Epithelium In Colitis Research Articles

Vanillic acid restores homeostasis of intestinal epithelium in colitis through inhibiting CA9/STIM1-mediated ferroptosis

The damage of integrated epithelial epithelium is a key pathogenic factor and closely associated with the recurrence of ulcerative colitis (UC). Here, we reported that vanillic acid (VA) exerted potent therapeutic effects on DSS-induced colitis by restoring intestinal epithelium homeostasis via the inhibition of ferroptosis. By the CETSA assay and DARTS assay, we identified carbonic anhydrase IX (CAIX, CA9) as the direct target of VA. The binding of VA to CA9 causes insulin-induced gene-2 (INSIG2) to interact with stromal interaction molecule 1 (STIM1), rather than SREBP cleavage-activating protein (SCAP), leading to the translocation of SCAP-SREBP1 from the endoplasmic reticulum (ER) to the Golgi apparatus for cleavage into mature SREBP1. The activation of SREBP1 induced by VA then significantly facilitated the transcription of stearoyl-CoA desaturase 1 (SCD1) to exert an inhibitory effect on ferroptosis. By inhibiting the excessive death of intestinal epithelial cells caused by ferroptosis, VA effectively preserved the integrity of intestinal barrier and prevented the progression of unresolved inflammation. In conclusion, our study demonstrated that VA could alleviate colitis by restoring intestinal epithelium homeostasis through CA9/STIM1-mediated inhibition of ferroptosis, providing a promising therapeutic candidate for UC.

TRPM8 inhibits substance P release from primary sensory neurons via PKA/GSK-3beta to protect colonic epithelium in colitis

Transient receptor potential melastatin 8 (TRPM8) is a cold sensory receptor in primary sensory neurons that regulates various neuronal functions. Substance P (SP) is a pro-inflammatory neuropeptide secreted by the neurons, and it aggravates colitis. However, the regulatory role of TRPM8 in SP release is still unclear. Our study aimed to investigate TRPM8’s role in SP release from primary sensory neurons during colitis and clarify the effect of SP on colonic epithelium. We analyzed inflammatory bowel disease patients’ data from the Gene Expression Omnibus dataset. Dextran sulfate sodium (DSS, 2.5%)-induced colitis in mice, mouse dorsal root ganglion (DRG) neurons, ND7/23 cell line, and mouse or human colonic organoids were used for this experiment. Our study found that TRPM8, TAC1 and WNT3A expression were significantly correlated with the severity of ulcerative colitis in patients and DSS-induced colitis in mice. The TRPM8 agonist (menthol) and the SP receptor antagonist (Aprepitant) can attenuate colitis in mice, but the effects were not additive. Menthol promoted calcium ion influx in mouse DRG neurons and inhibited the combination and phosphorylation of PKAca from the cAMP signaling pathway and GSK-3β from the Wnt/β-catenin signaling pathway, thereby inhibiting the effect of Wnt3a-driven β-catenin on promoting SP release in ND7/23 cells. Long-term stimulation with SP inhibited proliferation and enhanced apoptosis in both mouse and human colonic organoids. Conclusively, TRPM8 inhibits SP release from primary sensory neurons by inhibiting the interaction between PKAca and GSK-3β, thereby inhibiting the role of SP in promoting colonic epithelial apoptosis and relieving colitis.

Использование кишечных органоидов человека при воспалительных заболеваниях кишечника: от экспериментальной модели к регенеративной терапии

Despite advances in the treatment of inflammatory bowel disease (IBD), current therapies have limited efficacy and can cause serious adverse reactions, which not only reduces the quality of life but also results in shorter life expectancy for patients. Therefore, it is necessary to develop new methods of diagnosis and find more effective ways of treatment. A significant breakthrough in the study of intestinal epithelial cells in various pathologies, including IBS, has been achieved by the establishment of three-dimensional (3D) organoid cultures that in vitro recreate intestinal epithelial tissue. Organoid cultures are derived from either somatic stem cells or pluripotent stem cells. These cells, grown under appropriate conditions, self-organize into specific spherical tissue structures with the properties and functions of gastrointestinal tissues. It has been shown that intestinal organoids may be a promising tool for assessing the molecular and functional impact on the intestinal epithelium of genetic and epigenetic changes associated with the gastrointestinal tract. To objectively evaluate the functional status of the intestinal epithelium in IBD, one needs to present organoid models together with the intestinal microbiota in order to determine the specific effect of bacteria on the epithelial barrier. Intestinal organoids are also being used to explore novel strategies for both direct drug exposure to the intestinal epithelium and targeted drug delivery, including nanoparticle and microparticle-based approaches, thereby reducing the limitations of systemic treatment. Finally, organoids serve as the basis of regenerative medicine for the treatment of IBD. New treatment techniques can be applied in patients with IBD in combination with immunoregulatory therapy and contribute to the achievement of prolonged clinical remission. Key words: inflammatory bowel disease, Crohn’s disease, ulcerative colitis, organoid cultures, diagnosis, stem cells, regenerative medicine

Patient-derived Colonoids From Disease-spared Tissue Retain Inflammatory Bowel Disease-specific Transcriptomic Signatures.

