bluuebunny/crossref_metadata_2025 huggingface dataset:
DOP81 Unaffected ileal transcriptomics in Crohn's disease is associated with the response to anti-TNF therapy
Authors: Lee H S , Lee Y , Baek J , Kim Y , Park S , Jung S , Kim K K , Hwang S W , Lee J L , Park S H , Yang S K , Han B , Song K , Yoon Y S , Ye B D
Published: January 2023

Background

We aimed to assess the gene expression profiles of uninflamed small bowels in patients with Crohn’s disease (CD) to identify its accompanying molecular alterations.

Methods

We performed RNA sequencing of the uninflamed small bowel tissues obtained from 70 patients with ileal CD, and 9 patients with colon cancer (non-CD controls) during bowel resection. Differentially expressed gene (DEG) analyses were performed using DESeq2. Gene set enrichment, correlation, and cell deconvolution analyses were applied to identify modules and functionally enriched transcriptional signatures of CD.

Results

A comparison of CD patients and non-CD controls revealed that of the 372 DEGs, 49 protein-coding genes and 5 lncRNAs overlapped with the IBD susceptibility loci. The pathways related to immune and inflammatory reactions were upregulated in CD, while metabolic pathways were downregulated in CD. Compared with non-CD controls, CD patients had significantly higher proportions of immune cells, including plasma cells (P=1.15×10-4), and a lower proportion of epithelial cells (P=1.12×10-4). Co-upregulated genes (M14 module) and co-downregulated genes (M9 module) were identified in CD patients. The M14 module was enriched in immune-related genes and significantly associated with the responses to anti-TNF therapy. Intestinal tissue transcriptomic analysis of 3 independent cohorts, identified M14 module upregulation in non-responders at baseline, which was predictive of response with acceptable discrimination ability (area under the curve of 77~83%).

Conclusion

The differences in gene expression and cellular composition between CD patients and non-CD controls imply significant molecular alterations, which are associated with the response to anti-TNF treatment.
DOP85 Crohn's Disease Strictures Respond to Drug Treatment and Treat-to-Target Intense Combination Therapy is More Effective than Standard Anti-TNF Therapy. Two year results of the STRIDENT Randomised Controlled Trial.
Authors: Schulberg J , Hamilton A , Wright E K , Holt B , Lovett G , Sutherland T , Ross A , Vogrin S , Kamm M
Published: January 2023

Background

The randomised STRIDENT (Stricture Definition and Treatment) trial showed that Crohn’s disease symptomatic strictures are responsive to anti-TNF therapy with most patients clinically improved after 12 months treatment with adalimumab +/- thiopurine. Treat to target intensification resulted in greater stricture morphology improvement. We present here 2 year (from study entry) follow up to assess response durability and risk of surgery.

Methods

Patients with symptomatic Crohn’s disease strictures and associated inflammation (elevated faecal calprotectin and CRP) were assessed with imaging (intestinal ultrasound and MRI) and ileo-colonoscopy. The Obstructive Symptom Score (OSS) was used for clinical assessment. Patients were randomised 2:1 to high dose adalimumab induction (160mg weekly for 4 weeks) followed by 40mg fortnightly plus thiopurine, with adalimumab dose increase at 4 and/or 8 months if evidence of ongoing inflammation, versus standard dose adalimumab monotherapy. The primary endpoint was improved OSS at 12 months. Patient interview was conducted at 24 months, assessing drug treatment, symptoms, need for endoscopic therapy, hospitalisation and surgery.

Results

In the initial 12 months study 52 patients were randomised to the intensive and 25 to the standard treatment arm. 64/77 (83%) patients recorded an outcome at 12 months (13/77 (17%) withdrawn: 8 surgery, 5 other) 74/77 (96.1%) patients were followed up at a mean of 25.3 (IQR 24.4-26.3) months. Of these, 59 (79.7%) remained on adalimumab, 10 (13.5%) had switched to another biologic and 5 (6.7%) had ceased biologic therapy. For patients who were on adalimumab at 12 months the risk of surgery by 24 months was 15%. Of patients on adalimumab 40mg fortnightly at 12 months, 12/48 (25%) had been dose escalated by 24 months. Endoscopic balloon dilatation, hospitalisation or surgery was required in 6 (8.1%), 14 (18.9%) and 13 (17.5%) respectively; rates were not different between the standard and intensive treatment groups (P = 0.982). Median time to intestinal resection was 12.2 (IQR 6.9-18.6) months. Clinical responders at 12 months, compared to non-responders, had a reduced likelihood of surgery at 24 months (9% vs 42%, P=0.003).

Conclusion

Clinical response to adalimumab for structuring Crohn’s disease is durable in a majority of patients, with most patients remaining on adalimumab and avoiding surgery at two years. Rates of surgery were not different between the standard and intensive therapy arms. Clinical response to adalimumab at 12 months is an important predictor of future surgical risk.
P001 Obefazimod upregulates miR-124 and downregulates the expression of some cytokines in blood and rectal biopsies of patients with moderate-to-severe ulcerative colitis.
Authors: Santo J , Gineste P , Scherrer D , Nitcheu J , Ehrlich H , Sands B E , Vermeire S
Published: January 2023

Background

The oral small molecule obefazimod (ABX464) has demonstrated efficacy and safety in patients with moderate-to-severe ulcerative colitis (UC) in a randomized, placebo-controlled, 8-week phase 2b induction study1. We examined the effects of obefazimod on miR-124 and cytokines levels in blood (baseline, weeks 4 and 8) and rectal biopsies (baseline, week 8) of these patients.

