Subepithelial Myofibroblasts Research Articles

Oncostatin M Induces a Pro-inflammatory Phenotype in Intestinal Subepithelial Myofibroblasts.

Oncostatin-M (OSM) is associated with antitumor necrosis factor (anti-TNF)-α 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. 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)-1α/TNF-α. RNAseq data of mucosal biopsies were also analyzed. 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-1α/TNF-α (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-1α/TNF-α (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). 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.

Intestinal Stromal Cells in the Turmoil of Inflammation and Defective Connective Tissue Remodeling in Inflammatory Bowel Disease.

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.

UPREGULATION OF SERPINE1/PAI-1 INDUCED BY IL-33 CONTRIBUTES TO INTESTINAL FIBROSIS IN A CROHN’S DISEASE-LIKE ILEITIS MODEL

Abstract IL-33 is a pleiotropic cytokine known to possess dichotomous roles during gut health and disease. We previously described that IL-33 promotes epithelial restitution/repair in otherwise healthy (C57BL/6) mice that have been challenged with dextran sodium sulphate (DSS) to induce acute colitis, with the end result of efficient resolution of inflammation. However, in SAMP1/YitFc (SAMP) mice that spontaneously develop Crohn’s disease (CD)-like ileitis, elevated IL-33 levels persist as disease progresses, perpetuating chronic gut inflammation and severe fibrosis. While IL-33 has been implicated in the development of inflammation-associated fibrosis, the precise mechanism(s) by which this occurs remains unclear. The aim of this study was to determine IL-33-dependent events leading to intestinal fibrosis in ileitis-prone SAMP mice. Our results show IHC co-localization of IL-33 with fibrotic lesions, specifically in cells morphologically-consistent with both macrophages and subepithelial myofibroblasts (SEMFs). Bulk RNA-Seq analysis of the human SEMF cell line, CCD-18Co, stimulated +/- IL-33 identifies one of the highest-expressing transcripts as SERPINE1, encoding for plasminogen activator inhibitor-1 (PAI-1), which has previously been reported as highly-enriched in IBD patients with active disease that do not respond to anti-TNF therapy. Analysis of publicly-available scRNA-Seq data confirms the increased expression of SERPINE1 in IBD patients that localizes to mesenchymal cell populations. Of note, robust upregulation of SERPINE1 is detected in activated fibroblasts from involved vs. non-involved areas of CD patients. Furthermore, spatial transcriptomics of SAMP ilea reveal a progressive and concomitant increase in both Il33 and Serpine1, with strong clusterization compared to healthy AKR controls, particularly during later time points when fibrosis is evident. Currently, results are pending on the treatment of SAMP mice with the PAI-1 inhibitor, MDI-2268, to determine its direct effect(s) on the development of inflammation-associated intestinal fibrosis. Taken together, these findings suggest IL-33-dependent regulation of Serpine1/PAI-1 that promotes intestinal fibrosis, commonly observed in CD patients, and may provide a novel target to treat IBD patients with fibrostenoic disease.

Molecular Mechanisms of Reelin in the Enteric Nervous System and the Microbiota-Gut-Brain Axis: Implications for Depression and Antidepressant Therapy.

Current pharmacological treatments for depression fail to produce adequate remission in a significant proportion of patients. Increasingly, other systems, such as the microbiome-gut-brain axis, are being looked at as putative novel avenues for depression treatment. Dysbiosis and dysregulation along this axis are highly comorbid with the severity of depression symptoms. The endogenous extracellular matrix protein reelin is present in all intestinal layers as well as in myenteric and submucosal ganglia, and its receptors are also present in the gut. Reelin secretion from subepithelial myofibroblasts regulates cellular migration along the crypt-villus axis in the small intestine and colon. Reelin brain expression is downregulated in mood and psychotic disorders, and reelin injections have fast antidepressant-like effects in animal models of depression. This review seeks to discuss the roles of reelin in the gastrointestinal system and propose a putative role for reelin actions in the microbiota-gut-brain axis in the pathogenesis and treatment of depression, primarily reflecting on alterations in gut epithelial cell renewal and in the clustering of serotonin transporters.

