ABSTRACT Photopharmacology offers powerful opportunities for the spatiotemporal control of biological processes, yet the rational design of photoswitchable enzyme inhibitors remains challenging. Here, we report a target‐guided strategy for the development of diazo‐based photoswitchable inhibitors of human 15‐lipoxygenase‐1 (15‐LOX‐1), a key enzyme in inflammatory signaling, ferroptosis, and cancer. Guided by the structural features of known ligands, we developed three complementary photoswitch classes: reversible azobenzenes (ABs), azo‐heteroarenes (HAs), and covalent azo‐bis‐alkynes (BAs). These compounds exhibit efficient E / Z photoisomerization and high bistability, supported by single‐crystal x‐ray diffraction and density functional theory calculations. Enzymatic inhibitory and kinetic studies revealed distinct activity and selectivity profiles within the tested substrates/isoenzyme: AB and HA derivatives function as E ‐ON/Z‐OFF inhibitors, whereas BA derivatives display Z ‐ON/ E ‐OFF behavior, enabling programmable light‐controlled modulation. We validated 15‐LOX‐1 as a therapeutic target in cellular and in vivo mouse models of colonic inflammation, where inhibition suppressed IL‐8 expression. Finally, using our reversible and covalent photoswitches, we demonstrate photoisomer‐dependent suppression of IL‐8. Beyond 15‐LOX‐1, this work establishes a generalizable framework for the rational development of selective photoswitchable inhibitors with tunable biological outcomes.

Background Sphingosine-1-phosphate (S1P) receptor (S1PR) modulators (S1PRMs) block chemotaxis of cells of immunity to the locus of inflammation. S1PRM effectiveness depends on the extent of the pathway activation. We explored the contribution of colonic stromal cells (CSC) to S1P production in patients with ulcerative colitis (UC) and healthy controls (HC). Methods We looked into the two S1P-producing kinases (SPHK1, SPHK2), the transporter exocytosing S1P (SPNS2) and the lyase degrading it (SGPL1). Basal mRNA transcription in primary CSC in culture from UC patients and from HC was assayed with quantitative reverse-transcription PCR. The effect of each one of the principal T helper (Th) 1 (ΤNF-α, IFN-γ), Th2 (IL-4, IL-13) or regulatory T (Treg; TGF-β, IL-10) cytokines was also tested. Medians of ΔΔCT and statistical significance between groups of unpaired, paired or between scale variables with Mann-Whitney, Wilcoxon or Spearman’s rho test, respectively, are reported. Results Eleven patients with UC (mean age: 49 years; 9 males; 7 with left-sided and 4 with extensive disease; mean UC duration: 180 months) and 9 HC were included. CSC from both UC and HC had a basal expression of SPHK1, slightly lower in UC (0.56x10-3 vs 2.18x10-3, p<0.04), and of SPHK2 (8.04x10-4, 1.05x10-3, respectively). However, CSC from UC patients with an endoscopic Mayo component of 3 vs 2 and from UC patients with higher erythrocyte sedimentation rate expressed more SPHK1 (3.69x10-3 vs 0.45x10-3, p<0.05; r 0.762, p<0.04). Similarly, both UC and HC CSC expressed SPNS2 (5.96x10-5, 3.46x10-4, respectively) and SGPL1, with the latter overexpressed in UC (5.41x10-2 versus 2.68x10-3, p<0.001). CSC from UC, in sharp contrast with those from HC, were responsive to proinflammatory cytokines. In detail, SPHK1 was upregulated by TNF-α (x5 times, p<0.034; Figure 1A). SPHK2 was downregulated by TGF-β (/3 times, p<0.042). SPNS2 was downregulated by IFN-γ (/18 times, p<0.001; Figure 2B) and by TGF-β (/8 times, p<0.02; Figure 2B). SGPL1 was downregulated by IFN-γ (/16 times, p<0.001; Figure 2C) and by IL-10 (/7 times, p<0.04; Figure 2C). Conclusion In health and UC, CSC express the machinery synthetizing, exocytosing and catabolizing S1P. In UC the transcription of these genes is controlled by cytokines. Hence, activity of the pathway in CSC should be further investigated as a parameter to the direction of personalised treatment with S1PRMs in UC.