Methods

Absolute quantification (QuantaSoft Pro) of the miR-124 copy number was performed by using droplet digital PCR technology on 644 rectal biopsy samples and 1104 whole blood samples from patients receiving obefazimod (25mg, 50mg or 100mg once daily) or placebo. A total of 15 cytokines (IL1b, IL13, IL17, IL6, IL10, IL23, MCP-1, IL34, CXCL-12, IL22, LIF, TGFb, GM-CSF, IFNg and TNFa) were examined by immunohistochemistry (IHC) on 208 rectal biopsies. For each cytokine, antibodies were tested for the chromogenic IHC detection on paraffin tissue sections; repeatability and reproducibility tests were conducted to validate protocols with robust signal. IHC signal was digitized and quantified with Halo® image analysis software. IL17 was also measured in blood samples using an ELLA immunoassay (Bio-Techne).

Results

The three tested doses of obefazimod (25mg, 50mg or 100mg once daily) significantly upregulated miR-124 in blood at week 1 (A) and 8 (B), and in rectal tissue at week 8 (C) (from p<0.001 to p<0.0001 vs. placeb

The three doses of obefazimod significantly reduced IL17 levels in blood of patients at week 1, 4 and 8 (from p<0.01 to p<0.0001 vs. placebo).

IHC protocols in rectal tissue were validated for 4 out of 15 cytokines: IL17, IL23, CXCL-12, INFg. Compared with baseline levels, an 8-week obefazimod treatment significantly down-regulated the expression of IL17 (at all doses, (from p<0.05 to p<0.001), IL23 (p<0.01 at 50mg, p<0.001 at 100 mg), and INFg (p<0.001 at 50 mg).

Within obefazimod treated subjects, clinical responders showed higher reduction of IL-17A and IL-23 expression in rectal tissue than non-clinical responders at week 8 (reduction in MMS ≥2 points and ≥30% from baseline + decrease in rectal bleeding subscore ≥1 or absolute rectal bleeding subscore ≤1 point). No significant change was observed with CXCL-12.

Conclusion

The 8-week efficacy of obefazimod in patients with moderate-to-severe UC is associated with an increase in miR-124 expression in blood and tissue and with a decrease of IL17, IL23 and INFg in the colorectal tissue.

References:

1. Vermeire S, et al. ABX464 (obefazimod) for moderate to severe active ulcerative colitis: a randomised, placebo controlled phase 2b induction trial and 48 week extension. The Lancet Gastroenterology & Hepatology. 2022. https://doi.org/10.1016/S2468-1253(22)00233-3
P004 Untargeted serum metabolome shows Crohn Disease (CD) associated signature, signals that correlate with disease activity and CRP, and signals that may precede disease flare
Authors: Levhar N , Hadar R , Braun T , Efroni G , Abramovich I , Gottlieb E , Granot M , Sandra N , Selinger L , Picard O , Yavzori M , Lahat A , Eliakim R , Weiss B , Kopylov U , Ben-Horin S , Amir A , Haberman Ziv Y
Published: January 2023

Background

The serum metabolome contains potential disease biomarkers, some of which are produced endogenously, derived from the diet, or formed by the gut microbiome. We aimed to characterize metabolomics profiles linked with Crohn Disease (CD), with active CD, and with remitting pre-flare CD.

Methods

Serum untargeted metabolomics was performed using liquid chromatography Mass Spectrometer (LC/MS). PERMANOVA on beta diversity was used to define factors affecting serum metabolome. Multivariate analyses in MaAsLin2 were applied to identify differentially abundant metabolites between CD and controls, between active CD and those in remission, and between those that subsequently flared and those that remained in remission within 24 months of follow-up.

Results

The cohort included 202 samples from 53 CD patients and 31 healthy controls (156 samples from CD and 46 from controls; Table 1). 545 metabolites were included in our predefined library and were subsequently analyzed. PERMANOVA on the serum metabolomic data indicated that fasting, age, gender, disease, and subject are significant factors contributing to metabolomics variations. Using multivariate analyses and controlling for fasting, age, gender, and subject, we identified 34 specific metabolites that were significantly higher in CD and 26 that were significantly higher in Controls (FDR<0.25; Fig. 1). CD samples were significantly enriched for purine derivatives, simple sugars, and histidine, compared to healthy controls. In contrast, amino-acids, caffeine, and TCA cycle related metabolites were consistently downregulated in CD. 37 metabolites were higher in active CD and 24 in quiescent CD. Saturated fatty acids and acetylated amino-acids were positively correlated with active CD, while histidine and other standard amino-acids were downregulated in active samples compared to remission. Interestingly, acetylated amino acids, asparagine and phenylalanine, were also significantly correlated with CRP. A sub-cohort of 25 CD subjects was enrolled during remission and monitored every 3 months over 2 years. During that prospective follow-up, 11 flared. Focusing on the pre-flare samples, patients who eventually flared showed a significant reduction in N-acetyl glycine and reduction in two ketogenic derivates, acetoacetate and 3-hydroxybutyric acid, in comparison to patients who remained in remission throughout (FDR<0.15).

Conclusion

CD, CD activity, and a subsequent flare in CD patients with longitudinal data were associated with distinct serum metabolic signature after controlling for confounders like age, gender, and fasting. Additional validation is needed to test if these can be used as predictive biomarkers.
P005 0190Molecular-based classification of ulcerative colitis and its dynamic
Authors: Seyed Tabib N S , Verstockt S , Verstockt B , Ferrante M , Sabino J , Vermeire S
Published: January 2023

Background

A better characterization of inflammatory bowel disease is needed. We previously developed a molecular classification tool for Crohn’s disease, based on a combination of metagenomics (dysbiosis score - MDI), transcriptomics (barrier integrity score - BIS, autophagy score – ATS, unfolded protein response score - UPRS), serum proteomics (inflammation score - IPS) and genetics (polygenic risk score - GRS)1. We here investigated if this score could also apply for ulcerative colitis (UC) and how the individual components correlate with clinical outcomes.