Colonic mucosa barrier defects in collagenous and ischemic colitis.

The subepithelial myofibroblasts (SEMFs) and the subepithelial band of macrophages (SEBM) are major components of the colonic mucosa barrier. Although their role in homeostasis is widely recognized, their contribution to disease states is largely unknown. Our aim was to explore histological characteristics of SEMFs and SEBM in collagenous and ischemic colitis in order to identify specific changes in distinct mucosa backgrounds lacking significant inflammation. SEMFs, SEBM and lamina propria (LP) macrophages were identified immunohistochemically by alpha smooth muscle Actin and Cluster of Differentiation 68 respectively in 38 colonic biopsies [14 collagenous colitis (CC), 14 ischemic colitis (IC), 10 normal mucosa]. In CC, SEMFs were rarely detectable in the collagenous band while aSMA-negative pericryptal fibroblast-like cells appeared. In lower LP interconnecting SEMFs processes were formed. SEBM was preserved in areas with a collagenous layer up to 20 μm. In thicker layers, it was fragmented and gradually disappeared in parallel with engulfment of enlarged macrophages. LP macrophages were usually increased. In IC, slight SEMFs changes preceded discernible epithelial alterations. Rounding, disintegration and extinction of SEMFs constituted successive alterations coinciding with crypt shrinkage and denudation. SEBM displayed total or almost total abolishment in areas with crypt damage but also in sites with minimal changes and in adjacent normal mucosa. Our findings provide evidence of impairment of both mucosa barrier constituents in CC and IC. In CC, histological alterations are closely related to the collagenous layer which seems to affect SEMFs differentiation and migration as well as SEBM integrity. The early extinction of SEBM in IC is indicative of its high sensitivity to hypoxia and hypoperfusion.

Role of PDGFRA+ cells and a CD55+ PDGFRALo fraction in the gastric mesenchymal niche

PDGFRA-expressing mesenchyme supports intestinal stem cells. Stomach epithelia have related niche dependencies, but their enabling mesenchymal cell populations are unknown, in part because previous studies pooled the gastric antrum and corpus. Our high-resolution imaging, transcriptional profiling, and organoid assays identify regional subpopulations and supportive capacities of purified mouse corpus and antral PDGFRA+ cells. Sub-epithelial PDGFRAHi myofibroblasts are principal sources of BMP ligands and two molecularly distinct pools distribute asymmetrically along antral glands but together fail to support epithelial growth in vitro. In contrast, PDGFRALo CD55+ cells strategically positioned beneath gastric glands promote epithelial expansion in the absence of other cells or factors. This population encompasses a small fraction expressing the BMP antagonist Grem1. Although Grem1+ cell ablation in vivo impairs intestinal stem cells, gastric stem cells are spared, implying that CD55+ cell activity in epithelial self-renewal derives from other subpopulations. Our findings shed light on spatial, molecular, and functional organization of gastric mesenchyme and the spectrum of signaling sources for epithelial support.

SAT030 HM15912, A Novel Long-Acting GLP-2 Analog, Improves Intestinal Growth And Absorption Capacity In Rat Model Of Short Bowel Syndrome

