bluuebunny/crossref_metadata_2025_split huggingface dataset:
P030 Distinct molecular profiles between idiopathic cryptoglandular and Crohn-related perianal fistulas
Authors: Verstockt B , Verstockt S , Bislenghi G , Sabino J , Ferrante M , D’Hoore A , Vermeire S 
Published: January 2022

Abstract

Background
Perianal fistula can originate idiopathically (cryptoglandular fistula) or can be a representation of underlying Crohn’s disease (CD). In case of idiopathic fistula, the cryptoglandular theory suggests their development from the anal glands. In contrast, the pathophysiology of CD-related fistula is very poorly understood. Hence, we molecularly characterised and compared the fistula tract in both conditions.

Methods
We collected surgical biopsies from the fistula tract in 70 CD patients with active draining perianal fistula and in 12 patients with cryptoglandular non-CD related, active draining fistula, all requiring surgical examination under anaesthesia. RNA was sequenced using Illumina HiSeq4000, and these data were analysed through differential gene expression analysis (DESeq2). A false discovery rate (FDR) of 0.05 and a |log 2-fold change (log2FC)| &gt;1 was considered significant. Pathway analysis was performed using IPA (Qiagen). In addition, cellular deconvolution methods (xCell) were applied to study the cellular composition.

Results
Gene expression analysis identified 1087 genes being differentially expressed between CD-related and cryptoglandular fistula (716 up, 371 down). Top differentially expressed genes encode proteins implicated in IBD pathogenesis including CARD18 (log2FC= 23.2, p=3.9E-21), BATF2 (log2FC=2.8, p=1.8E-8), ETV7 (log2FC=2.1, p=2.0E-7), DSG1 (log2FC=7.8, p=2.7E-6) and IL22RA1 (log2FC=5.3, p=4.6E-6). Additional pathway analysis highlighted various proinflammatory processes in CD fistula (as compared to cryptoglandular fistula), including upregulation of antigen presentation and Th1/Th2 activation (p&lt;1.0E-9). Intriguingly, CD fistula showed a significant downregulation of wound healing signaling pathways (p=6.5E-6), emphasising the refractory character of this debilitating condition. Upstream analyses showed an increased activation of top regulators IFNγ, TNF, LPS and STAT1 (p&lt;1.0E-24). Cellular deconvolution identified significant differences between both fistula types, with a predominance of inflammatory cells in CD including Th1 cells, dendritic cells, naïve CD4 T cells, memory B cells and central memory CD8 and CD4 T cells (p&lt;5.0E-2). In contrast, cryptoglandular fistula were characterised by a significant enrichment of myocytes, smooth muscle cells and neurons (p&lt;3.0E-3). Presence of neutrophils, wound healing and pro-inflammatory macrophages did not differ between both fistula types.

Conclusion
CD fistulas have a strong proinflammatory fingerprint, but seem to have lower wound healing capacity as compared to cryptoglandular fistula. An aggressive medical and surgical treatment is therefore required, including the search for novel, powerful anti-inflammatory and wound healing compounds.
P037 MicroRNA-31 and microRNA-200 family reflect disease activity in Crohn’s disease: results from the BIOMIR study
Authors: Cristina T , Mateescu B , Mitroi A , Cozaru G , Brinzan C , State M , Dumitru A , Rafti R , Dina E , Alexandrescu L , Dumitru E 
Published: January 2022

Abstract

Background
Accurate assessment of disease activity in inflammatory bowel disease (IBD) is of paramount importance in decisions regarding treatment strategies. To apply a treat-to-target strategy, a tight assessment of activity is mandatory. MicroRNAs (miRNAs) are small, non-coding short (ribonucleic acid) RNAs that have a role in the regulation of genes and protein expression. MiRNAs have altered expression in multiple autoimmune disorders including IBD. The aim of the study was to assess the tissue and circulating miR-31, miR-200b, and miR-200c expression levels as potential biomarkers for intestinal disease activity in patients with Crohn’s disease (CD).