Methods

For a total of 173 UC patients and 181 controls (CO), we collected faecal and blood samples, as well as colon biopsies (Table). Faecal microbiota 16S-sequencing, intestinal RNA-sequencing, and serum proteomics (OLINK inflammatory panel) were performed. Using proteomics and RNA expression data, IPS, ATS, UPRS and BIS were calculated. MDI was calculated as previously described1. Based on these findings, patients were categorized into quartiles, ranging from Q1 (the least dysfunctional state) to Q4 (the most dysfunctional state). The individual scores were correlated with time to first biological or biological switch, as well as correlations with C-reactive protein (CRP), faecal calprotectin (FC), and the Montreal classification. The therapeutic value of MDI score was studied in a small dietary intervention pilot study (n=8)2.

Results

None of the individual components of the score were associated with disease extent. Patients diagnosed at a later age had increased ATS and BIS levels. BIS and UPRS showed a positive correlation with disease duration (r=0.36, r=0.54 respectively, p<0.001) (Figure2). MDI (spearman r=0.27, r=0.44, r=0.46 respectively, all p<0.0001) and IPS (r=0.5, r=0.7, r=0.6 respectively, all p<0.0001) correlated with CRP and FC levels, and time to first biologic. UPRS, ATS, MDI, BIS, and IPS were considerably higher in patients who required a switch in biologic therapy (all p<0.001). Finally, we observed a decreasing trend in MDI in response to dietary modification (Figure3).

Conclusion

In UC patients, we created a multifaceted scoring system to molecularly describe disease by the degree of dysbiosis, dysregulation of the immune proteome, and intestinal barrier integrity. We also demonstrated the dynamic of MDI of this score in relation to dietary intervention. Therefore, molecular profiling of patients may represent a new, individualized strategy to the management of UC.

References:

1. OP30 ECCO 2020, JCC 14(Supplement_1):S028-S0302.

2. P767 ECCO 2017, JCC 11(suppl_1):S473-S473.
P008 Fibrostricturing Crohn’s disease is characterised by an imbalance in active eosinophils, Th1, Th2 and regulatory T cells
Authors: Jacobs I , Ke B J , Cremer J , D’Hoore A , Bislenghi G , Matteoli G , De Hertogh G , Sabino J , Ferrante M , Vermeire S , Breynaert C , Vanuytsel T , Verstockt B
Published: January 2023

Background

About one third of patients with Crohn’s disease (CD) develop strictures during their disease course requiring surgical resection. The immune landscape involved in this process is poorly understood. Therefore, we aimed to characterise the fibroblast phenotype, immune cells and their mediators involved in intestinal strictures.

Methods

We included 25 CD patients with stricturing disease in the terminal ileum (TI) and 10 controls with colorectal cancer (CRC), all undergoing an ileocolonic resection. Transmural samples from the resection specimen of the TI were obtained. Macroscopically, CD tissue was divided into unaffected, fibrostenotic and inflamed regions by an experienced histopathologist. Next, mucosa was separated from deeper layers, after which single cells were isolated and fluorescently stained for flow cytometry. Protein levels were determined via the MesoScale Discovery (MSD) platform in the corresponding samples. Comparisons between CRC controls and CD patients were performed via an unpaired t-test or Mann-Whitney analysis and corrected for multiple testing.

Results

An increase in active fibroblasts and decrease in inactive fibroblasts in the fibrotic and inflamed mucosa (p=0.0002 and p<0.0001) and deeper layers (p=0.003 and p=0.02) when compared to the CRC controls was observed, confirming ongoing tissue remodelling. An enrichment of active eosinophils was only seen in the fibrotic deeper layers (p=0.02), although an increase in T helper 2 (Th2) cells was observed in both the fibrotic and inflamed deeper layers (p=0.02 and p=0.04). In contrast, T helper 1 (Th1) cells were decreased in both fibrotic and inflamed mucosa (p=0.03 and p=0.02) and deeper layers (p=0.01 for both). Regulatory T cells were significantly enriched in both fibrotic and inflamed mucosa (p<0.0001 and p=0.0005) and deeper layers (p=0.01 and p=0.006) (figure 1). Protein quantification confirmed a significant increase in transforming growth factor-β3 (TGF-β3) in the fibrotic (p=0.007) and inflamed (p=0.0002) layers, but not in the more superficial mucosa. Comparably, IL-1β was increased in the fibrotic (p=0.05) and inflamed (p=0.05) deeper layers. A similar observation was made for basic fibroblast growth factor (bFGF) (p=0.004), although only a trend could be seen in the fibrotic deeper layers (p=0.08) (figure 2).

Conclusion

The fibrotic and inflamed tissue of CD patients is characterized by increased activated eosinophils, Th2 and regulatory T cells and decreased Th1 cells, as well as many of their mediator cytokines. The current immunological characterisation can help to prioritise potential anti-fibrotic targets for stricturing CD.
P011 Patient-derived organoids as an epithelial model for preclinical IBD research and drug development.
Authors: Frias J , Derksen M , Verissimo C S , van Dooremalen W T M , Oldenburg B , Vries R G J , Boj S F , Pourfarzad F , Pott J
Published: January 2023

Background

The study of the intestinal epithelial barrier is hampered by the lack of a suitable in vitro model system. Patient-derived organoids (PDOs) recapitulate the cellular diversity and patient heterogeneity and provide a relevant platform to study the intestinal epithelium and its response to microbial or inflammatory challenges.

