bluuebunny/crossref_metadata_2025 huggingface dataset:
DOP39 Constructing butyrate-producing yeast as a cell-dependent treatment option for Inflammatory Bowel Disease
Authors: Wu J , Kang G , Wang L , Gao M , Meng S , Cao X , Li B , Huang H
Published: January 2024

Background

Intestinal microbiota dysbiosis and metabolic disruption are essential characteristics of inflammatory bowel disorders (IBD). Butyrate has been proven to regulate immune response and gut microbiota. It has been confirmed that the butyrate content in the intestinal cavity of IBD patients decreases (Fig. 1A).

Methods

In this study, gene editing was used to reconstruct the butyrate synthesis pathway and regulate related metabolic pathways in Saccharomyces cerevisiae to produce butyrate and enrich its synthesis in anaerobic environments to cope with complex intestinal conditions. A series of engineered strains were co-cultured with gut microbiota samples from six patients with mild-to-moderate ulcerative colitis and 16S ribosomal RNA sequencing was used to analyze the regulatory effect of engineered strains on intestinal microbiota in vitro. The TNBS-induced IBD mice model was used to explore the protective effect of butyrate-engineered yeasts in vivo.

Results

A series of engineered strains with different butyrate synthesis abilities was constructed to meet the needs of different patients. S. cerevisiae was engineered by introducing an exogenous butyrate synthesis pathway and regulating yeast metabolism to increase the availability of the precursors. Engineered yeast can produce up to 1.8 g/L butyrate (Fig. 1B). The modification of related metabolic pathways also focused on enhancing the synthesis of butyrate under anaerobic conditions to enable engineered yeasts to adapt to the intestinal environment. This series of engineered yeasts with different butyrate synthesis abilities regulated the intestinal microbiota of IBD patients to varying degrees, enhancing the abundance of related probiotics such as Lactobacillus and Bifidobacterium and reducing the abundance of harmful bacteria like Candidatus Bacilloplasma (Fig. 1C). Meanwhile, strains improved TNBS-induced colitis in mice by restoring butyrate content. Remarkably, the engineered strain with the second-highest butyrate yield showed the most significant therapeutic effect (Fig. 1D).

Conclusion

This study realized butyrate synthesis in S. cerevisiae and regulated related metabolic modules to adjust the butyrate production to achieve the best therapeutic effect on colitis in vivo and in vitro. In addition, the strain with the second-highest butyrate production has the best therapeutic effect. This highlights the dual role of butyrate in epithelial cell repair and suggests a therapeutic window for IBD treatment through butyrate supplementation. Selecting the most suitable strain for each patient's condition is essential for restoring butyrate levels, regulating microbiota, and promoting mucosal repair.
DOP43 Histologic improvement, attenuation of inflammation and microbiome modulation by engineered high acetate producing Saccharomyces boulardii in DSS-induced colitis
Authors: Deleu S , Trindade de Carvalho B , Jacobs I , Vazquez Castellanos J , Verstockt S , Verstockt B , Arnauts K , Deprez L , Vissers E , Lenfant M , De Hertogh G , Huys G , Thevelein J , Raes J , Vermeire S
Published: January 2024

Background

The probiotic Saccharomyces cerevisiae var. boulardii (Sb) may be optimized for therapeutic use in ulcerative colitis by enhancing its acetate production, as acetate is a key factor for its probiotic efficacy and was shown to have anti-inflammatory and barrier-protective effects in vitro (Deleu, 2023). Therefore, higher acetate producing Sb strains have been engineered which were preclinically evaluated in an acute DSS mice model of colitis.

Methods

Nine-week-old female C57/Bl6 mice (N=120) were allocated to 12 treatment groups receiving drinking water or 2.75% DSS in combination with PBS (control), Baker’s yeast (non-probiotic control), SDH1 (no-acetate Sb), ENT (transient acetate strain-probiotic Enterol control), SbP (high acetate Sb) and ENT3 (extra high acetate Sb). Disease activity including weight loss, diarrhoea and the presence of occult blood were scored daily. On day 7, the DSS groups were transferred to regular drinking water and on day 14 mice were sacrificed. Colonic tissue and faeces (0, 9, 14d) were collected for resp. histology, RNA, and 16S rRNA sequencing.

Results

Disease activity in DSS subgroups (Fig.A), determined by the area under the curve, was lower for SbP compared to PBS and Baker’s yeast (both p<0.05). Sb SDH1 showed higher disease activity compared to the Sb strains ENT, SbP and ENT3 (all p<0.05). At sacrifice, the macroscopic damage score in DSS was lower for SbP and ENT3 (both p<0.05) compared to Sb SDH1, and the colon weight/length-ratio was decreased for ENT and SbP compared to Sb SDH1 (resp. p=0.06 and p=0.08). Higher histologic inflammation was noted in the non- or transient-acetate producing strains on DSS compared to healthy PBS control (all p<0.05), whereas this increase was not observed for both high-acetate producing strains SbP and ENT3 on DSS (p=NS) indicating a production dependent response. Anti-inflammatory findings were confirmed by transcriptomic analysis of differentially expressed genes e.g. decreased expression of Tnf, Il1b and S100a8/9, predominantly expressed as calprotectin, upon stimulation with the highest acetate producing strain ENT3 (all adj.p<0.1; except PBS adj.p=0.16). Subsequent, microbial analysis showed a superior effect of ENT3 in maintaining a stable alpha diversity (p=0.58, Fig.E), which was not observed for the other strains.

