bluuebunny/crossref_metadata_2025 huggingface dataset:
P007 Neutrophil function in lesional biopsies is a strong determinant of IBD severity and may relate to an altered functional state already present in circulating cells
Authors: Pascutti M F , Calado B , Nugteren S , Simons-Oosterhuis Y , Smits W K , Tuk B , Hulleman-van Haaften D H , Barendregt D M H , Heredia M , Klomberg R C W , de Ridder L , Samsom J N
Published: January 2024

Background

Neutrophils clear luminal bacteria from the intestinal lamina propria and have a protective role in Inflammatory Bowel Disease (IBD). However, their production of inflammatory cytokines, matrix metalloproteases and radical oxygen species causes irreversible tissue damage. Histological assessment of infiltrating neutrophils and indirect measurement of neutrophil-derived calprotectin in feces are used to assess disease severity in patients. Since IBD is a heterogeneous disease in terms of presentation, severity and course, we hypothesize that different patients have a different composition of their neutrophil compartment that leads to a different balance between protection and damage. We aim to study the variability in neutrophil phenotype and function and relate it to patient heterogeneity.

Methods

We analyzed endoscopic biopsies and peripheral blood samples from children diagnosed with IBD within the PIBD-SETQ cohort. Paired biopsies obtained at diagnosis were scored by a pathologist and analyzed by IHC and RNAseq. RNAseq was also performed on purified circulatory neutrophil at diagnosis and their phenotype was analyzed by whole-blood flow cytometry.

Results

At diagnosis, high numbers of infiltrating neutrophils in intestinal tissue differentiated IBD patients with high clinical disease activity, more severe endoscopic and histological disease and high plasma concentrations of IL-17. RNA sequencing of lesional intestinal biopsies revealed differential expression of genes linked to neutrophil degranulation and IL-17 signaling in the colon of IBD patients with high neutrophil infiltration. Moreover, these biopsies had high mRNA expression for Oncostatin M, a neutrophil product known to associate with therapy resistance.

To assess whether circulating neutrophils were altered in IBD before entering the intestinal tissue, we performed bulk RNAseq analysis of purified circulating neutrophils from non-IBD and CD patients. Neutrophils from CD patients had increased expression of genes related to granule formation and filling (MMP9, S100A8, S100A9) and pathogen sensing (TLR5, NLRC4) but not cytokine production (IL1B, OSM), relative to non-IBD patients. Expression of CD10 was decreased in circulating neutrophils from UC and CD patients while CD16 expression was lower in UC-derived neutrophils, suggesting these cells have a more immature state.

Conclusion

We have detected changes in the maturity of neutrophils in the circulation of some IBD patients that could have functional consequences in terms of bacterial clearance and tissue damage in the intestinal mucosa. Uncovering different functional profiles in neutrophils may help dissect patient heterogeneity and provide novel targets for therapy.
P041 Assessment of Serum Biomarkers associated with Vedolizumab Therapy Response in Inflammatory Bowel Disease
Authors: O'Connell J , Argue R , Dunne M , O'Connell F , Breslin N , Cullen G , Doherty G , Dunne C , Hartery K , Ismail M S , MacCarthy F , McDonagh P , McKiernan S , McNamara D , Mulcahy H , O'Connor A , Ryan B , Sheridan J , O'Sullivan J , Kevans D
Published: January 2024

Background

Vedolizumab (VDZ) is an anti α4β7-integrin inflammatory bowel disease (IBD) therapy postulated to reduce immune cell trafficking to the intestine. A significant proportion of patients fail to respond to VDZ. We aimed to assess the effect of VDZ on a panel serum inflammatory proteins (IPs) during induction therapy for IBD with the aim of identifying a serum biomarker of VDZ therapy response. We also aimed to gain further insights in VDZ mechanism of action by evaluating changes in serum IP concentrations during induction.

Methods

Patients commencing VDZ for IBD were prospectively recruited. All received standard VDZ induction. Baseline and week 6 serum concentrations of 54 IPs were measured by ELISA (Meso Scale Discovery, USA). Clinical outcomes were evaluated at weeks 14 and 30 of VDZ therapy. Corticosteroid-free remission (SFR) was defined as persistence of VDZ therapy, absence of corticosteroid therapy, a partial Mayo score ≤1 Ulcerative Colitis (UC) or HBI <5 Crohn’s Disease (CD). The associations between baseline serum IP concentrations and VDZ therapy outcome at weeks 14 and 30 were evaluated. Changes in IP concentrations from week 0 to week 6 of VDZ therapy were assessed. P values <0.05 following multiple test correction were considered significant in all analyses.

Results

Thirty-nine patients were included: 51% male, 51% UC, median [range]: age 52 [18.2-75.8] years; disease duration 13.4 [0.4–40.8] years; clinical Mayo subscore 4 [0–9]; and HBI 7 [1–20]. 28(72%) had received prior anti-TNF therapy. In univariate analysis, at higher baseline serum TNF-β concentration and lower IL-22, VEGF-C, and IL-7 concentrations were associated with week 14 SFR, p values p=0.003, 0.034, 0.042, and 0.045 respectively. Higher baseline serum IL-4, MDC and MCP-4 concentrations and lower baseline IL-10 concentration were associated with week 30 SFR, p=0.011, 0.026, 0.028 and 0.03 respectively. Significance was not maintained after correction for multiple testing. From baseline to week 6 post VDZ initiation, significant increases were observed in the concentration of six IPs: bFGF, eotaxin, eotaxin-3, MIP-1β, TARC and TNF-β, p=0.034, 0.0012, 0.0013, 0.043, 0.015 and 0.0077 respectively; and a significant decrease in one IP, IL-15, p=0.0004. The significant changes observed in IL-15, eotaxin, and eotaxin-3 concentrations from baseline to week 6 following VDZ initiation remained significant after multiple test correction.