Here, we present the characterization of an inflammatory bowel disease (IBD) organoid biobank derived from Crohn’s disease and Ulcerative Colitis patients and its application for IBD drug development efforts.

Methods

PDOs from different regions of the gastrointestinal tract of 14 IBD patients were established. To recapitulate the full cellular diversity, specialized medium formulations were defined that support the differentiation of distinct epithelial cell lineages as confirmed by transcriptional profiles and histology.

To study the inflammation driven loss of barrier integrity observed in the mucosa of IBD patients, we employed organoid derived monolayers and mimicked the inflammatory environment by cytokine challenge (TNF, IFNγ).

Results

In response to differentiation media, intestinal organoids and monolayers displayed gene expression patterns consistent with maturation of epithelial cell types found in the human gut.

The cytokine challenge robustly induces loss of barrier integrity across seven patient-derived monolayers. The barrier breakdown was ameliorated in all models by treatment with the IBD therapy Tofacitinib.

Conclusion

Intestinal PDOs were generated from IBD patients and were capable to differentiate into mature epithelial cell types. PDO-derived monolayers built trans-epithelial electrical resistance (TEER) and are a relevant model to study epithelial barrier function and inflammation mediated barrier breakdown. Proof-of-concept studies with Tofacitinib demonstrated the suitability of the assay for IBD drug development efforts.
P012 The administration of mm-miR-378a-3p exacerbates chronic inflammation and fibrosis in a murine intestinal model
Authors: Seco-Cervera M , Argilés-Arberola D , Bauset C , Lis-López L , Coll S , Cosín-Roger J , Ortiz-Masià D , Calatayud S , Barrachina M D
Published: January 2023

Background

Fibrosis constitute an important complication of CD. MicroRNAs (miRNAs), which are small RNA molecules that regulate gene expression, have been shown to participate in the molecular interactions of both inflammation and fibrosis. Lower levels of miR-378a-3p have been reported associated to murine liver fibrosis (Hyun et al. DOI: 10.1038/ncomms10993) and we analyze here the relevance of miR-378a-3p on intestinal fibrosis.

Methods

B57BL/6 mice were intravenously injected with 2,5mg/kg of negative control (NC) or mm-miR-378a-3p mimic, twice a week and received vehicle or Dextran Sulfate Sodium (DSS) for 2 cycles (7 days drinking DSS 2% in water solution followed by 10 days drinking water). Body weight and DAI score was obtained every day and the colon was collected after sacrifice. Sirius and hematoxylin-eosin dyes were employed to determine the fibrosis and structural state in 5µm slides of intestinal tissue. Gene expression and miRNA profiles were analyzed by RT-qPCR. Human small intestinal fibroblasts (HSIF; P10760, Innoprot, Spain) were transfected with 20nM of NC or hsa-miR-378a-3p mimic during 24h.

Results

No significant changes in body weight, DAI score, and colon length were detected between mice receiving NC and those receiving mimic, all along the two DSS cycles. Colon of mice treated with DSS, exhibited a significant diminution in the mRNA expression of mm-miR-378a-3p compared with naïve samples. In DSS-treated mice, the iv administration of the mimic compared with the NC: a) significantly increased levels of miR-378a-3p in the colon; b) increased the number of neutrophils, and heightened changes in the glandular epithelia and architectural distortion (figure 1a) C) decreased the number of lymphoid follicles (figure 1a); d) slightly increased collagen deposition, as analyzed by sirius red (figure 1b) and e) significantly increased the mRNA expression of Tgfb1, Il1b, and Mmp2. Treatment of human intestinal fibroblasts with hsa-miR-378a-3p mimic did not significant modify the mRNA expression of markers of fibrosis but it significantly increased the mRNA expression of two antiapoptotic molecules BCL2 and MCL1.

Conclusion

Levels of mm-miR-378a-3p are diminished in a murine model of intestinal fibrosis; The exogenous administration of miR-378a-3p, increased the acute inflammatory response and the expression of fibrosis markers in murine fibrotic colon and the expression of antiapoptotic molecules in human intestinal fibroblasts.
P017 Fecal wash host transcriptomics identifies inflammation throughout the colon and terminal ileum
Authors: Dan S , Ungar B , Ben-Moshe S , Bahar Halpern K , Yavzori M , Fudim E , Picard O , Abitbol C M , Harnik S , Kopylov U , Ben-Horin S , Itzkovitz S
Published: January 2023

Background

Non-invasive modalities for assessing active endoscopic and histological inflammation in Crohn’s disease and Ulcerative Colitis patients are critically needed. Fecal wash host transcriptomics has been shown to be a robust classifier of endoscopic and histological inflammation in inflammatory bowel disease (IBD) patients with distal colitis. Whether such fecal washes can inform on inflammatory processes occurring in more proximal intestinal segments is currently unknown.

Methods

Crohn's disease and Ulcerative Colitis patients as well as healthy controls were prospectively enrolled. Host transcriptomes of biopsies and fecal washes obtained during colonoscopy at predefined locations throughout the colon and terminal ileum were analyzed and associated with same time-point clinical, endoscopic and histological parameters.

