Non-inflamed Regions Research Articles

P0147 Regulation of Inflammatory Pathways by miR-338-3p and miR-378a-3p: A Novel Approach for Crohn’s Disease Treatment

Abstract Background MicroRNAs (miRNAs), key regulators of gene expression, are promising biomarkers and therapeutic targets in inflammatory bowel disease (IBD) due to their roles in immune response and tissue integrity. This study explores miRNA roles in IBD, focusing on Crohn's disease (CD) pathogenesis and potential therapies. We analyzed epithelial cell-derived miRNA expression in CD patients versus healthy controls and investigated how these miRNAs modulate inflammation in experimental IBD models. Methods Terminal ileum samples were prospectively collected via ileocolonoscopy from non-IBD healthy controls (n=26), CD patients in remission (n=34), and active CD patients (n=42). Clinical remission was defined as a Crohn’s Disease Activity Index score below 150 with endoscopic mucosal healing. For active CD cases, samples were taken from inflamed and non-inflamed regions. miRNA levels were quantified by small-RNA sequencing and validated by qRT-PCR. Based on these results, specific miRNAs were selected, and single-stranded mimics were administered in a 2.5% DSS-induced colitis model to evaluate their effects. Histological grading, immunohistochemistry, flow cytometry, and microbiome analysis using mouse fecal samples were performed. Results Small-RNA sequencing revealed distinct miRNA expression patterns in CD patients, both in remission and in active states (including inflamed and non-inflamed tissues), compared to healthy controls. qRT-PCR validation showed significantly reduced expression of miR-141-3p, miR-338-3p, miR-378a-3p, and miR-200b-3p, along with significantly increased levels of miR-125b-5p, miR-29b-3p, and miR-155-5p in inflamed tissue of active CD patients. Receiver operating characteristic curve indicated diagnostic potential for these miRNAs in relation to disease activity and endoscopic inflammation. In a DSS-induced colitis mouse model, miR-338-3p and miR-378a-3p showed anti-inflammatory effects, reducing body weight loss, colon inflammation, and immune cell markers. Target analyses identified MACC1 and IL-33 as key mRNA targets. Immunohistochemistry indicated an IL-33 pathway-dependent anti-inflammatory response via miR-378-3p, while miR-338-3p appeared to regulate gut inflammation by targeting MACC1 gene expression. In vitro analyses revealed that IL-33 activates MACC1, a process also reduced by miR-378a-3p treatment. Fecal microbiome analysis identified several bacterial species including Akkermansia muciniphilia and certain Bacteroides, associated with beneficial effects in the groups treated with both miRNA mimics. Conclusion This study identifies miR-338-3p and miR-378a-3p as modulators of inflammation in an IL-33-dependent manner in CD patients, underscoring their potential as promising targets for treatment of CD.

OP37 Delineating molecular and microbial signatures across disease severity in Inflammatory Bowel Disease in adult treatment naive patients through single-cell transcriptomic and microbiome profiling.

Abstract Background Inflammatory Bowel Disease (IBD), including Crohn's disease (CD) and Ulcerative Colitis (UC), is a chronic inflammatory condition with complex etiology involving genetic, immune, microbiome, and environmental factors. This study aims to elucidate the cellular and microbial landscape of adult treatment-naive IBD patients with varying disease severity to identify potential disease mechanisms and therapeutic targets, presenting one of the largest single-clinic, IBD single-cell datasets to date. Methods Tissue biopsies were collected from inflamed and non-inflamed regions of the colon in UC patients and colon + ileum in CD patients. Using 10x Genomics single-cell RNA sequencing (scRNA-seq), we analyzed 1.4 million cells from 68 treatment-naive IBD patients (38 CD, 30 UC) and 70 symptomatic healthy controls. Shotgun metagenomic sequencing was performed on 36 IBD patients with matched scRNA-seq data to explore host-microbe interactions, and a further 49 patients with paired clinical data. At presentation, standard clinical metadata, Patient Reported Outcomes, post-biopsy treatment, and 6/12-month treatment response were collected for stratification across disease severity and response. Results Our analysis revealed distinct cellular populations and gene expression profiles between IBD patients and healthy controls along the disease severity axis. Specific immune cell subsets were significantly expanded in IBD patients, with inflammatory monocytes particularly prominent in those with higher disease severity, exhibiting upregulated expression of pro-inflammatory cytokines and chemokines. Additionally, a shift from IgA to IgG antibody-producing cells was observed along the disease severity axis, indicating a dysregulated immune response associated with disease progression. Fecal microbial community typing based on pathway abundance revealed two clusters defined by low or high abundance of Bacteroidetes phylum taxa. Integration with clinical metadata and transcriptional profiles showed that Bacteroidetes-high UC patients displayed lower levels of inflammation and up-regulation of interferon signaling in B cells and myeloid cells. Conclusion This comprehensive profiling of adult treatment-naive IBD patients provides valuable insights into the cellular and microbial factors contributing to IBD prior to treatment perturbation. Our findings offer a deeper understanding of the underlying disease pathology of IBD patients at the time of presentation along the axis of disease severity, expanding the dynamic range of IBD patient profiling and informing future therapeutic strategies that may more quickly put patients on the right treatment and better address the causes of IBD in addition to the inflammatory response alone.

