeks after UST treatment, and we analyzed the proportion of CD4 T cells by flow cytometry. Clinical information and laboratory data were obtained at 0, 8, and 16 weeks. Patients We evaluated 13 patients with UC who received UST for the induction of remission between July 2020 and August 2021. Results The median partial Mayo score improved from 4 (1-7) to 0 (0-6) (p<0.001) with UST. Among serological parameters, albumin concentrations, C-reactive protein concentrations, the sedimentation rate, and leucine-rich alpha 2 glycoprotein concentrations showed significant improvement with UST. A flow cytometric analysis of circulating CD4 T cells showed that the percentage of Th17 cells was significantly decreased by UST treatment in all patients (1.85% to 0.98%, p<0.0001). Th1 cells were significantly increased by UST treatment (9.52% to 10.4%, p<0.05), but Th2 and regulatory T cells were not significantly different. The high-Th17 subgroup had a significantly better partial Mayo score than the low-Th17 subgroup at 16 weeks after UST treatment (0 vs. 1, p=0.028). Conclusion Treatment with UST decreases circulating Th17 cells, suggesting that this change may be related to the anti-inflammatory effect of UC.

Increased ACE2 and TMPRSS2 expression in ulcerative colitis

Ulcerative colitis (UC) is a cryptogenic inflammatory bowel disease, and there is an urgent need to elucidate its pathogenesis. ACE2 and TMPRSS2, the entry molecules of SARS-CoV-2, are reportedly associated with the disease; however, no consensus has been reached yet. In this study, we examined the expression of ACE2 and TMPRSS2 in colon and rectal specimens of UC. We collected colorectal specimens from 60 patients (30 patients with UC and 30 controls from 2018 to 2021) and analyzed the proportion and intensity of ACE2 and TMPRSS2 using immunohistochemistry. The results revealed a significant increase in the proportion of ACE2 expression and the intensity of TMPRSS2 expression in patients with UC. ACE2 and TMPRSS2 expression in UC remained unaffected by the COVID-19 pandemic. We demonstrated that ACE2 and TMPRSS2 are likely involved in the pathogenesis of UC.