Results

59 IBD patients and 50 controls were prospectively enrolled. Biospies and fecal washes from the distal, proximal colon and terminal ileum were compared. We find that host transcriptomics of distal fecal washes robustly classify histological inflammation in ileal and proximal colonic Crohn’s disease, even without distal colonic involvement. Receiver operating characteristic (ROC) curve analysis demonstrated significantly higher classification power based on the fecal wash transcriptomics compared to biopsies: Area Under the Curve, AUC=0.94+-0.07 for UC distal fecal washes and AUC=0.94+-0.09 for distal fecal washes of CD patients without distal colonic involvement, as compared to all distal biopsies AUC= 0.82+-0.20. We further demonstrate that fecal washes consist of modules of co-expressed immune, stromal and epithelial genes. Fecal wash expression of gene modules previously associated with lack of response to biological therapies correlates with disease severity.

Figure 1 - Host transcriptomics of fecal washes from different intestinal segments captures information that is distinct from same – segment biopsy transcriptomics. A) Experimental layout. B) Clustergram of transcriptomes of biopsies (purple) and fecal washes (yellow). C) Principal component analysis (PCA) of biopsies (purple) and fecal washes (yellow). Black circles highlight inflamed samples. D) Spearman correlations between the transcriptomes of pairs of either biopsies or fecal washes that are both annotated as inflamed and mixed pairs (one annotated as inflamed and the other not). (A) Created with BioRender.com. TI – terminal ileum, P – proximal colon, D – distal colon.

Conclusion

Our study establishes the power of colonic fecal washes for identifying pathological processes throughout the ileal-colonic axis.
P018 Expression of annexin A1 and NLRP3 inflamassome in Chron’s disease
Authors: Koga Ito H T , Lice I , Sasso G R S , Artigiani R , Gil C D
Published: January 2023

Background

Despite annexin A1 (ANXA1), an anti-inflammatory and pro-resolving protein, regulates the NLRP3 inflammasome activation in inflammatory cells, this relationship has not been explored in inflammatory bowel disease. Then, this study evaluated the expression patterns of ANXA1 and NLRP3 inflammasome in Crohn’s disease (CD).

Methods

Paraffin-embedded human intestine biopsies, obtained from CD (n=20) and control (n=8) patients were processed for histological and immunohistochemical analyses. The study was approved by the UNIFESP Ethics Committee (CEP 4.491.836/2020). GSE179285 study, which features ileum (Control - n=8, CD non-inflamed - n=48, CD inflamed - n=33) and colon (Control - n=12, CD non-inflamed – n=74, CD inflamed - n=14) transcriptome, was selected from the Gene Expression Omnibus repository. Datasets were analyzed using the GEO2R tool to detect ANXA1, CASP1, NLRP3, and PYCARD expressions.

Results

Bioinformatics analysis of GSE179285 samples showed a marked increase in the ANXA1 expression in the CD inflamed ileum and colon compared to CD non-inflamed and controls samples (Fig.1A). Regarding the genes associated with the NLRP3 inflammasome activation, transcriptome analysis also revealed a significant increase in NLRP3 and CASP1 in the CD inflamed intestine compared to the other ones (Fig.1B,C). PYCARD expression did not show alterations between the different conditions (Fig.1D). Immunohistochemical analyzes showed intense immunoreactivity for ANXA1 and NLRP3 proteins in the CD ileum and colon, especially in inflammatory infiltrate and epithelium of the mucosal layer (Fig.2C,D,G,H), compared to controls (Fig. 2A,B,E,F). These findings corroborate the transcriptome analyses.

Conclusion

ANXA1 and NLRP3 expression is increased in CD-inflamed intestines. High levels of ANXA1 may contribute to controlling inflammation and repairing intestinal injuries. High levels of NLRP3 evidence dysfunction in the pro-inflammatory activity of DC, contributing to the release of IL-1β and IL-18. Support: FAPESP (grants 20/03565-2, 21/01541-1 and 21/06059-3).

Figure 1. ANXA1, NLRP3, CASP1 and PYCARD expressions. Analysis performed in the healthy or CD terminal ileum and ascending/descending colon of human biopsies from GSE179285 study. Data represent mean ± S.E.M. of the gene expression values. **p<0.01; ***p<0.001; ****p<0.0001 (Kruskal-Wallis, Dunn’s post-test).

Figure 2. ANXA1 and NLRP3 expression in human intestine. Representative images of ileum (A, C, E, G) and colon (B, D, F, H) of control (A,B,E,F) and CD patients (C,D,G,H). ANXA1 and NLRP3 expression detected in intestinal epithelium (arrows) and inflammatory infiltrate (asterisks). Counterstaining: Carazzi’s Hematoxylin. Bars: 50 µm.
P019 Host-Enterobacteriaceae interactions and mitochondrial dysfunction in Crohn’s colitis
Authors: Holani R , Bar-Yoseph H , Krekhno Z , Moon K M , Stacey R G , Serapio-Palacios A , Bressler B , Donald K , Foster L J , Finlay B B
Published: January 2023

Background

Crohn’s disease (CD) is characterized by chronic gastrointestinal inflammation and unfavourable changes in the composition of intestinal microbiota (‘dysbiosis’). Members of Enterobacteriaceae such as K. pneumoniae and E. coli are often over-represented in colonic mucosa of CD patients. These bacteria contribute to vicious circle of dysbiosis and inflammation by expressing multiple virulence factors. Further, mitochondrial dysfunction in Paneth cells has recently been reported in ileal CD. In agreement, high-fat diet and repeated use of antibiotics increases susceptibility to colitis by damaging mitochondria. However, it remains unknown whether impaired mitochondrial bioenergetics could enhance mucosal colonization by Enterobacteriaceae and thus, contribute to the pathophysiology of colonic CD. Furthermore, the molecular mechanism leading to mitochondrial damage in colonic CD are incompletely understood.

