bluuebunny/crossref_metadata_2025 huggingface dataset:
P0103 Systems biology analysis of cytokine signalling in IBD highlights the importance of TYK2-dependent pathways
Authors: Desch N , Petrakis I , Singh N , Tang J , Sirota-Madi A , Schuster R , Davidzohn N , Wyrobnik I , Shen-Orr S S , Chowers Y , Panchal P , Wennbo H , Thakker P , Arunachalam V
Published: January 2025

Background

Ulcerative colitis (UC) and Crohn’s disease (CD), collectively inflammatory bowel disease (IBD), are characterized by chronic gastrointestinal tract inflammation. Recently, there has been interest in the use of selective oral tyrosine kinase 2 (TYK2) inhibitors for IBD treatment, which inhibit signalling downstream of multiple pro-inflammatory cytokines and may have an improved safety profile relative to non-specific Janus kinase (JAK) inhibition. Here, we use a transcriptomic approach to assess the involvement of TYK2-dependent signalling pathways in IBD, and how these are impacted by currently available treatments.

Methods

TYK2-dependent gene signatures were characterized using cytokine stimulation studies and co-expression analysis. Involvement of signature modules in IBD was assessed using disease model data generated from 4059 biopsy samples from 2993 unique patients in 15 UC and 38 CD clinical studies, including assessments of golimumab, infliximab, ustekinumab, and vedolizumab. Pathway differential expression analysis was performed using Gene Set Enrichment Analysis (GSEA) and single-sample GSEA methods.1,2

Results

Seven TYK2-dependent gene signature modules were defined, including two for type I interferon (IFN), three for IL-23, and two for IL-12. Type I IFN signatures were strongly enriched in both UC and CD, while enrichment of IL-23 was stronger for UC than for CD, and enrichment of IL-12 was stronger for CD than for UC. In each case, gene signature module enrichment was associated with greater disease severity. Pathway enrichment analysis of data from individual clinical studies demonstrated that specific type I IFN and IL-12 modules were significantly reduced, but not completely ameliorated, following treatment with golimumab, infliximab, ustekinumab, or vedolizumab. Further assessment of response to therapy in these studies showed that enrichment of type I IFN, IL-23, and IL-12 signature modules was associated with non-response to golimumab, infliximab, and vedolizumab.

Conclusion

Enrichment of TYK2-dependent gene signatures, including type I IFN, IL-23, and IL-12, is associated with both UC and CD. These TYK2-dependent enrichments are not fully mitigated through currently available treatments, and may be associated with non-response to treatment. It is important that all three signalling pathways are considered in the development of future treatment options.

References

1.Barbie DA, Tamayo P, Boehm JS, et al. Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1. Nature. Nov 5 2009;462(7269):108-12. doi:10.1038/nature08460

2.Subramanian A, Tamayo P, Mootha VK, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. Oct 25 2005;102(43):15545-50. doi:10.1073/pnas.0506580102
P0112 Cannabidiol (CBD) targeting of colorectal cancer cells is mutation-independent and via CB2 receptor
Authors: Moniruzzaman M , Janjua T , Begun J , Popat A
Published: January 2025

Background

Cannabidiol (CBD) is a non-neurotoxic, phytocannabinoid from cannabis with reported medicinal properties including anti-epileptic and anti-inflammatory activity. Several in vitro and in vivo studies have shown that CBD has antitumour potential against colorectal cancer (CRC), the third deadliest cancer in the world. However, as different mutations influence the antitumour effects and CBD can bind a variety of receptors, it is yet to be determined whether specific CRC mutations affect CBD’s efficacy in treatment of CRC.

Methods

To investigate this, we selected four CRC cell lines, including HCT116, HT-29, LS174T, and LS153 which harbour distinct mutations. Cells were treated with a range of concentrations of CBD to evaluate its cytotoxic effects and impact on cell proliferation, migration, and invasion using a live-cell imaging system. IC50 values were then calculated for each parameter. The level of endoplasmic reticulum (ER) stress pathway markers was also measured using qRTPCR. The requirements for CB1 or CB2 receptor-medicated signalling was investigated using the selective inhibitors AM251 and SR144528, respectively.

Results

Our results demonstrate that CBD induces apoptosis and halts proliferation, migration, and invasion of CRC cells in a concentration dependent manner. CBD showed potent antitumour effects in the tested cell lines with no obvious effect of different mutations such as KRAS, BRAF, APC, PTEN etc. CBD also induced ER stress in CRC cells but not the healthy intestinal organoids. Co-treatment with SR144528 inhibited the effects of indicating involvement of CB2 receptor activation in the anticancer effects of CBD.

Conclusion

Together, these results demonstrated that CBD could be effective for CRC regardless of the underlying mutation through CB2 receptor activation.
P0114 Tracking Crohn’s Disease Progression through Extracellular Matrix Remodeling: A Longitudinal Study of Pre- and Post-Resection Samples
Authors: Poulsen A , Alexdóttir M S , Buhl Riis L , Ovesen P D , Rasmussen J , Wewer M D , Lin V , Lagström R M B , Al-Sheikh M , Dahl E , Ries A , Porsbjerg L , Katranara Tsapanou T , Pehrsson M , Gögenur I , Krarup P M , Burisch J , Mortensen J H , Seidelin J B
Published: January 2025

Background

Crohn's disease (CD) is a chronic inflammatory condition that can lead to irreversible intestinal damage. Fibrosis, caused by imbalanced extracellular matrix (ECM) remodeling, is difficult to manage, as current anti-inflammatory treatments do not prevent or reverse these processes. ECM fragments released during remodeling may serve as biomarkers for transmural disease activity and fibrosis burden. This study evaluated the correlation of serum C3M, PRO-C3, and PRO-C16 levels and their potential to differentiate between stenosing and luminal CD.

