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Background Tissue factor (TF) plays an important role on blood clotting and the risk of thromboembolic events is increased in ulcerative colitis (UC). Tofacitinib, recently introduced in UC therapeutics, may further increase the risk. To delineate thrombosis pathophysiology in this context, TF expression in primary human colonic mesenchymal cells (PHCMC) of patients with active UC was correlated with clinical parameters, serum markers of inflammation and endoscopy. We then treated those PHCMC with tofacitinib with or without cytokines to better mimic the in vivo milieu they are exposed to. Methods PHCMC from endoscopic biopsies of the inflamed mucosa of 10 UC patients with an endoscopic Mayo score ≥2 were treated with all major T helper (Th)1 (TNF-α, IFN-γ), Th2 (IL-4, IL-13) or T regulatory (Treg; TGF-β, IL-10) cytokines with or without tofacitinib. Cells were lysed, RNA was isolated and reverse-transcribed to cDNA. TF and RPL4 (housekeeping) cDNAs were quantified with real-time PCR. Wilcoxon and Mann-Whitney U tests were used to compare TF ΔΔCT for paired or unpaired values, respectively, and Spearman’s rho to correlate TF ΔΔCT with scale clinical variables and continuous laboratory values. Results Increased TF mRNA abundance in PHCMC was correlated to increased partial Mayo score (Spearman's rho 0.661, p < 0.044), reduced serum albumin (Spearman's rho -0.723, p < 0.05) and to more severe endoscopic lesions: cells originating from UC patients of an endoscopic Mayo score 3 expressed 20 times more TF than those from an endoscopic Mayo score 2 (p < 0.017; Figure 1). Moreover, PHCMC from difficult-to-treat patients, defined as requiring >1 biologics, expressed 9 times more TF (Figure 2). On the other hand, treatment with tofacitinib did not upregulate TF (Figure 3). PHCMC expressed receptors and responded to treatment with all major Th1 (TNF-α, IFN-γ), Th2 (IL-4, IL-13) or Treg (TGF-β, IL-10) cytokines by further upregulating TF by 5.5-7.5 times (p 0.001-0.035). IL-13 had the maximal effect (Figure 4). Even when tofacitinib was added together with the aforementioned cytokines, it did not further upregulate TF; instead, it tended to partially inhibit their effects. For example, it decreased upregulation of TF by IL-13 by 40%. Conclusion The expression of TF mRNA in active UC is significantly associated with clinically and endoscopically severe disease and resistance to treatment. Tofacitinib per se does not increase TF. Instead, it may limit the upregulating effect of pro-inflammatory cytokines.

Background Inflammatory Bowel Disease (IBD) is a chronic inflammatory condition of the alimentary tract, primarily manifested as Crohn’s disease and Ulcerative colitis. Dysbiosis of the intestinal microbial microflora is a well-established characteristic of IBD. The microbiota metabolism ends in the production of several metabolites that participate in interactions with the host and ultimately influence its physiology. Our aim is to investigate whether metabolite receptor expression differs between IBD and healthy individuals and unravel their possible interactions with inflammatory and fibrotic pathways in the intestine. Methods RNA-sequencing data from over 2500 intestinal biopsy samples were collected from publicly available datasets via the Sequence Read Archive. Bioinformatics analysis of metabolite receptor genes was performed using the RaNA-seq online platform which included normalization, alignment and differential expression. In addition, correlation analysis between the target receptor genes and inflammatory/fibrotic genes as well as functional analysis was performed via a R based pipeline to elucidate molecular interactions during IBD. Results The expression of 22 receptors responding to the major metabolite ligand classes bile acids, short chain fatty acids and indole derivatives from tryptophan metabolism was targeted. Several receptors of bile acids as well as receptors of short chain fatty acids, such as the vitamin D receptor (VDR), the peroxisome proliferator-activated receptor alpha (PPARα) and the hydroxycarboxylic acid receptor 2 (HCAR2), were found differentially expressed in intestinal biopsies of CD patients as compared to healthy individuals. Negative and positive correlation of these receptors with genes of known involvement in IBD was explored, along with their simultaneous contributions to inflammatory and fibrotic processes. Conclusion Our results demonstrate the interplay between the intestinal microbiota and the host on a metabolite-receptor level and identify disease related interactions with inflammation and fibrosis.