P0132 ACSS2 inhibition alleviates inflammatory signaling and improves epithelial integrity in Inflammatory Bowel Disease in vitro models

Abstract Background Inflammatory bowel disease (IBD), encompassing Ulcerative Colitis (UC) and Crohn’s Disease (CD), is marked by chronic gastrointestinal inflammation stemming from genetic, environmental, immunological, and microbiota factors1. Acetyl-CoA Synthetase Short Chain Family Member 2 (ACSS2) converts acetate into acetyl-CoA and is primarily cytosolic, but translocates to the nucleus under metabolic stress, where it regulates gene transcription and cellular processes2. Although the gastrointestinal microbiota influences acetate levels, the role of ACSS2 in colitis and intestinal inflammation remains poorly understood3. Methods To evaluate ACSS2's role in IBD, we employed in vitro IBD models using Caco-2 and HT-29 intestinal epithelial cell lines. ACSS2 activity was inhibited with an ACSS2 inhibitor, followed by inflammatory stimulation with 100 ng/ml Tumor Necrosis Factor-α (TNF-α) for 30 minutes or 1% Dextran Sodium Sulfate (DSS) for 6 hours. Protein expression levels were assessed via Western blot and immunohistochemistry (IHC) on paraffin-embedded samples from UC patients. The effects of ACSS2 inhibition on inflammatory signaling pathways and cell integrity were assessed using RT-qPCR, immunoblotting, and transepithelial electrical resistance (TEER) measurements. Results Treatment of Caco-2 and HT-29 cells with DSS or TNF-α induced upregulation of ACSS2 protein levels compared to controls. IHC analysis of 30 paired paraffin-embedded colon samples from UC patients revealed higher ACSS2 expression and nuclear localization in inflamed sections compared to non-inflamed regions. Inhibition of ACSS2 activity in stimulated Caco-2 and HT29 cells led to a significant decrease in the gene expression levels of pro-inflammatory cytokines such as Interleukin 8 (IL-8), IL-1β and TNF-α. Additionally, immunoblotting demonstrated reduced phosphorylation of p65 and ERK/MEK, indicating suppression of NF-κB and MAPK inflammatory signaling pathways. To assess the effect of ACSS2 inhibition on cell integrity, TEER assays revealed that ACSS2 inhibition preserved cell integrity in DSS-treated monolayers, accompanied by increased expression of tight junction proteins Occludin and ZO-1, suggesting improved epithelial integrity in the inflammatory context. Conclusion Our findings demonstrate a pro-inflammatory role for ACSS2 in IBD, mediated through its involvement in inflammatory signaling and epithelial integrity. These results highlight ACSS2 as a potential therapeutic target for IBD and warrant further investigations in more advanced models, including human-derived intestinal organoids and in vivo systems, to validate its role in disease pathogenesis and therapeutic potential.

P0130 Assessment of macrophage types in the intestinal tissue of treatment naïve Inflammatory Bowel Disease patients in relation to their disease and activation of the CCL2/CCR2 pathway

Abstract Background Resident intestinal macrophages play a key role in maintaining gut homeostasis and exhibit a hypoactive tolerogenic phenotype. Recently, several subsets of intestinal macrophages have been identified based on CD11c expression which differ in their inflammatory and functional profiles. Notably, pro-inflammatory CD11c+ monocyte-like macrophages are found elevated in patients with inflammatory bowel disease (IBD), specifically Ulcerative colitis (UC) and Crohn’s disease (CD). These cells are recruited from blood monocytes via the CCR2/CCL2 chemokine pathway. Our study is focused on observing the patterns of CCL2-driven blood monocyte migration and their translation into subsets of intestinal tissue macrophages that can determine the degree of gut inflammation in newly diagnosed patients. Methods Patients referred with suspected IBD were seen in an inception clinic. Intestinal biopsies and matched blood samples were obtained from pre-treatment UC and CD patients during their index colonoscopy. Those with inflammation excluded during this procedure formed a ‘non-IBD’ control group. Biopsy segments were cultured to generate conditioned media and also digested for flow cytometry analysis. Plasma concentrations of CCL2 and intestinal secretome were measured using ELISA or Olink proteomic analysis respectively. Efficacy of blood monocyte chemotaxis towards CCL2 was assessed in the presence or absence of CCL2/CCR2 antagonists and the profile of monocyte subsets in whole blood were determined using whole blood flow cytometry analysis. Results As previously described, we observed distinct CD11c+ and CD11c- macrophage populations in IBD intestinal tissue, with expansion of the CD11c+ subset within inflamed regions which expressed higher levels of the chemokine receptors CCR2 and CX3CR1. Higher concentrations of CCL2 were observed in conditioned media from inflamed IBD tissue compared to non-inflamed regions and this correlated with elevated numbers of circulating blood monocytes. Chemotaxis assays revealed a corresponding increase in CCL2-induced monocyte migration in patients with IBD compared to the ‘non-IBD’ control group. Conclusion Our study provides further insights into the role of the CCL2/CCR2 chemokine axis in driving monocyte recruitment and their differentiation into pro-inflammatory intestinal macrophages in IBD. We demonstrate that inflamed intestinal regions exhibit elevated levels of CCL2 correlating with an expansion of the CD11c⁺ macrophage subset and increased CCL2-driven monocyte migration in these subjects. These findings underscore the significance of targeting the CCL2/CCR2 pathway and that it might be an important driver in the development IBD.