A key histopathological feature of inflammatory bowel disease is damage to the mucosa, including breakdown of the epithelial barrier. Human enteroids and colonoids are a critical bench-to-bedside tool for studying the epithelium in inflammatory bowel disease. The goal of the current study was to define transcriptional differences in healthy versus diseased subjects that are sustained in enteroids and colonoids, including from disease-spared tissue. Biopsies and matching enteroid or colonoid cultures from pediatric patients with ileal Crohn disease (N = 6) and control subjects (N = 17) were subjected to RNA sequencing followed by bioinformatic and machine learning analyses. Late passage enteroids were exposed to cytokines to assess durable transcriptional differences. We observed substantial overlap of pathways upregulated in Crohn disease in enteroids and ileal biopsies, as well as colonoids and rectal biopsies. KEGG pathways for cytokine-cytokine receptor interaction, chemokine signaling, protein export, and Toll-like receptor signaling were upregulated in both ileal and rectal biopsies, as well as enteroids and colonoids. In vitro cytokine exposure reactivated genes previously increased in biopsies. Machine learning predicted biopsy location (100% accuracy) and donor disease status (83% accuracy). A random forest classifier generated using ileal enteroids identified rectal colonoids from ileal Crohn disease subjects with 80% accuracy. We confirmed transcriptional profiles of Crohn disease biopsies are expressed in enteroids and colonoids. Furthermore, transcriptomic data from disease-spared rectal tissue can identify patients with ileal Crohn disease. Our data support the use of patient enteroids and colonoids as critical translational tools for the study of inflammatory bowel disease.

Group 3 innate lymphoid cells produce the growth factor HB-EGF to protect the intestine from TNF-mediated inflammation.

Tumor necrosis factor (TNF) drives chronic inflammation and cell death in the intestine, and blocking TNF is a therapeutic approach in inflammatory bowel disease (IBD). Despite this knowledge, the pathways that protect the intestine from TNF are incompletely understood. Here we demonstrate that group 3 innate lymphoid cells (ILC3s) protect the intestinal epithelium from TNF-induced cell death. This occurs independent of interleukin-22 (IL-22), and we identify that ILC3s are a dominant source of heparin-binding epidermal growth factor-like growth factor (HB-EGF). ILC3s produce HB-EGF in response to prostaglandin E2 (PGE2) and engagement of the EP2 receptor. Mice lacking ILC3-derived HB-EGF exhibit increased susceptibility to TNF-mediated epithelial cell death and experimental intestinal inflammation. Finally, human ILC3s produce HB-EGF and are reduced from the inflamed intestine. These results define an essential role for ILC3-derived HB-EGF in protecting the intestine from TNF and indicate that disruption of this pathway contributes to IBD.

P031 Intestinal lymphoepithelial interactions in Crohn’s disease: modeling alphaE-beta7 and NKG2D blockade

