bluuebunny/crossref_metadata_2025 huggingface dataset:
DOP50 Oral α4β7 integrin inhibitor MORF-057 demonstrates exposure driven biomarker response in non-human primates
Authors: Wong J , Lee D , Mangada M , Yadav V , Troast D , Zhong C , Hahn K , Wang W , Ge X , Sun L , Jiang S , Wang B , Zhang J , Yan L , Nguyen H , Cui D , Bursavich M G , Lippa B , Rogers B N , Ray A S
Published: January 2022

Background

Disruption of immune cell trafficking via integrins is a proven and effective mechanism for treating inflammatory bowel disease. When α4β7 integrin is inhibited through pharmacological intervention, immune cells destined for the gut tissue become sequestered in blood circulation and these alterations can be detected through several methods. MORF-057 is a novel, oral, selective, small molecule inhibitor of α4β7 integrin developed for treating IBD. MORF-057 demonstrated favorable tolerability, pharmacokinetic and pharmacodynamic profiles including saturating receptor occupancy and corresponding evidence for proof of biology based on effects on circulating cells during a Phase 1 clinical trial in healthy volunteers (Ray, ECCO 2021). Here we demonstrate an exposure:response relationship of α4β7 related biomarkers examined among MORF-057 treated non-human primates (NHPs) as a means for pre-clinical testing of inhibitors of this pathway.

Methods

Single-cell RNA sequencing (scRNAseq) was performed on NHP CD45+ blood cells to determine baseline populations potentially impacted through exposure to MORF-057. 40 Naïve cynomolgus monkeys were enrolled over 5 separate studies and dosed orally BID with MORF-057 over 2–7 days using several dose levels to examine biomarker dynamics over a wide range of exposures. Peripheral blood was sampled at various timepoints (n= 125) and assayed using: mass spectroscopy, flow cytometry (FACS), and mRNA quantification to determine MORF-057 exposure levels, on-target receptor occupancy (RO), immune cell subset changes, and CCR9 mRNA levels.

Results

MORF-057 Ctrough ranged from 3.3–429 ng/ml. In samples tested for RO, MORF-057 achieved >95% saturation of α4β7 even with the lowest Ctrough of 4.5 ng/ml. MORF-057 treatment led to significantly sustained increases in circulating β7high CD4+ T memory cells detectable as early as 24h post-exposure. A maximal effect where β7high cells accounted for approximately 60% of the T memory population was achieved at saturating receptor occupancy (Fig., left). CCR9 mRNA values demonstrated increases upon drug exposure (Fig., right).

Conclusion

In NHP, acute changes in circulating β7high T memory cells was a sensitive biomarker demonstrating a dose-dependent response to MORF-057 exposure. CCR9 mRNA levels also showed similar exposure related changes reflecting its expression on a subset of β7high cell types. Analysis of scRNAseq shows expression of α4β7 on other cell types beyond T memory cells including: NK, NKT, B cells, plasmablasts, monocytes, and eosinophils. Pharmacodynamic changes in NHP are consistent with human Phase 1 data in healthy volunteers.
DOP51 Loss of PTPN23 in the intestinal epithelium results in epithelial hyperproliferation and lethal diarrhea in a microbiota dependent manner
Authors: Spalinger M , Sanchez Alvarez R , Gottier C , Montalban-Arques A , Schwarzfischer M , Niechcial A , Scharl M
Published: January 2022

Background

Protein tyrosine phosphtase non-receptor type 23 (PTPN23) plays a critical role in regulating epidermal growth factor (EGF) receptor signaling and its loss has been associated with aberrant cell proliferation and promotes onset of epithelial cancers. However, the role of PTPN23 in the intestinal epithelium has not been investigated yet. Here, we investigated how PTPN23 deletion in intestinal epithelial cells (IEC) affects intestinal homeostasis.

Methods

To study the role of PTPN23 in the intestinal epithelium, we crossed mice with a LoxP flanked PTPN23 gene (PTPN23fl/fl mice) to mice expressing the CreERT construct under the villin promoter (PTPN23-VilCreERT mice). PTPN23 was deleted in IEC in these mice by tamoxifen-injections on five consecutive days. PTPN23 fl/fl mice injected with tamoxifen served as control.