Abstract Disclosure: J. Choi: Employee; Self; Hanmi Pharm. Co., Ltd. C. Park: Employee; Self; Hanmi Pharm. Co., Ltd. E. Park: Employee; Self; Hanmi Pharm. Co., Ltd. H. Kwon: Employee; Self; Hanmi Pharm. Co., Ltd. S. Bae: Employee; Self; Hanmi Pharm. Co., Ltd. D. Kim: Employee; Self; Hanmi Pharm. Co., Ltd. S. Lee: Employee; Self; Hanmi Pharm. Co., Ltd. I. Choi: Employee; Self; Hanmi Pharm. Co., Ltd. Short bowel syndrome (SBS) with intestinal failure requires partial or total parenteral nutrition (PN) to maintain health and growth. However, long-term use of PN may lead to life-threatening complications. Although teduglutide was firstly approved, the widespread use of it is still limited due to insufficient efficacy and leading to a significant burden for patients by daily administration. Hence, there is a medical unmet needs for more effective and longer lasting GLP-2 analog drugs. Here, we investigated the potential therapeutic effect of HM15912, which is currently under clinical development for once a month use, in small bowel resected rat model of SBS. To evaluate in vitro activity of HM15912, cAMP accumulation in CHO cells overexpressed human GLP-2 receptor (GLP-2R) was measured, and HM15912 was potently activated GLP-2R with a full agonistic activity as native human GLP-2 (EC50= 0.327 vs 0.173 nM, relative activity= 52.5%). Next, to demonstrate that GLP-2R stimulation by HM15912 results in the production and release of insulin-like growth factor-1 (IGF-1) as a known mechanism of GLP-2, HM15912 was treated to mice primary intestinal subepithelial myofibroblasts. mRNA transcription and protein secretion levels of IGF-1 was dose-dependently increased by treatment of HM15912 (p &amp;lt; 0.05 and p &amp;lt; 0.001, respectively). Longer-lasting property was evaluated in rats after single administration, and HM15912 exhibited 70-fold extended elimination half-life (42.4 h) compared to teduglutide (0.6 h). Based on this prolonged mode of action with the potent in vitro activity, intestinotrophic effect of HM15912 was investigated in 80% of small intestine (SI) resected jejuno-ileal anastomosis model rats. The SBS rats given HM15912 every week for 2 weeks significantly increased wet weight of jejunum compared to resection vehicle or b.i.d treatment of teduglutide. This result was well-correlated with histological analysis such as villus height, crypt depth, and mucosal area. In addition, HM15912 treated group was associated with a significant increase in absorption capacity of SI such as plasma D-xylose concentrations. Furthermore, the mice given HM15912 with various administration intervals for 2 weeks significantly increased SI weight compared to b.i.d treatment of teduglutide (35% over vehicle). In Q2D, Q4D and Q1W dosing intervals of HM15912, SI weights were dose-dependently increased 66∼112% (p &amp;lt; 0.001), 91∼103% (p &amp;lt; 0.001) and 55∼74% over vehicle (p &amp;lt; 0.05∼0.001), respectively. The results supported that small bowel hypertrophic effect of HM15912 are well-correlated with functional improvement of SI in short bowel condition, and superior efficacy to teduglutide was still observed even after once weekly administration in mice. Therefore, HM15912 could be a novel therapeutic option for SBS by providing remarkable small bowel tropic effect with extended administration interval. Presentation: Saturday, June 17, 2023

Niclosamide Attenuates Inflammation-Associated Profibrotic Responses in Human Subepithelial Lung Myofibroblasts.

Niclosamide is a commonly used helminthicidic drug for the treatment of human parasitosis by helminths. Recently, efforts have been focusing on repurposing this drug for the treatment of other diseases, such as idiopathic pulmonary fibrosis. Subepithelial lung myofibroblasts (SELMs) isolated from tissue biopsies of patients undergoing surgery for lung cancer were stimulated with TNF-α (50 ng/mL), IL-1α (5 ng/mL), added alone or in combination, and TGF-β1 (5 ng/mL). After treatment with niclosamide at 30 nM and 100 nM concentrations, expression of collagen type I, collagen type III, and fibronectin was studied by total RNA isolation and qRT-PCR and protein collagen secretion with the use of Sircol collagen assay. The migration of SELMs was assessed by a wound-healing assay. Niclosamide had no effect on baseline SELM fibrotic factor expression. When stimulated with TGF-β1, IL-1α, and/or TNF-α, SELM expression of collagen type I, type III, and fibronectin were upregulated, as was the secretion of total collagen in the culture medium. Treatment with niclosamide attenuated the effects of cytokine stimulation leading to a notable decrease in the mRNA expression of collagen type I, type III, and fibronectin in a concentration-dependent manner. SELM collagen secretion was also reduced by niclosamide at 100 nM concentration when examined at the protein level. Migration of both TGF-β1 stimulated and unstimulated SELMs was also inhibited by niclosamide. In this study, we highlight the anti-fibrotic properties of niclosamide on SELMs under stimulation with pro-fibrotic and pro-inflammatory cytokines, thus proposing this compound as a possible new therapeutic agent against lung fibrosis.