Impaired activation of succinate-induced type 2 immunity and secretory cell production in the small intestines of Ptk6−/− male mice

Protein tyrosine kinase 6 (PTK6) is an intracellular tyrosine kinase that is distantly related to the SRC family of tyrosine kinases. It is expressed in epithelial linings and regulates regeneration and repair of the intestinal epithelium. Analysis of publicly available datasets showed Ptk6 is upregulated in tuft cells upon activation of type 2 immunity. We found that disruption of Ptk6 influences gene expression involved in intestinal immune responses. Administration of succinate, which mimics infection and activates tuft cells, revealed PTK6-dependent activation of innate immune responses in male but not female mice. In contrast to all wild type and Ptk6−/− female mice, Ptk6−/− male mice do not activate innate immunity or upregulate differentiation of the tuft and goblet secretory cell lineages following succinate treatment. Mechanistically, we found that PTK6 regulates Il25 and Irag2, genes that are required for tuft cell effector functions and activation of type 2 innate immunity, in organoids derived from intestines of male but not female mice. In patients with Crohn’s disease, PTK6 is upregulated in tuft cells in noninflamed regions of intestine. These data highlight roles for PTK6 in contributing to sex differences in intestinal innate immunity and provide new insights into the regulation of IL-25.

A199 BIOFILMS IN THE APPENDIX AND OTHER NON-INFLAMED SECTIONS OF THE COLON IN PEDIATRIC PATIENTS WITH INFLAMMATORY BOWEL DISEASES

Abstract Background Biofilms, aggregated bacteria colonizing the extracellular polymeric substances matrix, are associated with the mucosa of inflammatory bowel diseases (IBD) patients with some studies showing a mean density of the mucosal biofilms 2-fold higher in IBD patients than in controls. The appendix, which is a highly immune organ, seems to be involved in IBD pathogenesis. Purpose In this study we aimed to evaluate biofilms in the appendix and other non-inflamed regions of the colon in pediatric IBD patients. We hypothesized that biofilms composed of pathobionts in these sections could drive inflammation in IBD patients. Method Tissues from the appendix, peri-appendicular region, cecum, and ascending colon (ASC), collected from the resected colons of 9 pediatric IBD patients and one pediatric non-IBD patient, preserved in methanol Carnoy’s solution were processed and paraffin embedded. Combined fluorescence in situ hybridization (FISH) for biofilms (probe: EUB338) and immunofluorescence (IF) mucin staining (MUC2) identified biofilms and their location. Biofilm formation capacity of culturable bacteria (identified through 16S DNA Sanger sequencing) from these sections was measured. Interleukin (IL)-8 (pro-inflammatory chemokine) and IL-10 (anti-inflammatory cytokine) expressions were assessed by tissue qPCR. Result(s) FISH demonstrated biofilms in these sections, in close proximity to epithelial cells. We used a biofilm formation assay to assess the ability of the identified bacteria from these sections to form biofilms, illustrating their potential to colonize and evade host defense and potential antibiotics. We found that Enterococcus avium has a significantly higher ability to form biofilms than the negative control. IL-8 and IL-10 both had the highest expression level in the appendix and peri-appendicular region and the lowest in the ASC among all the patients, suggesting an active immune response. Mechanistic experiments are in progress to investigate the type of bacteria involved in the biofilms and their effects on the gut barrier integrity. Conclusion(s) Biofilms adjacent to the epithelium, especially in the appendix and peri-appendix, could interact with or invade epithelial cells. The elevated chemokine transcript level in the appendix could reflect the recruitment of immune cells to this section following bacterial invasion, resulting in the activation of the immune system. Identifying the bacteria involved in the biofilms and clarifying their characteristics will aid us in developing novel microbe-altering treatment strategies or personalized medicine. Disclosure of Interest None Declared

P039 The disappearance of cell death brakes in inflammatory bowel disease: a step towards molecular healing

Abstract Background The core mechanistic driver underpinning the relapsing and progressive nature of inflammatory bowel disease (IBD) remains unknown with current therapies targeting immune or inflammatory pathways. Our in-depth analysis of regulated death pathways remarkably reveals that cell death brakes are removed in intestinal IBD tissue, indicating cells remain “primed” to die regardless of endoscopic and histologic healing. Characterisation of cell death is essential to confirm our hypothesis that disinhibition of programmed cell death is a rudimentary molecular defect in IBD. Restoration of cell death brakes may be intrinsic to molecular healing and novel drug development. Methods Intestinal biopsies are collected from inflamed, marginal and non-inflamed regions during endoscopy for patients with IBD and matched with those without IBD (“controls”). Tissue is processed to quantify cell death signals and changes to the local microbiome using immunoblotting, immunohistochemistry, immunofluorescence, organoid preparation and RNA sequencing (Fig. 1). Laboratory data are correlated with clinical, biochemical, endoscopic and histologic parameters. Results To date, 59 patients have been recruited: 29 controls; 31 IBD (13 Crohn’s disease, 16 ulcerative colitis, 2 pouchitis). Interim analyses of 20 patients show loss of cell death brakes (cIAP1, cIAP2, cFLIP) suggesting disinhibition of cell death even in endoscopically and histologically normal IBD tissue (Fig. 2). Concurrently, there is increased apoptosis (characterised by cleaved caspase-3) in IBD intestinal tissue regardless of inflammatory activity (Fig. 3). Necroptosis, a programmed lytic form of cell death, is active in steady state conditions in controls. Additionally, necroptosis appears markedly elevated in stricturing and penetrating Crohn’s disease compared to other subsets of IBD (Fig. 2). Conclusion This study demonstrates for the first time that cellular death brakes are removed in intestinal tissue in IBD, despite deep remission with seemingly effective biologic therapy. Disinhibition of programmed cell death is likely intrinsic to the inflammatory sequence underlying disease recurrence and progression. Restoration of cell death brakes may hence be key to achieving long-term, relapse-free remission and should be considered an aspirational goal for both drug development and achievement of molecular healing. Our results challenge current dogma based on murine data that necroptosis occurs in the absence of apoptosis and that necroptosis is universally increased in all forms of IBD. Instead, we propose that a balance of apoptosis and necroptosis is essential in intestinal homeostasis with endotyping of disease based on cell death profile a feasible goal towards precision medicine in IBD.