Background We have previously shown that human subepithelial myofibroblasts (SEMFs), which are of mesenchymal origin, express the receptor of Oncostatin M (OSM), which is further induced when SEMFs are previously exposed to IL-1α and TNF-α. Human Intestinal Organoids (HIOs), derived from embryonic stem cells (ESC), form epithelial crypts consisting of several subtypes of epithelial cells and are surrounded by cells of mesenchymal origin. Recently, we reported that the mesenchymal component of HIOs is gradually reduced, as culture passages increase. The aim of our study was to examine the effect of exogenous OSM on fibrotic and pro-inflammatory marker expression of HIOs, with and without the combined presence of IL-1α and TNF-α. Methods The human ESC line (H1)-derived HIOs were developed using a commercially available kit and characterized by immunofluorescence in all differentiation stages. HIOs from passage 2 were stimulated with either 100ng/ml OSM for 12 hours or 5ng/ml IL-1α and 50ng/ml TNF-α for 24 hours and then with 100ng/ml OSM for 12 hours. mRNA expression of fibrotic markers, Collagen Type I, III, and Fibronectin, and pro-inflammatory markers, CCL2, CXCL10 and CXCL11 were examined by reverse transcription quantitative PCR. Results OSM alone significantly downregulated both Collagen Type I and III mRNA expression, and had no effect on Fibronectin expression. When HIOs were previously exposed to IL-1α and TNF-α, however, OSM stimulation resulted in significant upregulation of Collagen Type I and Fibronectin, and had no effect on Collagen Type III. Regarding the pro-inflammatory marker expression, OSM alone induced the expression of CCL2, CXCL10 and CXCL11, with a greater effect observed when HIOs were previously exposed to IL-1α and TNF-α. Conclusion Our findings indicate that OSM induces the expression of both fibrotic and pro-inflammatory factors in early passages of HIOs, previously exposed to IL-1a and TNF-a, as has been previously observed on SEMFs. Since the mesenchymal cell component of HIOs is strongly present in early passages and OSM’s receptor is mainly found to be expressed in these cells, our results suggest OSM affects HIOs via the cells that are of mesenchymal origin.

Background Oncostatin M (OSM) may play an important role in Inflammatory Bowel Disease (IBD) pathogenesis. Specifically, both OSM and its receptor are upregulated in inflamed colonic regions of IBD patients, and high OSM expression has been associated with failure to respond to anti-TNF therapy. Our aim was to investigate the effect of OSM in fibrotic factors and chemokine expression on primary colonic subepithelial myofibroblasts (SEMFs) from healthy individuals (HI). Methods Primary SEMFs were isolated from endoscopically-obtained colonic biopsies from HI. SEMFs were stimulated with 1, 10, or 100ng/ml OSM for 6 hours, with or without pre-stimulation with 5ng/ml IL-1α plus 50ng/ml TNF-α for 24h. Total RNA was collected and mRNA transcripts for collagen type I, type III, fibronectin, and the chemokines CCL2, CXCL9, CXCL10 and CXCL11 were measured by reverse transcription quantitative PCR. Results Unstimulated SEMFs had a basal expression of collagen type I, III, fibronectin, CCL2, CXCL9, CXCL10 and CXCL11. OSM stimulation augmented chemokine mRNA expression in a dose-dependent manner (Table 1) but had no effect on fibrotic factors expression. Pre-stimulation of myofibroblasts with TNF-α and IL-1α resulted in augmented expression of collagens I and III and fibronectin, in addition to further increases in chemokine expression in response to subsequent stimulation by OSM (Table 2). Conclusion Our results show that stimulation with OSM induces fibrotic and chemokine responses by SEMFs. Our findings further support the hypothesis that SEMFs may play a key role in regulating chronic intestinal inflammation and response to biological therapy.