Conclusion

Engineered high acetate producing Sb strains attenuated DSS-induced colitis, with its anti-inflammatory effects further supported by transcriptomic analysis and superiority in maintaining a stable microbiome composition. Together with our previous in vitro work in patient-derived organoids, these data indicate a role for high-acetate producing Sb- in attenuating intestinal inflammation.
DOP64 Steroid-sparing effect of risankizumab vs ustekinumab in patients with moderately to severely active Crohn’s Disease: Post hoc results from the phase 3b SEQUENCE trial
Authors: Raine T , Danese S , Schreiber S , Gao X , Hanauer S , Torres J , Kligys K , Huang X , Neimark E , Anschutz T , Crowley J , Cross R K
Published: January 2024

Background

Corticosteroid (CS) dependency and/or resistance occurs in approximately 50% of patients with Crohn’s disease (CD).1 In SEQUENCE, risankizumab (RZB; interleukin [IL]-23 inhibitor) demonstrated superiority over ustekinumab (UST; IL-12/IL-23 inhibitor) for the ranked secondary endpoints of CS-free clinical remission and CS-free endoscopic remission at week 48. In this post hoc analysis, we evaluate the CS-sparing effects of RZB and UST over 48 weeks in patients taking CS at baseline (BL).

Methods

SEQUENCE (NCT04524611) was an open-label, multicenter, randomised, efficacy assessment-blinded study in adults with moderately to severely active CD who previously failed ≥ 1 anti-tumor necrosis factor (TNF) therapy. Patients in the primary efficacy analysis were randomised 1:1 to receive RZB (intravenous [IV] 600 mg induction dose at weeks 0, 4, and 8, followed by a subcutaneous [SC] 360 mg maintenance dose every 8 weeks starting at week 12) or UST (single weight-based IV induction dose, followed by a SC 90 mg maintenance dose every 8 weeks starting at week 8) up to week 48. A mandatory CS taper started at week 2. CS-free outcomes (clinical remission or endoscopic remission and no CS at the assessed visit) were evaluated among patients taking CS at BL. Nominal P values were reported.

Results

The primary efficacy analysis included 520 patients (RZB, n = 255; UST, n = 265), and 24.8% (129/520) of patients were taking CS at BL. Demographics and disease characteristics were generally similar between treatment groups and between patients with and without CS use at BL. The mean (SD) daily prednisone-equivalent dose at BL was 21.4 (12.9) mg (RZB) and 19.5 (10.7) mg (UST), respectively. By week 8, more patients treated with RZB vs UST discontinued CS (75.9% vs 59.2%; nominal P < .05). Although a similar proportion of patients in either treatment group discontinued CS at week 24, more patients treated with RZB vs UST discontinued CS at week 48 (86.2% vs 57.7%; nominal P < .001). Of the patients taking CS at BL, RZB vs UST treatment resulted in higher rates of CS-free clinical remission (week 24: RZB, 44.8% vs UST, 33.8%; week 48: RZB, 56.9% vs UST, 31.0%) and CS-free endoscopic remission (week 24: RZB, 34.5% vs UST, 11.3%; week 48: RZB, 32.8% vs UST, 12.7%) (Table 1). Most patients who achieved CS-free outcomes at week 48 were CS-free for 90 days before week 48.

Conclusion

In patients with CD who previously failed anti-TNF therapy, RZB was more effective than UST for discontinuing CS use and achieving CS-free clinical and endoscopic remission.

References:

1. Lichtenstein G., et al. Am J Gastroenterol. 2018;113:481−517
DOP66 INSPIRE: Preliminary data from an observational post-marketing registry on the effectiveness and safety of darvadstrocel in patients with Crohn’s Disease and complex perianal fistulas
Authors: Fathallah N , Zmora O , Baumgart D C , Faubion W , Ferrante M , Gecse K , Wright E , McKay C , Ren K , Punchard M , Zhang B , Panés J
Published: January 2024

Background

Darvadstrocel (DVS), a suspension of expanded allogeneic adipose-derived mesenchymal stem cells, is approved for the treatment of complex Crohn’s perianal fistulas (CPF). INSPIRE (EUPAS24267) is a European observational post-approval study evaluating real-world effectiveness and safety of DVS in patients with complex CPF for up to 36 months. Here we report preliminary clinical and safety outcomes from INSPIRE.

Methods

Patients with complex CPF who had received DVS according to local standard practice were eligible to enrol. Clinical response (closure of ≥50% of external openings [EOs]) and clinical remission (closure of all EOs) of DVS-treated fistulas that were draining at baseline, were evaluated in the all-treated (AT; received ≥1 dose of DVS with an evaluation for fistula response after 6 months) and per-protocol (PP; received DVS as per label-approved guidelines) cohorts. Treatment-emergent adverse events (TEAEs), serious TEAEs and AEs of special interest were reported for the safety cohort (all patients treated with DVS).