Conclusion

No serum inflammatory protein was identified as a biomarker of SFR in patients receiving VDZ therapy for IBD. VDZ was observed to significantly affect serum IPs known to be involved in chemotaxis. Further study is required to identify biomarkers of VDZ therapy response.
P052 Mucosal IgG-like naive B-cell expansion and maturation contribute to ulcerative colitis pathogenesis
Authors: Fung I , Koelink P J , Li Yim A Y F , Mulders L G M , Admiraal I , Verseijden C , Verhoeff J , Patel A , Wildenberg M E , Luo Y , D’Haens G R , de Jonge W J
Published: January 2024

Background

A higher abundance of colonic naive B-cells and IgG plasma cells is implicated in the pathogenesis of ulcerative colitis (UC) during active inflammatory episodes. However, B-cell inhibition by targeting CD20 was found to be ineffective in achieving remission in UC patients indicating that the causal role of the B-cell expansion in UC is not well understood. In this study, we performed single cell RNA-sequencing (scRNA-seq) on mucosal tissue where we interrogated the B-cells in inflamed and non-inflamed UC colon biopsies. We subsequently explored the pathogenic role of B-cells in colitis mouse models.

Methods

We collected biopsies from the colon of 5 UC patients with active inflammation and 5 UC patients showing no inflamed mucosa. Three mucosal biopsies were collected from the same colon location for each patient and processed for scRNA-seq. We acquired usable transcriptomes of 21.475 cells. In a T-cell transfer colitis mice model, we evaluated the effects of the co-transfer of naive B-cells. Both CD4+ T and naive B-cells were transferred into SCID mice through intraperitoneal injection in ratios of 2:1, 1:1 and 1:2 (T:B ratio). The colitis course was followed for 35 days.

Results

Differential abundance analysis relative to all immune cells confirmed a higher abundance of naive B-cells (p= 0.03) and IgG plasma B-cells (p= 0.002) in inflamed relative to non-inflamed UC patient tissue, whereas no differences in abundance of transitional B-cells and memory B-cells were found. Pseudo-time analysis indicated a higher rate of maturation in the naive B-cells in inflamed tissue. This was evidenced by higher expression of IGHG1 (p= 0.025) and IGHG3 (p< 0.001) in naive B-cells in inflamed tissue. In a T-cell transfer model, we co-transferred naive B-cells to the CD4+ T-cell pool into SCID mice in various ratios (2:1, 1:1 and 1:2 T to B-cells respectively) and T-cell only control. Significant increase in disease severity was observed in a 1:2 T:B ratio based on body weight loss, increase of colon density, mouse colitis histology index and histological changes by disrupted mucosa. This observation was associated to significant elevated IgG concentration in serum and colon and B-cell presence in colon through immunohistochemistry.

Conclusion

We observed significantly higher abundance of naive B-cells and IgG plasma cells in inflamed relative to non-inflamed UC colon tissue, where naive B-cells were shown to be more mature and IgG-like in inflamed condition. Notably, transferring increased ratio of naive B-cells resulted in elevated disease severity in a colitis model, demonstrating its pathogenic role. Our findings suggest that specific targeting of the IgG-like naive B-cell may be beneficial for UC patients.
P066 Serum amyloid A – A Novel Biomarker of Ulcerative Colitis Disease Activity
Authors: McShane C , O'Connell F , Corcoran R , Buckley C , Lynam-Lennon N , Kevans D , O'Sullivan J
Published: January 2024

Background

Despite recent advancements in the treatment of ulcerative colitis (UC), a substantial number of patients fail to achieve long-term remission. Persistent histological activity has been linked with poorer treatment outcomes. Histological remission is now an accepted treatment target; however there remains significant variability in the interpretation of UC histology. As such, there is a need for novel biomarker identification to aid assessment and ultimately predict disease relapse. Serum amyloid A (SAA) is an acute-phase protein, of which serum levels have shown promise as a biomarker in IBD1. This study aims to explore the utility of SAA levels in UC colonic tissue as a diagnostic biomarker for disease activity and progression.

Methods

Two cohorts were prospectively recruited, including healthy controls and UC patients. Sigmoid biopsies were collected and tissue explants generated. Tissue-conditioned media from these explants was collected and secreted SAA quantified using 54 V-plex ELISA. Demographic information, disease characteristics, endoscopic Mayo scores and disease progression were documented. Endoscopic remission was defined as a Mayo endoscopic sub-score ≤1. Disease progression was defined as the requirement for corticosteroid therapy, UC-related hospitalisation, UC-related surgery or the introduction of a new immunomodulatory agent in follow-up period. P values <0.05 were considered significant in analyses.

Results

The two cohorts included 11 healthy controls and 16 UC patients (endoscopic remission n=6). Active UC patients demonstrated significantly higher SAA concentrations than healthy controls (p=0.0013) and those in endoscopic remission (p=0.02). There was no significant difference in SAA concentrations between healthy controls and UC patients in remission (p=NS). UC patients in the lowest SAA concentration quartile had a significantly longer time to disease progression (p=0.0462) (Figure 1).

Conclusion

Quantification of SAA secretion in IBD ex-plants has potential as a biomarker of UC activity and progression. Further investigation of SAA as a biomarker in IBD is warranted.