Modulation of Indoleamine 2,3-Dioxygenase 1 During Inflammatory Bowel Disease Activity in Humans and Mice.

Indoleamine 2,3 dioxygenase-1 (IDO1), a key enzyme in tryptophan metabolism, is strongly up-regulated both in human inflammatory bowel disease (IBD) and animal models of colitis, however its role in the pathogenesis is still controversial. In this study, we investigated IDO1 expression and activity in a mouse model of DSS-induced chronic colitis as well as in colon biopsies and sera from IBD patients. Chronic colitis was induced in mice through the oral administration of dextran sodium sulfate (DSS), and IDO1 activity was induced by i.p. treatment with N-acetyl serotonin (NAS). IDO1 expression and catalytic activity (measured as Kyn/Trp ratio) was evaluated in sera and tissue samples collected from mice and 93 IBD patients under immunotherapy with Vedolizumab (VDZ) or Ustekinumab (UST). Strong up-regulation of IDO1 was found in colons of mice with acute colitis, which follows disease activity. Enhanced IDO1 activity by NAS treatment protects the intestinal mucosa during the recovery phase of chronic colitis. In IBD patients, IDO1 expression and activity correlate with the severity of mucosal inflammation with inflamed regions showing higher IDO1 expression compared to non-inflamed regions within the same patient. Endoscopic response to VDZ/UST treatment is associated with decreased expression of IDO1. This is the first study demonstrating immunomodulatory activity of IDO1 in a chronic mouse model of DSS-induced colitis. As its expression and catalytic activity correlate with the grade of mucosal inflammation and treatment response, IDO1 could represent a promising biomarker for disease severity and treatment monitoring in IBD.

Tryptophan Metabolism 'Hub' Gene Expression Associates with Increased Inflammation and Severe Disease Outcomes in COVID-19 Infection and Inflammatory Bowel Disease.

The epithelial barrier's primary role is to protect against entry of foreign and pathogenic elements. Both COVID-19 and Inflammatory Bowel Disease (IBD) show commonalities in symptoms and treatment with sensitization of the epithelial barrier inviting an immune response. In this study we use a multi-omics strategy to identify a common signature of immune disease that may be able to predict for more severe patient outcomes. Global proteomic approaches were applied to transcriptome and proteome. Further semi- and relative- quantitative targeted mass spectrometry methods were developed to substantiate the proteomic and metabolomics changes in nasal swabs from healthy, COVID-19 (24 h and 3 weeks post infection); serums from Crohn's disease patients (scored for epithelial leak), terminal ileum tissue biopsies (patient matched inflamed and non-inflamed regions, and controls). We found that the tryptophan/kynurenine metabolism pathway is a 'hub' regulator of canonical and non-canonical transcription, macrophage release of cytokines and significant changes in the immune and metabolic status with increasing severity and disease course. Significantly modified pathways include stress response regulator EIF2 signaling (p = 1 × 10-3); energy metabolism, KYNU (p = 4 × 10-4), WARS (p = 1 × 10-7); inflammation, and IDO activity (p = 1 × 10-6). Heightened levels of PARP1, WARS and KYNU are predictive at the acute stage of infection for resilience, while in contrast, levels remained high and are predictive of persistent and more severe outcomes in COVID disease. Generation of a targeted marker profile showed these changes in immune disease underlay resolution of epithelial barrier function and have the potential to define disease trajectory and more severe patient outcomes.

The protective impact of adapted trimebutine maleate-loaded nanostructured lipid carriers for alleviating the severity of acute colitis

Nanoparticles for colon-drug delivery were designed and evaluated to solve many discrepancy issues such as high adverse effects of released drugs, insufficient drug amount at diseased areas, and unintentionally premature drug release to noninflamed GIT regions. Herein, the goal of this work was to convert trimebutine maleate (TMB) into nanostructured lipid carriers (NLC) in order to improve its protective effects in ulcerative colitis. NLC of TMB was prepared by the hot homogenization followed by ultra-sonication method. A full 42-factorial design was used to estimate the produced TMB-NLC. The study design included the exploration of the impact of two independent variables namely lipid mix amount and ratio (glyceryl mono stearate and capryol 90), surfactant concentration (0.5, 1, 1.5, and 2%), on the particle size, polydispersity index, and the entrapment efficiency (EE%). The protective activity of F9 was examined through macroscopical scores, histopathological changes, immunohistochemical localization of tumor necrosis factor-α (TNF-α) and examination of oxidative stress such as reduced glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) against acetic acid-induced colitis in rats. Consistent with our expectations, the orally administered optimized formula (F9) alleviated the severity of colitis in acetic acid-induced rat model of colitis likely owing to the controlled release compared to free TMB. We aimed to develop TMB-loaded NLC for the treatment of acute colitis with the goal of providing a superior drug safety profile over long-term remission and maintenance therapy.