Results

PTPN23 deletion in IEC resulted in drastic loss of weight starting around 10-14days after the first tamoxifen injection, eventually leading to death within 3–5 weeks due to severe wasting disease with severe diarrhea. Histology revealed massive hyper-proliferation of the colonic epithelium accompanied with elevated immune cell infiltration. In line with previous reports on the function of PTPN23, we observed highly elevated levels of EGF receptor, possibly accounting for the massive epithelial hyper-proliferation and aberrant water secretion/defective water resorption in these mice. Furthermore, we observed bacterial translocation to the spleen and the liver, indicating defective anti-bacterial defense in the intestinal epithelium of PTPN23-VilCreERT mice. In line with this, 16S sequencing revealed significant differences in the intestinal microbiome with an overgrowth of bacteria that have been reported to be heavily coated with IgA. PTPN23-VilCreERT mice showed reduced apical presence of the poly Ig receptor, a receptor that transports IgA from the lamina propria through IEC into the gut lumen. Notably, while serum IgA levels were normal, its levels in the stool were reduced, confirming defective transport of IgA into the gut lumen. In addition, autophagy was significantly reduced in PTPN23-VilCreERT mice when compared to their littermates. Of interest, antibiotic treatment was sufficient to prevent epithelial hyper-proliferation, diarrhea and death in PTPN23VilCreERT mice, indicating that this drastic phenotype was microbiota-dependent and likely the result of defects in bacterial handling.

Conclusion

Our results demonstrate that PTPN23 is indispensible for normal IEC function and its loss renders the intestinal epithelium unable to cope with invading bacteria. This uncovers a novel, so far unknown role of PTPN23 for regulating pathways involved in bacterial handling.
DOP60 Self-reported treatment effectiveness for Crohn’s Disease using a novel crowdsourcing web-based platform
Authors: Engel T , Dotan E , Ben-Horin S , Kopylov U
Published: January 2022

Background

The world wide web and social media platforms have become an unprecedented source for sharing self-experience, potentially allowing the collection and integration of health data with patient experience

Methods

StuffThatWorks (STW) is an online open platform that applies machine learning and the power of crowdsourcing where patients with chronic medical conditions can self-report and compare their individual outcomes using a structured online questionnaire. The present study analyzed de-identified self-reported personalized comparative treatments’ effectiveness for CD. The design was a cross-sectional, international, crowdsourcing, questionnaire and AI web-based study of patients with Crohn’s self-reporting their outcomes by 06/11/21. A proprietary STW Bayesian inference model was built to measures the level of improvement in condition severity and clinical indicators for each treatment and ranks treatment effectiveness. A linear regression model was used to examine co-variate association with the current condition severity as the outcome. Finally, the effectiveness of first-line biological treatments was analyzed by multiple treatment comparisons model and by calculating odds ratio and 95% confidence intervals for each treatment pair.

Results

A total of 5898 self-reported CD patients were included for the analysis. Most participants were female (76.13%) and from English speaking countries (91%). Overall, anti TNF drugs were the most reported tried treatment (71.97%) followed by steroids (46.22%) and diet (43.8%). Among Biologic therapy (BT) tried by STW CD users, Infliximab (IFX) and Adalimumab (ADA) were ranked most effective by the STW effectiveness model, by change in condition severity on 1–5 scale (mean change of 1.19 points, estimated lower-upper bounds 1.10–1.28), followed by Ustekinumab (UST) (mean 1.07, bounds 0.88–1.26), and Vedolizumab (VDZ) (mean 0.96, bounds 0.74–1.17). Bowel surgery (mean 1.19, bounds 0.98–1.40), and cannabis (mean 1.01, bounds 0.72–1.31) were also among the top 5 most effective treatments together with BT. The odds ratio was calculated for each BT pair, with IFX more effective than ADA, UST and VDZ (OR 2.34 (CI 1.88 – 2.80), 3.08 (CI 2.44 – 3,73), 7.16 (CI 6.53 – 7.80), respectively), ADA more effective than UST and VDZ (OR 1.32 (CI 0.74 - 1.9), 3.06 (CI 2.49 - 3.63), respectively), and UST more effective than VDZ (OR 2.32 (CI 1.6 - 3.05)).