References

1. Chen R, Chen Q, Zheng J, Zeng Z, Chen M, Li L, et al. Serum amyloid protein A in inflammatory bowel disease: from bench to bedside. Cell Death Discov. 2023;9(1):154.
P072 LDN-071, a novel first-in-class amino acid-based PAI-1 inhibitor, significantly reduces the severity of experimental colitis in mice
Authors: Jójárt B , Csákány-Papp N , Laub H , Vasas B , Barta B , Szabó V , Bagyánszki M , Mándity I , Heltovics G , Pallagi P , Maléth J
Published: January 2024

Background

Inflammatory Bowel Diseases (IBD) are chronic inflammatory disorders characterised by alterations of acute relapses and remission. The past decades have brought substantial advances in the pharmacological management of IBD by wide range of medicines. Although, the sustained efficacy of the currently available anti-inflammatory therapies is still far from optimal. Therefore, the development of novel therapies represents a major unmet clinical need in this field. Recently the enrichment of the coagulation pathway genes and among these, Plasminogen activator inhibitor 1 (PAI-1) was described in IBD, whereas PAI-1 emerged as a link between the epithelium and inflammation. Moreover, the genetic deletion of PAI-1 impaired the severity of experimental colitis in mice. Based on these, our aim was to develop and test novel PAI-1 inhibitors for the treatment of IBD.

Methods

Novel, amino acid-based PAI-1 inhibitors were synthetized by A3SMO® platform. The inhibitory potential and cytotoxicity effects of the peptides were determined by in vitro assays. Based on the in vitro results LDN-071 was selected for further analysis. Experimental colitis was induced in mice with 3% DSS and after the induction of colitis 10 mg/bwkg LDN-071 was administered per os for four consecutive days. The changes of body weight, spleen weight and colon length were measured. Haematoxylin eosin (HE) and Periodic acid-Schiff (PAS) staining were performed for histology. ELISA and qRT-PCR measurements were applied to analyse the mucosal cytokine expressions. In vivo toxicity of LDN-071 was determined in mice.

Results

LDN-071 significantly inhibited PAI-1 in vitro without decreasing the cell viability in a concentration range of 1µM-1mM. The administration of 10 mg/bwkg LDN-071 in the DSS-treated mice prevented the body weight loss, the development of bloody diarrhoea, the decrease of colon length and increase of the spleen weight, which were displayed by the DSS+vehicle-treated animals. The histological analysis of the colon revealed that DSS treatment induced a severe transmural inflammation, ulceration, loss of crypt structures and goblet cell depletion. Importantly, all of these parameters were significantly impaired by the administration of 10 mg/bwkg LDN-071. Additionally, mucosal gene and protein expressions of TNF-α, IL-1β and IL-6 were significantly impaired by PAI-1 inhibition. Importantly, no signs of toxicity were LDN-071 observed in mice exposed to 100 mg/bwkg LDN-071 for 7 days.

Conclusion

Our results showed that LDN-071, a novel amino acid-based inhibitor of PAI-1. It significantly reduces the severity of experimental colitis in mice. We propose that the inhibition of PAI-1 could be a potential novel therapeutic strategy in IBD.
P075 Early intestinal ultrasound non-response is a prediction tool of complicated disease outcome in Crohn’s disease patients treated with infliximab
Authors: Bárbara A , Bravo C , Reves J , Nascimento C , Gomes C , Torres J , Palmela C
Published: January 2024

Background

The ultimate goal of Crohn’s disease (CD) treatment is to modify the natural course of disease and prevent complications. We aim to assess the role of intestinal ultrasound (IUS) as predictor tool for complicated disease.

Methods

Prospective longitudinal study including patients with active CD starting infliximab. Clinical, laboratorial and IUS parameters were assessed at week(W) 0, 14, 30, and 54. The IBUS-SAS score was used to assess ultrasonographic disease activity and includes, among others, bowel wall thickness (BWT) measurement. IUS response was defined as reduction in 25% in BWT and IUS remission as the normalization of BWT (≤3mm), doppler sign (≤1), stratification (BWS), and inflammatory fat in the most affected segment. Ileocolonoscopy was done at W0 and 54. Endoscopic healing (EH) was defined as SES-CD <3. We defined a composite outcome(CO) for complicated disease including development of stricturing or penetrating disease, need for bowel surgery, CD-related hospitalization, or the need to switching due to loss of response to therapy.

Results

We included 48 patients (48% male; median age 31 years, IQR 26-44). The median follow-up time was 23.5 months(M) (IQR11-38.25). The median time until the development of the outcome was 12M, IQR 9.75-15.25. During follow-up, 37.5% developed the CO (27.1% with stricturing or penetrating disease, 12,5% needed bowel surgery, 18,8% needed hospitalization and 6% switched therapy). Baseline disease behavior (B1:21,2% vs B2/B3:73.3% p<0.001) and a higher IBUS-SAS score (70.34 vs 58.80 p=0.042) were associated with development of CO during follow-up. Patients who developed the CO had a numerically higher BWT throughout the first year of therapy (W14: 5.00mm vs 3.65mm, p=0.007; W30: 4.78mm vs 3.43mm, p=0.007); a higher IBUS-SAS score was also seen at W14, W30 and W54 (57.00 vs 40.35, p= 0.03; 54.74 vs 31.00, p=0.004 and 29.84 vs 23.39, p=0.026, respectively). On survival analysis, IUS remission at W30 (HR 0.27 95% CI (0.76-0.94), p=0.04) and IUS response at W30 (HR 0.27 95% CI (0.09-0.94), p =0.015) were protective for the development of the CO. In multivariate Cox regression, the absence of IUS response at W30 (HR 4.80, 95% CI 1.03-22.45, p=0.046), and EH at W54 (HR 5.70, 95% CI 1.33-24.45, p=0.02) were independent predictors of worse outcome. The best cut-off at W30 to predict the CO was IBUS-SAS >52 (AUC 0.74) and BWT > 4.11 mm (AUC 0.75).