Methods

In this prospective cohort study 62 patients with stenosing CD requiring resection without residual disease and 49 with luminal CD managed medically were included. Phenotype was assessed by diagnostic imaging and/or endoscopy at inclusion. Serum levels of C3M, PRO-C3, and PRO-C16 were measured pre- and post-resection, Neo-epitope specific biomarkers of type III collagen degradation (C3M) and type III and XVI collagen formation (PRO-C3, PRO-C16) were measured in serum using a competitive ELISA. To compare the stenotic and luminal groups, the Wilcoxon signed-rank test was used, and for pre- and post-resection ECM marker values, one-way ANOVA with Tukey’s post hoc was applied.

Results

Baseline levels of PRO-C3 were significantly higher in patients with stenosing CD compared to those with luminal CD (57.3 (IQR: 47.7-75.7)) vs 43.4 (36.0-52.9) ng/ml, p<0.001). Conversely, PRO-C16 levels were higher in the luminal group compared to the stenotic group (2961.5 (2780.8-3264.3) vs 2677.0 (2407.5-2904.5), p<0.001). C3M levels showed no difference at baseline between stenotic and luminal patients (596.0 (546.0-677.5) vs 585.0 (538.5-643.3), p=0.394). Post-resection, C3M and PRO-C3 levels decreased initially (p=0.003 and <0.001), then showed an increase at one month, stabilizing from three to twelve months. The median PRO-C16 value decreased significantly the day after surgery and subsequently increased throughout the first year to levels exceeding pre-resection values (Figure 1).

Conclusion

Baseline biomarker differences between stenosing and luminal CD suggest their potential to distinguish disease phenotypes. However, a sustained post-operative change in ECM markers linked to fibrostenotic phenotype was not observed in patients, challenging this explanation. Day one post-resection ECM marker changes likely reflected wound healing, while subsequent dynamics may indicate collagen processes relevant to stenosis formation without being primary local drivers. These findings underscore the need for longitudinal and cross-sectional studies to clarify ECM biomarkers' role in tissue repair and fibrosis in CD.
P0124 Therapeutic Potential of Adipose-Derived Extracellular Vesicles for Inflammation Modulation and Mucosal Healing in Crohn’s Disease
Authors: Clua L , Suau R , Monfort-Ferré D , Boronat-Toscano A , Menacho M , Caro A , Mañas M J , Mañosa M , Domènech E , Manyé J , Serena C
Published: January 2025

Background

Crohn’s disease (CD) is a chronic, idiopathic inflammatory bowel disease characterised by discontinuous lesions and transmural inflammation that can affect the entire gastrointestinal tract. Despite recent therapeutic advancements, around 50% of patients with CD require at least one surgical intervention within ten years of diagnosis, with 80% of these patients developing new lesions following surgery.1 Emerging evidence highlights that the therapeutic efficacy of mesenchymal stem cells (MSCs) is predominantly attributed to the extracellular vesicles (EVs) they secrete.2 These nanovesicles have gained recognition as critical mediators of anti-inflammatory responses and tissue repair. This research seeks to develop MSC-derived EV-based acellular therapy to regulate the immune response, heal the intestinal mucosa, restore gut microbiota balance, and ultimately reduce disease severity and recurrence.

Methods

Inclusion criteria for selecting healthy donors for obtaining EVs derived from subcutaneous adipose tissue MSCs include sex, age <40 years, and BMI <25 kg/m². Exclusion criteria include smokers, alcohol consumers, and patients with cancer or other chronic conditions. EV isolation was performed using size-exclusion chromatography, and characterisation was conducted via flow cytometry and electron microscopy (Cryo-TEM). To assess the therapeutic properties of the EVs, biopsies of inflamed perilesional adipose tissue and intestinal mucosa from CD patients were treated with a dose of 150 µg/mL for 20 hours. Finally, the gene expression profile of pro-inflammatory markers (TNFα, IL1B, IL6, and CCL2) and anti-inflammatory markers (IL10) was analysed by qPCR.

Results

The isolation and characterization of EVs were successfully demonstrated, along with their functional efficacy in both adipose and intestinal explant models. Explants treatment with EVs resulted in a significant reduction in pro-inflammatory cytokines, with IL-1β and TNF-α expression decreased by up to 200-fold compared to untreated explants, while also promoting a notable increase in the anti-inflammatory marker IL-10.

Conclusion

Adipose tissue-derived EVs could regulate inflammation and promote the regeneration of the intestinal barrier, thus reducing the severity of CD and lowering the risk of post-surgical recurrence.

References

1Golovics PA, Mandel MD, Lovasz BD, Lakatos PL. Inflammatory bowel disease course in Crohn’s disease: Is the natural history changing? World J Gastroenterol. 2014;20(12):3198-3207.

2.Zhou Y, Yamamoto Y, Xiao Z, Ochiya T. The immunomodulatory functions of mesenchymal stromal/stem cells mediated via paracrine activity. J Clin Med. 2019;8(1)
P0126 Single-cell analysis reveals enrichment of mucosal Th17-polarized T cells in Acute Severe Ulcerative Colitis
Authors: Bourgonje A R , Meringer H , Roudko V , Jha D , Canales-Herrerias P , Livanos A E , Cossarini F , Tankelevich M , D'Souza D , Nie K , Kim-Schulze S , Petralia F , Kayal M , Colombel J F , Mehandru S
Published: January 2025

Background

Acute severe ulcerative colitis (ASUC), a severe and potentially life-threatening manifestation of ulcerative colitis (UC) affects up to 25% of patients over their lifetime and requires urgent medical intervention. T cell subsets and myeloid cells play a critical role in the pathogenesis of mucosal inflammation. In this study, we aimed to perform an unbiased analysis of mucosal immune cell populations in patients with ASUC and to identify ASUC-specific cell type alterations.