Conclusion

We present the first online crowdsourcing platform-based study of treatment self-reported outcomes in CD. Net-based crowdsourcing patient-reported outcomes’ platforms can potentially help both clinicians and patients select the best treatment for their condition.
P003 Identification of an intestine-derived ex-Trm population in the blood of healthy individuals and patients with Inflammatory Bowel Disease
Authors: Rodger B , Gordon H , Hoti I , Stagg A , Lindsay J
Published: January 2022

Background

Tissue-resident memory T cells (Trm) persist in peripheral tissues and contribute to pathogen clearance and inflammation. Trm can re-enter the circulation (ex-Trm) and give rise to new effector and Trm populations. Skin-derived ex-Trm can be identified in human blood based on co-expression of the residency marker CD103 and cutaneous leukocyte antigen (CLA), a skin-tropism marker. The existence of ex-Trm derived from the gut would have implications for inflammatory bowel disease (IBD) and its treatment targeting the recruitment of circulating gut-homing cells.

Methods

Peripheral blood and colonic biopsies were taken from healthy volunteers and patients with IBD (Crohn’s disease or ulcerative colitis). PBMCs and cells isolated from biopsies by enzymatic digestion were analysed by multi-colour flow cytometry.

Results

More than 80% of colonic αβT cells were Trm, as defined by CD69 expression, in health and IBD; there was no significant increase in cells with a non-resident phenotype in inflamed tissue. Few CD4+ Trm co-expressed CD103. In contrast, CD8+ Trm comprised CD103+ and CD103- subsets, and CD69+CD103- cells were significantly reduced in IBD. Increased staining for KLRG1 and the cytotoxicity-associated protein perforin, indicated a more effector-like Trm phenotype in IBD. Putative gut-derived ex-Trm were identified amongst TCRαβ+CD45RA- blood cells as a β7++CD103+ population, indicative of cells expressing both α4β7 and CD103(α E)β7 integrin complexes. A separate CD103β7+α4β7 population defined by 1:1 expression of CD103 and β7 contained CLA+ skin ex-Trm. Gut ex-Trm comprised 0.3% of circulating CD8+ T cells (range 0.02–1.4%), and 1.2% of CD4+ T cells (range 0.3–3%). Gut and skin ex-Trm were phenotypically similar; both expressed the residency associated markers CD9 and CD101 but lacked CD69 expression. Gut ex-Trm were phenotypically distinct from both the traditional gut-trophic population (α4β7+CD103-CD45RA) and from naïve T cells. The proportion of gut ex-Trm did not differ between health and IBD. However, gut-derived ex-Trm were significantly reduced, relative to skin-derived ex-Trm, in Crohn’s disease, but not ulcerative colitis, when compared with health.

Conclusion

A putative gut-derived ex-Trm population can be identified in both healthy and IBD peripheral blood, with IBD-associated changes identified in this population and intestinal Trm. Circulating ex-Trm could link discreet areas of intestinal inflammation in Crohn’s disease and there is a selective loss of the gut ex-Trm population from the blood of these patients. The role of ex-Trm in IBD merits further study.
P016 Colonoids derived from UC patients retain higher transcriptional response to inflammatory triggers in-vitro, even after several passages
Authors: Abbas Egbariya H , Ben-Shoshan M , Braun Z , Berger T , Granot M , Loberman-Nachum N , Vais B , Gal Mor O , Amir A , Haberman Y
Published: January 2022

Background

Ulcerative colitis (UC) patients show high variations in disease phenotype and response to therapy. Intestinal epithelia play a key role in UC pathogenesis, and therefore patient-derived epithelial model to study this variation are warranted. Few studies suggest that colonoids derived from UC patients retain some disease-related transcriptional and epigenetic changes, but they also raise questions regarding their persistence in culture. Here, we aimed to characterize if UC epithelial dysfunctions and response to inflammatory signals retained in colonoid culture.