Methods

Right colonic biopsies were collected from 74 adult CD patients and 28 healthy participants at St. Paul’s Hospital (Vancouver, BC, Canada). Microbes in colonic biopsies were characterized by 16S rDNA sequencing using Illumina MiSeq platform, followed by analysis of differentially abundant families via ANCOM-BC methodology. Further, high-throughput data-independent acquisition (DIA) proteomics was performed in colonic biopsies (Bruker Daltonics TimsTof Pro2 LC-MS/MS), and later analyzed via DIA-neural network software. Spearman's rank correlation approach was used for host proteomics- Enterobacteriaceae correlational analysis. Microbiome and proteomic data were corrected for false discovery rate (FDR; < 0.5 for statistical significance). For validation studies, human colonic TC7 epithelial cells and patient-derived organoid cultures were infected with CD-patient derived E. coli ± C75, a chemical inhibitor of fatty acid synthase (FASN; de novo fatty acid synthesis enzyme).

Results

We found an increased abundance of Enterobacteriaceae members in colonic mucosa of CD patients compared to healthy controls (Fig A). Next, our proteomics data revealed a significant downregulation in a group of proteins which were functionally related to mitochondrial health and activity, in CD patients compared to controls (Fig B). Further, Spearman’s analysis showed a positive correlation between Enterobacteriaceae and host FASN enzyme (Fig C), a protein directly related to mitochondrial metabolism. Interestingly, we found that blocking FASN with chemical inhibitor (C75) significantly enhanced colonization by E. coli in TC7 cells (Fig D).

Conclusion

Mechanistically, our study reports that aberrant activity of host FASN enzyme may cause mitochondrial dysfunction and thus, facilitates intestinal colonization by Enterobacteriaceae in CD patients.
P029 Histopathological characterization of a chronic DSS-induced mouse model of IBD with intestinal fibrosis
Authors: Nielsen M H , Zachariassen L F , Sárvári A K , Marstrand-Jørgensen A B , Hansen H H , Feigh M
Published: January 2023

Background

Inflammatory bowel disease (IBD) comprises a group of intestinal disorders, including ulcerative colitis and Crohn's disease. Intestinal fibrosis, as result of chronic inflammation, is a common complication in IBD. High treatment failure rates associated with existing interventions indicate a high unmet need for more effective drugs to improve the management and outcomes of IBD. Consequently, translational animal models of IBD demonstrating chronic, progressive colonic fibrosis are important tools in preclinical drug discovery for IBD. The aim of the present study was to characterize intestinal histopathology in a chronic DSS-induced mouse model of IBD.

Methods

Ten-weeks-old male C57BL/6JRj mice were randomized into study groups based on body weight and received 3 cycles of DSS (2.5 % w/v) in the drinking water (DSS-IBD) or normal water (CTRL). Terminal endpoints included colon morphometry and quantitative histological markers of inflammation and fibrosis assessed in proximal, middle and distal colonic segments. Using RNA sequencing, transcriptome signatures were profiled in the middle colon segment.

Results

Compared to healthy controls, the chronic DSS-IBD mouse model demonstrated hallmarks of IBD, including mild-to-moderate weight loss and colonic hypertrophy. Histopathological analysis indicated increased deposition of inflammatory (CD45 and CD11b), fibrogenesis (alpha-smooth muscle actin) and fibrosis markers (collagen 1a1, Picrosirius red) in the middle and distal colon. Severe fibrosis was observed throughout all layers of the middle colon. Histological findings were supported by significantly upregulated gene expression markers of inflammation and fibrosis.

Conclusion

The DSS-IBD mouse is a translational preclinical model suitable for testing novel drug therapies for IBD with intestinal fibrosis.
P034 A common IL2RA polymorphism alters natural killer cell immunophenotype and function in Crohn’s disease, with implications for IL-2 based therapy
Authors: Clough J , Roberts L , Goldberg R , Lord G , Irving P
Published: January 2023

Background

The single nucleotide polymorphism (SNP) rs61839660 lies within the IL2RA (CD25) intronic enhancer and has been identified as a causal variant in Crohn’s disease (CD), with a heterozygote frequency of 16.8% in a European population. CD25 is the high-affinity receptor for IL-2, and is constitutively expressed on regulatory T cells (Tregs). Low-dose IL-2 (LD-IL2) is under investigation in IBD, under the premise that it will expand a group of tolerising Tregs. However, finding a therapeutic dose which activates Tregs without activating effector T cells (Teffs) has proved challenging and activation of CD56dim NK cells has been linked to adverse events in trial subjects. We have demonstrated that the rs61839660 risk (T) allele carriers exhibit higher CD4+ T cell CD25 expression, enhanced pSTAT5 activation on stimulation with IL-2, and greater Teff release of pro-inflammatory cytokines. We sought to understand the impact of the risk allele on NK cell immunophenotype and function.

Methods

We recruited CD patients homozygous major allele (CC), or heterozygous (CT) at the rs61839660 locus. We extracted peripheral blood mononuclear cells and flow-sorted CD56dim (cytotoxic) and CD56bright (regulatory) NK subsets. Cells were stimulated with IL-2, then stained for intracellular pSTAT5. To assess cytotoxicity, NK cells were rested in media either with or without IL-2, before plating with CFSE-labelled K562 target cells. The proportion of K562 cells positive for dead cell marker was assessed for each condition.