A non-invasive strategy for suppressing asthmatic airway inflammation and remodeling: Inhalation of nebulized hypoxic hUCMSC-derived extracellular vesicles.

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) are extremely promising nanoscale cell-free therapeutic agents. We previously identified that intravenous administration (IV) of human umbilical cord MSC-EVs (hUCMSC-EVs), especially hypoxic hUCMSC-EVs (Hypo-EVs), could suppress allergic airway inflammation and remodeling. Here, we further investigated the therapeutic effects of Hypo-EVs administration by atomizing inhalation (INH), which is a non-invasive and efficient drug delivery method for lung diseases. We found that nebulized Hypo-EVs produced by the atomization system (medical/household air compressor and nebulizer) maintained excellent structural integrity. Nebulized Dir-labeled Hypo-EVs inhaled by mice were mainly restricted to lungs. INH administration of Hypo-EVs significantly reduced the airway inflammatory infiltration, decreased the levels of IL-4, IL-5 and IL-13 in bronchoalveolar lavage fluid (BALF), declined the content of OVA-specific IgE in serum, attenuated the goblet cell metaplasia, and the expressions of subepithelial collagen-1 and α-smooth muscle actin (α-SMA). Notably, Hypo-EV INH administration was generally more potent than Hypo-EV IV in suppressing IL-13 levels and collagen-1 and α-SMA expressions. RNA sequencing revealed that various biological processes, such as cell adhesion, innate immune response, B cell activation, and extracellular space, were associated with the activity of Hypo-EV INH against asthma mice. In addition, Hypo-EVs could load exogenous miR-146a-5p (miR-146a-5p-EVs). Furthermore, INH administration of miR-146a-5p-EVs resulted in a significantly increased expression of miR-146a-5p mostly in lungs, and offered greater protection against the OVA-induced increase in airway inflammation, subepithelial collagen accumulation and myofibroblast compared with nebulized Hypo-EVs. Overall, nebulized Hypo-EVs effectively attenuated allergic airway inflammation and remodeling, potentially creating a non-invasive route for the use of MSC-EVs in asthma treatment.

Smooth muscle contributes to the development and function of a layered intestinal stem cell niche.

Wnt and Rspondin (RSPO) signaling drives proliferation, and bone morphogenetic protein inhibitors (BMPi) impede differentiation, of intestinal stem cells (ISCs). Here, we identify the mouse ISC niche as a complex, multi-layered structure that encompasses distinct mesenchymal and smooth muscle populations. In young and adult mice, diverse sub-cryptal cells provide redundant ISC-supportive factors; few of these are restricted to single cell types. Niche functions refine during postnatal crypt morphogenesis, in part to oppose the dense aggregation of differentiation-promoting BMP+ sub-epithelial myofibroblasts at crypt-villus junctions. Muscularis mucosae, a specialized muscle layer, first appears during this period and supplements neighboring RSPO and BMPi sources. Components of this developing niche are conserved in human fetuses. The invivo ablation of mouse postnatal smooth muscle increases BMP signaling activity, potently limiting a pre-weaning burst of crypt fission. Thus, distinct and progressively specialized mesenchymal cells together create the milieu that is required to propagate crypts during rapid organ growth and to sustain adult ISCs.

P099 IL-33 activates CD73-expressing cells promoting tumorigenesis during colitis-associated colorectal cancer