Abstract Background T resident memory (Trm) cells in the intestinal mucosa and in particular subpopulations expressing phenotypic markers such as alphaE-beta7 or NKG2D have been associated with chronic inflammatory bowel disease (IBD) activity. We hypothesize that these populations may have a direct deleterious impact on the intestinal epithelium in IBD. Methods The phenotypic study of mucosal lymphocytes was performed in two prospective cohorts: ELYP including patients with active IBD before initiation of biotherapy and REMIND including patients with ileal Crohn’s disease (CD) requiring ileocaecal resection. These analyses were performed before initiation of treatment and one year after continuous therapy in the ELYP cohort and on the resection specimen in the REMIND cohort. An innovative ex vivo autologous organoid-mucosal T cell coculture model was developed using the REMIND cohort specimens for patients and healthy ileum from individuals without IBD for controls (Figure 1). T cell infiltration within the organoid and epithelial cell death were assessed by confocal microscopy. A panel of 30 cytokines was quantified in the supernatants of cocultures. Figure 1 Results In the ELYP cohort, before the start of treatment, IBD patients had lower expression of alphaE-beta7 and NKG2D on mucosal CD8 T cells. While alphaE-beta7 and NKG2D expression on mucosal CD8 T cells had returned to normal in endoscopic responders, it remained decreased in non-responders. Similarly, the inflammatory mucosa of the REMIND surgical specimens had lower levels of CD4 and CD8 T cells expressing alphaE-beta7 and NKG2D compared with controls and non-inflamed regions. We developed a coculture model between mucosal lymphocytes and organoids generated under autologous conditions. We showed an increase in apoptotic cell death in epithelial cells from CD patients which was not found in control cocultures. There was a significant correlation between the degree of epithelial cell death and T cell infiltration (Figure 2). Various proinflammatory cytokines such as IFNgamma, TNFalpha, IL-6 and IL- 17a were also increased in the supernatants. The use of antibodies blocking the alphaE-beta7 and NKG2D pathways of interest inhibited this effect by different mechanisms (Figure 3) While anti-beta7 and anti-NKG2D had comparable effects in terms of cell death inhibition, only anti-beta7 reduced T cell infiltration. Anti-NKG2D had no effect on cell infiltration but a reduction of perforin was observed in the supernatants. Figure 2 Figure 3 CC= Coculture Conclusion These data demonstrate for the first time the direct cytotoxic deleterious effect of mucosal T cells on the epithelium of CD patients using a novel coculture model. AlphaE-beta7 and NKG2D pathways appear to be relevant in this process.

Development of Physiologically Responsive Human iPSC-Derived Intestinal Epithelium to Study Barrier Dysfunction in IBD.

In inflammatory bowel disease (IBD), the intestinal epithelium is characterized by increased permeability both in active disease and remission states. The genetic underpinnings of this increased intestinal permeability are largely unstudied, in part due to a lack of appropriate modelling systems. Our aim is to develop an in vitro model of intestinal permeability using induced pluripotent stem cell (iPSC)-derived human intestinal organoids (HIOs) and human colonic organoids (HCOs) to study barrier dysfunction. iPSCs were generated from healthy controls, adult onset IBD, and very early onset IBD (VEO-IBD) patients and differentiated into HIOs and HCOs. EpCAM+ selected cells were seeded onto Transwell inserts and barrier integrity studies were carried out in the presence or absence of pro-inflammatory cytokines TNFα and IFNγ. Quantitative real-time PCR (qRT-PCR), transmission electron microscopy (TEM), and immunofluorescence were used to determine altered tight and adherens junction protein expression or localization. Differentiation to HCO indicated an increased gene expression of CDX2, CD147, and CA2, and increased basal transepithelial electrical resistance compared to HIO. Permeability studies were carried out in HIO- and HCO-derived epithelium, and permeability of FD4 was significantly increased when exposed to TNFα and IFNγ. TEM and immunofluorescence imaging indicated a mislocalization of E-cadherin and ZO-1 in TNFα and IFNγ challenged organoids with a corresponding decrease in mRNA expression. Comparisons between HIO- and HCO-epithelium show a difference in gene expression, electrophysiology, and morphology: both are responsive to TNFα and IFNγ stimulation resulting in enhanced permeability, and changes in tight and adherens junction architecture. This data indicate that iPSC-derived HIOs and HCOs constitute an appropriate physiologically responsive model to study barrier dysfunction and the role of the epithelium in IBD and VEO-IBD.

P099 Neutrophil gelatinase-associated lipocalin (NGAL) as biomarker in collagenous colitis