Methods
The study included 45 patients with histopathologically confirmed CD and active disease (defined as fecal calprotectin (fCal) &gt; 50 ug/g and Short Endoscopic Score of Crohn’s Disease (SES-CD) &gt; 3), and 21 subjects as controls for the validation cohort. Demographic and clinical data, biomarkers (fCal), endoscopy data, the expression levels of miR-31, miR-200b, and miR-200c in tissue and serum were assessed (by RT-PCR). Receiver operating characteristic (ROC) analysis was performed to assess the miR-31, miR-200b, and miR-200c expression levels as potential biomarkers for active CD.

Results
Mean fCal was 1540 +/- 890 ug/g. Mean SES-CD was 8.9 +/- 4.2. Tissue and circulating miR-31 was significantly correlated with fCal (r = 0.81, r = 0.83, p &lt; 0.01) and with SES-CD (r = 0.82, r = 0.79, p &lt; 0.01). The expression level of miR-31 was significantly upregulated in CD tissue cases compared to the control tissue samples (6.24 ± 1.57 vs. 3.70 ± 1.44; p &lt; 0.01). Similarly, serum miR-31 expression levels in CD patients were significantly upregulated than in control serum samples (-2.07 ± 1.00 vs. 0.78 ± 0.42; p &lt; 0.01). The expression levels of tissue miR-200b and miR-200c were significantly upregulated in CD tissue cases compared to the control tissue samples (-5.25 ± 0.93 vs. -4.69 ± 0.80, p = 0.03 for miR-200b, and -0.86 ± 0.96 vs. 0.39 ± 0.66, p &lt; 0.01 for miR-200c). Similarly, serum miR-200b and miR-200c expression levels in CD patients were significantly upregulated than in the control serum samples (p &lt; 0.05). ROC analysis revealed that the expression levels of the selected miRNAs could help to discriminate active CD patients from healthy controls with very good specificity and sensitivity.

Conclusion
Tissue and circulating miR-31, miR-200b, and miR-200c reflect disease activity in CD patients and can be used as biomarkers for active CD. Furthermore, circulating miR-31, miR-200b, and miR-200c may be used as non-invasive biomarkers for active CD patients.
P053 The intestinal complement system and its role in dysregulation of the intestinal B-cell compartment in Crohn’s Disease patients
Authors: Raschdorf A , Preisker S , Brethack A K , Bokemeyer A , Bettenworth D , Sina C , Derer S 
Published: January 2022

Abstract

Background
For several decades, there is growing evidence that a hyperactive B-cell compartment may be involved in the disturbed mucosal immune response in Crohn’s disease (CD) patients. This is reflected by approximately one third of all CD patients displaying an excessive production of autoantibodies against the intestinal M-cell expressed FimH receptor glycoprotein 2 (GP2) which inhibits induction of adaptive immunity against adherent-invasive flagellated bacteria. However, underlying mechanisms of an imbalanced B-cell compartment in CD patients still remain elusive. Given that the complement system plays a critical role in the detection and clearance of bacteria and promotes B-cell immunity, we asked if the complement system contributes to a dysregulated B-cell response in CD.

Methods
Systematical expression analyses of the intestinal complement components were performed in IBD patients, including CD and ulcerative colitis (UC) patients, versus non-IBD controls. Additionally, activity of the three activation pathways of complement system was studied in serum samples of CD patients and controls. Further, immunoglobulin levels in intestinal biopsies, sera and fecal samples were analyzed.

Results
CD patients, but no UC patients, showed increased expression of the early classical complement pathway components C1QA, C1QB, C1S, C2, C3, C4A, CR1 and CR2 in sigmoidal biopsy samples that does not differ significantly between remission and active disease state. This could be verified by immunohistochemistry, displaying an over-representation of mucosal C1q, C3 and B-cell expressed CR2 (also CD21) in colon biopsies of CD patients in remission. In line with this, CD patients in remission present with enhanced activity of the classical as well as alternative pathway of complement activation in serum compared to non-IBD controls. Accordingly, high protein levels of C3 cleavage fragments were detected in fecal samples from CD patients in contrast to non-IBD controls. While there were no differences in systemic immunoglobulin levels in serum, intestinal IgM was increased in CD patients in remission. Notably, fecal IgA levels were strongly diminished in these patients, while expression of the polymeric immunoglobulin receptor was not affected.