A237 VIRULENCE POTENTIAL OF BACTERIA ISOLATED FROM NON-INFLAMED COLON IN PATIENTS WITH INFLAMMATORY BOWEL DISEASES

BackgroundAs inflammation can impact microbes, our lab has focused on non-inflamed bowel sections of patients with inflammatory bowel diseases (IBD) and found bacterial alterations in the terminal ileum. We are also interested in the appendix given the involvement of the peri-appendicular region and effects of appendectomy in ulcerative colitis.AimsIn this study, we aimed to identify and evaluate microbes from the appendix and non-inflamed regions of the colon in IBD patients. We hypothesized that microbes originating from the non-inflamed sections of the colon or appendix are more invasive and could trigger inflammation in IBD patients.Methods16S DNA Sanger sequencing was performed on bacteria, aerobically and anaerobically cultured from tissues of the appendix, peri-appendicular region, cecum, and ascending colon, collected from the resected colons of 5 pediatric IBD patients. The invasive capacity of the identified bacteria was evaluated by gentamicin protection assays on Caco-2 intestinal epithelial cells in vitro. Presence of select invasive or adhesive genes in the bacteria was assessed by PCR.Results Escherichia coli was the most frequently cultured species in the aerobic cultures; additional aerobic species included Enterococcus and Klebsiella. Bifidobacterium and Erysipelatoclostridium were identified among the anaerobic cultures. Gentamicin protection assays indicated that Klebsiella isolated from the peri-appendicular region was significantly more invasive (mean ~3000 CFU/ml) than HB101 (~1000 CFU/ml; non-invasive control; P<0.05). Enterococcus avium and Enterococcus faecalis were also more invasive than HB101 in the peri-appendicular region (~10000 CFU/ml) and cecum (~4000 CFU/ml; P<0.05), respectively. E. coli isolated from the appendix showed a higher invasive potential (~3000 CFU/ml) than E. coli isolated from other sections. Additionally, PCR showed that E. coli obtained from different sections, except the ascending colon of one the patients, had the fimH gene while the other types of bacteria did not. None of the isolated bacteria had Hemolysin ( hlyA) or attaching and effacing ( eaeA) genes.ConclusionsBacteria, especially E. coli, from non-inflamed bowel in IBD (including the appendix) appear to have increased invasive potential. This suggests that the microenvironment in these regions could be altered, resulting in increased invasion of bacteria or the gut harboring more invasive microbial populations in IBD patients. As a result, inflammation could be triggered or exacerbated through this reservoir of pathobionts.Funding AgenciesCIHR

P033 Single-cell analysis of gut mucosal- and peripheral blood cells in ulcerative colitis patients undergoing vedolizumab treatment

Abstract Background Vedolizumab (VDZ), a monoclonal antibody that targets α4β7 integrin, was approved to treat moderate-to-severe ulcerative colitis (UC) based on the presumption that it blocks T cell recruitment to the inflamed intestinal mucosa. The clinical evidence suggests that up to 50% of UC patients do not achieve disease remission under VDZ treatment. This study aims to identify changes in cell abundances and molecular pathways associated with VDZ response in UC. To this end, we included anti-tumor necrosis factor (anti-TNF)-naïve and anti-TNF-exposed patients with active UC, and utilized single-cell RNA sequencing (scRNAseq) and high-dimensional flow cytometry (Cytek) to assess the peripheral blood and the gut mucosal compartments. Methods Gut mucosal biopsies from inflamed and non-inflamed regions, and peripheral blood mononuclear cells (PBMCs) were obtained from UC patients 2 wks before (t0) and 14 wks after (t4) the start of VDZ administration. Response to treatment was prospectively evaluated based on endoscopic assessment (defined as a decrease in total Mayo score between t0 and t4) and physician global assessment (PGA) that incorporates disease activity score and biochemical measurements. Results A total of 25 UC patients (pts) were included: 44% anti-TNF-naïve. Endoscopic response to VDZ was observed in 32% of UC pts, while 56% of pts showed response based on PGA. The VDZ response rate (by PGA) was higher in anti-TNF-naïve pts vs anti-TNF-exposed pts (82% vs 36% responders, respectively). A preliminary analysis was performed on samples from 8 (out of 25) UC pts, profiling &amp;gt;70,000 gut mucosal cells and &amp;gt;25,000 PBMCs. Within the mucosal compartment, at t0 we identified immune cells (50% of all captured cells), stromal cells (10%), and epithelial cells (40%). Upon inflammation, the proportion of immune cells increased to 70%, stromal cells to 20%, while epithelial cells depleted to 10%. Notably, all main identified immune cell lineages – T cells, B cells and myeloid cells – contributed to the expansion of the immune cell compartment in inflamed mucosa. In line with scRNAseq data, we identified all major immune cell populations and detected expression of both the classic gut-directed and the redundant trafficking integrins by Cytek. Conclusion The preliminary results substantiate our current understanding of VDZ biology in UC. We confirm that anti-TNF-naïve pts have a higher response rate to VDZ vs anti-TNF-exposed pts. With this unique cohort, our study has the power to further explore molecular mechanisms and pathways that underlie VDZ response at the single-cell level.