Conclusion

Early assessment of ultrasonographic response at W30 of therapy can help to predict prognosis in CD. Our results highlight that in the absence of significant IUS response after 6 months of therapy, we should consider treatment intensification and close monitoring to prevent complications.
P089 Development and preliminary evaluation of a suicidal risk assessment protocol in a research trial using the Patient Health Questionnaire (PHQ-9)
Authors: Wileman V , McGuinness S , Sweeney L , Miller L , Stagg I , O'Carroll R , Moss-Morris R , Norton C
Published: January 2024

Background

People with Inflammatory Bowel Disease (IBD) have an increased risk of depression and self-harm. Participants in IBD research trials often disclose severe depression symptoms, including thoughts of self-harm and suicidal ideation, in validated self-administered questionnaires such as the Patient Health Questionnaire-9 (PHQ-9). However, there is no standard protocol for responding to such disclosure in research trials and the opportunity to support people at risk is potentially missed. We developed and evaluated a risk assessment protocol for the IBD-BOOST randomised controlled trial (ISRCTN71618461).

Methods

Participants completed the PHQ-9 scale at baseline, six-month and 12-month follow-up. Responding to item 9: ‘Thoughts that you would be better off dead or of hurting yourself in some way’, if a participant indicated ‘More than half the days’ or ‘Nearly every day’, the trial database automatically alerted the research team to risk assess the participant. Trial risk assessors, trained in the protocol, contacted participants by telephone, completed the risk assessment protocol (Figure 1) and signposted participants to appropriate professional services. Risk assessments were completed at the first occurrence of a PHQ-9 item 9 response. Risk assessments were reviewed by the Chief Investigator and summarised to trial governing committees.

Results

780 participants were randomised. Overall, there were 58 PHQ-9 risk alerts during the trial (baseline, six- or 12-month follow-up), 41 of which were new alerts and required risk assessment. One participant declined assessment so 40 risk assessments were completed. 24 participants were assessed as low-risk and 16 participants as medium-risk, with 12 declaring previous suicide attempts. No participants were rated as high-risk. The mean time for the risk assessment phone call was 14 minutes (range 5-36 minutes) and total time spent on calls was approximately nine hours. Trial participants who received a risk assessment expressed appreciation for being contacted and all except two wished to receive information about professional support services. In a focus group, trial risk assessors reported positive experiences of conducting the risk assessment with suggestions for improvement, which resulted in minor modifications to the protocol.

Conclusion

Our evaluation demonstrated that it was viable for a research trial team to successfully conduct a risk-assessment protocol for trial participants reporting thoughts of self-harm, with training and support from senior colleagues. Resources are required for training and delivery, but it is not unduly onerous. Trial participants appeared to find completing the assessment acceptable. The revised protocol will be made available for researchers to use.
P094 Lack of DNA methyltransferase DNMT3A in fibroblasts impairs cell growth and wound healing
Authors: Montague J , Rosenstiel P , Escudero-Hernández C
Published: January 2024

Background

Fibrotic scarring is an untreatable pathological wound-healing complication that invalidates correct organ function. However, pathomechanisms leading to intestinal fibrosis during inflammatory bowel disease (IBD) are not fully understood. Since the de novo DNA methyltransferase DNMT3A has been associated with increased risk for IBD and alters epithelial homeostasis, we sought to characterize how DNMT3A loss might affect fibroblasts, the key mesenchymal cells involved in wound healing.

Methods

We stably depleted Dnmt3a in 3T3 fibroblast cell line using CRISPR-Cas9 technology and subsequently selected GFP-positive clones using FACS sorting. Cell cultures were performed in vessels with or without 0.2% gelatin coat. Reverse transcription quantitative PCR, western blot, and immunofluorescence were used to confirm Dnmt3a gene deletion and characterize wild-type (WT) and knockout cells (KO). Wound healing assays in the presence or absence of tissue repair cytokine TGF-β served to quantify and assess cell capacity of wound closure.

Results

3T3 fibroblasts lacking Dnmt3a acquire an activated myofibroblast phenotype (characterized by increased α-SMA expression) that also overexpress tissue remodelling matrix metalloproteinase Mmp3, and the inflammation-induced necroptosis marker RIPK3 when compared to their WT counterparts. In addition, Dnmt3a-depleted 3T3 cells grow overlapped in tightly aggregated foci (fig. 1), and when seeded in vessels without the gelatin coat, they form spheroids. To assess healing properties, the closure of a wound (gap) in a cell monolayer was monitored. While WT 3T3 fibroblasts closed the gap within 48h, Dnmt3a KO 3T3 cells were unable to refill the empty space and formed aggregated foci in the vicinity of the wound (fig. 1). When the wound healing assay was repeated in the presence of TGF-β, WT cells nearly closed the gap within 24h whereas Dnmt3a KO 3T3 still failed to grow.