Abstract Background Colorectal cancer associated to chronic colitis (CAC) has a different pathogenesis compared to sporadic or familial colorectal cancer and represents the major complication of IBD. IL-33 and its receptor, ST2, are important factors in the pathogenesis of IBD. Emerging evidence suggests the potential contribution to inflammation-driven tumorigenesis that can lead to CAC. The aim of our study was to characterize the precise contribution of IL-33/ST2 axis in the azoxymethane (AOM)/dextran sodium sulfate (DSS) model of CAC. Methods C57/BL6 wild-type (WT), Il33-/-, T1/St2-/- and Nt5e-/- mice were given a single dose of AOM followed by two cycles of 3% DSS for 7d in drinking water. Vehicle-treated WT mice served as controls and were sacrificed at the same time points. Another group of WT followed the same protocol and received CD73 inhibitor i.p. treatment or vehicle. Disease Activity Index, endoscopic, stereomicroscopic and histological evaluations of colons were performed. IHC, immunofluorescence (IF), qPCR, and ELISA were done on full-thickness colons. RNA-Seq was performed on whole tissues from AOM/DSS treated WT, Il33-/- and T1/St2-/-. qPCR analysis was done on isolated polyps from WT, Il33-/- and T1/St2-/- for Nt5e (CD73) and adenosine pathway targets. Results Il33, Ilrl1(ST2L), and Ilrl1(sST2) mRNA transcripts, as well as IL-33 and total ST2 proteins were dramatically elevated in AOM/DSS-treated WT mice vs. controls. IHC and IF of treated WT mice revealed localization of IL-33 to the colonic epithelium and to cells within the polyp LP morphologically consistent with stromal and mast cells. Using IF, IL-33 co-localized with sub-epithelial myofibroblast markers Actin and Vimentin, or with mast cell markers Tryptase and MCPT1. AOM/DSS treatment in Il33-/- and T1/St2-/- mice resulted in a significant decreased polyp number and size vs. WT, with colonoscopy revealing the development of protruding lesions with abnormal vascular patterns, suggesting tumorous lesions in WT mice, while all deficient mice showed their absence with a more impressive mucosal inflammation, likely due to reduced epithelial proliferation and repair caused by the deficiency of IL-33 signaling. RNA-Seq identified a significant reduction of Nt5e and adenosine pathway targets in Il33-/- and T1/St2-/-vs. WT. qPCR on isolated polyps confirmed this observation. AOM/DSS-treated Nt5e-/- showed a significant decreased polyp number and size vs. controls. Therapeutic inhibition of CD73 produced similar results. Conclusion Our results suggest that the IL-33/ST2 axis promotes tumorigenesis in CAC through the activation of CD73. Further studies are underway to determine mechanisms of action that support these findings.

Amphiregulin Derived from Multiple Sources Is Important for Barrier Function of the Intestine Following Hematopoietic Stem Cell Transplantation

Conditioning therapy prior to allogeneic hematopoietic stem cell transplantation (allo-SCT) causes damage to the intestinal epithelia, leading to the loss of barrier function. Amphiregulin (AREG), a member of the epidermal growth factor (EGF) family. The EGF family plays an important role in the proliferation and differentiation of the intestinal epithelia1. We have previously shown that co-transplantation of activated type 2 innate lymphoid cells (ILC2) reduced mortality in murine acute GvHD, improved intestinal barrier function following allo-SCT and that ILC2 production of AREG was critical for intestinal repair and restoration of intestinal barrier2. Here we show that isolated intestinal stem cell crypts (ISC) grow and expand significantly when co-cultured with activated ILC2s compared to untreated. We see similar growth in cultures containing media that was conditioned with ILC2 cells for 48 hours prior to use in the organoid cultures. However, both ILC2 culture conditions as well as cultures containing EGF itself. In other experiments ISC co-cultures containing media conditioned with AREG deficient ILC2, IL-13 deficient ILC2 and AREG / IL-13 deficient ILC2 there is significant reduction of organoid growth. These findings further support the role of ILC2 cells during intestinal repair following allo-SCT. ILC2 cells are not the only source of AREG along the GI tract. Intestinal subepithelial myofibroblasts (ISEMF) have been shown to produce AREG in response to radiation treatment in mice.1 To investigate the role of AREG derived from ISEMFs we used a conditional knockout Myh11Cre+/Aregfl/fl strain3 in a B10.BR in to B6 model of acute GvHD. While Myh11Cre+/Aregfl/fl recipients of bone marrow alone all survived long term, Myh11Cre+/Aregfl/fl recipients that also received T cells had significantly increased mortality compared to WT B6 following allo-SCT with a median survival of 10 days and 21 days respectively. Myh11Cre+/Aregfl/fl BM+T recipients also had significantly higher clinical scores than WT B6 controls. Even though Myh11Cre+/Aregfl/fl recipients of BM only did survive long term they did have significantly increased clinical scores in the first 15 days when compared to WT B6. These findings indicate that ISEMF derived AREG is extremely important for early tissue repair following conditioning therapy and allo-SCT. In summary, ILC2s represent a cell population that produce AREG and are required for recovery of GI tract homeostasis following allo-SCT. ISEMFs are also an important source of AREG following transplantation. Additional investigation is needed to fully understand the role of AREG in intestinal health and recovery following allo-SCT.