Conclusion
In summary, our findings demonstrate an augmented activation of the intestinal complement system that may potentially be involved in the etiology of CD. Expansion of mucosal IgM+/CR2+ B cells together with a reduced fecal IgA level, points to a locally disturbed induction of intestinal IgA secreting plasma cells in CD patients.
P054 LHFPL3-AS2 long non-coding (lncRNA) is reduced in mucosal biopsies of Crohn Disease, and its reduction in tissue culture disrupts epithelial polarity
Authors: Sosnovski K , BenShoShan M , Braun T , Barshack I , Denson L , Haberman Y 
Published: January 2022

Abstract

Background
Disruption of apico-basal polarity attenuate Intestinal epithelia barrier and is part of the Epithelial–mesenchymal transition process linked with Crohn Disease (CD). We have defined widespread dysregulation of long non-coding RNAs (lncRNA), known to have cellular regulatory role, in treatment naïve CD ileum including reduction of LHFPL3-AS2.

Aim
We aimed to explore the effect of LHFPL3-AS2 reduced expression in the context of CD pathogenesis.

Methods
dCas9/CRISPRi was used for LHFPL3-AS2 knock-down in Caco-2 cells. Transcriptomics, qPCR, western blots, and confocal microscopy was used to evaluate the effect of LHFPL3-AS2 knock-down.

Results
LHFPL3-AS2 expression in isolated ileum and ascending colon epithelia from controls was significantly higher than in the sigmoid colon, with LHFPL3-AS2 reduction in the ileum of CD cases vs. controls (p&lt;0.01). LHFPL3-AS2 co-expression analyses showed enrichment for genes linked with cytoskeleton and tight junction. Reducing LHFPL3-AS2 expression by 2 gRNAs, strikingly resulted in abnormal cell morphology. Unlike control cells that developed polarized cysts in 3D, the LHFPL3-AS2 knockdown cells failed to form the luminal cavity within the cyst, which was coupled with abnormal distribution of F-actin throughout the cell membrane, rather than primarily in the luminal area (Fig. 1). mRNA-seq analysis on LHFPL3-AS2 knockdown and controls detected 584 differentially expressed genes (fold change&gt;=1.5 and FDR corrected p&lt;0.05). Similarly, the down regulated genes showed enrichment for pathways (p&lt;E-4) linked with apical part of cell membrane, actin filament bundle, microvillus, regulation of morphogenesis of an epithelium, adherent junctions, and b-catenine-TCF4 complex. Staining for CTNNB1, JAM-A, e-Cadherin (CDH1), and CDH11 showed abnormal organization and formation of no lumen or multiple smaller lumens within the cysts, overall suggesting abnormal epithelial polarity organization. Using String network analyses tool on the down-regulated genes, we identified CTNNB1, a key downstream component of the canonical Wnt signaling pathway, as a central node in the network suggesting potential attenuation of downstream Wnt signaling (Fig. 2). Activating the Wnt/β-catenin pathway, via LiCl, showed more substantial reduction of CTNNB1, and tight junction proteins - CDH1, CDH11, indicating that the LHFPL3-AS2 knock-down further inhibited the ability of the cells to respond to WNT activation.