Conclusion

We conclude that Dnmt3a is crucial for fibroblast homeostasis and correct wound healing. Furthermore, TGF-β signalling is impaired when Dnmt3a is lacking. Thus, additional characterization of Dnmt3a depletion in fibroblasts will deepen the understanding of fibrosis and point towards new therapeutic approaches for this untreatable complication.
P097 Spatially resolved insights into fistulating Crohn's disease pathogenesis: Unveiling molecular heterogeneity
Authors: Mcgregor C , Yin Z , Bridges E , Gupta T , Aulicino A , Siejka-Zielinska P , Lee C H J , Bornschein J , Bignell M , George B , Vieth M , Tandon R , Fryer E , Antanaviciute A , Simmons A
Published: January 2024

Background

Crohn's disease (CD) frequently results in fistula development in approximately 40% of patients due to sustained, transmural inflammation within the bowel wall. Despite advanced treatments, recurrence of fistulae affects one third of patients. Understanding its pathogenesis is crucial for targeted treatments, yet remains poorly defined. This study employs spatial transcriptomic and single-cell RNA-sequencing (scRNA-seq) technologies to characterise human fistulating CD tissue pathology.

Methods

Unbiased FFPE spatial transcriptomics (10x Visium) were applied to surgically resected, full-thickness (FT) tissue from 20 CD-associated fistulae cases and 15 controls. Subsequently, selected cases underwent further analysis using custom 500-plex MERFISH subcellular resolution spatial transcriptomics (Vizgen MERSCOPE). Additionally, a reference single-cell cohort encompassing patients with fistulating, stricturing, and inflammatory CD phenotypes, along with healthy controls, was generated. Optimisation of scRNA-seq for resected FT ileal tissue enabled the isolation and profiling of epithelial, immune, and stromal populations (10x Chromium). Computational analysis facilitated the spatial localisation of single-cell clusters enriched in fistula CD tissue, identifying key parameters linked to fistula development.

Results

The scRNA-seq data revealed diverse CD-specific cellular states adopted by intestinal fibroblasts, with IL11+ fibroblasts prominently expressing CD82, COL7A1, MMP1, CHI3L1, suggestive of pro-fibrotic signalling and regenerative morphogen pathways. This coincided with the expansion of a CD-specific subpopulation of pericytes (CCL19/CCL21) involved in leucocyte migration. Spatial profiling of CD fistula tracts by Visium and MERSCOPE unveiled an enrichment of key epithelial developmental transcription factors (e.g., GRHL3) and localised the pro-fibrotic signature displaying increased IL11 and MMP expression, along with perturbed WNT signalling within the fistula stroma. Active proliferation (HOPX, MKI67) was observed at the base of fistula tracts and within the stroma. Transcriptomic characterisation depicted a gradual loss in stem cell signature (LGR5, ASCL2, SMOC2) and supporting telocytes (POSTN) toward the leading edge of the fistula, signifying abnormal epithelium loss.

Conclusion

This pioneering study integrates multi-modal spatial transcriptomics and single-cell profiling to unveil the intricate cellular and molecular landscape in FT, fistulating CD pathology. Spatial analyses elucidate specific mechanisms involved in epithelial loss, stromal remodelling, and perturbed WNT signalling pathways within fistulating tissue, outlining a course of disrupted regenerative processes in fistulating CD.
P104 Per- and poly-fluoroalkyl substances (PFAS) exposure in early life is associated with intestinal inflammation
Authors: Agrawal M , Midya V , Picker M , Rendon A , Valvi D , Sabino J , Dolios G , Barupal D , Torres J , Petrick L , Colombel J F , Peter I
Published: January 2024

Background

Early life environmental exposures are linked with intestinal inflammation and inflammatory bowel disease (IBD) later in life. Per- and poly-fluoroalkyl substances (PFAS) are a large class of fluorinated organic chemicals used widely in consumer products that may be risk factors for IBD; however, the association between exposure to PFAS in early life and intestinal inflammation has not yet been studied. Dried blood spots (DBS) are archived neonatal capillary blood samples collected and stored as part of the New York State neonatal screening program and a cost-effective early life environmental exposure screening resource.

Methods

We measured 135 PFAS in DBS collected at birth from offsprings of women with and without IBD [n=37 (44%) and 47 (56%), respectively] enrolled in the MEChanisms Of disease traNsmission In Utero through the Microbiome (MECONIUM) cohort between January 2015 and November 2020, using untargeted metabolomics with liquid chromatography high-resolution mass spectrometry. In a subsample (n = 46), we also measured offspring fecal calprotectin (FC) at one year. We fitted exposome-wide regression models to estimate the association between each PFAS signal in the DBS and continuous FC at one year of age. For the significant associations (nominal p-value <0.05), we further explored potential effect modification by stratifying by maternal IBD status. All models were adjusted for relevant confounders.

Results

The mean (SE) of FC at age one year was 213.9 (23.8) ug/gm. The PFAS chemicals perfluoroundecanoic acid (PFUnDA) and N-ethyl-perfluorooctane sulfonamido acetic acid (N-Et-FOSAA) were significantly associated with increased scaled and log-transformed FC per decile increase in exposures [estimate (95% CI) = 0.52 (0.13,0.91) and 0.34 (0.05,0.63), Figure A]. Associations tended to be stronger in offspring of women with IBD relative to offspring of women without IBD (Figure B). estimate (95% CI) for the associations between PFUnDA and FC were 0.86 (0.34,1.39) and 0.12 (-0.47,0.71), respectively, and between N-Et-FOSAA and FC, these were 0.54 (-0.01,1.08) and 0.31(-0.07,0.68), respectively.

Conclusion

DBS can be used to measure early life exposure to toxins, and prenatal PFAS exposure is associated with elevated FC, with a slightly stronger effect in the offspring of women with IBD. Larger prospective studies are needed to validate these associations and understand potential underlying mechanisms through which early life PFAS exposures may contribute to IBD etiology.
P112 Lymphocytic colitis can be transcriptionally divided into channelopathic and inflammatory lymphocytic colitis
Authors: Bhardwaj A , Münch A , Montague J , Koch S , Rosenstiel P , Escudero-Hernández C
Published: January 2024

Background

The pathobiology of the non-destructive inflammatory bowel disease (IBD) lymphocytic colitis (LC) is poorly understood. We aimed to define an LC-specific mucosal transcriptome to gain insight into LC pathology, identify unique genomic signatures, and uncover potentially druggable disease pathways.