Methods

Rectal biopsies were used to generate epithelial organoids (colonoids) that were grown through 5–10 passages using L-WRN conditioned medium. Media and total RNA were collected for measuring CXCL1 secretion using ELISA and qPCR, respectively.

Results

3 control and 5 UC patients-derived colonoids were generated. Median age was 17 years and 75% were females. Control patients were evaluated for loose stool, abdominal pain, and anemia, but had normal rectal mucosa endoscopically and histologically. UC included newly diagnosed and established patients. Colonoids were allowed to differentiate for 48 hours before applying different inflammatory cocktails to induce inflammation for 24 h with IFNγ+LPS, IFNγ+TNFα, and TNFα+LPS (20 ng/ml each trigger). UC organoids secreted significant higher levels of CXCL1 as detected in the media at baseline (p=0.02) and with IFNy+TNFa (p=0.01), but no difference was noted with TNFa+LPS (Fig. 1). In addition, CXCL1 mRNA showed significant higher levels after inflammatory triggering in UC vs. controls (Fig. 1) with IFNγ +LPS, (p=0.02), IFNγ+TNFα (p=0.003), and TNFα+LPS (p=0.016). Similarly, several other genes showed significant and substantial induction in UC organoids vs. controls, in some or all the inflammatory triggers (Fig. 2). The tight junction-associated protein, ZO1 showed substantially higher induction in UC compared to controls with the 3 inflammatory cocktails [IFNγ +LPS, (p=0.03), IFNγ+TNFα (p=0.02), and TNFα+LPS (p=0.005)]. Significantly higher expression in UC organoid was further noted in comparison to control in IL8 levels upon IFNγ +LPS (p=0.002), in interferon inducible IDO1 with IFNγ+TNFα (p=0.001), and in goblet associated MUC2 (p=0.04) and bacterial sensing DUOX2 (p=0.001) with TNFα+LPS.

Conclusion

Our data suggest that UC colonoids retain some inflammatory response activity in-vitro even after several passages. We show that UC organoids secrete higher level of CXCL1. Additionally, we note higher transcriptional response to different inflammatory signals in UC organoids potentially implying an “immune” epithelial transcriptional memory that may contribute to UC chronicity.
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

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)| >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<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<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<5.0E-2). In contrast, cryptoglandular fistula were characterised by a significant enrichment of myocytes, smooth muscle cells and neurons (p<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

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) > 50 ug/g and Short Endoscopic Score of Crohn’s Disease (SES-CD) > 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 < 0.01) and with SES-CD (r = 0.82, r = 0.79, p < 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 < 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 < 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 < 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 < 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

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

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<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>=1.5 and FDR corrected p<0.05). Similarly, the down regulated genes showed enrichment for pathways (p<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

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

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<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<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

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

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<0.001), type III (102cfu/ml: 1.7-fold, 104cfu/ml: 1.8-fold; p<0.0001), Fibronectin (102 cfu/ml, 1.5-fold, 104cfu/ml 1.5-fold; p<0.01), Tissue Factor (102cfu/ml: 1.4-fold, p<0.0001; 104cfu/ml: 1.3-fold, p<0.001) as well as SEMFs’ migration rate (102cfu/ml: 128.3%, 104cfu/ml: 124.1%; p<0.05). On the other hand, S. boulardii stimulation resulted in downregulated Collagen type I (102cfu/ml: 0.9-fold, p<0.01) and type III mRNAs (102cfu/ml: 0.8-fold, p<0.01), upregulated Tissue Factor mRNA (102cfu/ml: 1.2-fold, p<0.0001; 104cfu/ml: 1.1-fold, p<0.01), increased protein collagen production (104cfu/ml: by 211.6%, p<0.001) and increased SEMFs’ migration rate (104cfu/ml: 130.2%, p<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.
P095 Anti-inflammatory effect of high acetate concentration on organoid-derived epithelial monolayer from patients with Ulcerative Colitis
Authors: Deleu S , Arnauts K , Machiels K , Huys G R B , Thevelein J , Raes J , Vermeire S
Published: January 2022