P031 Intestinal lymphoepithelial interactions in Crohn’s disease: modeling alphaE-beta7 and NKG2D blockade

Abstract Background T resident memory (Trm) cells in the intestinal mucosa and in particular subpopulations expressing phenotypic markers such as alphaE-beta7 or NKG2D have been associated with chronic inflammatory bowel disease (IBD) activity. We hypothesize that these populations may have a direct deleterious impact on the intestinal epithelium in IBD. Methods The phenotypic study of mucosal lymphocytes was performed in two prospective cohorts: ELYP including patients with active IBD before initiation of biotherapy and REMIND including patients with ileal Crohn’s disease (CD) requiring ileocaecal resection. These analyses were performed before initiation of treatment and one year after continuous therapy in the ELYP cohort and on the resection specimen in the REMIND cohort. An innovative ex vivo autologous organoid-mucosal T cell coculture model was developed using the REMIND cohort specimens for patients and healthy ileum from individuals without IBD for controls (Figure 1). T cell infiltration within the organoid and epithelial cell death were assessed by confocal microscopy. A panel of 30 cytokines was quantified in the supernatants of cocultures. Figure 1 Results In the ELYP cohort, before the start of treatment, IBD patients had lower expression of alphaE-beta7 and NKG2D on mucosal CD8 T cells. While alphaE-beta7 and NKG2D expression on mucosal CD8 T cells had returned to normal in endoscopic responders, it remained decreased in non-responders. Similarly, the inflammatory mucosa of the REMIND surgical specimens had lower levels of CD4 and CD8 T cells expressing alphaE-beta7 and NKG2D compared with controls and non-inflamed regions. We developed a coculture model between mucosal lymphocytes and organoids generated under autologous conditions. We showed an increase in apoptotic cell death in epithelial cells from CD patients which was not found in control cocultures. There was a significant correlation between the degree of epithelial cell death and T cell infiltration (Figure 2). Various proinflammatory cytokines such as IFNgamma, TNFalpha, IL-6 and IL- 17a were also increased in the supernatants. The use of antibodies blocking the alphaE-beta7 and NKG2D pathways of interest inhibited this effect by different mechanisms (Figure 3) While anti-beta7 and anti-NKG2D had comparable effects in terms of cell death inhibition, only anti-beta7 reduced T cell infiltration. Anti-NKG2D had no effect on cell infiltration but a reduction of perforin was observed in the supernatants. Figure 2 Figure 3 CC= Coculture Conclusion These data demonstrate for the first time the direct cytotoxic deleterious effect of mucosal T cells on the epithelium of CD patients using a novel coculture model. AlphaE-beta7 and NKG2D pathways appear to be relevant in this process.

Associations between Cellular Energy and Pediatric Inflammatory Bowel Disease Patient Response to Treatment.

Inflammatory bowel diseases (IBDs), including Crohn's disease (CD) and ulcerative colitis, are chronic diseases of the gastrointestinal tract, with an unknown etiology, that affect over 6.8 million people worldwide. To characterize disease pathogenesis, proteomic and bioinformatic analyses were performed on colon biopsies collected during diagnostic endoscopy from 119 treatment-naïve pediatric patients, including from 78 IBD patients and 41 non-IBD patients who served as controls. Due to the presence of noninflamed and/or inflamed regions in IBD patients, up to two biopsies were obtained from IBD patients as compared to a single noninflamed biopsy from non-IBD pediatric control patients. Additional biopsies were obtained and analyzed from 33 of the IBD patients after IBD-directed therapeutic intervention for comparison of pre- and post-treatment proteomes. SuperSILAC was utilized to perform quantitative analysis of homogenized tissues, which were processed by filter-aided sample preparation. Hierarchical clustering and principal component analyses revealed proteomic patterns that distinguished inflamed from noninflamed tissues independent of therapy. Gene ontology revealed that proteins downregulated in inflammation are associated with metabolism, whereas upregulated proteins contribute to protein processing. A comparison of pre- and post-treatment proteomes from CD patients identified over 100 proteins that are significantly different between patients who responded and those who did not respond to therapy, including creatine kinase B and basigin.

P082 Assessment of anti-inflammatory effect of high acetate administration in UC patient-derived epithelial monolayer cultures