The Probiotic Strains Bifidοbacterium lactis, Lactobacillus acidophilus, Lactiplantibacillus plantarum and Saccharomyces boulardii Regulate Wound Healing and Chemokine Responses in Human Intestinal Subepithelial Myofibroblasts

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.

Amniotic Fluid-Derived Mesenchymal Stem/Stromal Cell-Derived Secretome and Exosomes Improve Inflammation in Human Intestinal Subepithelial Myofibroblasts.

Inflammatory Bowel Diseases (IBDs) are characterized by chronic relapsing inflammation of the gastrointestinal tract. The mesenchymal stem/stromal cell-derived secretome and secreted extracellular vesicles may offer novel therapeutic opportunities in patients with IBD. Thus, exosomes may be utilized as a novel cell-free approach for IBD therapy. The aim of our study was to examine the possible anti-inflammatory effects of secretome/exosomes on an IBD-relevant, in vitro model of LPS-induced inflammation in human intestinal SubEpithelial MyoFibroblasts (SEMFs). The tested CM (Conditioned Media)/exosomes derived from a specific population of second-trimester amniotic fluid mesenchymal stem/stromal cells, the spindle-shaped amniotic fluid MSCs (SS-AF-MSCs), and specifically, their secreted exosomes could be utilized as a novel cell-free approach for IBD therapy. Therefore, we studied the effect of SS-AF-MSCs CM and exosomes on LPS-induced inflammation in SEMF cells. SS-AF-MSCs CM and exosomes were collected, concentrated, and then delivered into the cell cultures. Administration of both secretome and exosomes derived from SS-AF-MSCs reduced the severity of LPS-induced inflammation. Specifically, IL-1β, IL-6, TNF-α, and TLR-4 mRNA expression was decreased, while the anti-inflammatory IL-10 was elevated. Our results were also verified at the protein level, as secretion of IL-1β was significantly reduced. Overall, our results highlight a cell-free and anti-inflammatory therapeutic agent for potential use in IBD therapy.

Inflammatory and Proliferative Pathway Activation in Human Esophageal Myofibroblasts Treated with Acidic Bile Salts.

Subepithelial human esophageal myofibroblasts (HEMFs) in gastroesophageal reflux disease (GERD) are exposed to luminal contents via impaired squamous epithelium barrier integrity. The supernatant of HEMFs treated with acidic bile salts reflective of in vivo reflux increases squamous epithelial thickness. We aimed to identify the involved mechanisms using an unbiased approach. Acidic-bile-salt-treated primary HEMF cultures (n = 4) were submitted for RNA-Seq and analyzed with Partek Flow followed by Ingenuity Pathway Analysis (IPA). A total of 1165 molecules (579 downregulated, 586 upregulated) were differentially expressed, with most top regulated molecules either extracellular or in the plasma membrane. Increases in HEMF CXCL-8, IL-6, AREG, and EREG mRNA, and protein secretion were confirmed. Top identified canonical pathways were agranulocyte and granulocyte adhesion and diapedesis, PI3K/AKT signaling, CCR5 signaling in macrophages, and the STAT3 pathway. Top diseases and biological functions were cellular growth and development, hematopoiesis, immune cell trafficking, and cell-mediated response. The targets of the top upstream regulator ErbB2 included CXCL-8, IL-6, and AREG and the inhibition of CXCL-8 in the HEMF supernatant decreased squamous epithelial proliferation. Our work shows an inflammatory/immune cell and proliferative pathways activation in HEMFs in the GERD environment and identifies CXCL-8 as a HEMF-derived chemokine with paracrine proliferative effects on squamous epithelium.