Results

As of April 2023, 652 patients had enrolled (Table 1). Complete data to evaluate 12-month clinical response and remission were available for 134 (AT cohort) and 124 (PP cohort) patients, of whom 79.9% (AT, n = 107) and 79.8% (PP, n = 99) had a clinical response and 76.1% (AT, n = 102) and 76.6% (PP, n = 95) had clinical remission (Figure 1). From baseline to Month 12, there was a decrease in the mean (standard deviation [SD]) Perianal Disease Activity Index score (–3.5 [3.3]; n/N = 96/302 and –3.7 [3.2]; n/N = 89/249) and a decrease in the mean (SD) Harvey–Bradshaw Index for Crohn’s disease activity (-0.3 [2.8]; n/N = 102/302 and -0.4 [2.9]; n/N = 95/249) in the AT and PP cohorts, respectively. Complete safety data were available for 619 patients (median [min–max] follow-up 480 [6–1595]) days. Overall, 26% (n = 162) had ≥1 TEAE and 10% (n = 63) ≥1 serious TEAE. The most frequent TEAEs were anal abscess (6.9%), COVID-19 (1.9%) and proctalgia (1.1%). No deaths were reported. One breast cancer lymph node metastasis and one ovarian epithelial cancer were reported; both events were confounded by immunosuppressant treatment and were not considered related to DVS treatment.

Conclusion

These preliminary data are consistent with the pivotal ADMIRE-CD study and demonstrate a single dose of DVS in patients with complex CPF is associated with positive clinical outcomes in up to 80% of patients, and a favourable safety profile, both of which are maintained after 12 months. The inherent limitations of registry-based studies mean these data should be interpreted with caution. However, the ongoing INSPIRE study will provide longer follow-up data to confirm these observations with respect to any study limitations.
DOP73 Early biological use in a Belgian, prospective inception cohort of patients with Inflammatory Bowel Disease: the PANTHER cohort
Authors: Verstockt S , Glorieus E , De Wolf M , Lenfant M , Barbaraci M , Sabino J , Ferrante M , Geldof J , Verstockt B , Laukens D , Cleynen I , Vandermeulen L , Lobaton T , Vermeire S
Published: January 2024

Background

The growing number of advanced therapies has revolutionized the management of inflammatory bowel disease (IBD). Although early use of biological therapies is associated with better long-term outcomes, no data exist for the Belgian population. To this end, we evaluated treatment patterns in biological use and persistence in a Belgian inception cohort.

Methods

The PANTHER (Prognostic biobANk of paTients witH Early cRohn’s or colitis) cohort consists of adult IBD patients recruited in 3 Belgian IBD referral centres. Patients are included within 3 months after diagnosis and are naïve for immunosuppressives and biologicals, and without previous IBD-related surgery. Treatment use and outcomes are prospectively collected, and time trends for biological use were analysed using log-rank tests and Cox regression (R 4.3.2).

Results

Between 2015 and 2023, a total of 473 newly-diagnosed IBD patients were recruited (270 Crohn’s disease (CD) [57%]; 199 ulcerative colitis (UC) [42%]; 4 [1%] IBD type unclassified) (Table 1). During a median (IQR) follow-up of 2.6 (1.3-4.3) years, 64 patients (14%) required surgery (n=10 colectomy; n=54 ileocecal/small bowel resection); and 250 patients (53%) received biological therapy within the 1st year after diagnosis. Most patients were treated with anti-TNF (CD 67%; UC 55%) as first-line biological, followed by anti-integrins (CD 24%; UC 43%) and anti-IL12/23 (CD 9%; UC 2%). Time series analysis showed a significant increase in biological use within the 1st year after diagnosis when comparing patients diagnosed between 2015-2017 (44%) to those between 2018-2020 (57%), and to 2021-2023 (66%) (p=0.03) (Fig. 1A). Factors associated to this early biological use were younger age (HR=0.99 [95%CI: 0.98-0.99]), a diagnosis of CD (HR=2.2 [95%CI: 1.6-2.8]); and perianal disease in CD (HR=2.8 [95%CI: 1.8-12.8]). Within this early biological exposure group, 26 patients (10%) needed a resection later on. Therapy persistence over time was higher with early exposure rates in patients diagnosed in 2021-2023 (82%) and 2018-2021 (71%), as compared to 2015-2017 (63%) (p=0.08) (Fig.1B). The mode-of-action of first-line biological did not show any association with persistence (HR=1.0 [95%CI: 0.4-3.0]). Overall, only 26% of patients had to switch to a second-line, with a switch [anti-TNF >anti-IL12/23] being the most frequent in CD (50%); and from [anti-TNF >anti-integrins] (46%) or vice versa (40%) in UC.

Conclusion

In this Belgian inception cohort, two thirds of patients are currently initiated with biological therapy within the first year after diagnosis. This increased biological use is associated with high therapy persistence rates of >80% after a median follow-up of 1.5 years, and with low rates of surgical resections.
DOP87 Characterization of pathologic fibroblast and macrophage cell populations in colonic Crohn’s Disease with spatial transcriptomics
Authors: Gudiño V , Acera M , Corraliza A M , Garrido-Trigo A , Sanzo Á , Heyn H , Mereu E , Salas A
Published: January 2024

Background

Single-cell transcriptomic data of the human intestine has shed new light on the cellular composition and heterogeneity present in healthy and diseased tissues. Nonetheless, little information is currently available on their spatial distribution within tissue structures and cellular neighborhoods, which is particularly relevant to infer the function of disease-specific cell types. Here, we apply spatial transcriptomics to healthy control (HC) and Crohn’s disease (CD) intestinal samples and provide a map of cell types across healthy and diseased colon.

Methods

Fresh frozen colonic sections from HC (n=1) and CD (n=3) patients were placed onto 10X Visium slides, which feature thousands of spatially barcoded spots, each capable of capturing RNA from 10-15 cells. To retrieve single-cell resolution, spots were deconvoluted according to the Cell2location approach using our previously generated human single cell RNA sequencing (scRNAseq) dataset1 as a reference. For validation and further analysis at single-cell resolution, a panel of 100 genes was measured using single molecule fluorescent in situ hybridization (Molecular Cartography) (Fig. 1A).