Methods

Ten adult ASUC patients (defined by the Truelove and Witts criteria), 7 patients with active UC (not fulfilling ASUC criteria), and 5 normal volunteers (NV) were recruited (Fig. 1A). Intestinal mononuclear cells were isolated from endoscopically obtained intestinal biopsies and analyzed by single-cell RNA-sequencing (scRNA-seq). Linear mixed-effects models were applied to compare lineage-normalized cell type fractions across groups, while adjusting for relevant covariates including age, sex, and body mass index.

Results

Unsupervised clustering of 127,795 cells, expressing 38,224 unique genes, identified 45 distinct cell types (Fig. 1B). Among the salient findings specific to lymphoid cells, re-clustering of the T cell/NK cell compartment identified 9 cell subsets (Fig. 1C-D). Notably, TH17 cells (RORA, CTSH, CXCR6, and CCL20) and cycling TH17 cells (MKI67, PCLAF, SMC4, NUSAP1, and RRM2) were significantly enriched in patients with ASUC compared to those with active UC (adjusted P=0.019 and P=0.027, respectively) and NV (adjusted P<0.001 for both) (Fig. 1E-F). Among myeloid cells, of the 11 delineated subsets, patients with ASUC showed a significant reduction in lineage-normalized fractions of type 2 conventional dendritic cells (cDC2s) (CD1C, FCER1A) relative to both active UC and NV (adjusted P=0.017 for both) (Fig. 1G). Additionally, a cluster of CD14+ monocytes (CD14, FCN1, and LYZ) was significantly elevated in patients with ASUC compared to NV (adjusted P=0.032).

Conclusion

Elevated TH17 cell frequencies distinguish ASUC from UC. While further investigations, including protein-level validation of transcriptomic data and an assessment of the impact of cellular perturbation on treatment outcome, are ongoing, these findings shed light on a potentially underappreciated role for TH17-polarized T cells in ASUC.
P0127 Fistula-derived fibroblasts express genes associated with a dysfunctional wound healing process
Authors: Van Os B , Su J , Bruckner R , Zhou Z , van der Meulen-de Jong A , Hawinkels L , Barnhoorn M
Published: January 2025

Background

Approximately a third of patients with Crohn’s Disease will develop perianal fistulas. Perianal fistulas greatly impact the quality of life by causing pain, discharge and faecal incontinence. Current drug treatments in combination with surgery are not very effective in curing fistulas, and only a low number of patients with complex fistulas reach complete remission. One of the main reasons for the lack of treatment options is our limited understanding of the formation of these fistulas. In this study we investigated the role of fibroblasts in the formation of perianal fistulas by evaluating their expression profiles compared to healthy colonic fibroblasts.

Methods

To gain more insight into the characteristics of fistula fibroblasts, fibroblasts were isolated from fistula scrapings derived during drainage surgery from both CD (n = 8) and non-CD patients (n = 6) and from healthy colon tissue of non-IBD patients (n = 18). After expanding the fibroblasts in vitro, they were subjected to mRNA sequencing. After sequence alignment, gene set enrichment and pathway activation analysis was performed. Differentially regulated processes were further investigated in vitro to confirm these results.

Results

Remarkably, fibroblasts isolated from CD and non-CD fistulas share a similar transcriptional profile, with only 25 differentially expressed genes (DEGs) found between them. Comparing fistula derived fibroblasts with healthy colon fibroblasts revealed 3606 DEGs. Pathway analysis revealed increased extracellular matrix remodelling and proliferation but reduced inflammation in fistula fibroblasts compared to healthy colon. Strikingly, although collagen type 1 production was increased, myofibroblast-associated genes such as ACTA2, LRRC17, LRRC32, NKX2-3 and RSPO3 were decreased in fistula fibroblasts. We confirmed these observations in vitro.

Conclusion

Currently, CD and non-CD fistulas are treated differently. Whereas surgical removal is the main treatment for non-CD fistulas, CD fistulas are primarily treated with the combination of surgery and medication targeting inflammation. However, our results suggest that at least the fibroblasts isolated these fistulas are very much similar on a transcriptional level. We found these fibroblasts display a signature that seems to point at aberrant wound-healing. Further research in 3D co-cultures, comparing the role of fistula and healthy fibroblasts, will elucidate the role of these cells in the formation of fistulas.
P0128 IL-27 signalling, but not IL-1B, is functionally relevant in the pathophysiology of immune checkpoint inhibitor induced colitis
Authors: Ibraheim H , Cozzetto D , Goldin R , Kudo H , Zhou X , Lo J W , Powell N
Published: January 2025

Background

Immune checkpoint inhibitors (CPI) augment T-cell responses to tumour cells and have transformed treatment paradigms in cancer. However, CPI frequently trigger autoimmune like events, including potentially life-threatening colitis. Current immunosuppressive treatments for CPI- colitis (CPI-c) incur significant side effects and may even reduce CPI efficacy. New treatments are urgently needed.