Conclusion
Reduced LHFPL3-AS2 in CD epithelia may contribute to attenuated epithelial polarity. Boosting LHFPL3-AS2 lncRNA expression to enable proper epithelial polarity may reverse the initial step in the CD-associated Epithelial–mesenchymal transition process.
P060 Unravelling the microbial-derived metabolic alterations in a T-bet-/- x RAG2-/- mouse colitis model
Authors: Liu Z , Lo J , Alexander J , Cozzetto D , Powell N 
Published: January 2022

Abstract

Background
T-bet (Tbx21) is a T-box transcription factor that can regulate immune cells’ differentiation and function. Loss of T-bet leads to the absence of adaptive immunity. T-bet-/- x RAG2-/- mice develop spontaneous ulcerative colitis (TRUC) that resembles human colitis, thus they are widely used as a colitis model in recent years. While many researchers studied the host and gut microbiota interaction in immune dysregulation, how the host and bacteria metabolites regulate the immune response is less understood.

Methods
TRUC and Rag2-/- (as a control group) mice models were employed. When TRUC mice developed anorectal prolapse and rectal inflammation, mice colon tissue and faeces were collected for gene expression analysis and metabolic profiling, respectively. Metabolites were measured by Nuclear Magnetic Resonance and Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) was performed to determine differential metabolites. Metabolite enrichment analysis was done using MetaboAnalyst. RNA-seq was employed for colon gene expression. Differential genes were generated with DESeq2 package and enrichment analysis was conducted with clusterProfiler package.

Results
Principle component analysis of the metabolic profile showed a clear separation between Rag2-/- and TRUC group (Fig 1A). The OPLS-DA model between the two groups is robust (R2 = 0.55, Q2 = 0.89, p-value = 0.03). 12 metabolites were significantly changed. Alanine, butyrate, beta-glucose, glycerol, lysine, pyruvate, gamma-aminobutyrate, dimethylamine, galactose, hypoxanthine and aspartate were significantly increased in TRUC mice compared to Rag2-/- mice, while tyramine was decreased (Fig 1B&C). Metabolite enrichment analysis showed that glycolysis, glucose-alanine cycle, gluconeogenesis, etc. were among the most enriched pathways (Fig 1D). KEGG module over-representation analysis also identified these glucose-related metabolic pathways as top enriched pathways with many genes increased in TRUC mice (Fig 2). In addition to metabolic pathway perturbance, gene set enrichment analysis suggested that cytokine production and regulation were notably altered in TRUC mice, as well as the response to molecules of bacterial origin (Fig 3).

Conclusion
TRUC mice with ulcerative colitis have significantly affected gut-host metabolism. The up-regulation of glucose-related metabolic pathways suggested increased energy consumption. Increase of butyrate, dimethylamine and enriched genes in response to bacteria implied the host-bacteria interaction process. Further investigation of gut microbiota alteration and integration of bacteria-metabolites-host immune response axis is warranted to determine whether targeting certain pathways can be harnessed as a novel therapy to colitis.
P086 Mucosal microbiota modulate host intestinal immune signatures in Inflammatory Bowel Disease
Authors: Hu S , Bourgonje A R , Gacesa R , Jansen B H , Bangma A , Hidding I , Festen E A M , van Dullemen H M , Visschedijk M C , Dijkstra G , Harmsen H J M , Vich Vila A , Spekhorst L M , Weersma R K 
Published: January 2022

Abstract

Background
Host intestinal immune gene signatures and microbial dysregulations expose potential mechanisms in the pathogenesis of inflammatory bowel diseases (IBD). Profiling of mucosa-attached microbiota allows the understanding of locally present microbial communities and their immediate impact on the host. This study evaluated interactions between host mucosal gene expression and intestinal mucosa-attached microbiota in IBD.

Methods
Intestinal mucosal bulk RNA-sequencing data was combined with mucosal 16S rRNA gene sequencing data from 696 intestinal biopsies derived from 337 patients with IBD (181 with Crohn’s disease [CD] and 156 with ulcerative colitis [UC]) and 16 non-IBD controls. Hierarchical all-against-all associations testing (HAllA) was used to assess factors affecting host gene expressions and microbiota. Mucosal cell enrichments were predicted by deconvolution. Linear mixed interaction models were used to investigate host-microbiota interactions, adjusting for age, sex, BMI and batch effects. Variation explanation analysis was performed by Lasso regression.