Results

Deconvolution analysis revealed the cellular composition of each spot across all four different tissue sections including epithelial, stromal and immune cell types. Here, we focused on studying the location and diversity of fibroblasts and macrophages given their plasticity during inflammation1,2. The most abundant fibroblast cell type in CD were the inflammation-associate (IAFs) and the GREM1+ subepithelial fibroblasts. These two subsets showed a distinct gene signature and location, suggesting different functions (Fig. 1B). Most GREM1+ fibroblasts co-expressed FGFR1 and were in proximity with endothelial cells (VWF+) expressing the VEGF receptor 2 KDR, highlighting a potential pro-angiogenic role of GREM1+ fibroblasts3 (Fig. 1C). Remarkably, GREM1 was abundantly expressed by CCL19+ fibroblastic reticular cells and TNC+ stromal cells, underlying the potential heterogeneity of GREM1+ fibroblasts. Regarding macrophages, we could identify an inflammation-specific population expressing PLA2G2D and PTGDS. These macrophages were mainly localized within granulomas surrounding M1 macrophages (Fig. 1D). While their function is unknown, their signature suggests they might be modulating inflammation through the production of anti-inflammatory lipid mediators4.

Conclusion

Using two different ST technologies, we resolved the tissue distribution of cell populations previously identified by scRNAseq and reveal their potential function based on their spatial location.
P015 iNKT cells immunomodulation and mucosal healing by microbiota-derived lactate
Authors: Strati F
Published: January 2024

Background

Invariant natural killer T (iNKT) cells are unconventional, CD1d-restricted, T lymphocytes playing a critical role in mucosal immune homeostasis. iNKT cells plastically adapt their functional phenotypes to the surrounding environment and are involved in IBD pathogenesis. Although iNKT cells release proinflammatory cytokines in response to the altered gut microbiota of IBD patients, exposure to microbiota-derived metabolites can promote homeostatic IL10-mediated iNKT responses resulting in better clinical outcomes in Crohn's disease (CD) patients. Thus, understanding the mechanisms leading to iNKT cells’ functional shaping by microbiota-derived metabolites is important to design novel therapeutic approaches for IBD patients. We hypothesised that iNKT cells, serving as sentinels of tissue integrity, are the primary immune cells sensing microbiota-derived metabolic signals promoting the resolution of inflammation in IBD. In particular, here we show that microbiota-derived lactate can tightly control iNKT cell functions promoting iNKT cell-mediated mucosal tolerance while preventing T cell-mediated overt inflammation and tissue injury.

Methods

We performed immunophenotyping of iNKT cells by multiparametric flow-cytometry from surgical specimens of CD patients and correlated NKT10 responses with intra-colonic levels of lactate, gut microbiome composition and tissue injury/healing. We studied the molecular pathway determining the NKT10 phenotype in a lactate-enriched microenvironment by RNA-seq and CHIP-seq. We validated the role of NKT10 responses by microbiota-derived lactate in-vivo by using experimental colitis models.

Results

Our data suggest that microbiota-derived lactate plays a role in the epigenetic control of anti-inflammatory iNKT cell functions through histone lysine lactylation, thereby protecting from intestinal inflammation.

Conclusion

On overall our data show i) how microbiota-derived lactate modulates the immunophenotype of iNKT cells, ii) the mechanisms underlying the sensing of microbiota-derived lactate by iNKT cells, iii) iNKT cells role in inflammation resolution.

This work is supported by an ECCO grant '22 to Francesco Strati
P023 Serological markers of intestinal barrier function in patients with primary sclerosing cholangitis after liver transplantation
Authors: Drastich P , Hlavaty M , Fabian O , Brezina J , Wohl P , Spicak J , Cahova M , Bajer L
Published: January 2024

Background

Primary sclerosing cholangitis (PSC) is a chronic cholestatic disease associated with inflammatory bowel disease. Liver transplantation (LT) is the only curative treatment for most patients with PSC. Recurrence of PSC (rPSC) is a common long-term complication after liver transplantation in this patient group, affecting graft survival and overall patient survival. There are several lines of evidence indicating that dysregulation of the microbiome and gut barrier dysfunction are key etiologic factors in the pathogenesis of PSC. The aim of this study was to evaluate serological markers of intestinal barrier function in patients with PSC after LT and in control group, to determine potential biomarkers for rPSC.

Methods

This is a cross-sectional study of patients after LT for PSC at the Institute for Clinical and Experimental Medicine, Prague. The control group consists of patients who underwent liver transplantation for alcohol-related liver disease and/or hepatocellular carcinoma. The rPSC was assessed by MRCP and/or liver biopsy according to the Mayo criteria. IBD status was assessed by endoscopy, biopsy, and faecal calprotectin. We performed ELISA for Zonulin and Reg3a. The Kruskal-Wallis and Mann-Whitney test were used to evaluate the statistical differences.

Results

A total of 85 patients were included in the study after LT for PSC and 56 controls. rPSC was detected in 18 (21.18%) patients. IBD was diagnosed in 77 (90.5%) patients, of which 3 (3.9%) were categorised as Crohn’s disease. The remainder, 82 patients, were classified as ulcerative colitis, 41.9% with right-sided predominance and 24.3% with back-wash ileitis. The IBD treatment consisted of 5-ASA in 58 patients, azathioprine in 12 patients, vedolizumab in 5 patients, anti-TNFa in 1 patient and 9 patients were on 10 mg or more of prednisone or equivalent. MayoDAI was evaluated at the time of the visit with mean 1.74 (SD ±2.06). There was a significant difference in Reg3a levels between patients with IBD and control subjects (p<0.05, Figure 1.). No significant difference in Reg3a levels was found between rPSC patients and controls (p=0.3). Interestingly, Zonulin levels were significantly higher in the control group than in PSC patients with or without IBD (p<0.001). No significant difference in Reg3a and Zonulin was found between PSC and rPSC patients and between patients in remission and with active IBD.