Methods

Changes at gene level, pathway level (GSEA) and predicted upstream regulators (Ingenuity Pathway Analysis-IPA), and enrichment scores (Gene Set Variation Analysis-GSVA) were investigated in colonic biopsies (RNA-sequencing) from patients with CPI-c (n=12), CPI (no colitis) (n=6), healthy controls (n=13) and ulcerative colitis (UC) (n=12). Potentially relevant pathways were functionally tested in preclinical disease models. CPI-c was induced in mice transplanted with a pro-inflammatory microbiota and administered anti-PD-1 and anti-CTLA-4 intraperitoneally (i.p). Anti-IL1R (anakinra), MCC950 (inflammasome inhibitor) and anti-IL27 were administered i.p. Colonic leukocytes were extracted to interrogate myeloid and lymphocyte compartments by flow cytometry. Whole colon segments were processed for histology.

Results

In CPI-c patients, IL-1B was the top differentially expressed cytokine at gene level with interleukin-1 signalling significantly enriched at pathway level. IPA also identified IL-1B and IL-27 as predicted upstream activators. Interestingly, in vivo blockade of IL-1B, or the inflammasome (MCC950), had no impact on colitis.

Administration of IL-27-neutralising antibodies at the onset of CPI-colitis in mice, reduced disease susceptibility and improved histological colitis scores. This was associated with decreased infiltration of colonic lamina propria neutrophils, Ly6C+ inflammatory monocytes and the number of CD8+ IFNg+ T-cells. Conversely, administering neutralising anti-IL27 during established CPI-colitis did not ameliorate disease and was linked to an enhanced CD4+ effector response characterised by heightened production of TNFa and IFNg.

Further analysis of colonic biopsies of CPI-c patients confirmed enrichment of IL27-responsive genes which we localised to CD8+ resident memory and CD8+ cytotoxic cells in single cell RNA-seq analyses. Additionally, serum IL-27 levels were significantly elevated in CPI-c patients.

Conclusion

IL-1B signalling was shown to be dispensable to disease in mice. However, IL-27 signalling has a context specific role, serving as a functionally important activator of innate immune responses, while inhibiting later phases of acquired immunity via suppressing cytotoxic lymphocyte responses. Targeting IL-27 presents an attractive therapeutic approach for CPI-c given its potential tumourogenic effects.
P0131 Characterisation of a human multi-organ-on-chip model to unravel gut-blood-brain communication
Authors: Maes L , Van Welden S , Vandenbroucke R , Laukens D ,   IBD Research Unit
Published: January 2025

Background

Persistent fatigue is a frequently reported symptom among patients with Inflammatory Bowel Disease (IBD), significantly diminishing quality of life. Due to the lack of understanding how IBD-associated fatigue originates, currently available treatments do not efficiently tackle this symptom. Developing targeted therapies requires tools to unravel the mechanisms behind central nervous system dysfunction in response to chronic intestinal inflammation. We are therefore establishing and characterising a human multi-organ-on-chip model to recapitulate key physiological elements of gut-brain communication via the systemic circulation.

Methods

In our model, a gut-blood barrier, comprised of Caco-2 (enterocytes) and HT29-MTX cells (goblet cells), is connected to a blood-cerebrospinal fluid (CSF) barrier, composed of HIBCPP and iHCPEnC cells (choroid plexus epithelial and endothelial cells respectively), and to a brain compartment (SH-SY5Y, neurons) via recirculation of cell culture medium. Gut inflammation was mimicked by the addition of the pro-inflammatory cytokines TNFα and IFNγ to the intestinal epithelial cells. Barrier integrity was measured by transepithelial/endothelial resistance (TEER) and by paracellular permeability assays with 4 kDa FITC-dextran. In addition, we performed a bead-based immunoassay to measure secreted IL-8 levels and RT-qPCR to analyse differential gene expression.

Results

The gut-blood barrier under low perfusion (3,6*10-4 mPa) exhibited increased expression of differentiation marker genes compared to static culture, such as LYZ, SOX9 and CYP3A4, indicating an improvement of cell differentiation. Moreover, low perfusion induced the formation of 3D villi-like structures, mucus production and the reduction of barrier strength towards more physiological relevant levels (TEER: 81,6±44,8 Ω.cm2; Papp: 1.10-5±4.10-6 cm/s). The gut-blood barrier was responsive to the addition of TNFα and IFNγ and high IL-8 levels could be measured in the circulatory blood flow. The barrier properties of the blood-CSF barrier under low perfusion (endothelial: 2,52 mPa, epithelial: 4,6*10-4 mPa) were not significantly different from a static setup, but less variance was observed in TEER. Neurons differentiated with BDNF and retinoic acid in our model displayed long projections and expressed mature neuronal marker genes (SYP, SNAP25, TUBB3).

Conclusion

We have successfully established and characterised each organ compartment of the gut-blood-brain model. Moreover, we have proven that low perfusion improves the physiological relevance of our model, especially of the gut-blood barrier. We are now exploring phenotypical and transcriptional alterations across the blood-CSF barrier and the brain compartment when gut inflammation is triggered.
P0134 Human umbilical cord-derived mesenchymal stem cells promote intestinal mucosal repair by activating fibroblasts-epithelial interactions
Authors: Huang Z , Yang H , Chao K , Gao X
Published: January 2025

Background

Mesenchymal stem cells (MSC) have shown promising potential for treating inflammatory bowel disease (IBD). The interactions between fibroblasts and epithelial cells play a crucial role in initiating the intestinal mucosal repair process. This study explored whether MSC therapy promoted intestinal mucosal repair via regulating fibroblast-epithelial interactions.

Methods

MSCs were isolated from human umbilical cord tissue. MSCs (2x106 cells/mouse) were injected intraperitoneally into mice treated with dextran sulfate sodium (DSS). The rate of mucosal proliferation, as indicated by Ki67 expression, was analyzed using immunofluorescence techniques. The co-culture of CCD-18co with Caco-2 was used to model the epithelial-fibroblast interaction. Wound healing assays and EdU assays were used to assess proliferative response, and RNA-sequencing to explore the mechanism.