Methods

We performed bulk RNA-sequencing of LC and collagenous colitis (CC) colonic mucosa from patients with active disease, and healthy controls (n=4-10 per cohort). Differential gene expression was analyzed by gene-set enrichment and deconvolution analyses to identify pathologically relevant pathways and cells, respectively, altered in LC. Key findings were validated using reverse transcription quantitative PCR and/or immunohistochemistry. Finally, we compared our data to a previous cohort of ulcerative colitis and Crohn’s disease patients (n=4 per group) to distinguish non-destructive from classic IBD.

Results

LC can be subdivided into channelopathic LC, which is governed by organic acid and ion transport dysregulation; and inflammatory LC, which is driven by microbial immune responses. Inflammatory LC displays an innate and adaptive immunity that is limited compared to CC and classic IBD (Fig. 1). Conversely, we noted a distinct induction of regulatory non-coding RNA species in inflammatory LC samples. Moreover, compared to CC, water channel and cell adhesion molecule gene expression decreased in channelopathic LC, whereas it was accentuated in inflammatory LC and associated with reduced intestinal epithelial cell proliferation.

Conclusion

We conclude that LC can be subdivided into channelopathic LC and inflammatory LC that could be pathomechanistically distinct subtypes despite their shared clinical presentation. Inflammatory LC exhibits a dampened immune response compared to CC and classic IBDs. Our results point to regulatory micro-RNAs as a potential disease-specific feature that may be amenable to therapeutic intervention.
P116 Gastro-resistant microparticles for the oral delivery of Phycocyanin in the Inflammatory Bowel Disease therapy
Authors: Terracina F , Cicio A , Scirè S , Serio R , Licciardi M , Zizzo M G
Published: January 2024

Background

Inflammatory Bowel Disease (IBD) is a chronically relapsing inflammation of the gastrointestinal tract, with ambiguous etiology.

Oral route is the most common and acceptable approach for drug administration in the IBD treatment. However, it remains a challenge to maintain the stability of the drugs during oral treatment, particularly in the gastric environment, due its harsh conditions. In this context, the antioxidant and antiinflagmmatory properties of various natural compounds, as Phycocyanin (PC), a water-soluble bioactive molecule found in algae, can be strongly affected by factors as pH and temperature, inducing PC degradation and attenuating the curative effects.

In this work, we produced a gastro-resistant microparticles (MPs) as controlled release systems for PC, and we aimed to compare its efficacy with free PC effects, on rat model of IBD.

Methods

The novel pharmaceutical system (SPC) was produced by microencapsulation technique, the spray-drying technique, using soy proteins (SPs) as natural excipients, aiming to improve the stability of the active ingredient and to ensure a modified release profile of PC.

Rats were divided into groups and colitis was induced by intracolonic instillation of Dinitrobenzene sulfonic acid (DNBS, 20 mg/rat). Effects of preventive oral treatment of free PC or SPC were investigated, bioavailability was assessed and then body weight loss, stool consistency, colon weight/length index, myeloperoxidase activity (MPO), macroscopic damage and disease activity index (DAI) were determined.

Results

The SPC microparticle systems showed enhanced bioavailability of Phycocyanin, resulting in increased blood concentrations. The SPC system achieved the maximum AUC value and a modified release profile, particularly for gastro-resistant types, with a delayed Tmax of 5 hours, indicating their potential as a gastro-resistant formulation.

Both PC and SPC pretreatments significantly ameliorated the severity of DNBS-induced colitis, however SPC pretreatment demonstrated enhanced activity vs PC.

SPC rats showed a reduction of DAI, body weight loss and diarrhea incidence >15%, compared to PC (P<0.05); the ability of preventing the DNBS-induced macroscopic colonic damage and the MPO increase in SPC group were significantly higher than PC group (P<0.05) and also a tendency to improvement of colon weight/length index was observed.

Conclusion

In conclusion, the results of our research demonstrated that gastro-resistant microparticles (SPC) exhibit significantly improved therapeutic efficacy compared to orally administered free PC and can be developed as an effective new oral drug delivery system with controlled release profile in the treatment of IBD.
P118 Fecal content from IBD patients disturbs epithelial functions in two model systems
Authors: Abbas Egbariya H , Elwahidi L , Braun T , Sosnovski K E , Granot M , Krauthammer A , Loberman-Nachum N , Weiss B , Gal-Mor O , Denson L , VanDussen K , Amir A , Haberman Ziv Y
Published: January 2024

Background

Inflammatory Bowel Diseases (IBD) result from abnormal interactions between the immune system, epithelial barrier, and gut microbiota. However, understanding the abnormal interaction between aerobic epithelia and mainly anaerobic microbiota is complicated and is limited in part by the lack of human-relevant models.

Methods

We developed a model system that is based on whole human fecal samples and human-derived epithelia to study the interactions between the gut bacteria and the epithelia. To capture patient heterogeneity, we prioritized and pooled fecal material from 10 Crohn disease (CD), 10 ulcerative colitis (UC), and 10 healthy subjects. Prioritization of samples was based on our published gut microbial health index (PMID: 35197084), matching gender and age. Monolayered epithelial cells (Caco2 cells and patient-derived colonoids) were used for the co-culturing. Measurable outputs included cell viability, epithelial integrity, and secretion of CXCL1.