Results
In total, 15,934 intestinal genes and 113 microbial taxa were identified and included in subsequent analyses. Host intestinal gene expressions were characterized by tissue- and inflammation-specificity, whereas intraindividual variability of the mucosal microbiota dominated over disease location and inflammation effects. We observed forty associations between the mucosal expression of genes and the abundance of specific microbes independent of dysbiosis (FDR&lt;0.05). Examples include a positive association between aryl hydrocarbon receptor (AHR) and Bifidobacterium, and a negative association between interleukin 18 receptor 1 (IL18R1) and Lachnoclostridium. Furthermore, 112 gene-microbiota interactions changed in patients with microbial dysbiosis compared to non-dysbiosis (FDR&lt;0.05). These interactions were enriched in immune-related and extracellular matrix organization pathways. For example, the IL1R1 gene was positively associated with Collinsella abundance in non-dysbiotic patients, whereas an inverse association was observed in high dysbiosis. Finally, the presence of mucosal microbial taxa explained up to 10% of the variation in cell type enrichment, affecting epithelial cells, macrophages and regulatory T-cells.

Conclusion
Interactions between host intestinal gene expressions and mucosa-attached microbiota are disrupted in patients with IBD. Furthermore, mucosal microbiota are highly personalized and potentially contribute to intestinal cell type alterations. Our study unravels key immune-mediated molecular pathways and relevant bacteria in intestinal tissue, which may guide drug development and precision medicine in IBD.
P091 A single-cell RNAseq approach to understand and predict the efficacy of tofacitinib in Ulcerative Colitis
Authors: Melon E , Corraliza A M , Garrido A , Veny M , Masamunt M C , Giner A , Ordás I , Fernández A , Ricart E , Panés J , Salas A 
Published: January 2022

Abstract

Background
The variability and unpredictability of drug efficacy in ulcerative colitis (UC) patients, including tofacitinib, an oral JAK1 and JAK3 inhibitor, remains a perplexing reality. The aim of this study is to identify the relevant cellular subsets and genes involved in response and/or resistance to tofacitinib using single-cell RNA sequencing (scRNA-seq).

Methods
Colon biopsies from UC patients starting tofacitinib treatment (10 mg twice daily) were collected from the involved region at baseline (n=9), week 8 (n=7) and week 16 (n=5). Endoscopic response was evaluated at week 16 and patients were classified as responders or non-responders based on whether they had a decrease in the endoscopic Mayo score of at least 1 point from baseline. Biopsies were immediately disaggregated and single-cell suspensions were processed using 10x Genomics Next-GEM Single Cell kit assays (Chromium 3’ v3.0). After QC filtering, a total of 61,646 cells were obtained. Additional biopsies were collected at all timepoints for validating scRNA-seq results by bulk RNA analysis (qPCR).

Results
Four out of nine patients enrolled in this study responded to tofacitinib treatment. Analysis of the proportions of mucosal cells identified by scRNAseq revealed a partial recovery of the epithelial and stromal cell compartments and a decrease in the total number of plasma and myeloid cells in responding patients at week 8, which was sustained at week 16 (Table 1).

scRNA-seq analysis revealed a significant downregulation of SOCS1 in the CD4 and B cell populations, and of both SOCS3 and IRF1 in the neutrophil and B cell populations in all patients receiving tofacitinib treatment irrespective of endoscopic response. These results were validated by qPCR of bulk biopsy RNA (p=0.0245, p=0.0299 and p=0.0230 respectively). In contrast, S100A8 and CHI3L1 were significantly downregulated only in responders, correlating with a decrease in the proportion of mucosal inflammatory cells including neutrophils, monocytes, and inflammatory fibroblasts.
Regarding prediction of outcome, a scRNA-seq comparative analysis showed that high levels of IL17A-expressing T cells at baseline significatively correlated with failure to achieve response upon tofacitinib treatment. This result was validated by qPCR of bulk biopsy RNA (p=0.0059).