Conclusion

Reg3a was associated with PSC-IBD in patients with PSC after LT, but its association with rPSC is unclear and should be investigated in a prospective setting. Zonulin reached higher levels in the control group.
P040 Prediction of endoscopic features of disease from single-cell RNA sequencing data in sigmoid colon of patients with ulcerative colitis
Authors: Riva A , Primas C , Wiekowski M , Badalamenti S , Reinisch W , Busslinger G
Published: January 2024

Background

Patients suffering from ulcerative colitis (UC) have reduced life qualities and an increased risk of developing cancer. Current treatment options are limited and have a ceiling effect with remission rates of ~35%. A possible reason is the multi-factorial aetiology, which are associated with an overactivation of the immune system and a dysbiosis of the gut microbiome. An important step towards a better understanding is the characterization of epithelial lesions. Their severity is classified by the Mayo score system based on their macroscopic appearance. While previous studies analysed cellular changes within the colon in UC patients, a detailed characterization of different UC-associated lesions are still missing, which is the aim of our study in collaboration with Sanofi Pharmaceuticals.

Methods

We recruit patients with active disease at our endoscopy unit and collect different biopsies from multiple regions within these patients to compare the reproducibility of single cell RNA sequencing (scRNAseq) results of similar appearing lesions within and between patients. For the sample preparation, we include a cellular enrichment step for immune, epithelial and stromal cells before performing 10x scRNAseq to obtain equal numbers for each cell fraction from every sampled area.

Results

Our cellular isolation strategy allows successful recovery of fibroblast, epithelial and immune cell fractions from each lesion. All expected epithelial cell types as previously described are detected in our dataset as well. Moreover, our experimental setup to obtain comparable numbers of fibroblasts, epithelial and immune cells enables us now to investigate cell-cell communication differences within different Mayo score lesions. For example, CXCL signal increased with the severity of macroscopic lesions and involves multiple cell types.

Conclusion

Our preliminary results promise exciting insights into cell-cell communications at an unprecedented depth, which identifies aberrant signalling pathway that may contribute to treatment failure in the clinics.
P045 MMP12 stimulates colonic afferents, and nociceptors through the activation of PAR1
Authors: Gupta R , Higham J , Raine T , Wright P , Bulmer D
Published: January 2024

Background

Abdominal pain is a cause of significant morbidity for people with colitis. We have previously shown that biopsy samples from people with Crohn’s disease (CD) are pro-nociceptive suggesting that peripheral nociceptor activation is a cause of pain in colitis. Furthermore, these effects were correlated with biopsy expression of macrophage metalloproteinase (MMP12), which was shown to stimulate mouse and human colonic nociceptors highlighting the potential utility of MMP12 inhibitors to act as visceral analgesics. Following on from this work we sought to determine the contribution of proteinase-activated receptor 1 (PAR1) to MMP12 mediated nociceptor activation.

Methods

MMP12 mediated sensory neuron activation: changes in the intracellular [Ca2+]i within thoracolumbar (T12-L5 spinal segments) dorsal root ganglia sensory neurons were examined in cells cultured overnight and loaded with Fluo-4-AM (30min) prior to imaging. Responses were measured to MMP12 or the PAR1 receptor agonists TRAP6 following pre-treatment with vehicle, the MMP12 inhibitor MMP408 or the PAR1 antagonist SCH79797. Afterwards cells were exposed to capsaicin (1 µM) followed by 50 mM KCl, to confirm the presence of nociceptors and neurons respectively by positive response.

MMP12 mediated colonic nociceptor activation: electrophysiological recordings were made of lumbar splanchnic nerve activity and of the isolated colorectum (ex-vivo) perfused with carbogenated Krebs solution, and responses determined following application with MMP12 alone or in the presence of respective MMP12 or PAR1 inhibitors.

All experiments were performed using tissue from male C57/B6 mice euthanised by rising concentration of CO2followed by exsanguination in accordance with Schedule 1 of the UK Animals Scientific Procedures act (1986).

Results

Application of MMP12 or TRAP6 produced a robust increase in [Ca2+]i levels in DRG sensory neurons classified as nociceptors by their co-sensitivity to capsaicin. These effects were significantly attenuated by pre-treatment with respective MMP12 or PAR1 inhibitors, with the response to MMP12 also being inhibited by PAR1 antagonist pre-treatment. Consistent with this pattern of nociceptor stimulation, the application of MMP12 also a robust activation of colonic afferents that was abolished by inhibition of MMP12 or PAR1 activity.