Background

Short-chain fatty acids (SCFA), such as acetate and butyrate, have gained increasing interest for their potential to suppress IBD inflammation1,4. Most data have been generated for butyrate, showing beneficial effects on the gut microbiome composition, intestinal barrier function and immune system1. The effects of acetate are less known, despite its lower toxicity on epithelial cells and its ability to support growth of butyrate-producing bacteria by metabolic cross-feeding2. Furthermore, the probiotic effect of the biotherapeutic yeast Saccharomyces cerevisiae var. boulardii might be linked to its high acetic acid production3. Here, we studied the effect of acetate on inflammatory markers and barrier integrity using an organoid-derived epithelial monolayer system obtained from Ulcerative Colitis (UC) patients.

Methods

Confluent Transwell® organoid-derived monolayers evaluated by Transepithelial electrical resistance (TEER) of 8 UC patients were basolaterally stimulated with control medium (CTRL) or with an inflammatory mix (INFL) [100 ng/ml TNF-α, 20 ng/ml IL-1β, 1 µg/ml Flagellin] (Fig. 1). After 24h, the monolayer was stimulated apically with control medium or with high acetate (HA) [100mM] and TEER was measured at 0, 24, and 48h post-stimulation. After 48h, RNA extraction of the epithelial cell monolayers and qPCR for selected target genes (IL8, TNFα, CLDN1, CLDN2, HIF1A, ZO1, OCLDN, MUC2 and MKI67) was performed. Cytokine measurement was performed using Mesoscale Discovery (Proinflammatory panel 1) on apical and basolateral media. Data were analyzed using GraphPad Prism 9 (D’Agostino & Pearson test for normality followed by Wilcoxon tests).

Results

Transcriptomic analysis of the monolayer cells showed a decrease in expression of IL8 (p=0.03), TNFα (p=0.08) and increase of HIF1A (p=0.03) upon HA administration in inflamed conditions (Fig. 2) whereas the proliferation marker MKI-67 was upregulated (p=0,03). Moreover, these findings were supported by significant decreases in pro-inflammatory cytokines measured apically after HA stimulation (Fig. 3A) such as IFNγ, TNFα, IL6, IL8 and IL1β (all p=<0.01). Basolaterally, IL8 (p=0.02) and IL13 (p=<0.01) increased significantly upon HA stimulation (Fig.3B). Administration of HA was associated with a numerical though non-significant increase in TEER-values (p=0.15) (Fig. 4) suggesting a potential for improved barrier integrity.

Conclusion

In this UC patient-derived epithelial cell culture model, high acetate administration positively affected barrier integrity gene expression and TEER-values and suppressed expression and production of pro-inflammatory cytokines.

References

1. Venegas, 2019

2. Gill, 2018

3. Offei, 2019

4. Deleu, 2021
P100 Serum inflammatory protein profiling to corroborate selection of therapy naïve moderate-to-severe pediatric Crohn’s disease patients eligible for first-line infliximab treatment
Authors: Jongsma M M E , Costes L M M , Tindemans I , Cozijnsen M A , Raatgreep R H C , van Pieterson M , Li Y , Escher J C , de Ridder L , Samsom J N
Published: January 2022

Background

Early intervention with biologic therapy, especially with anti-Tumor Necrosis Factor-a, is very effective and now more commonly used to prevent disease progression and complications in Crohn’s disease (CD). However, a rational approach to decide which patient should be treated First-line Infliximab (FL-IFX) is lacking. In this study, using serum from a randomized control trial, we assessed how changes in immune profiles prior to therapy and after the first standardized induction treatment with either FL-IFX or conventional treatment (CONV) related to maintaining disease remission at week 52.