Background Various probiotic strains play a positive role in Inflammatory Bowel Disease and might support mucosal healing. Colonic Subepithelial Myofibroblasts (SEMFs) have a pivotal role in mucosal healing and fibrosis, by migrating to the trauma region and secreting collagen. Our study aimed to investigate whether different strains of probiotics can affect the migration capacity and the fibrotic phenotype of SEMFs isolated from healthy individuals. Methods The probiotic mix of Lactobacillus acidophilus, Lactobacillus plantarum, Bifidobacterium lactis and Saccharomyces boulardii was reconstituted in SEMFs culture medium, they were 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, 104 or 108 cfu/ml of the mix for 6h or 48h; in 6h total RNA was collected and mRNA expression of collagen type I, III, fibronectin, tissue factor (TF) and α-SMA was measured by quantitative PCR, and in 48h their effect on migration rate through the Wound Healing Assay and collagen secretion via Sircol was assessed. Results Trypan Blue confirmed that the majority of the probiotic mix was alive, and all three bacterial strains and the yeast were stained Gram positive. Stimulation of SEMFs with the probiotic mix resulted in different effects depending on its concentration; 102 cfu/ml downregulated protein collagen secretion (3.4μg/ml, IQR: 2.7–5.0, p Conclusion This study provides evidence that the probiotic strains L. plantarum, L. acidophillus, B. lactis and S. boulardii remain viable in SEMFs’ culture medium and have the ability to interact with and alter the behavior of SEMFs. Moreover, the effect of the probiotic mix on SEMFs fibrotic phenotype is dependent of the concentration of the probiotics used. Further preclinical research is needed in order to identify the effect of each strain, as well as the suitable dose and combinations in various pathological conditions.

Background The inflammatory bowel disease (iBD) etiology remain elusive and both genetic and environmental factors have been implicated in the establishment of systemic inflammatory reactions that typify IBD. A basic, modifiable environmental factor is the gut microbiota and dysbiosis seems to be a key factor in IBD pathogenesis. Nowadays, it is well known that anti-TNF therapy significantly improves the rates of remission in patients with inflammatory bowel disease (IBD), however, there is a subgroup of patients who do not respond to treatment. The aim of the present study is to profile changes in the mucosal gut microbiome before and after anti-TNF therapy and investigate its possibility to predict patient response at baseline. Methods Mucosal biopsy samples from IBD patients [14 Crohn’s disease patients (CD), 6 ulcerative colitis (UC) patients] and healthy controls (HC) were examined for differences in microbiota composition (16S rRNA gene sequencing) at baseline and upon completion of anti-TNF treatment, accordingly, via an in silico pipeline. Results Although no statistically significant differences at phylum level were noted, a relative reduction in Bacteroidetes and an increase in Actinobacteria, Fusobacteria and Chloroflexi were detected in IBD versus HC biopsies, however the microbiomes of UC, CD and HC display qualitative differences at the OTU level. At genus level, statistically significant differential abundance between the HC and IBD groups was observed. The genera Parabacteroides, Barnesiella, Butyricimonas, Ruminococcus_1, Ruminococcaceae_UCG013, Phascolarctobacterium, Ruminoclostridium_6, Paraprevotella as well as members of the Eubacterium_ruminantium group appear reduced in IBD, whereas Dialister abundance is increased. The genus Collinsella exhibits an induction during UC versus both HC and CD. Regarding anti-TNF treatment on the microbiome, Alpha-diversity is reduced after anti-TNF treatment. Beta-diversity indicates differences before and after treatment as well as between responders and non-responders. By using the biomarker discovery tool LEfSe, we identified microbial genera associated mainly with anti-TNF responders. Conclusion In the present study, we showed that the response to anti-TNF therapy is related to specific microbiota profiles in the gut mucosa. Correlation analysis between these parameters of response at baseline showed that the assessment of microbial taxa can further distinguish responders from non-responders.

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 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.