In steady state, intestinal subepithelial myofibroblasts form a thin layer below the basement membrane. Unlike the rest of the stromal cells in the lamina propria, they express tensile proteins, guide epithelial regeneration, and sense luminal microbiota. Upon inflammation in inflammatory bowel disease (IBD), they express activation markers, accept trophic signaling by infiltrating neutrophils and macrophages, and are activated by cytokines from helper T cells to produce a narrow spectrum of cytokines and a wider spectrum of chemokines, attract cells of innate and adaptive immunity, orchestrate inflammatory responses, and qualitatively and quantitatively modify the extracellular matrix. Thus, beyond being structural tissue components, they assume active roles in the pathogenesis of complicated IBD. Discrimination between myofibroblasts and fibroblasts may be an oversimplification in light of single-cell sequencing data unveiling the complexity of multiple phenotypes of stromal cells with distinct roles and plasticity. Spatial transcriptomics revealed distinct phenotypes by histologic localization and, more intriguingly, the assembly of mucosal neighborhoods that support spatially distinct functions. Current IBD treatments target inflammation but fail in fibrostenotic or fistulizing disease. Baseline and recent findings on stromal cells, molecules, and pathways involved in disrupted extracellular matrix homeostasis are reviewed to provide relevant pharmacologic targets.

Metabolites produced by dysbiotic intestinal microbiota can influence disease pathophysiology by participating in ligand–receptor interactions. Our aim was to investigate the differential expression of metabolite receptor (MR) genes between inflammatory bowel disease (IBD), healthy individuals (HIs), and disease controls in order to identify possible interactions with inflammatory and fibrotic pathways in the intestine. RNA-sequencing datasets containing 643 Crohn’s disease (CD) patients, 467 ulcerative colitis (UC) patients and 295 HIs, and 4 Campylobacter jejuni-infected individuals were retrieved from the Sequence Read Archive, and differential expression was performed using the RaNA-seq online platform. The identified differentially expressed MR genes were used for correlation analysis with up- and downregulated genes in IBD, as well as functional enrichment analysis using a R based pipeline. Overall, 15 MR genes exhibited dysregulated expression in IBD. In inflamed CD, the hydroxycarboxylic acid receptors 2 and 3 (HCAR2, HCAR3) were upregulated and were associated with the recruitment of innate immune cells, while, in the non-inflamed CD ileum, the cannabinoid receptor 1 (CNR1) and the sphingosine-1-phospate receptor 4 (S1PR4) were downregulated and were involved in the regulation of B-cell activation. In inflamed UC, the upregulated receptors HCAR2 and HCAR3 were more closely associated with the process of TH-17 cell differentiation, while the pregnane X receptor (NR1I2) and the transient receptor potential vanilloid 1 (TRPV1) were downregulated and were involved in epithelial barrier maintenance. Our results elucidate the landscape of metabolite receptor expression in IBD, highlighting associations with disease-related functions that could guide the development of new targeted therapies.