Conclusion

Findings demonstrate that MMP12 stimulates colonic afferents, and nociceptors through the activation of PAR1.
P074 Inflammatory Environment-Induced Transcriptomic Alterations in Crohn's Disease Adipose Stem Cells
Authors: Montfort-Ferré D , Boronat-Toscano A , Sánchez-Herrero J F , Caro A , Menacho M , Vañó-Segarra I , Martí M , Espina B , Pluvinet R , Cabrinety L , Abadia C , Ejarque M , Nuñez-Roa C , Maymo-Masip E , Sumoy L , Vendrell J , Fernández-Veledo S , Serena C
Published: January 2024

Background

Crohn’s disease (CD) is characterised by expansion of mesenteric adipose tissue, called creeping fat, which seems to be directly related to disease activity. Adipose stem cells (ASCs) isolated from the creeping fat of CD patients exhibit dysfunction, featuring impaired adipogenesis and an intensely pro-inflammatory phenotype. This study aims to explore the transcriptome of ASCs isolated from active and inactive CD subjects in comparison with healthy subjects, seeking key markers of this dysregulation.

Methods

Patients were recruited at Hospital Joan XXIII of Tarragona and Hospital Vall d’Hebron of Barcelona in accordance with the principles of the Helsinki Declaration. Transcriptome profiling was performed in ASCs isolated from adipose-tissue biopsies of visceral origin: CF and hMES in Crohn subjects (n=7, each) and hMES in inactive CD patients (n=7) and healthy control subjects (n=7). Groups were matched by age, gender, and BMI. Finally, we examined the pathways involving the differentially expressed gene (DEGs) in ASCs isolated from patients with CD with different clinical activity by gene set enrichment analysis (GSEA).

Results

Patients with CD across different clinical activity stages exhibited similar transcriptome patterns, indicating persistent ASC dysregulation even during remission state (Fig 1A). ASCs isolated from both creeping fat and mesenteric adipose tissue distant from intestinal damage displayed similar dysregulation, implying a global dysregulation of all mesenteric fat in CD. Specifically, transcriptomic analysis identified significant dysregulation of the NK2 Homeobox 3 (NKX2-3) gene in ASCs from active CD patients. Although higher NKX2-3 expression has been associated with B cells and intestinal tissues in CD1-3, our study is the first to link elevated expression of this gene also to creeping fat. Pathway enrichment analysis revealed significant enrichment in oxidative stress and immune response pathways in active CD (Fig 1B). Furthermore, our findings indicated also an elevated NKX2-3 expression in ASCs of inactive CD compared to controls, with oxidative stress pathway up-regulation (Fig 1C). Notably, no significant DEGs were found between ASCs from active and inactive CD groups (Fig 1D), suggesting a similar profile in both states.

Conclusion

Our study reveals profound ASC dysregulation in CD, in both active and inactive phases. Elevated NKX2-3 expression, previously associated with increased risk of complications in IBD, is now demonstrated in the mesenteric adipose tissue, persisting even in inactive CD patients.

Ref:

1. Yamazaki K, et al Gut. 2009 Feb;58(2):228-32.

2. Yu W, et al. J Crohn’s Colitis. 2009 Sep;3(3):189-95.

3. Hong SN et al. Gut. 2016 May;65(5):788-96.
P107 Comparing the Colonic Tissue Proteome of Active and Inactive Ulcerative Colitis
Authors: Penner R , de Almeida L , Rosentreter R , Hirota S , Dufour A , Lu C
Published: January 2024

Background

Ulcerative colitis (UC) is an inflammatory bowel disease that begins at the rectum and can advance proximally and continuously throughout the colon. In this pilot study, we aim to differentiate the protein expression between active and inactive patients, comparing the same tissue region of the colon pairwise between active and inactive UC. Additionally, we are looking to build on previous work to determine which proteins are differentially proteolyzed in active UC and postulate whether this alternative post-translational processing significantly correlates to disease activity. Here, we aim to use a multi -omics approach to spatially profile the entire large intestine.

Methods

We used an N-terminomics proteomic approach to determine whether proteins are differentially expressed and proteolytically processed in all colon segments of patients with active and inactive UC. Colon tissues were retrieved from patients and classified according to disease activity. Five active and five inactive UC patients were compared from each of the colonic regions (rectum, sigmoid, transverse, ascending colon). Samples were quantified by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Peptides were identified and matched at a 5% false discovery rate (FDR). Boxplot analysis was used to determine the upper and lower threshold for protein fold changes between conditions, and various bioinformatic software (MetaScape, StringDB, TopFINDer) were used to analyze the data.

Results

Primary analysis discovered a combined 36 statistically changing proteins in patients with active disease, including several that have previously been published and others that are direct interactors with known biomarkers for active UC, including calmodulin and S100A9. Interestingly, S100A9 was translated significantly more in the transverse colon of patients with active UC but was not in other colonic regions. Protease activity was noticeably different in active and inactive UC, in addition to having unique activity dependent on the region of the colon from which the samples were acquired. For example, delta-catenin was processed more in active UC in the rectum and transverse colon, whereas it’s processing was enriched in inactive UC in the sigmoid colon.

Conclusion

While this pilot study is incomplete, the promising initial results exhibit the power of a proteomics and N-terminomics approach to elucidate the mechanisms that contribute to UC pathogenesis. There appears to be a pattern in how proteins and their altered proteolysis leads to an increased likelihood of active UC.
P109 Organic Cation Transporter (OCTN)-1 variants shape innate immunity and predict individual response to vedolizumab In ulcerative colitis patients
Authors: Capobianco I , Puca P , Masi L , Petito V , Pafundi P C , De Felice M , Fidaleo M , Di Vincenzo F , Del Chierico F , Delogu G , Palucci I , Laterza L , Pizzoferrato M , Schiavoni E , Napolitano D , Turchini L , Amatucci V , Strazzeri M , Pugliese D , Putignani L , Armuzzi A , Lopetuso L R , Gasbarrini A , Pani G , Scaldaferri F
Published: January 2024

Background

Limited evidence is available about predictors of response to specific medications for ulcerative colitis (UC). OCTN1, an organic cation transporter, with its variants, could modulate the inflammatory response and potentially modify drug response to immune-modulatory therapy (anti-TNFα and vedolizumab), perhaps predicting individual response.