Results

The MSC treatment reduced intestinal ulcer size in colitic mice, which was accompanied by an increase in the proliferation of epithelial cells and fibroblasts. Conditioned medium (CM) from MSCs induced a pro-wound-healing transcriptional program in CCD-18co, resulting in increased EdU-positive rates and enhanced cell migration. In the co-culture model, MSC-CM-treated CCD-18co enhanced Caco-2 proliferation by increasing EdU-positive rates and promoted wound healing through improved cell migration. Transcriptional analyses revealed that several processes associated with cell proliferation and the WNT signaling pathway were activated.

Conclusion

MSC treatment enhanced fibroblast-epithelial interactions to promote the intestinal mucosal repair.
P0139 Dynamics of the species structure of the gut microbial community in patients with ulcerative colitis
Authors: Tarasova G , Zubova A
Published: January 2025

Background

The potential role of microbiome in the pathogenesis, progression and treatment of ulcerative colitis (UC) is becoming a subject of considerable interest and research. The study of the microbial ecosystem of the colon in patients with UC in different stages of the disease provides an opportunity to identify key taxon-biomarkers for non-invasive monitoring of the severity of UC and the effectiveness of treatment.

Methods

The study included 21 patients (ages from 20 to 41, 13 men and 8 women) in the relapse stage of UC - left-sided UC with mild to moderate activity (Truelov-Witts scale, Mayo index); and 20 patients (ages from 26 to 52, 10 men and 10 women) in the stage of clinical endoscopic remission. The study of the coprofiltrate microbiota was carried out by NGS sequencing of the structure of the bacterial 16S ribosomal RNA gene using the Illumina MiSeq platform.

Results

Non-zero values of representation were found in 273 species in patients of the studied groups, however, differences in the presence of microorganisms were statistically significant only for 10 species presented in Table 1.

A higher level of Pelomonas_aquatica, Granulicatella_adiacens / para-adiacens, Gemella_morbillorum, Escherichia/Shigella_coli, Enterococcus_azikeevi / casseliflavus / durans / faecalis / faecium / hirae / lactis / mundtii / raffinosus / ratti / rivorum / thailandicus / villorum, Solobacterium_moorei, Streptococcus_australis / parasanguinis and lower levels of Roseburia_inulinivorans, Ruminococcus_bicirculans, Ruminococcus_callidus were found in the relapse group.

Conclusion

The differences identified in the taxonomic structure at the species level among patients in the relapse and clinical-endoscopic remission stages of UC determine the potential of the colon microbiota in non-invasive assessment of the severity of the disease.
P0146 Phosphoglycerate dehydrogenase plays a vital role in ER-stress-related intestinal inflammation
Authors: Yang G , Xiang H , Welz L , Bhardwaj A , Treitz C , Sima K , Koncina E , Kim N M , Kugler J , Tran F , Nikolaus S , Sievers L K , Liu Z , He Q , Schreiber S , Rosenstiel P , Aden K
Published: January 2025

Background

Inflammatory Bowel Disease (IBD) is characterized by chronic inflammation and metabolic dysregulation in the intestinal epithelium. Serine, a non-essential amino acid synthesized via the rate-limiting enzyme phosphoglycerate dehydrogenase (PHGDH) maintains cellular redox balance. Serine metabolism is upregulated in cancer and immune cells, supporting survival and growth. Our previous story shows ER stress-mediated rewiring of serine metabolism is a key molecular feature in IBD. However, the mechanism of how it influences ER stress in IBD remains unclear.

Methods

To explore the molecular regulation of de novo serine synthesis via PHGDH, we used murine intestinal epithelial cells (Mode-K cells) and murine intestinal organoids. WB and IHC were performed in vivo to analyze ER stress and PHGDH on DSS colitis model. Mode-K cells were subjected to serine starvation and co-treated with LPS. Proinflammatory cytokines (Cxcl1, Tnfα, Il6) were evaluated by qPCR and ELISA. Metabolic supplement formate and hypoxanthine were tested to mitigate hyperinflammation. Total starvation was assessed by using serine deprivation medium and PHGDH inhibitor BI-4916. Tunicamycin, NAC, Y27632, and rapamycin were used to study the biofunction under ER stress effect by using qPCR, WB, and IF. Cell viability was performed by CellTiter-Glo® 2.0 Cell Viability Assay. RNA sequencing and proteomic analyses were performed to investigate molecular mechanisms. Clinical analysis of PHGDH was assessed in two cross-sectional IBD cohorts correlating PHGDH expression with endoscopic or clinical disease activity.

Results

We show elevated ER stress and de novo serine synthesis in DSS colitis model. Using total starvation to completely block serine pathway, we observed increased ER stress which subsequently leads to dramatically enhanced inflammation signature under LPS treatment. ER stress induced by total starvation also leads to mitochondria dysfunction as assessed by Seahorse and JC-1 assay. By using cell viability assay and IF, we show ER stress induces AIF-mediated anoikis. Autophagy induced by ROS under ER stress conditions can protect cells from anoikis and inflammation. Combining RNAseq and proteomic analysis, we identified CEBPB as the critical regulator in mediating cellular anoikis and inflammation during ER stress which were further confirmed by IF and qPCR. Finally, we observed unregulated PHGDH in IBD patients showing a strong association between serine metabolism and IBD severity.