Results

There was no difference in CD phenotype or severity between groups, in terms of age at diagnosis, sex distribution, presence of perianal disease or the proportion requiring surgery. CD25 expression was significantly enhanced on CD56Bright NK cells in CT subjects compared to CC subjects (1b). Enhanced CD25 expression was also seen in CD56Dim NK cells, though this did not reach statistical significance (1a). The concentration of IL-2 required to activate 50% intracellular STAT5 was lower in CT subjects’ CD56Bright NK cells compared to those from CC subjects (21.5IU/ml vs. 132.4IU/ml, p=0.039). The cytotoxicity of CT subjects’ CD56dim NK cells was significantly enhanced following exposure to IL-2, compared to CC subjects (1c).

Conclusion

Our findings suggest that risk (T) allele carriers have enhanced responsiveness to IL-2 signalling in NK cells, mediated by increased expression of CD25. We hypothesise that these subjects would be at greater risk of adverse events on exposure to LD-IL2, through enhanced CD56Dim NK cytotoxicity. Our results suggest that screening of subjects entering trials of IL-2-based therapy for the rs61839660 risk variant should be considered, as the threshold for adverse events in these subjects may be lower.
P039 The disappearance of cell death brakes in inflammatory bowel disease: a step towards molecular healing
Authors: Al-Ani A , Samson A , Rickard J , Christensen B , Murphy J
Published: January 2023

Background

The core mechanistic driver underpinning the relapsing and progressive nature of inflammatory bowel disease (IBD) remains unknown with current therapies targeting immune or inflammatory pathways. Our in-depth analysis of regulated death pathways remarkably reveals that cell death brakes are removed in intestinal IBD tissue, indicating cells remain “primed” to die regardless of endoscopic and histologic healing. Characterisation of cell death is essential to confirm our hypothesis that disinhibition of programmed cell death is a rudimentary molecular defect in IBD. Restoration of cell death brakes may be intrinsic to molecular healing and novel drug development.

Methods

Intestinal biopsies are collected from inflamed, marginal and non-inflamed regions during endoscopy for patients with IBD and matched with those without IBD (“controls”). Tissue is processed to quantify cell death signals and changes to the local microbiome using immunoblotting, immunohistochemistry, immunofluorescence, organoid preparation and RNA sequencing (Fig. 1). Laboratory data are correlated with clinical, biochemical, endoscopic and histologic parameters.

Results

To date, 59 patients have been recruited: 29 controls; 31 IBD (13 Crohn’s disease, 16 ulcerative colitis, 2 pouchitis). Interim analyses of 20 patients show loss of cell death brakes (cIAP1, cIAP2, cFLIP) suggesting disinhibition of cell death even in endoscopically and histologically normal IBD tissue (Fig. 2). Concurrently, there is increased apoptosis (characterised by cleaved caspase-3) in IBD intestinal tissue regardless of inflammatory activity (Fig. 3). Necroptosis, a programmed lytic form of cell death, is active in steady state conditions in controls. Additionally, necroptosis appears markedly elevated in stricturing and penetrating Crohn’s disease compared to other subsets of IBD (Fig. 2).

Conclusion

This study demonstrates for the first time that cellular death brakes are removed in intestinal tissue in IBD, despite deep remission with seemingly effective biologic therapy. Disinhibition of programmed cell death is likely intrinsic to the inflammatory sequence underlying disease recurrence and progression. Restoration of cell death brakes may hence be key to achieving long-term, relapse-free remission and should be considered an aspirational goal for both drug development and achievement of molecular healing. Our results challenge current dogma based on murine data that necroptosis occurs in the absence of apoptosis and that necroptosis is universally increased in all forms of IBD. Instead, we propose that a balance of apoptosis and necroptosis is essential in intestinal homeostasis with endotyping of disease based on cell death profile a feasible goal towards precision medicine in IBD.
P044 Ex vivo effects of infliximab on the long non-coding RNAs expression levels in Crohn′s disease
Authors: Baldan-Martin M , Rubín de Célix C , Orejudo M , Ortega L , Fernández-Tomé S , Soleto I , Ramírez C , Arroyo R , Fernández P , Santander C , Moreno-Monteagudo J A , Casanova M J , Casals F , Casabona S , Becerro I , Marigorta U M , Aransay A M , Bernardo D , Chaparro M , Gisbert J P
Published: January 2023

Background

In recent years, evidence shows that long non-coding RNAs (lncRNAs) are key regulators of gene transcription and play important roles in the pathogenesis of inflammatory bowel diseases. LncRNA are involved in regulation of intestinal epithelial cell apoptosis, cell-cell interactions, enhancing inflammation, among others. Biological therapies, which are considered the most potent for disease control, only benefit one-third of patients. For this reason, a deeper understanding regarding the mechanisms by which biological drugs elicit their effect on intestinal mucosal is needed. Hence, we aimed to unravel the ex vivo modulator effect of infliximab on the lncRNAs expression in intestinal biopsies from patients with Crohn′s Disease (CD).

Methods

We performed an unbiased transcriptomic analysis of intestinal biopsies from the ileum and colon from 30 patients [active CD = 10, quiescent CD = 10, healthy controls (HC) = 10] to identify lncRNA differentially expressed in the setting of infliximab modulation (Figure 1). Endoscopic biopsies were cultured with or without infliximab, and the transcriptome was determined by stranded total-RNAseq (reagents from Illumina Inc.). We used different databases (Ensembl Biomart, RNAcentral and ToppGene) to search for non-annotated lncRNA information and data on the location (cellular component), biological process and molecular function of differentially expressed lncRNAs.