Retinoic Acid Promotes the In Vitro Growth, Patterning and Improves the Cellular Composition of Human Pluripotent Stem-Cell-Derived Intestinal Organoids.

Human intestinal organoids (HIOs) generated from human pluripotent stem cells hold great promise for modeling human development and as a possible source of tissue for transplantation. HIOs generate all of the main epithelial and mesenchymal cell types found in the developing human intestine and mature into intestinal tissue with crypts and villi following transplantation into immunocompromised mice. However, incomplete in vitro patterning and the presence of contaminating neurons could hinder their use for regenerative medicine in humans. Based on studies in model organisms, we hypothesized that the treatment of HIOs with all trans retinoic acid (ATRA) would improve their in vitro growth and patterning. We found that ATRA not only improved the patterning of HIOs, ATRA also increased organoid forming efficiency, improved epithelial growth, enriched intestinal subepithelial myofibroblasts (ISEMFs) and reduced neuronal contamination in HIOs. Taken together, our studies demonstrate how the manipulation of a single developmental signaling pathway can be used to improve the survival, patterning and cellular composition of HIOs.

Anti-Inflammatory and Anti-Fibrotic Effect of Immortalized Mesenchymal-Stem-Cell-Derived Conditioned Medium on Human Lung Myofibroblasts and Epithelial Cells.

Idiopathic pulmonary fibrosis (IPF) is caused by progressive lung tissue impairment due to extended chronic fibrosis, and it has no known effective treatment. The use of conditioned media (CM) from an immortalized human adipose mesenchymal stem cell line could be a promising therapeutic strategy, as it can reduce both fibrotic and inflammatory responses. We aimed to investigate the anti-inflammatory and anti-fibrotic effect of CM on human pulmonary subepithelial myofibroblasts (hPSM) and on A549 pulmonary epithelial cells, treated with pro-inflammatory or pro-fibrotic mediators. CM inhibited the proinflammatory cytokine-induced mRNA and protein production of various chemokines in both hPSMs and A549 cells. It also downregulated the mRNA expression of IL-1α, but upregulated IL-1β and IL-6 mRNA production in both cell types. CM downregulated the pro-fibrotic-induced mRNA expression of collagen Type III and the migration rate of hPSMs, but upregulated fibronectin mRNA production and the total protein collagen secretion. CM’s direct effect on the chemotaxis and cell recruitment of immune-associated cells, and its indirect effect on fibrosis through the significant decrease in the migration capacity of hPSMs, makes it a plausible candidate for further development towards a therapeutic treatment for IPF.

P093 Probiotic strains Lactobacillus plantarum and Saccharomyces boulardii influence the fibrotic responses of human intestinal subepithelial myofibroblasts