Conclusion

In this study, we demonstrate the critical roles of PHGDH and Serine metabolism in regulating cellular inflammatory responses under ER stress. Notably, we reveal a protective role of ROS in ER stress and inflammation, providing new insights into ER stress and IBD severity.
P0147 Regulation of Inflammatory Pathways by miR-338-3p and miR-378a-3p: A Novel Approach for Crohn’s Disease Treatment
Authors: Choi C H , Moon J M , Kim K U , Lim E , Hwang H W , Kyuwon K , Seo J , Shin S Y , Lim H , Min H
Published: January 2025

Background

MicroRNAs (miRNAs), key regulators of gene expression, are promising biomarkers and therapeutic targets in inflammatory bowel disease (IBD) due to their roles in immune response and tissue integrity. This study explores miRNA roles in IBD, focusing on Crohn's disease (CD) pathogenesis and potential therapies. We analyzed epithelial cell-derived miRNA expression in CD patients versus healthy controls and investigated how these miRNAs modulate inflammation in experimental IBD models.

Methods

Terminal ileum samples were prospectively collected via ileocolonoscopy from non-IBD healthy controls (n=26), CD patients in remission (n=34), and active CD patients (n=42). Clinical remission was defined as a Crohn’s Disease Activity Index score below 150 with endoscopic mucosal healing. For active CD cases, samples were taken from inflamed and non-inflamed regions. miRNA levels were quantified by small-RNA sequencing and validated by qRT-PCR. Based on these results, specific miRNAs were selected, and single-stranded mimics were administered in a 2.5% DSS-induced colitis model to evaluate their effects. Histological grading, immunohistochemistry, flow cytometry, and microbiome analysis using mouse fecal samples were performed.

Results

Small-RNA sequencing revealed distinct miRNA expression patterns in CD patients, both in remission and in active states (including inflamed and non-inflamed tissues), compared to healthy controls. qRT-PCR validation showed significantly reduced expression of miR-141-3p, miR-338-3p, miR-378a-3p, and miR-200b-3p, along with significantly increased levels of miR-125b-5p, miR-29b-3p, and miR-155-5p in inflamed tissue of active CD patients. Receiver operating characteristic curve indicated diagnostic potential for these miRNAs in relation to disease activity and endoscopic inflammation. In a DSS-induced colitis mouse model, miR-338-3p and miR-378a-3p showed anti-inflammatory effects, reducing body weight loss, colon inflammation, and immune cell markers. Target analyses identified MACC1 and IL-33 as key mRNA targets. Immunohistochemistry indicated an IL-33 pathway-dependent anti-inflammatory response via miR-378-3p, while miR-338-3p appeared to regulate gut inflammation by targeting MACC1 gene expression. In vitro analyses revealed that IL-33 activates MACC1, a process also reduced by miR-378a-3p treatment. Fecal microbiome analysis identified several bacterial species including Akkermansia muciniphilia and certain Bacteroides, associated with beneficial effects in the groups treated with both miRNA mimics.

Conclusion

This study identifies miR-338-3p and miR-378a-3p as modulators of inflammation in an IL-33-dependent manner in CD patients, underscoring their potential as promising targets for treatment of CD.
P0148 Studying the role of adipose tissue in intestinal inflammation using acquired generalized lipodystrophy
Authors: Letizia M , Omar T , Weidner P , Siegmund B , Weidinger C
Published: January 2025

Background

Crohn’s disease (CD) is associated with creeping fat, characterized by hyperplasia of mesenteric adipocytes and increased secretion of adipokines, including leptin (1). However, limited models currently exist to study the effect of adipose tissue on intestinal inflammation. Therefore, acquired generalized lipodystrophy and Crohn’s disease (AGLCD), a rare metabolic disorder characterized by the complete loss of fat tissue and intestinal inflammation, could provide a unique model to further elucidate the contribution of adipose tissue to the pathogenesis of inflammatory bowel disease.

Methods

To decipher the role of adipose tissue, we characterized the immune cell compartment in a patient with AGLCD using mass cytometry, whole exome sequencing and single cell sequencing. Samples collected for the analysis include lamina propria mononuclear cells (LPMCs) isolated from ileum biopsies and peripheral blood mononuclear cells (PBMCs) from blood obtained from 6 CD patients, one AGLCD patient, and 4 non-inflamed controls.

Results

Using mass cytometry, we were able to identify 17 intestinal immune cell clusters in the LPMCs. Namely, there was a higher frequency of CD8+ CD45RO+ T cells, CD11c+ CD11b+ myeloid cells and CD19+ CCR7+ B cells in the AGLCD sample compared to the fat-proficient CD controls. In addition, single cell sequencing of CD3+ T cells isolated from the blood of the AGLCD patient showed an expansion of CD8+ and CD4+ effector memory T cells compared to a healthy donor, suggesting that adipose tissue plays a role in T cell homeostasis in CD. To confirm that leptin plays a role in the regulation of T cells, we analyzed PBMCs from the AGLCD patient pre and post treatment with recombinant leptin. An increase in the expression of IL-17 producing CD4 T cells were present after treatment with leptin. To further investigate the expansion of CD8+ T cells, analysis of whole exome sequencing of the AGLCD showed a mutation in the NRAS gene, an essential regulator of hematopoietic development. The mutation was confirmed only in CD8 and CD4 T cells in the blood and intestinal biopsies of the AGLCD patient using Sanger sequencing.

Conclusion

These observations demonstrate that AGLCD is a unique model that can be used to further study the effects of adipose tissue in intestinal inflammation.