Conclusion
Our study demonstrates the potential of using scRNAseq of intestinal biopsies not only to characterize the mechanism of action of tofacitinib, but also to describe the cellular events associated with response to this therapeutic intervention. Moreover, we show the potential of scRNAseq to identify predictors of response to tofacitinib in patients with UC.
P093 Probiotic strains Lactobacillus plantarum and Saccharomyces boulardii influence the fibrotic responses of human intestinal subepithelial myofibroblasts
Authors: Tarapatzi G , Filidou E , Kandilogiannakis L , Spathakis M , Arvanitidis K , Kotzampassi K , Kolios G , Vradelis S 
Published: January 2022

Abstract

Background
The strains Lactobacillus plantarum and Saccharomyces boulardii, widely used among probiotic supplements, seem to ameliorate Inflammatory Bowel Disease (IBD) progression and to promote mucosal healing. Intestinal Subepithelial Myofibroblasts (SEMFs) are key mediators in mucosal healing and fibrosis, mainly by migrating to the trauma region and secreting collagen. The aim of this study was to investigate the different effects of the aforementioned probiotic strains in the migration rate and the fibrotic responses of SEMFs.

Methods
L. plantarum and S. boulardii were reconstituted in SEMFs culture medium, identified by Gram staining and their viability was assessed by Trypan Blue staining. Primary SEMFs were isolated from colonic biopsies from healthy individuals and stimulated with 102 and 104 cfu/ml of each strain for: a) 6h, when mRNA expression of collagen type I, III, fibronectin, Tissue Factor and α-SMA was assessed by quantitative PCR, and b) 48h, when SEMFs’ migration rate was examined via Wound Healing Assay and protein collagen was measured via Sircol Assay.

Results
Trypan Blue staining confirmed the viability of the studied probiotics, and both the bacteria and the yeasts were stained Gram positive. Stimulation of SEMFs with L. plantarum resulted in upregulated mRNAs of Collagen type I (102cfu/ml: 1.6-fold, 104cfu/ml: 1.5-fold; p&lt;0.001), type III (102cfu/ml: 1.7-fold, 104cfu/ml: 1.8-fold; p&lt;0.0001), Fibronectin (102 cfu/ml, 1.5-fold, 104cfu/ml 1.5-fold; p&lt;0.01), Tissue Factor (102cfu/ml: 1.4-fold, p&lt;0.0001; 104cfu/ml: 1.3-fold, p&lt;0.001) as well as SEMFs’ migration rate (102cfu/ml: 128.3%, 104cfu/ml: 124.1%; p&lt;0.05). On the other hand, S. boulardii stimulation resulted in downregulated Collagen type I (102cfu/ml: 0.9-fold, p&lt;0.01) and type III mRNAs (102cfu/ml: 0.8-fold, p&lt;0.01), upregulated Tissue Factor mRNA (102cfu/ml: 1.2-fold, p&lt;0.0001; 104cfu/ml: 1.1-fold, p&lt;0.01), increased protein collagen production (104cfu/ml: by 211.6%, p&lt;0.001) and increased SEMFs’ migration rate (104cfu/ml: 130.2%, p&lt;0.05).

Conclusion
This study provides evidence that not only both probiotic strains remain viable in SEMFs’ culture medium but are also able to interact with SEMFs and alter their fibrotic behaviour. Moreover, it was shown that L. plantarum may promote mucosal healing, while S. boulardii could reduce profibrotic responses. Further preclinical research is needed to identify the optimal dosages and combinations of probiotic strains as potential auxiliary treatment of IBD.
The study was supported by «Strategic expansion of the Greek Biobanking Infrastructure» (BBMRI-GR) in the framework of the Action “ENHANCING RESEARCH AND INNOVATION INFRASTRUCTURE - SECOND CYCLE” (NSRF 2014–2020) and “AMKE KLEON TSETIS” Foundation, Athens, Greece.