Abstract Background Neutrophil gelatinase-associated lipocalin (NGAL) is upregulated in the intestinal epithelium in inflammatory bowel disease and is a biomarker with sensitivity and specificity comparable to calprotectin. Microscopic colitis (MC) is a common cause of chronic, watery diarrhoea and represents an inflammatory bowel disease with unknown aetiology and pathogenesis. Diagnosis depends on histological evaluation of colonic biopsies. There is a need for non-invasive diagnostic tools, and this study examines the potential of NGAL as a biomarker in collagenous colitis (CC) as one of the two main histological forms of MC. Methods Gene expression of colonic biopsies (n = 9/group) from active CC, budesonide treated CC in clinical remission and healthy controls (HC) was explored by RNA-sequencing analysis. An extended set of colonic biopsies (n = 17–29/group) was also examined by immunohistochemistry (IHC) and in situ hybridisation (ISH). Faecal samples from the same patient groups, in addition to samples from patients with irritable bowel disease diarrhoea (IBS-D), were assayed for NGAL and calprotectin (Calpro) by ELISA. Results The NGAL gene, LCN2, was significantly upregulated in active CC vs. HC (log2 2.694, fold change 6.471) adjusted p-value 0.005) and in pairs of active CC vs. treated CC LCN2 was significantly downregulated (log2 0.345, fold change −1.536), adjusted p-value 0.04). Both immunohistochemical staining and in situ hybridisation identified increased NGAL expression localised to the mucosal epithelial cells in active CC, compared with an almost absent and scarce expression in HC and treated CC, respectively. There were great individual differences in faecal concentrations of NGAL particularly in the active CC group, but the NGAL concentrations were significantly increased compared with HC, IBS-D and treated CC. Conclusion NGAL is upregulated and located mainly to the colonic epithelium of active CC and reduced in clinical remission after budesonide treatment. This is also reflected in the faecal concentrations. We propose NGAL as a valuable biomarker in evaluating the inflammatory activity related to CC and a potential faecal biomarker discriminating CC from IBS-D.

Potential Use of Human Stem Cell-Derived Intestinal Organoids to Study Inflammatory Bowel Diseases.

Inflammatory bowel disease (IBD) is a chronic remitting disorder with increasing incidence worldwide. The intestinal epithelial barrier plays a major role in IBD, contributing to its pathogenesis, evolution, and perpetuation over time. Until recently, studies focused on exploring the role of the intestinal epithelium in IBD were hampered by the lack of techniques for the long-term culturing of human primary epithelial cells ex vivo. Recently, however, a methodology for generating stable human 3D epithelial cultures directly from adult intestinal stem cells was established. These long-term cultures, called organoids, mimic the tissue of origin and can be generated from small-size intestinal tissue samples, making it a promising tool for modeling the course of IBD.In this review, we provide an overview of the versatility of human organoid cultures in IBD modeling. We discuss recent advances and current limitations in the application of this tool for modeling the contribution of the intestinal epithelium alone and in combination with other key cellular and molecular players in the context of IBD pathophysiology. Finally, we outline the pressing need for technically standardizing the laboratory manipulation of human epithelial organoids for their broader implementation in clinically oriented IBD studies.

G protein-coupled receptor 35 contributes to mucosal repair in mice via migration of colonic epithelial cells

G protein-coupled receptor 35 (GPR35), a receptor for lysophosphatidic acid, is highly expressed in the gastrointestinal tract. Recently, GPR35 has been implicated in the onset of inflammatory bowel disease (IBD), but its role in physiological and pathological processes in the colon remains undefined. In this study, we investigated the contribution of GPR35-mediated signalling to mucosal repair of colonic epithelium in IBD. GPR35 function was examined in a wound healing model, using young adult mouse colon epithelium (YAMC) cells, and in a dextran sulphate sodium (DSS)-induced mouse model of colitis. Cell proliferation, mRNA expression, extracellular signal-regulated kinase (ERK) activation, and protein localization were determined by MTT assay, quantitative RT-PCR, western blotting, and immunohistochemistry, respectively. GPR35 agonists (YE120, zaprinast, and pamoic acid) promoted wound repair in a concentration-dependent manner independently of cell proliferation, whereas a specific GPR35 antagonist CID2745687, forskolin, and pertussis toxin reversed the YE120-induced effect. YE120 increased the mRNA expression of fibronectin and its receptor integrin α5, and ERK1/2 phosphorylation, but these responses were attenuated by CID2745687 and forskolin. Furthermore, the severity of DSS-induced colitis was significantly reduced by daily injections of pamoic acid via upregulation of fibronectin and integrin α5 in the colonic epithelium. GPR35 signalling promotes mucosal repair by inducing fibronectin and integrin α5 expression, coupling to Gi protein, and activating ERK1/2 in colonic epithelial cells. These findings define GPR35 as a candidate therapeutic target in IBD.

P020 Wnt5b could contribute to regeneration of the epithelium in inflammatory bowel disease by potentiating epithelial mesenchymal transition

P020 Wnt5b could contribute to regeneration of the epithelium in inflammatory bowel disease by potentiating epithelial mesenchymal transition

When Insult Is Added to Injury: Cross Talk between ILCs and Intestinal Epithelium in IBD.