Abstract Background The strains Lactobacillus plantarum and Saccharomyces boulardii, widely used among probiotic supplements, seem to ameliorate Inflammatory Bowel Disease (IBD) progression and to promote mucosal healing. Intestinal Subepithelial Myofibroblasts (SEMFs) are key mediators in mucosal healing and fibrosis, mainly by migrating to the trauma region and secreting collagen. The aim of this study was to investigate the different effects of the aforementioned probiotic strains in the migration rate and the fibrotic responses of SEMFs. Methods L. plantarum and S. boulardii were reconstituted in SEMFs culture medium, identified by Gram staining and their viability was assessed by Trypan Blue staining. Primary SEMFs were isolated from colonic biopsies from healthy individuals and stimulated with 102 and 104 cfu/ml of each strain for: a) 6h, when mRNA expression of collagen type I, III, fibronectin, Tissue Factor and α-SMA was assessed by quantitative PCR, and b) 48h, when SEMFs’ migration rate was examined via Wound Healing Assay and protein collagen was measured via Sircol Assay. Results Trypan Blue staining confirmed the viability of the studied probiotics, and both the bacteria and the yeasts were stained Gram positive. Stimulation of SEMFs with L. plantarum resulted in upregulated mRNAs of Collagen type I (102cfu/ml: 1.6-fold, 104cfu/ml: 1.5-fold; p&amp;lt;0.001), type III (102cfu/ml: 1.7-fold, 104cfu/ml: 1.8-fold; p&amp;lt;0.0001), Fibronectin (102 cfu/ml, 1.5-fold, 104cfu/ml 1.5-fold; p&amp;lt;0.01), Tissue Factor (102cfu/ml: 1.4-fold, p&amp;lt;0.0001; 104cfu/ml: 1.3-fold, p&amp;lt;0.001) as well as SEMFs’ migration rate (102cfu/ml: 128.3%, 104cfu/ml: 124.1%; p&amp;lt;0.05). On the other hand, S. boulardii stimulation resulted in downregulated Collagen type I (102cfu/ml: 0.9-fold, p&amp;lt;0.01) and type III mRNAs (102cfu/ml: 0.8-fold, p&amp;lt;0.01), upregulated Tissue Factor mRNA (102cfu/ml: 1.2-fold, p&amp;lt;0.0001; 104cfu/ml: 1.1-fold, p&amp;lt;0.01), increased protein collagen production (104cfu/ml: by 211.6%, p&amp;lt;0.001) and increased SEMFs’ migration rate (104cfu/ml: 130.2%, p&amp;lt;0.05). Conclusion This study provides evidence that not only both probiotic strains remain viable in SEMFs’ culture medium but are also able to interact with SEMFs and alter their fibrotic behaviour. Moreover, it was shown that L. plantarum may promote mucosal healing, while S. boulardii could reduce profibrotic responses. Further preclinical research is needed to identify the optimal dosages and combinations of probiotic strains as potential auxiliary treatment of IBD. The study was supported by «Strategic expansion of the Greek Biobanking Infrastructure» (BBMRI-GR) in the framework of the Action “ENHANCING RESEARCH AND INNOVATION INFRASTRUCTURE - SECOND CYCLE” (NSRF 2014–2020) and “AMKE KLEON TSETIS” Foundation, Athens, Greece.

P012 The effect of Oncostatin M on human intestinal organoids

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.

Novel agonist and antagonist radioligands for the GLP-2 receptor. Useful tools for studies of basic GLP-2 receptor pharmacology.

BackgroundGlucagon‐like peptide‐2 (GLP‐2) is a pro‐glucagon‐derived hormone secreted from intestinal enteroendocrine L cells with actions on gut and bones. GLP‐2(1–33) is cleaved by DPP‐4, forming GLP‐2(3–33), having low intrinsic activity and competitive antagonism properties at GLP‐2 receptors. We created radioligands based on these two molecules.Experimental approachThe methionine in position 10 of GLP‐2(1–33) and GLP‐2(3–33) was substituted with tyrosine (M10Y) enabling oxidative iodination, creating [125I]‐hGLP‐2(1–33,M10Y) and [125I]‐hGLP‐2(3–33,M10Y). Both were characterized by competition binding, on‐and‐off‐rate determination and receptor activation. Receptor expression was determined by target‐tissue autoradiography and immunohistochemistry.Key resultsBoth M10Y‐substituted peptides induced cAMP production via the GLP‐2 receptor comparable to the wildtype peptides. GLP‐2(3–33,M10Y) maintained the antagonistic properties of GLP‐2(3–33). However, hGLP‐2(1–33,M10Y) had lower arrestin recruitment than hGLP‐2(1–33). High affinities for the hGLP‐2 receptor were observed using [125I]‐hGLP‐2(1–33,M10Y) and [125I]‐hGLP‐2(3–33,M10Y) with K D values of 59.3 and 40.6 nM. The latter (with antagonistic properties) had higher B max and faster on and off rates compared to the former (full agonist). Both bound the hGLP‐1 receptor with low affinity (K i of 130 and 330 nM, respectively). Autoradiography in wildtype mice revealed strong labelling of subepithelial myofibroblasts, confirmed by immunohistochemistry using a GLP‐2 receptor specific antibody that in turn was confirmed in GLP‐2 receptor knock‐out mice.Conclusion and implicationsTwo new radioligands with different binding kinetics, one a full agonist and the other a weak partial agonist with antagonistic properties were developed and subepithelial myofibroblasts identified as a major site for GLP‐2 receptor expression.