Methods

Serum samples from therapy naive moderate to severe CD patients from the TISKIDS study1 were used at time of diagnosis and at 10–14 weeks after induction therapy with FL-IFX (n=48) and CONV (n=43). FL-IFX consisted of 5 IFX infusions (5 mg/kg) combined with azathioprine (AZA) maintenance. CONV consisted of induction therapy with Exclusive Enteral Nutrition or oral prednisolone combined with AZA maintenance. Concentrations of 92 inflammatory proteins were determined with Olink Proteomics®. Significant differences in mean protein NPX after FDR adjustment with log2fold > 0.5 were considered meaningful.

Results

After 10–14 weeks of induction therapy, FL-IFX suppressed a larger number inflammatory proteins and induced stronger suppression compared to CONV. Of the 30 significantly modulated FL-IFX target proteins 18 were differentially regulated by FL-IFX only. Hierarchical clustering of patients based on their baseline profiles of the 30 IFX-modulated proteins revealed 2 patient clusters, a CD-hi cluster with significantly higher baseline clinical disease activity, CRP and blood neutrophil concentrations compared to the CD-lo cluster. The CD-hi cluster had an increased abundance of 23/30 proteins amongst which Oncostatin-M, TNFSF14, HGF and TGF-alpha compared to the CD-lo cluster (Figure 1A). Of the CD-lo patients treated with FL-IFX, 58% reached clinical disease remission without treatment escalation at week 52, while this was only 24% for CD-hi patients treated with FL-IFX. Only 20% of the CD-lo patients treated with CONV reached remission while this was only 6% for the CONV treated patients in the CD-hi group (Figure 1B). Strikingly, similar clustering using the immune profiles after 10–14 weeks induction therapy did not predict remission at week 52.

Conclusion

FL-IFX achieves stronger reduction and modulates more inflammatory serum immune protein concentrations than CONV. Stratification of patients according to profiles of IFX-modulated proteins prior to therapy identifies patients with a lower chance of reaching clinical remission without treatment escalation at week 52.

Reference

1. Jongsma MME et al. Gut. 2020 Dec; DOI:10.1136/ PMID:33384335
P102 Changes in serum immune protein profiles following vedolizumab therapy in Inflammatory Bowel Disease
Authors: O’Connell J , O’Connell F , O’Sullivan J , Kevans D
Published: January 2022

Background

Vedolizumab(VDZ) is a monoclonal antibody targeting α4β7-integrin which has demonstrated efficacy as induction and maintenance therapy for inflammatory bowel disease(IBD). We aimed to characterise changes in serum immune protein profiles following VDZ induction therapy and evaluate the association between serum immune protein profiles and outcome of VDZ therapy.

Methods

IBD patients commencing VDZ induction therapy were prospectively recruited. Patients received VDZ induction as per standard protocol. Week 0 and week 6 pre-infusion serum samples were collected from each patient. Serum samples were analysed using human multiplex panels (Meso Scale Discovery, Rockville, MD, USA) which quantifies 54 discrete inflammatory proteins. Corticosteroid-free remission(SFR) was assessed at week 14 and 30. Changes in serum immune protein profile were compared between baseline(week 0) and following two VDZ infusions(week 6). Serum immune proteins which demonstrated a significant week 0 to 6 change were evaluated as candidate biomarkers of week 14 and 30 SFR. Continuous variable are presented as median[range]. P values < 0.05 were considered significant.

Results

N=39 IBD patients were included: age 50.4[18.2–75.8] years, UC phenotype 51%, male 51%; disease duration 13.4[0.4 – 40.8] years, clinical Mayo subscore 4[0 – 9] and HBI 7[1 – 20]. 7 of 54 analytes demonstrated a significant change from baseline to week 6. Eotaxin(p=0.001), Eotaxin-3(p=0.001), TNF-b (p<0.008), TARC(p=0.015), bFGF(p=0.034) and MIP1-b(p=0.043) increased from baseline to week 6. IL-15(p<0.001) reduced from baseline to week 6. There was no association between any of these 7 immune proteins and VDZ therapy outcome at week 14 or 30.

Conclusion

Changes in serum immune protein concentrations with pro-inflammatory, immunoregulatory and chemotactic functions are observed following VDZ induction therapy. Further study is required to identify serum biomarkers of VDZ therapy response.