Results

Fecal samples were processed by separating the supernatant, heat-killing the bacteria, and rejoining the samples (Fig. 1A). 16S sequencing showed that bacterial composition and alpha diversity (Faith) were overall preserved after the heat-killing (Fig. 1B), as seen in the original samples. The gut microbial health index was higher among the control samples and pool, compared to the IBD samples (Fig. 1C), preserving the IBD pathogenic signals. Because previous studies indicated the enrichment of salivary bacteria in IBD feces, salivary samples were also processed for comparison. We confirmed that similar amounts of bacterial DNA (as a proxy of bacterial mass) were used for coculturing as indicated by qPCR of the 16S-V4 (Fig. 1D). Co-culturing the fecal and saliva pools with Caco-2 cells revealed a significant increase in CXCL1 proinflammatory chemokine secretion to the media in comparison to no-treatment (Fig. 2A). Moreover, the UC and CD pools significantly decreased epithelial integrity [transepithelial electrical resistance (TEER)], without affecting cellular viability (Fig. 2B-D). Finally, incubation of the fecal content with colon-derived organoids (from 2 controls and 1 UC) resulted in a substantial reduction of cell viability, which was more pronounced with fecal content derived from UC patients (Fig. 2E), and this effect was noted most with the UC fecal supernatants.

Conclusion

Using a novel co-culturing system, we demonstrated the effects of fecal contents from IBD and control subjects on CXCL1 secretion and epithelial integrity in Caco2 cells, and on colonoids viability. These models will be used to identify fecal factors and interventions relevant to IBD epithelial functions.
P124 Tissue Factor and Protease-Activated Receptor 2 associated genes are upregulated in patients with Inflammatory Bowel Disease
Authors: Qiu Y , Yao L , Taylor Meadows K , Rey D , Khalid M , Travis S
Published: January 2024

Background

Tissue Factor (TF) is well known for its role in coagulation, but it is also a key mediator of inflammatory cellular signaling relating to myeloid cell migration and kinase activation. TF is upregulated during inflammation and has been implicated in IBD pathology. Its role as a therapeutic target has not been well characterized, due to concerns that inhibition may induce a coagulopathy. When activated, TF cleaves protease-activated receptor 2 (PAR2) and induces multiple intracellular signaling pathways. Our aim was to investigate TF gene expression and PAR2 activated genes by colonic segment with inflamed tissue in patients with IBD compared to non-inflamed regions, and tissue from healthy controls.

Methods

RNA seq data from colon biopsy samples taken from The Mount Sinai Crohn's and Colitis Registry (MSCCR) cohort containing 1188 subjects (431 UC, 505 CD and 252 healthy controls) were downloaded from GEO (GSE193677) and preprocessed by the Endpoint Health RNA seq Analysis Pipeline. Tissues from patients with UC and CD were included in the analysis if the endoscopic score indicated moderate to severe inflammation. Differential gene expression analysis was performed using the Wilcoxon rank test. Gene set enrichment analyses (GSEA) was performed for PAR2 associated genes1.

Results

1,030 tissue samples, including 161 UC, 409 CD and 460 healthy control samples, were analyzed from 90, 174 and 252 subjects respectively. TF was found to be upregulated in inflamed tissue compared to non-inflamed tissue in patients with UC and CD, supporting a role for TF in inflamed lesions (Figure 1). Furthermore, PAR2-induced inflammatory related gene expression was observed in the inflamed tissue biopsies from patients with UC and CD compared to health control samples.Genes involved in inflammation, which include chemokines such as CXCL8 and chemokine receptors CXCR1, were consistently upregulated in all inflamed tissue regions isolated from patients with UC and CD (Table 1).

Conclusion

TF was found to be upregulated in inflamed tissue compared to non-inflamed tissue in patients with UC and CD, which may indicate TF contributing to tissue inflammation. TF represents a novel therapeutic target for IBD, but safety concerns with inhibiting coagulation have limited its clinical utility for inflammatory disease. To address this safety concern, EP004, a human IgG1 monoclonal antibody, targets the extracellular domain of human TF, inhibiting PAR2-induced inflammatory signaling while not altering coagulation activity. Clinical studies are required to elucidate its therapeutic effect.

1. Suen, J. Y. et al. 2010. Profiling Gene Expression Induced by Protease-Activated Receptor 2 (PAR2) Activation in Human Kidney Cells. PLOS ONE 5(11): e13809.
P139 A potential protective rule of Mannose Binding lectin (MBL) against SARS-COV2 infection in IBD patients
Authors: Albertini G , Pisani L F , Bolzoni A , Annunziata M L , Porta C , Pastorelli L
Published: January 2024

Background

Since the beginning of the COVID-19 pandemic, IBD patients have appeared to be less susceptible to a severe form of the disease, despite undergoing immunosuppressive treatments. Mannose binding lectin (MBL) is a humoral fluid-phase pattern recognition molecule (PRM) crucial for activating the lectin complement pathway. It is capable of binding to the spike protein and blocking SARS-CoV-2 infection. Low MBL expression has been linked to poor outcomes in both SARS and SARS- CoV-2 infections. In IBD patients, the role of MBL is controversial; low concentrations seem to predispose individuals to the development of IBD, while high concentrations have been observed in severe forms of Crohn's disease.

Methods

Since April 2020, 104 IBD patients have been enrolled, with 20% treated with mesalazine, 50% with Anti-TNFa, 20% with Vedolizumab, and 20% receiving no treatment. Additionally, 45 healthy controls (HC) from North Italy were included. Serum samples and colon biopsies from inflamed and non- inflamed mucosa were collected. Serum samples underwent an ELISA test to quantify circulating MBL concentration, and MBL gene expression in biopsies was analyzed using qPCR.