Methods

We first confirmed the ability of OCTN1 to differentially modulate IL1β secretion under different stimuli in a human leukemia monocytic cell line (THP-1) and in human peripheral blood mononuclear cells. We therefore prospectively enrolled a cohort of UC patients starting either anti-TNFα or vedolizumab, determined their OCTN1 genotype and evaluated their clinical features at baseline and clinical response to therapy or disease remission (according to clinical Mayo score) after six weeks (T1) and six months (T2). We finally enrolled a cohort of UC patients in clinical and endoscopic remission (according to full Mayo score) under immune-modulatory therapy (anti-TNFα or vedolizumab) for at least two years.

Results

OCTN1-deficient THP1 cells showed reduced IL-1β secretion in response to peptidoglycan (PGN) and Fusobacterium nucleatum (Fn), whilst primary monocytes bearing OCTN1 503F variant displayed an increased production of IL-1β in response to PGN and live bacteria. We therefore enrolled 90 patients, 50 starting anti-TNFα and 40 vedolizumab. Patients with 503F genotype showed higher rates of pancolitis as compared to 503L genotype. Among patients with 503L genotype, vedolizumab results highly associated with long-term response (OR 8.92, 95%CI 0.50-11.0; p=0.019), whilst conversely co-presence of 503F genotype and vedolizumab therapy disclosed an opposite role on the long-term response rate (OR 0.02, 95%CI 0.00-0.81; p=0.038).The population of UC patients in clinical and endoscopic remission was composed by 92 patients, 63 under anti-TNFα and 29 under vedolizumab. Of note, similar trends of disease extension were observed across genotypes: higher rates of pancolitis were observed in the 503F population as compared to 503L (62.5% vs 44%). Furthermore, among patients in remission with 503F genotype, the percentage of subjects receiving vedolizumab was sensibly lower than among patients with 503L genotype (87.5% anti-TNFα vs12.5% vedolizumab among 503F genotype; 56% anti-TNFα vs 44% vedolizumab among 503L genotype).

Conclusion

Mutated OCTN1 genotype (503F) favors an increased IL1β secretion from human stimulated leukocytes, thus suggesting a worse disease course with higher rate of pancolitis and lower response to vedolizumab as compared to 503L.
P113 Differentiation between active and quiescent Ulcerative Colitis using a novel wearable patch with self-adhesive dry electrodes: discriminating the role of heart rate variability
Authors: Metselaar P , van den Broek T J , Kamstra R J M , Uzunbajakava N , D'Haens G R , van den Brink W , te Velde A A , Löwenberg M
Published: January 2024

Background

The autonomic nervous system (ANS) maintains homeostasis of the gastrointestinal tract through the brain-gut axis. Previous studies have shown a dysregulated balance between the sympathetic and parasympathetic nervous system in Ulcerative Colitis (UC) patients, in particular during and preceding active disease. This leads to decreased vagal nerve activity and manifests in reduced heart rate variability (HRV). HRV could, therefore, potentially serve as quantitative non-invasive biomarker to differentiate active from quiescent UC. We aimed to increase our understanding of autonomic function in UC, by investigating a non-invasive wearable patch device measuring HRV to assess the difference in parasympathetic and sympathetic activity between active and quiescent UC, while correcting for HRV-modulating factors depression, anxiety and fatigue.

Methods

A proprietary vital signs patch with self-adhesive dry electrodes was used to acquire ECG (single lead) and bio-impedance (2 leads) in 26 UC patients for 72 hours continuous home monitoring, to calculate heart rate, respiratory rate and HRV. Fifteen patients had quiescent UC (SCCAI ≤ 2, CRP < 5mg/l, faecal calprotectin < 250µg/g) and eleven had active disease (SSCAI > 2, CRP > 5mg/l, faecal calprotectin > 250µg/g). Patients filled out the Multidimensional Fatigue Inventory and Hospital Anxiety and Depression Score questionnaires on fatigue, depression and anxiety, kept sleep diaries and provided feedback on patch usability. Time-domain (pNN50, RMSSD, for abbreviations see Figure 1), frequency-domain (high (HF), low (LF), LF/HF ratio), as well as non-linear (SD1, SD2, SD) HRV metrics were calculated for 5-minute segments during self-reported nocturnal sleep periods.

Results

No significant differences were found in fatigue, depression, and anxiety in the active and quiescent UC patient groups. Six HRV measures were significantly lower in active than in quiescent disease (p<0.001, Figure 1), indicating lower HRV and lower vagal parasympathetic tone. LF/HF ratio did not differ (p=0.242, Figure 1), reflecting no difference in ANS balance. The patch caused skin irritation in 72% of the patients. Nevertheless, 69% of patients indicated they would wear it again.