Abstract Background Human organoid-based intestinal monolayer cultures provide an interesting tool to preclinically assess new therapeutic properties in inflammatory bowel disease (IBD). Recently, short-chain fatty acids (SCFA) such as acetate and butyrate have gained interest for their potential beneficial effects in IBD therapy. Most studies have focused on butyrate, given its beneficial effects on gut microbiome composition, intestinal barrier function and the immune system1. Effects of acetate are less well known, despite its lower toxicity to epithelial cells and its potential to support growth of butyrate-producing bacteria by metabolic cross-feeding2. In fact, a recent report suggested that the probiotic potential of Saccharomyces cerevisiae var. boulardii might be related to its high acetic acid production3. We here studied the effect of acetate on organoid-derived epithelial monolayer cultures from ulcerative colitis (UC) patients. Methods Colonic biopsies were obtained from non-inflamed regions in 3 patients with UC. Crypts were isolated, cultured and expanded as 3D-organoids. These organoids were then dissociated and transferred to transwell inserts as monolayer cultures. Upon confluency of the monolayer, evaluated by Transepithelial electrical resistance (TEER), cells were basolaterally stimulated with control medium (CTRL) or an inflammatory mix (INFL) containing 100 ng/ml TNF-α, 20 ng/ml IL-1β and 1 µg/ml Flagellin (Fig. 1). After 24h, cells were apically stimulated with control medium or high acetate (HA) (100mM). TEER was measured at 0, 24 and 48h stimulation. After 48h, cells were subjected to RNA extraction followed by reverse transcriptase qPCR targeting a selection of key diagnostic marker genes. The apical and basolateral media were collected for cytokine determination by Mesoscale (Proinflammatory panel 1). Data were analyzed using GraphPad Prism 9 (D’Agostino &amp; Pearson test for normality followed by a Friedman test). Results A protective effect (Fig. 2) of high acetate administration on TEER values (HA vs INFL: p=0˒017) was observed, together with a trend towards decrease in IL8 (p=0˒11), TNFA (INFL vs INFL+HA: p=0˒68) and CLDN2 (CTRL vs HA: p=0˒03). Moreover, this was confirmed by a decrease of most proinflammatory cytokines (Fig. 3) in the apical and basolateral media upon HA stimulation e.g., IFN-γ (INFL vs INFL+HA: p=0˒25) and IL 10 (CTRL vs HA: p=0˒11). Conclusion In this patient-derived human epithelial cell culture model, a protective effect of high acetate administration on TEER-values, gene expression and cytokine production was observed. Ongoing experiments are expanding the number of patients to 10. References

Single-cell analyses of Crohn\u2019s disease tissues reveal intestinal intraepithelial T cells heterogeneity and altered subset distributions

Crohn’s disease (CD) is a chronic transmural inflammation of intestinal segments caused by dysregulated interaction between microbiome and gut immune system. Here, we profile, via multiple single-cell technologies, T cells purified from the intestinal epithelium and lamina propria (LP) from terminal ileum resections of adult severe CD cases. We find that intraepithelial lymphocytes (IEL) contain several unique T cell subsets, including NKp30+γδT cells expressing RORγt and producing IL-26 upon NKp30 engagement. Further analyses comparing tissues from non-inflamed and inflamed regions of patients with CD versus healthy controls show increased activated TH17 but decreased CD8+T, γδT, TFH and Treg cells in inflamed tissues. Similar analyses of LP find increased CD8+, as well as reduced CD4+T cells with an elevated TH17 over Treg/TFH ratio. Our analyses of CD tissues thus suggest a potential link, pending additional validations, between transmural inflammation, reduced IEL γδT cells and altered spatial distribution of IEL and LP T cell subsets.

Abstract PO-226: Localization of macrophages and neutrophils in the prostate tumor microenvironment and their association with prostate cancer racial disparities

Abstract African American (AA) men are two to three times more likely to die from prostate cancer (PCa) than European American (EA) men. This disparity may be due in part to socioeconomic status and discrepancies in access to quality care. Studies also suggest that differences in innate immune effector cells and function in the tumor microenvironment of AA men may promote PCa aggressiveness. Increased tumor- associated macrophages are associated with poorer outcomes and a high neutrophil- to-lymphocyte ratio is significantly associated with poorer survival in men with PCa. However, the prevalence of innate immune cells, their spatial localization, and their relationship to PCa racial disparities is largely unknown. We evaluated CD66ce+ neutrophils and CD68+ (pan), CD80+ (M1), and CD163+ (M2) macrophages using RNA in situ hybridization or immunohistochemistry on formalin-fixed paraffin-embedded whole tissue sections from prostate donor tissues with no cancer (n=4) and radical prostatectomy tissues from AA and EA men with low grade (Gleason ≤ 3+4) and higher grade (Gleason ≥ 4+3) PCa (n=38). Tissue microarray (TMA) sets containing radical prostatectomy tissues (n=932) or distant metastatic tissues obtained at autopsy (n=6) were also evaluated. Immune marker expression in TMAs was quantified using TMAJ and FrIDA software. CD66ce+ neutrophils were primarily localized to blood vessels in organ donor prostate specimens as well as normal appearing, non-inflamed regions of radical prostatectomy specimens. Increased CD66ce+ neutrophils were present in inflamed regions of benign tissues from both races. In analysis of TMAs, there was significantly increased CD66ce+ neutrophils in tissues from EA men compared to AA men (p &amp;lt; 0.0001). There was also increased CD66ce expression in cancer compared to benign tissue spots (p &amp;lt;0.01), in low grade compared to higher grade cancer (p &amp;lt; 0.0001), in cancer with PTEN loss versus intact PTEN (p &amp;lt; 0.0001), and in ERG positive versus ERG negative cancer (p = 0.0011). There was no association between CD66ce+ neutrophils and risk of metastasis or biochemical recurrence. CD68+ and CD163+ macrophages were abundant in organ donor specimens, while CD80 expression was minimal. Similar expression patterns were observed in benign regions of radical prostatectomy specimens. In analysis of TMAs, CD68, CD80 and CD163 were significantly increased in cancer compared to benign tissues (p &amp;lt; 0.0001). CD163 expression was comparable between races, while CD80 expression was significantly higher in benign tissues from AA men compared to EA men (p = 0.0004). CD68, CD80 and CD163 expression were all significantly increased in benign spots from ERG positive versus ERG negative cases. In conclusion, there is increased infiltration of innate immune cells in the prostate tumor microenvironment of both AA and EA men, however the composition of innate immune cell infiltration varies between races. Our future studies aim to determine the influence of innate immune effector cells on PCa aggressiveness and outcomes. Citation Format: Janielle P. Maynard, Jiayun Lu, Igor Vidal, Corinne E. Joshu, Angelo M. De Marzo, Karen S. Sfanos. Localization of macrophages and neutrophils in the prostate tumor microenvironment and their association with prostate cancer racial disparities [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-226.