References

(1) Coffey JC, O'Leary DP. The mesentery: structure, function, and role in disease. Lancet Gastroenterol Hepatol 2016;1:238-247.
P0149 Deep functional analysis of a defined sulphide-reducing diet (4-SURE diet) as a novel therapy for ulcerative colitis (UC) demonstrates all specific luminal micro environmental objectives were achieved
Authors: Day A , Slater R , Marcelino V , Wheeler R , Young R , Maddigan N , Forster S , Costello S , Uylaki W , Probert C , Andrews J , Yao C , Gibson P , Bryant R
Published: January 2025

Background

As a dietary approach to reducing inflammation in ulcerative colitis (UC), the 4-SURE diet was designed to correct pathogenic alterations of excessive protein fermentation and hydrogen sulphide (H2S) production in the distal colon. Specific dietary objectives were achieved (Day et al,J Nutr 2022;152:1690) but it is uncertain whether mechanistic objectives could be achieved with 8 weeks of diet. Therefore, we aimed to perform a deep functional analysis (microbial and metabolomic) of the faeces.

Methods

Faecal samples of 28 adults with mild-moderately active UC were collected at week 0 and 8 of diet intervention, subsampled and stored at−80 °C. Shotgun metagenomic sequencing was used to identify genes involved in H2S metabolism. Metagenomic reads were trimmed, reads mapping to the human genome removed. Gene identification was performed using Diamond v2.1.9 for alignment against a database of genes involved in global sulphur cycling. Gas-chromatography mass-spectrometry was used to characterise volatile organic compounds (VOCs) with specific analysis of products of protein fermentation. Microbiota capacity to produce H2S was assessed by anaerobic incubation of homogenates followed by spectrophotometric determination of total H2S production.

Results

Majority of microbiome members belonged to the Bacteroidota and Bacillota phyla, with no significant difference in bacterial diversity determined between time points (p=0.16). However, using a Random Forest Classifier, known H2S producers, Odoribacter and Peptostreptococcaceae, were identified as the most important taxa in discriminating between pre and post-diet samples, with abundances markedly lower after diet intervention. A shift in microbiota metagenomic profiles putatively involved in H2S metabolism was identified post-diet, with differences (p<0.05, Wilcoxon signed-rank with Benjamini-Hochberg correction) in 12 of 23 analysed genes involved in H2S cycling determined, including iscS, cysK (Fig), metC, luxS, asrC and sseA. 173 faecal VOCs were identified across all samples. Indole (a specific marker of protein fermentation) decreased from 0.42 [-0.25,0.61] at week 0 to -0.28 [-0.77,0.48] at week 8 (p=0.007, FDR correction; Fig). Faecal H2S reduced from median 2.61 (interquartile range 2–4.96)µmol to 1.41 (0.81-2.27)µmol of H2S/g stool (wet weight)(p=0.002)(Fig).

Conclusion

Deep functional analysis of the faeces demonstrated the micro-environmental objectives of the 4-SURE diet successfully reduced protein fermentation, changed microbial community sulphur-metabolic gene profile and reduced H2S production ex vivo. Applying functional analysis to a diet study is novel, highlighting exemplar framework for including biomarkers of pathogenic relevance in analysis of therapeutic diets.
P0153 The role of the proglucagon-derived peptide GLP-2 in resection-triggered growth and barrier response in mice
Authors: Mohebali N , Witte M , Berlin P , Lamprecht G , Reiner J
Published: January 2025

Background

Ulcerative colitis (UC) and Crohn’s disease (CD) of the colon may cause significant malnutrition by “inflammation-dependent”, and “inflammation-independent” mechanisms. Glucagon-like peptide 2 (GLP-2) is secreted by ileocolonic L cells in response to nutrient intake and promotes small intestinal barrier function. GLP-2 secretion is enhanced after small bowel resection. We hypothesized that loss of GLP-2 secretion by a) genetic ablation, and b) inflammatory ablation through severe DSS-colitis, impairs small intestinal barrier dysfunction in the mouse mid small bowel resection model.

Methods

The impact of GLP-2 loss on small intestinal barrier function was evaluated using two models. In the first model, gcg knockout (KO) and wild-type (WT) mice underwent 50% mid-small bowel resection (MSBR). In the second model, male C57BL/6J mice were divided into the following groups: non-operated controls +/- DSS, MSBR alone, MSBR with dextran sulfate sodium (MSBR+DSS), and MSBR with DSS plus teduglutide (GLP-2 analogue). Body weight changes, mucosal height, tight junction mRNA expression, and FITC-4kDa-dextran flux were assessed using Ussing chambers.

Results

The gcg-WT mice did not develop diarrhea during the experimental period, while gcg-KO mice showed significantly increased stool water content two days post-surgery (p < 0.05). Although no other major clinical differences were observed between gcg-WT and KO mice after MSBR, gcg-KO mice exhibited reduced mucosal height and increased FITC-dextran flux, indicating impaired barrier function (p < 0.05). DSS-colitis was less severe in the MSBR-model, compared to non-operated DSS-colitis. However, in the inflammatory model, MSBR+DSS mice experienced significant body weight loss, colon shortening, all of which were significantly mitigated by teduglutide treatment. Teduglutide also improved villus length (p < 0.001) and reduced FITC-dextran permeability (p < 0.05). Claudin-2 expression, which was markedly elevated in MSBR+DSS mice, was significantly reduced with teduglutide treatment.