Results

Transcriptomic results revealed a widespread dysregulation of lncRNAs in ileum biopsies from patients with active CD, quiescent CD and HC compared to the colon at baseline and after infliximab culture (Figure 2). These differentially expressed lncRNAs were enriched in such pathways as proliferation, apoptosis, migration, inflammatory response of fibroblasts, response to wounding, posttranscriptional regulation of inflammatory genes and activation of the mitogen-activated protein kinase signaling pathways. Regarding the effect of infliximab according to intestinal location, presence of disease and activity, no significant lncRNAs were identified in the different study comparatives.

Conclusion

We have characterized the basal transcriptomic landscape of lncRNAs in patients with CD (active and quiescent) and HC both in ileum and left colon. However, we have not found differential lncRNA expression due to the effect of infliximab, suggesting that the location (ileum or colon) is more relevant when analyzing differences in lncRNA expression in the intestinal tissue.
P049 Proteomic characterization of serum extracellular vesicles from newly diagnosed patients with inflammatory bowel disease
Authors: Soleto I , Baldan M , Ramírez C , Orejudo M , García S , Mercado J , Azkargorta M , Lloro I , Ortega Moreno L , Aldars Garcia L , Riestra S , Rivero M , Gutiérrez A , Rodríguez-Lago I , Fernández L , Ceballos D , Benítez J M , Aguas M , Bastón Rey I , Bermejo F , Casanova M J , Lorente R , Ber Y , Ginard D , Esteve M , Elortza F , Gisbert J P , Martin-Cofreces N , Chaparro M
Published: January 2023

Background

The etiology and pathogenesis of inflammatory bowel disease (IBD), Crohn’s disease (CD), and ulcerative colitis (UC) are complex and the mechanisms that lead to the development of these diseases remain unclear. Extracellular vesicles (EVs) are small particles covered with a cell membrane, originating from the emitting cell, excreted into the extracellular medium, and subsequently captured by receptor cells. EVs have a role in multiple diseases and they could also have a function in IBD pathogenesis. Therefore, the analysis of EVs isolated from the serum of newly diagnosed IBD patients (before starting any treatment) may represent an appropriate experimental approach to elucidate their role in IBD pathogenesis. We aimed to evaluate EVs’ composition and potential effects in the pathogenesis of IBD.

Methods

The protein content of EVs isolated by size exclusion chromatography from the 500 µl of serum from 100 patients with IBD (50 patients with CD and 50 patients with UC) recently diagnosed and 50 healthy controls (HC) was characterized by proteomics and their size and concentration in serum by Nanoparticle tracking analysis (NTA). The biological function of EVs and alterations in signaling pathways related to IBD were determined using Ingenuity Pathways Analysis (IPA) to analyze the OMICs results.

Results

A total of 1,100 proteins have been identified, of which 105 proteins were differentially expressed by patients with CD versus HC, 111 proteins by patients with UC versus HC, and 32 proteins by patients with UC versus CD. IPA analysis revealed that proteins carried in EVs are involved in the dysregulation of immune pathways such as the acute phase response (Figure 1), LXR/RXR activation pathway (Figure 2), and the complement pathway. These pathways were regulated differentially in IBD patients compared with HC, but also when the CU group was compared with the CD group (Figure 3) being upregulated nuclear receptors signaling and cytotoxicity pathways.

Conclusion

EVs carry proteins that can be involved in the dysregulation of the immune system in IBD; this effect would be different in UC and CD since their EVs show a differential profile. Consequently, EVs may play role in IBD pathogenesis and source of strong biomarkers candidates for diagnosis in UC and CD. Further studies on their specific function during IBD are warranted.
P051 Immune phenotypes in acute severe ulcerative colitis
Authors: Meringer H , Canales-Herrerias P , Jha D , Livanos A , Jougon J , Cossarini F , Tankelevich M , Kayal M , Colombel J F , Mehandru S
Published: January 2023

Background

Acute Severe Ulcerative Colitis (ASUC) may occur in up to 25% of patients with UC during their disease course. Despite intensive in-hospital management, some patients still require colectomy. We aimed to define the mucosal immune phenotypes related to ASUC that associate with treatment-outcomes.

Methods

Adult patients with ASUC (defined by Truelove and Witts criteria), (n=32) were recruited. UC patients (mild-moderate disease) (n=31) and normal volunteers (NV) (n=27) served as controls. Gastrointestinal (GI) biopsies from the left colon were analyzed by multiparameter flow cytometry (FC) for myeloid cells (classical and non-classical monocytes, dendritic cells), lymphoid cells (T cells, B cells) and innate (Natural Killer) cells. Therapeutic non-response was defined as surgery within 3 months of admission.

Results

Six of the ASUC patients (18.8%) required colectomy during index admission and 8 (25%) within 3 months of admission, i.e. non-responders. Only 14/32(44%) patients were bio naïve on admission. Clinical parameters significantly associated with treatment non-response included lower hemoglobin and albumin levels (Fig. 1A). Significantly higher frequency of classical (CD14+) and non-classical (CD16+) monocytes as well as proliferating CD4+ and CD8+ cells were seen in ASUC compared to NV and UC patients with Mayo 1, 2, 3 inflammation (Fig. 1B). Furthermore, ASUC patients who were non-responsive to medical treatment had higher frequencies of CD14+ monocytes and proliferating (Ki67+) CD4+ T cells in the colon compared to medical treatment responders (Fig 1C).

Conclusion

High frequencies of CD14+ monocytes and proliferating CD4+ T cells in the colon represent distinct immune signatures that relate to ASUC outcomes. Clinical trials targeting adverse clinical and immune phenotype-associated ASUC patients with combination therapies are warranted.