Mitochondrial impairment drives intestinal stem cell transition into dysfunctional Paneth cells predicting Crohn’s disease recurrence

ObjectiveReduced Paneth cell (PC) numbers are observed in inflammatory bowel diseases and impaired PC function contributes to the ileal pathogenesis of Crohn’s disease (CD). PCs reside in proximity to Lgr5+...

OP11 Exposure to an inflammatory mix re-induces inflammation in organoids of ulcerative colitis patients, independent of the inflammatory state of the tissue of origin

Abstract Background Patient-derived intestinal organoids provide a powerful tool to unravel mechanisms underlying inflammatory bowel disease (IBD). Recently, we showed that organoids derived from inflamed regions in ulcerative colitis (UC) patients lose their inflammatory phenotype during ex vivo culture and were indistinguishable from organoids of non-inflamed regions in these patients.1 To study UC in an ex vivo model, we hypothesised that inflammation should be re-induced towards levels corresponding to the in vivo situation. In addition, we aimed to elucidate if organoids derived from inflamed regions are more sensitive towards inflammatory stimulation, compared with organoids from non-inflamed regions of UC patients and non-IBD controls. Methods Biopsies were obtained from 8 patients with active UC (endoscopic Mayo score of ≥2), both in inflamed and non-inflamed regions, and in 8 non-IBD controls. Crypts were isolated and cultured as organoids for at least four weeks. Organoids were subjected to a predefined inflammatory mix (MIX: 100 ng/ml TNF-α, 20 ng/ml IL-1β, 1 µg/ml Flagellin) or medium only (CTRL) for 24 h (Figure 1). RNA was extracted from organoids for RNA sequencing by Lexogen QuantSeq for Illumina. Differential gene expression and pathways were studied through DESeq2 and ingenuity pathway analysis (false discovery rate &amp;lt;0.05). Results Prior to inflammatory stimulation, principal component analysis (PCA) demonstrated separate clustering between organoids derived from non-IBD controls and UC patients. Exposure to the inflammatory mix induced transcriptional activation of inflammatory genes (CXCL1, DUOXA2, IL1β, IL8, IL23α, etc., all p &amp;lt; 0.001) and pathways in all conditions (Figure 2). However, organoids of non-IBD controls clustered separate from organoids of UC patients. Within organoids of UC patients (inflamed vs. non-inflamed origin), we observed no differentially expressed genes after inflammatory stimulation but organoids clustered per patient instead (Figure 3). Inflammatory markers in UC organoids reached transcriptional expression levels (CXCL1, CXCL2, IFNGR1, IL1β, DUOXA2, etc.) and activated pathways (antigen presentation, interferon signalling, granulocyte adhesion and diapedesis) similar to those observed in crypts derived from inflamed biopsies. Conclusion Inflammation can efficiently be (re-)induced in organoids from both UC and non-IBD origin. However, a different response was observed between organoids of non-IBD and UC origin. Of note, in UC organoids, the state of inflammation in the source tissue was irrelevant. In conclusion, we showed that it is essential to re-induce inflammation in patient-specific organoids, but there is no need to obtain biopsies from inflamed regions. Reference

Budesonide-Loaded Eudragit S 100 Nanocapsules for the Treatment of Acetic Acid-Induced Colitis in Animal Model.

Nanoparticles for colon-drug delivery were designed and evaluated to solve many discrepancy issues as insufficient drug amount at diseased regions, high adverse effects of released drugs, and unintentionally premature drug release to noninflamed gastrointestinal regions. Herein, the prepared budesonide-loaded Eudragit S 100/Capryol 90 nanocapsules for the treatment of inflammatory bowel disease. Nanocapsules were prepared efficiently by nanoprecipitation technique and composed mainly of the pH-sensitive Eudragit S 100 polymeric coat with a semisynthetic Capryol 90 oily core. Full 31 × 21 factorial design was applied to obtain optimized nanocapsules. Optimal nanocapsules showed mean particle size of 171nm with lower polydispersity index indicating the production of monodispersed system and negative zeta-potential of - 37.6mV. Optimized nanocapsules showed high encapsulation efficiency of 83.4% with lower initial rapid release of 10% for first 2h and higher rapid cumulative release of 72% after 6h. The therapeutic activity of the prepared budesonide-loaded nanocapsules was evaluated using a rat colitis model. Disease activity score, macroscopical examination, blood glucose level, and histopathological assessment showed marked improvements over that free drug suspension. Obtained results demonstrate that the budesonide-loaded Eudragit S 100 nanocapsules are an effective colon-targeting nanosystem for the treatment of inflammatory bowel disease. Capryol 90 was found to be a successful, and even preferred, alternative to benzyl benzoate, which is commonly employed as the oil core of such nanocapsules.