Conclusion

Our findings demonstrate that DSS-induced colitis is less severe in the 50% MSBR model. Furthermore, genetic knockout of gcg, i.e. complete ablation of GLP-2 secretion capacity, diminishes resection-triggered barrier- and growth response, while DSS-colitis, i.e. incomplete ablation of GLP-2 secretion capacity, diminishes resection-triggered barrier-, but not growth response. Thus, GLP-2-deficiency with small intestinal barrier dysfunction may be an inflammation-independent mechanism ultimately contributing to malnutrition in severe colitis.
P0159 Effects of Transcranial Direct Current Stimulation (tDCS) on enteric nervous system (ENS) gene expression and intestinal barrier in chronic DSS colitis
Authors: Wiese J J , Manna S , Bubeck M , Patankar J , Siegmund B , Prüß M S , Hering N , Schumann M
Published: January 2025

Background

Chronic visceral pain is a clinical challenge in >50% of Inflammatory Bowel Disease (IBD) cases. A randomized controlled trial (RCT) revealed that transcranial direct current stimulation (tDCS) can modulate brain activity and effectively alleviate chronic abdominal pain associated with IBD. To elucidate the underlying mechanism of tDCS, we implemented this technique in a mouse model of inflammatory, chronic DSS colitis.

Methods

The tDCS protocol (10 stimulations) was deduced from human tDCS studies. We administered an anodal direct current (0.5 mA, 20 min) to both primary motor cortices over two separate periods (5 consecutive days of stimulation per period separated by a two-day break). The study was controlled by a sham-tDCS group.

For determining mucosal barrier integrity murine intestinal mucosa was mounted to Ussing chambers. Histology was done (H&E staining of FFPE colon sections). Murine activity after tDCS was assessed by mouse tracking in home cages (RFID-chipped mice). Myenteric plexus (MP) cells from the murine terminal ileum, were isolated and cultivated for 10 days. Immunostaining and bulk-RNA sequencing (NovaSeq Xplus system, read length 150 bp, read depth 30 M copies/run) were performed.

Results

DSS caused an inflammation-driven thickening of the colon wall, resulting in an increased transmural resistance. This effect was ameliorated by tDCS (mean±SEM DSS+sham-tDCS: 72.2±8.2 Ω·cm²; DSS+tDCS: 48.1±2.1 Ω·cm²; MWU: p=0.007). Induction of a DSS colitis was associated with a reduced murine activity, which was partially recovered by tDCS (mean±SEM DSS+sham-tDCS: 70.7±15.2 rel.dist.U/day; DSS+tDCS: 205.9 ± 40.4 rel.dist.U/day; MWU: p=0.029).

Neuronal cell identity was confirmed in isolated murine MP cells by immunostaining (b3-tubulin, S100B). Bulk-RNAseq significantly separated tDCS- from sham-tDCS treated DSS colitis mice as revealed by PCA. Consecutively, 739 differentially expressed genes were identified in tDCS-treated colitis mice compared to sham-tDCS-treated DSS colitic mice (622 upregulated, 117 downregulated genes; log2FC≥1.5, p-adj<0.05). KEGG and GO pathway analysis included neuroactive ligand-receptor interaction (p=7.94·10-3), gated channel activity (p=2.4·10-7), substrate-specific channel activity (p=1.99·10-7), leukocyte migration (p=3.99·10-7).

Conclusion

In colitis, tDCS has the capacity to modulate activity of DSS-treated mice and alters gene expression in MP cells. This reveals another layer of evidence for the impact of central pathways on the brain-gut-axis.

References

Neeb et al., Brain Stimulation, 2019
P0167 Circular extrachromosomal DNA (eccDNA): a potential diagnostic and prognostic tool to be introduced into the clinical practice of Inflammatory Bowel Disease
Authors: Foscarini E , Emoli V , Petito V , Di Vincenzo F , Masi L , Troisi S , Pane C , Piazzesi A , Turchini L , Pugliese D , Laterza L , Lopetuso L R , Gerovska D , Arauzo M , Putignani L , Regenberg B , Gasbarrini A , Scaldaferri F
Published: January 2025

Background

The diagnosis of Inflammatory Bowel Disease, which continues to increase year by year, is complex; these disorders can be confirmed by investigations such as fecal calprotectin, endoscopy, histology of biopsies, and CT scan; despite this, there is a lack of highly specific and minimally invasive techniques that allow not only diagnosis but also determination of the severity of the disease and its evolution. Given these challenges, the search for reliable and specific predictors that can direct the clinician toward personalized patient care is very important. In this context, can help extrachromosomal circular DNA (eccDNA) that are eukaryotic nuclear circle DNA molecules, independent from chromosomes, with a length ranging from a few hundred to a few thousand base pairs. In addition to its already known role in cancer, in IBD increased levels of eccDNA have been found compared with healthy controls. Circular DNA could represent a potential biomarker that can be isolated from peripheral blood or intestinal mucosa and is useful for making early diagnoses and assessing disease status.

Methods

We examined peripheral blood, collected by blood sampling . "Technical Tests" were performed in order to determine the right procedure for storing blood samples and optimize the results obtained in terms of eccDNA extraction . The samples will be divided into two groups, for the first group the procedure is to centrifuge the blood at 2000g for 15 minutes at different timepoints with or without storing the plasma at 4° C; for the second group the sample is centrifuged immediately after blood collection and the plasma is stored in the refrigerator at different timepoints [Table 1]. Then for both groups we proceed with eccDNA extraction by the "Circle-Seq" analysis.

Results

No significant differences are shown between the two groups in terms of the amount of eccDNA [Figure 1]. In particular, the non-immediate centrifugation and separation of plasma from whole blood does not negatively affect the extraction yield, highlighting that degradative factors are not present in whole blood. In addition, refrigerated storage for a longer period of time is a positive factor for eccDNA concentration, probably because in this way circular DNA found within microvesicles has time to be extruded, increasing its concentration in plasma.

Conclusion

Circulating eccDNAs could represent potential biomarkers to be detected in plasma by liquid biopsy, as the method is minimally invasive and easily reproducible in analytical laboratories, becoming a diagnostic and prognostic tool to be brought into clinical practice.

References

Fundings: European Union’s Horizon 2020 Research and Innovation Programme (899417)