Results

Both Crohn's disease and Ulcerative Colitis patients exhibited significantly higher circulating MBL levels than HC (411.6 ± 45.5 ng/ml vs. 160.9 ± 35.3 ng/ml, p < 0.05). Patients treated with Anti-TNFa showed significantly higher circulating MBL levels compared to those not undergoing therapy (530.1 ± 76.5 ng/ml vs. 225.4 ± 61.6 ng/ml, p < 0.05) and patients receiving Vedolizumab (530.1 ± 76.5 ng/ml vs. 155.3 ± 32.1 ng/ml, p < 0.05). MBL intestinal gene expression is higher in IBD patients than HC (5.55 ± 0.9 vs. 2.36 ± 0.6; p=0.591) and tended to be elevated at similar levels in inflamed and non-inflamed mucosa (5.14 ± 1.4 vs. 5.79 ± 1.1; p=0.337), suggesting a prevalent genetic determination of MBL concentration regardless of the inflammatory background.

Conclusion

MBL appears to play a key role in SARS-CoV-2 infection. We demonstrate that IBD patients have, on average, higher circulating MBL levels than HC independently of their inflammatory status. The highest MBL levels were observed in patients treated with Anti-TNFa. These findings contribute to understanding the reported low mortality and hospitalization for COVID-19 among IBD patients treated with Anti-TNFa.
P149 Bacteroides uniformis ameliorates pro-inflammatory diet-exacerbated colitis by targeting endoplasmic reticulum stress-mediated ferroptosis
Authors: Liu C , Zhuang X , Zhan S , Tian Z , Mao R , Zhang S , Chen M , Zeng Z
Published: January 2024

Background

Pro-inflammatory diet is positively associated with the risk and progression of inflammatory bowel diseases. Recently, ferroptosis was observed in patients with dietary pattern-associated colitis. However, the mechanisms underlying the effects of a pro-inflammatory diet and whether it mediates ferroptosis are unknown. This study aimed to clarify the mechanisms of pro-inflammatory diet in colitis.

Methods

Mice were fed a dietary inflammatory index-based pro-inflammatory diet for 12 weeks. Subsequently, colitis was chemically induced using 2.5% dextran sulfate sodium with or without a ferroptosis inhibitor (ferrostatin-1). Body weight, histological scores, immune response, and mucosal barrier function were evaluated to assess intestinal inflammation.Colon tissue transcriptomics, fecal microbiome, and serum metabolomics were applied to identify diet-microbe-host interactions. Dietary inflammatory index of 36 patients with Crohn’s disease were evaluated. Their intestinal specimens were also collected. The biological functions of Bacteroides uniformis were observed in vitro and in vivo.

Results

Pro-inflammatory diet induced low-grade intestinal inflammation and exacerbated colitis by activating glutathione peroxidase 4-associated ferroptosis in the endoplasmic reticulum stress-mediated pathway; ferrostatin-1 treatment reversed this pro-inflammatory potential. Additionally, the pro-inflammatory diet triggered colitis by modulating the gut microbiota and metabolites. Notably, supplementation with B. uniformis improved the pro-inflammatory diet-aggravated colitis by inhibiting endoplasmic reticulum stress-mediated ferroptosis. Co-culturing B. uniformis with intestinal epithelial cells revealed it is non-enterotoxigenic and non-enteroinvasive.

Conclusion

Pro-inflammatory diet drives colitis by targeting endoplasmic reticulum stress-mediated ferroptosis, possibly in a gut microbiota-dependent manner. Pro-inflammatory diet restriction and microbial-based therapies may be effective strategies for preventing and treating inflammatory bowel disease.
P156 Gut microbiota composition in Patients with Chron's Disease in Saudi Arabia
Authors: Alahdal H , Almuneef G , Alkhulaifi M M , Aldibasi O , Aljouie A , Alharbi O , Rejili M
Published: January 2024

Background

Crohn’s disease (CD) is a heterogeneous disease at the clinical, molecular, genetic, immunological, and microbial levels. The disease involves complex interactions among the host intestinal mucosa and gut microbiota, causing alterations in the microbial composition of the intestines, and leading to frequent clinical manifestations. Despite the challenge of diagnosing CD, lately, there has been an increase in the number of CD patients in Saudi Arabia. Large-scale metagenomics studies could enable the understanding of functional dysbiosis of the gut microbiome. Here, we aimed to compare the fecal microbiome of CD with the inactive form of the disease and with healthy subjects.

Methods

We analyzed 80 fecal samples from inactive CD and CD samples from Saudi patients using a 16S rRNA sequencing approach. Additionally, questionnaires were completed by CD patients, which covered multiple aspects of CD, including diagnosis, treatment, and impact on patient quality of life, further to standard demographic information.

Results

Dysbiosis was found significantly greater among CD patients compared to the inactive form of the disease, as shown with more reduced diversity. This research has also shown that certain types of bacteria (belonging to genera such as Filifactor, Peptoniphilus, and Sellimonas.) were characteristic of CD groups meanwhile, genera such as Defluviitalea, Papillibacter, and Petroclostridium) were characteristic of the control group. α. Diversity measures show that the CD groups have lower diversity and richness compared to the Control group. Smoking was found to be the most important factor affecting disease activity and microbiota dysbiosis. Age and gender were among the factors influencing the alteration of the microbiome.

Conclusion

In this project, we summarize the role of the gut microbiome and its manipulation in the development and management of Crohn's disease. This study will add great value to the amount of knowledge and help understand the relationship between microbiota and Crohn’s disease progress in Saudi Arabia.