Abstract Background Ulcerative colitis (UC) is associated with an increased risk of venous thromboembolism. We investigated the correlation of UC severity with latent activation of the extrinsic coagulation pathway and colonic stromal tissue factor (TF) expression. Methods Plasma exosomes, microparticles (EM), TF protein, activities of EM-bound TF (EM-TF) and FVIIa, as well as TF mRNA (F3) in primary colonic stromal cells in culture were measured. Disease extent by Montreal classification, medications, UC clinical activity by partial Mayo score (PMS), endoscopic severity by the MS endoscopic component (eMS; loss of response defines as ≥2), quality of life by the short IBD questionnaire- SIBDQ, complete blood count, CRP, ESR and albumin were retrieved. Statistical significance (p) was assessed with the following tests: Student’s t for 2 groups with normally distributed values, Mann-Whitney U if not, Wilcoxon test for paired data. Correlation of continuous against scale variables was assessed with Pearson’s or Spearman’s r and corresponding p values for linear or logarithmic continuous variables, respectively. Results Plasma was obtained from 38 UC patients (31 males, disease duration 151±25 months, 14 with extensive colitis, 5 current smokers) and 28 healthy blood donors (HBD; 20 males) who served as controls. TF mRNA (F3) was measured in primary colonic stromal cells in culture from 12 UC patients (11 males, disease duration 169±53 months, 8 with extensive colitis, 1 current smoker) and from 7 controls undergoing screening colonoscopy. UC patients had 4- and 3.7- times more exosomes and microparticles than controls, respectively (A). TF protein correlated with PMS (r 0.443), albumin (-0.362), ESR (0.353), PLT (0.575) and endoscopic UC severity (0.468; B). EM-TF activity was 1.6-times higher (C) and correlated to disease extent (E3: 1.9 x E2), albumin (-0.624), endoscopic severity (0.422; D) and SIBDQ (-0.64). Downstream, FVIIa activity was 2.2-times higher. Refractory-to-treatment patients had 5.8-, 1.5-, 2.1- times higher TF protein (E), MP-TF, FVIIa activity, respectively. Within responders, the need for steroids or biologics correlated with 2.2-times higher MP-TF activity. Colonic stromal cells from patients with UC maintained a 2.2-times higher F3 (F), which correlated to PMS (0.56), albumin (Spearman’s r -0.543), endoscopic severity (M3: 8.3 x M2). All the aforementioned were statistically significant with a < 0.05. Conclusion The extent of spontaneous activation of the extrinsic coagulation pathway is associated with clinical and endoscopic UC activity and response to treatment. TF in colonic stromal cells mirrors its systemic activity and indicates the possible contribution of such cells to increased TF plasma bioactivity.

Background Oncostatin-M (OSM) is associated with antitumor necrosis factor (anti-TNF)-alpha resistance in inflammatory bowel disease (IBD) and fibrosis in inflammatory diseases. We studied the expression of OSM and its receptors (OSMR, gp130) on intestinal subepithelial myofibroblasts (SEMFs) and the effect of OSM stimulation on SEMFs. Methods The mRNA and protein expression of OSM, OSMR, gp130, and several fibrotic and chemotactic factors were studied in mucosal biopsies and isolated human intestinal SEMFs of patients with IBD and healthy controls (HCs) and in a model of human intestinal organoids (HIOs). Subepithelial myofibroblasts and HIOs were stimulated with OSM and interleukin (IL)-1 alpha/TNF-alpha. RNAseq data of mucosal biopsies were also analyzed. Results Oncostatin-M receptors and gp130 were overexpressed in mucosal biopsies of patients with IBD (P < .05), especially in inflamed segments (P < .05). The expression of OSM, OSMR, and gp130 in SEMFs from HCs was increased after stimulation with IL-1 alpha/TNF-alpha (P < .001; P < .01; P < .01). The expression of CCL2, CXCL9, CXCL10, and CXCL11 was increased in SEMFs from patients with IBD and HCs after stimulation with OSM in a dose-dependent manner (P < .001; P < .05; P < .001; P < .001) and was further increased after prestimulation with IL-1 alpha/TNF-alpha (P < .01 vs OSM-alone). Similar results were yielded after stimulation of HIOs (P < .01). Oncostatin-M did not induce the expression of collagen I, III, and fibronectin. Oncostatin-M receptor expression was positively correlated with CCL2, CXCL9, CXCL10, and CXCL11 expression in mucosal biopsies (P < .001; P < .001; P = .045; P = .033). Conclusions Human SEMFs overexpress OSMR in an inflammatory microenvironment. Oncostatin-M may promote inflammation in IBD via its stimulatory effects on SEMFs, which primarily involve chemoattraction of immune cells to the intestinal mucosa.