Background Organoids are self-renewing, 3D structures, consisting of different cell types, with histology and physiology features very close to the physiology of the studied organ. Specifically, human Intestinal Organoids (HIOs) develop epithelial crypts consisting of all subtypes of intestinal epithelial cells which are surrounded by mesenchymal cells. Our aim was to develop 3D HIOs from human embryonic stem cells (hESCs) and examine the expression of fibrotic and mesenchymal factors during their maturation process. Additionally, we investigated the effect of the pro-inflammatory cytokines, IL-1α and TNF-α on the expression of fibrotic and inflammatory mediators in HIOs. Methods The human ESC line (H1) was cultured and then differentiated towards HIOs using commercially available kit. HIOs were characterized by immunofluorescence in all differentiation stages. In order to examine their maturation process, we compared the mRNA expression of fibrotic and mesenchymal markers from passages 1–10. In order to examine their functionality, HIOs from different passages were stimulated with 5ng/ml IL-1α and 50ng/ml TNF-α for 12 hours, total RNA was collected and the fibrotic and inflammatory mRNA expression was examined. The mRNA transcripts of CD90, collagen type I, III, fibronectin, CXCL8, CXCL10 and CXCL11 were measured by reverse transcription quantitative PCR. Results HIOs were successfully developed as they were stained positive for all tested markers throughout their developmental process. Regarding their maturation process, we observed high expression of CD90, collagen type I, type III and fibronectin that was gradually decreased during passages. As for the fibrotic and inflammatory responses from HIOs, we found that the IL-1α and TNF-α stimulation resulted in statistically significant upregulation of the fibrotic factors, fibronectin, collagen type I and type III in culture passages 2 and 4, but had no effect in culture passages 8 and 10. Similarly, IL-1α and TNF-α stimulation led to the statistically significant induction of the inflammatory chemokines CXCL8, CXCL10 and CXCL11 in culture passages 2 and 4, while no effect was observed in culture passages 8 and 10. Conclusion Our findings indicate that HIOs contain a functional mesenchymal component that is gradually diminished during passages. Inflammatory and fibrotic responses of HIOs seem to depend on the fitness of their mesenchyme. IBD studies using HIOs as in vitro models should be performed on early passages, when HIO’s mesenchymal component is still functional.

Background Fatigue is common in IBD patients, even in clinical remission and adversely affects the quality of life. For this reason, it has been suggested to use fatigue questionnaires as PROS (patient reported outcomes). We aimed to study the degree of fatigue in IBD patients and identify factors that are positively or negatively related to its presence. Methods Patients with IBD (men: 51.6%, Crohn’s Disease: 65.2%, age: 36.7 years median, 18.1–72 yrs range), with a regular f-up in our Department completed the Fatigue Severity Scale, IBD Fatigue self assessment 1 & 2, short IBDQ and Short Health Scale upon consensus. Two groups of patients were studied: Group A, patients attended the IBD outpatient clinic (121 patients / 144 questionnaires) and Group B, patients attended the infusion centre for biological therapy (55 patients / 414 questionnaires). The SPSS-23 statistical programme was used to identify correlations between fatigue and various clinical-laboratory parameters of patients. Results Problematic fatigue (FSS> 36) was reported by 46.5% of patients. No difference was observed between Crohn’s disease and Ulcerative Colitis or between the two groups of patients. There were statistically significant correlations between fatigue and disease activity (R = 0.428, P Conclusion In the group of IBD patients we studied, a significant correlation with various clinical characteristics was observed. The presence of fatigue is a factor associated with poor quality of life for patients.