Conclusion

A wearable vital signs patch, utilizing ECG, is able to distinguish active from quiescent UC in patients matched for fatigue, anxiety, and depression. HRV metrics across all three domains, except LF/HF ratio, were reduced in active UC, indicating reduced vagal nerve activity. Pending further longitudinal validation and clinical integration, it is envisioned that continuous HRV monitoring can support early and non-invasive detection of relapses in UC, allowing timely intervention and prevention of severe symptoms.
P115 Ustekinumab in DEVELOP: A safety analysis from an Inflammatory Bowel Disease multicenter, prospective, long-term registry of paediatric patients
Authors: Koletzko S , Veereman G , Hyams J , Dubinsky M , Godwin B , Busse C , Strauss R , Volger S , Wang Y , Griffiths A , Colletti R B , Kugathasan S , Markowitz J , Winter H S , Escher J , Baldassano R N , Ruemmele F , Faubion W , Gold B
Published: January 2024

Background

DEVELOP is a multi-center, global, prospective, observational, longitudinal registry of long-term safety of infliximab (and other treatments) in paediatric patients (pts) with inflammatory bowel disease (IBD) <18 years (yrs) at diagnosis. Over the course of follow-up, many pts required changes in therapy. Ustekinumab (UST) is approved for treatment of Crohn’s disease (CD) in adults and is currently being evaluated for safety/efficacy in paediatric pts. This report focuses on assessment of safety of UST in paediatric pts with CD from DEVELOP.

Methods

Data included paediatric pts treated with UST from 31 May 2007 through 30 June 2022; data are collected every 6 months. The analyzed population included: all paediatric pts (all pts), pts weighing <40kg (<40kg group), and pts weighing ≥40kg (≥40kg group). Assessments included medical and treatment history, UST exposure, adverse events (AEs), serious AEs (SAEs), CD-related SAEs leading to hospitalization, IBD surgeries, serious and opportunistic infections, malignancies, and deaths.

Results

A total of 150 pts received UST (49.3% females); 31 in the <40kg group and 119 in the ≥40kg group (Table 1). Most treated pts (92.0%) were 12-17yrs. The majority (91%) of pts were initially dosed with UST every 8 weeks (q8w); at the last known dose, 64% received UST q8w and 22% q4w. Mean (SD) age of initial UST exposure was 15yrs (2.19). Prior IBD surgery was reported in 65.3% pts. The majority of pts (98%) had prior biologic therapy and 72% received ≥2 biologics prior to UST. Overall, 37/150 (24.7%) of pts discontinued UST during follow-up, with a mean (SD) time to discontinuation of 16.7 (17.00) months; 84/150 (56.0%) were exposed to UST for ≥24 months.

Rates (events/100 pt-yrs) of AEs were higher in the ≥40kg group (177.62) than in the <40kg group (101.27). Rates of SAEs were slightly lower among the ≥40kg group (25.42) vs <40kg (32.67). There were no SAEs related to infusion or injection site reactions. Rates of serious infections and CD-related hospitalizations were low and similar between groups, and rates of IBD-related surgeries were similar between groups (Figure 1). One malignancy (malignant carcinoid tumor) was reported in a female (18yrs) in the ≥40kg group with a history of prior therapy with vedolizumab, methotrexate, and 6-MP. No deaths or opportunistic infections were reported.

Conclusion

The safety profile observed for this off-label use of UST in paediatric CD pts in DEVELOP was similar to that of the treated adult population, despite most paediatric pts having prior biologic exposure. No new safety signals are identified in this paediatric population.
P120 A novel population of classical monocytes is expanded in CD and defined by an upregulation of genes relating to mononuclear cell differentiation and response to microbes
Authors: Gadhok R , Hornsby E , Kok K B , Lindsay J O , Stagg A J
Published: January 2024

Background

In Crohn's disease (CD), differentiation of classical blood monocytes to intestinal macrophages in the mucosa generates inflammatory rather than regulatory cells. Microbial products and cytokines can systemically reprogramme monocytes via effects on myelopoiesis. We hypothesised that in CD transcriptional changes occur in monocytes prior to their recruitment to the intestine which predispose to pro-inflammatory signatures in the mucosa. Here, we used single cell RNAsequencing (scRNAseq) to explore monocyte heterogeneity and identify CD-associated changes in gene expression profiles.

Methods

PBMCs were isolated from newly diagnosed CD patients (n=10) and matched healthy controls (HC, n=10). scRNAseq was performed using the 10x Genomics Chromium 3’ assay at a target capture rate of 10,000 cells/sample and sequencing at ~50,000 reads/cell. A dataset was generated using Cell Ranger and following unsupervised clustering (using Seurat) the CD14+ and CD16+ monocyte populations were selected. Data were further analysed using Seurat, DeSEQ2, g:Profiler, GSEA and Cytoscape.

Results

In addition to non-classical and intermediate monocytes, five transcriptionally distinct clusters of classical monocytes were identified in both CD patients and controls. One cluster (‘cluster 3’) was significantly increased in abundance in CD; other clusters were present at similar frequency in health and CD. Cluster 3 was defined by gene ontology (GO) pathways related to response to bacteria, cell migration, lymphocyte proliferation and response to oxidative stress. In CD compared with health, the most upregulated genes included those encoding EGR (early growth response) proteins 1-3 that play a key role in the functional plasticity of monocytes, and the key inflammatory cytokine IL-1β, as well as cytokines and regulators of their signalling (e.g. CXCL2/3, SOCS3). Overall, in Cluster 3, GO pathways positively enriched in CD related to microbial response signalling, canonical WNT signalling and mononuclear cell differentiation.

Conclusion

Classical monocytes are heterogeneous and include a novel subset that is expanded in CD. In this population, the expression of genes and pathways related to functional plasticity, response to microbes and inflammatory activity are enhanced in CD. These data support a systemic reprogramming of monocytes in CD that predisposes to inflammatory activity upon recruitment to the intestine.