Background and aims: Monocyte recruitment in the lamina propria and inflammatory phenotype driven by the mucosal microenvironment is critical for the pathogenesis of inflammatory bowel disease. However, the stimuli responsible remain largely unknown. Recent works have focused on stromal cells, the main steady-state cellular component in tissue, as they produce pro-inflammatory chemokines that contribute to the treatment-resistant nature of IBD. Methods: We studied the regulation of these processes by examining the communication patterns between stromal and myeloid cells in ileal Crohn’s disease (CD) using a complete single-cell whole tissue sequencing analysis pipeline and in vitro experimentation in mesenchymal cells. Results: We report expansion of S4 stromal cells and monocyte-like inflammatory macrophages in the inflamed mucosa and describe interactions that may establish sustained local inflammation. These include expression of CCL2 by S1 fibroblasts to recruit and retain monocytes and macrophages in the mucosa, where they receive signals for proliferation, survival, and differentiation to inflammatory macrophages from S4 stromal cells through molecules such as MIF, IFNγ, and FN1. The overexpression of CCL2 in ileal CD and its stromal origin was further demonstrated in vitro by cultured mesenchymal cells and intestinal organoids in the context of an inflammatory milieu. Conclusions: Our findings outline an extensive cross-talk between stromal and myeloid cells, which may contribute to the onset and progression of inflammation in ileal Crohn’s disease. Understanding the mechanisms underlying monocyte recruitment and polarization, as well as the role of stromal cells in sustaining inflammation, can provide new avenues for developing targeted therapies to treat IBD.

Ulcerative colitis (UC) increases the risk for venous thromboembolism. Tissue factor (TF) initiates the extrinsic coagulation pathway (ECP). To investigate the correlation of UC severity with latent ECP activation and TF expression in primary colonic stromal cells (PCSC). In plasma of 38 UC patients (31 males, disease duration 151 ± 25 months) and 28 healthy controls, exosomes and microparticles (EM) were counted. Moreover, TF protein concentration, activities of EM-bound TF (EM-TFa) and coagulation factor VII (FVIIa) were assessed. In PCSC in culture, TF mRNA (F3) from 12 patients with active UC and 7 controls was evaluated. UC patients had 4– and 3.7– times more exosomes and microparticles, respectively, than controls. TF protein in UC was correlated with several disease severity indices, such as partial Mayo score (pMs; r 0.443), albumin (− 0.362), ESR (0.353), PLT (0.575), and endoscopic Ms (eMs 0.468). EM-TFa was also significantly higher in UC and was correlated to SIBDQ (− 0.64), albumin (− 0.624), disease extent and eMs (0.422). Refractory-to-treatment patients had significantly higher TF protein, EM-TFa and FVIIa. Even within responders, the need for steroids or biologics correlated with a 2.2–times higher EM-TFa. PCSC from active UC maintained higher F3 than controls, which was correlated to pMs (0.56), albumin (− 0.543) and eMs. Treatment with cytokines further upregulated F3. P for all comparisons was < 0.05. Low-grade activation of the ECP associates with clinical, endoscopic UC activity and response to treatment. TF in PCSC mirrors its systemic activity and points to them as a source.

EDITORIAL article Front. Immunol., 16 February 2023Sec. Mucosal Immunity Volume 14 - 2023 | https://doi.org/10.3389/fimmu.2023.1152140

Abstract 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.

Abstract 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.

Bifidobacterium lactis, Lactobacillus acidophilus, Lactiplantibacillus plantarum and Saccharomyces boulardii are common probiotic supplements. Colonic subepithelial myofibroblasts (cSEMFs) are actively involved in mucosal wound healing and inflammation. cSEMFs, isolated from healthy individuals, were stimulated with 102 or 104 cfu/mL of these probiotic strains alone and in combination, and their effect on chemokine and wound healing factor expression was assessed by qRT-PCR, ELISA and Sircol Assay, and on cSEMFs migration, by Wound Healing Assay. These strains remained viable and altered cSEMFs’ inflammatory and wound healing behavior, depending on the strain and concentration. cSEMFs treated with a combination of the four probiotics had a moderate, but statistically significant, increase in the mRNA and/or protein expression of chemokines CXCL1, CXCL2, CXCL4, CXCL8, CXCL10, CCL2 and CCL5, and healing factors, collagen type I and III, fibronectin and tissue factor. In contrast, when each strain was administered alone, different effects were observed, with greater increase or decrease in chemokine and healing factor expression, which was balanced by the mixture. Overall, this study highlights that the use of multiple probiotic strains can potentially alert the gut mucosal immune system and promote wound healing, having a better effect on mucosal immunity than the use of single probiotics.