Background Oncostatin M (OSM), a cytokine of the IL-6 family, has been implicated in the pathogenesis of inflammatory bowel disease (IBD). Specifically, OSM and its receptor, OSMR, are elevated in inflamed colonic regions of IBD patients; in addition, high OSM expression at baseline has been associated with failure to respond to anti-TNF. OSMR expression localised in stromal cells of the intestinal lamina propria. Our aim was to investigate the expression of OSM and its receptors subunits, OSMR, LIFR and gp-130 in primary subepithelial myofibroblasts (SEMFs) and test whether this expression is regulated by the innate cytokines, IL-1α and TNF-α. Methods Primary SEMFs were isolated from endoscopically-obtained colonic biopsies from healthy controls, set to culture and stimulated with 5ng/ml IL-1α and/or 50ng/ml TNF-α for 6 h. Total RNA was extracted and the mRNA transcripts for OSM, OSMR, LIFR and gp-130 were measured by reverse transcription-quantitative (RT-q) PCR. Results Unstimulated SEMFs had a basal expression of both OSM and its receptors subunits. IL-1α or TNF-α stimulations did not alter LIFR expression, but they induced a statistically significant upregulation of OSM, OSMR and gp-130. Specifically, the expression of OSM in SEMFs was significantly upregulated after stimulation with IL-1α alone or in combination with TNF-α (IL-1α: 11.48-fold increase, 8.29–26.05; IL-1α+TNF-α: 13.42-fold, 11.32–17.16; p < 0.0001). Regarding the OSMR and gp-130 subunits [which form the type II receptor of OSM], OSMR mRNA was induced by TNF-α alone or in combination with IL-1α (TNF-α: 1.75-fold, 1.27–2.02, p < 0.01; IL-1α+TNF-α: 2.06-fold, 1.83–2.34, p < 0.0001), whereas gp-130 mRNA expression was increased under all stimulatory conditions (IL-1α: 1.94-fold, 1.46–2.07; TNF-α: 1.81-fold, 1.46–2.25; IL-1α+TNF-α: 1.59-fold, 1.31–2.51; p < 0.01 for all comparisons). Conclusion Our results show that OSM and OSMR are expressed on primary human SEMFs and that they are upregulated under stimulation with innate pro-inflammatory cytokines. These data further support a potential role of this system of inflammatory mediators in the pathogenesis of intestinal inflammation.

Background Crohn’s disease (CD) and ulcerative colitis (UC), the two main entities of inflammatory bowel disease (IBD), are characterised by chronic and relapsing/remitting inflammation of the gastrointestinal tract, and occasionally ultimately result in debilitating intestinal fibrosis. Apart from their key role in fibrosis, there is evidence that subepithelial myofibroblasts (SEMFs) participate in the IBD inflammatory cascade, as they express various pro-inflammatory cytokine receptors. We examined the effect of pro-inflammatory IL-17—the hallmark cytokine of T-lymphocytes differentiated to Th17—on the expression of lymphocyte-chemotactic chemokines in SEMFs. Methods SEMFs were isolated from endoscopically obtained colonic biopsies from healthy controls, set to culture and stimulated with 100 ng/ml IL-17 for 6 h. Total RNA was extracted and mRNA expression of CCL5, CXCL1 and CXCL11 was assessed with reverse transcription quantitative (RT-q) PCR. Changes in cytokine mRNA are provided as medians (IQR). Results Untreated SEMFs had a basal expression of chemokines of the CCL and CXCL family groups. So far, our study has shown that the IL-17 stimulation leads to a statistically significant upregulation of CCL and CXCL chemokines in SEMFs (p < 0.001). In detail, CCL5 was upregulated 5.7-fold (4.9–7.5), CXCL1 72.9-fold (63.1–90.7) and CXCL11 25.4-fold (17.3–37.3). Conclusion IL-17 induced the expression of chemotactic factors in SEMFs. Our results further support a potential role of SEMFs in the shaping of intestinal mucosal immunity by serving as immunological intermediates that respond to cytokines of adaptive immunity and amplify the recruitment of immune cells via chemokine production.

P032 Pro-inflammatory cytokines induce the expression of TL1A/TNFSF15 in primary intestinal subepithelial myofibroblasts G. Bamias1 *, E. Filidou2, D. Goukos3, V. Valatas4, K. Arvanitidis2, M. Panagopoulou2, G. Kouklakis5, G. Daikos3, S. Ladas1, G. Kolios2. 1Laikon Hospital, Ethnikon & Kapodistriakon University of Athens, Academic Dpt. of Gastroenterology, Athens, Greece, 2Democritus University of Thrace, Laboratory of Pharmacology, Faculty of Medicine, Alexandroupolis, Greece, 3Laikon Hospital, Ethnikon & Kapodistriakon University of Athens, 1st. Dpt of Propaedeutic and Internal Medicine, Laboratory of Infectious Diseases, Athens, Greece, 4University of Crete, Laboratory of Gastroenterology, Faculty of Medicine, Heraklion, Greece, 5Democritus University of Thrace, Endoscopy Unit, Faculty of Medicine, University Hospital of Alexandroupolis, Alexandroupolis, Greece