Abstract 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.

IntroductionExtracellular matrix turnover, a ubiquitous dynamic biological process, can be diverted to fibrosis. The latter can affect the intestine as a serious complication of Inflammatory Bowel Diseases (IBD) and is resistant to current pharmacological interventions. It embosses the need for out-of-the-box approaches to identify and target molecular mechanisms of fibrosis.Methods and resultsIn this study, a novel mRNA sequencing dataset of 22 pairs of intestinal biopsies from the terminal ileum (TI) and the sigmoid of 7 patients with Crohn’s disease, 6 with ulcerative colitis and 9 control individuals (CI) served as a validation cohort of a core fibrotic transcriptomic signature (FIBSig), This signature, which was identified in publicly available data (839 samples from patients and healthy individuals) of 5 fibrotic disorders affecting different organs (GI tract, lung, skin, liver, kidney), encompasses 241 genes and the functional pathways which derive from their interactome. These genes were used in further bioinformatics co-expression analyses to elucidate the site-specific molecular background of intestinal fibrosis highlighting their involvement, particularly in the terminal ileum. We also confirmed different transcriptomic profiles of the sigmoid and terminal ileum in our validation cohort. Combining the results of these analyses we highlight 21 core hub genes within a larger single co-expression module, highly enriched in the terminal ileum of CD patients. Further pathway analysis revealed known and novel inflammation-regulated, fibrogenic pathways operating in the TI, such as IL-13 signaling and pyroptosis, respectively.DiscussionThese findings provide a rationale for the increased incidence of fibrosis at the terminal ileum of CD patients and highlight operating pathways in intestinal fibrosis for future evaluation with mechanistic and translational studies.

Inflammatory Bowel Diseases (IBDs) are characterized by chronic intestinal inflammation and fibrosis, the latter being the predominant denominator for long-term complications. Epithelial and mesenchymal 2D cultures are highly utilized in vitro models for the preclinical evaluation of anti-inflammatory and antifibrotic therapies. More recently, human intestinal organoids (HIOs), a new 3D in vitro model derived from pluripotent stem cells, have the advantage to closely resemble the architecture of the intestinal mucosa. However, the appropriate timing for the study of inflammatory and fibrotic responses, during HIO development, has not been adequately investigated. We developed HIOs from the human embryonic stem cell line, H1, and examined the expression of mesenchymal markers during their maturation process. We also investigated the effect of inflammatory stimuli on the expression of fibrotic and immunological mediators. Serial evaluation of the expression of mesenchymal and extracellular matrix (ECM) markers revealed that HIOs have an adequately developed mesenchymal component, which gradually declines through culture passages. Specifically, CD90, collagen type I, collagen type III, and fibronectin were highly expressed in early passages but gradually diminished in late passages. The proinflammatory cytokines IL-1α and TNF-α induced the mRNA expression of fibronectin, collagen types I and III, tissue factor (TF), and alpha-smooth muscle actin (α-SMA) primarily in early passages. Similarly, HIOs elicited strong mRNA and protein mesenchymal (CXCL10) and epithelial (CXCL1, CCL2, CXCL8, and CCL20) chemokine responses in early but not late passages. In contrast, the epithelial tight junction components, CLDN1 and JAMA, responded to inflammatory stimulation independently of the culture passage. Our findings indicate that this HIO model contains a functional mesenchymal component, during early passages, and underline the significance of the mesenchymal cells' fitness in inflammatory and fibrotic responses. Therefore, we propose that this model is suitable for the study of epithelial-mesenchymal interactions in early passages when the mesenchymal component is active.

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. 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. 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. 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.

Abstract 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.

Aims We aimed to investigate reasons for acute upper gastrointestinal bleeding(AUGIB) as well as to compare predictors and outcomes between portal(PH) and non-portal hypertension(NPH) related AUGIB in patients with cirrhosis.