Inflammatory bowel disease (IBD) is characterized by an impairment of the integrity of the mucosal epithelial barrier, which causes exacerbated inflammation of the intestine. The intestinal barrier is formed by different specialized epithelial cells, which separate the intestinal lumen from the lamina propria. In addition to its crucial role in protecting the body from invading pathogens, the intestinal epithelium contributes to intestinal homeostasis by its biochemical properties and communication to underlying immune cells. Innate lymphoid cells (ILCs) are a recently described population of lymphocytes that have been implicated in both mucosal homeostasis and inflammation. Recent findings indicate a critical feedback loop in which damaged epithelium activates these innate immune cells to restore epithelial barrier function. This review will focus on the signalling pathways between damaged epithelium and ILCs involved in repair of the epithelial barrier and tissue homeostasis and the relationship of these processes with the control of IBD.

Macrophage‐Specific ErbB4 is Induced by DSS Colitis and Regulates Macrophage Survival

The ErbB4 receptor tyrosine kinase is induced in the colonic epithelium in inflammatory bowel disease (IBD), and the ErbB4-specific ligand neuregulin-4 is protective in murine colitis models. Interestingly, ErbB4 is also robustly detectable in the submucosal stroma where immune cells accumulate during inflammation. Previous studies have shown ErbB4 expression on circulating human monocytes and neuronal macrophages (Mø), but its expression on intestinal Mø and role in Mø biology are unknown. HYPOTHESIS: As Mø play a significant role in the development of acute colitis, we hypothesized that receptor-mediated ErbB4 signaling may be an anti-inflammatory mechanism to limit Mø numbers during inflammation. METHODS: Mice (C57Bl6) were given 3% dextran sodium sulfate (DSS) in drinking water for 4 days (injury phase) followed by 3 days without DSS (recovery phase); colonic Mø populations were characterized by flow cytometry. Murine bone marrow-derived Mø (BM-Mø) were generated for in vitro activation and survival studies. RESULTS: DSS induced colonic recruitment of F480+/CD11b+/Ly6C+/ErbB4+ cells in injury and recovery phase mice compared to controls. In vitro, classical activation (IFNγ/LPS) of BM-Mø, but not alternative activation (IL-4), induced ErbB4 expression 10-fold (*p<0.01). Treatment of these cells with neuregulin-4 (100 ng/ml) reduced activated BM-Mø numbers by 40% (*p<0.05) after 48 hours, an effect blocked by ErbB4 neutralizing antibody (2 μg/ml). CONCLUSION Colitis induces recruitment of ErbB4+ Mø. ErbB4 signaling is a possible inhibitory feedback mechanism restricting Mø numbers, and thus represents a potential therapeutic target to regulate inflammation in IBD.

Expression of Toll-like receptor-3 is enhanced in active inflammatory bowel disease and mediates the excessive release of lipocalin 2

Anti-microbial peptides might influence the pathogenesis and course of inflammatory bowel disease (IBD). We sought to clarify the role of the anti-microbial glycoprotein lipocalin 2 (LCN2) in the colon by determining its localization and regulation in IBD. Following a microarray gene expression study of colonic biopsies from a large IBD population (n = 133), LCN2 was localized using immunohistochemistry and in-situ hybridization. Moreover, we examined the regulation of LCN2 in HT-29 cells with a panel of pattern recognition receptors (PRRs) and sought evidence by immunohistochemistry that the most relevant PRR, the Toll-like receptor (TLR)-3, was indeed expressed in colonic epithelium in IBD. LCN2 was among the 10 most up-regulated genes in both active ulcerative colitis (UCa) and active Crohn's disease (CDa) versus healthy controls. LCN2 protein was found in both epithelial cells and infiltrating neutrophils, while mRNA synthesis was located solely to epithelial cells, indicating that de-novo synthesis and thus regulation of LCN2 as measured in the gene expression analysis takes place in the mucosal epithelial cells. LCN2 is a putative biomarker in faeces for intestinal inflammation, different from calprotectin due to its epithelial site of synthesis. LCN2 release from the colonic epithelial cell line HT-29 was enhanced by both interleukin (IL)-1β and the TLR-3 ligand poly(I:C), and TLR-3 was shown to be expressed constitutively in colonic epithelial cells and markedly increased during inflammation.

Non‐apoptotic function of caspases in a cellular model of hydrogen peroxide‐associated colitis

Oxidative stress, caused by reactive oxygen species (ROS), is a major contributor to inflammatory bowel disease (IBD)-associated neoplasia. We mimicked ROS exposure of the epithelium in IBD using non-tumour human colonic epithelial cells (HCEC) and hydrogen peroxide (H2O2). A population of HCEC survived H2O2-induced oxidative stress via JNK-dependent cell cycle arrests. Caspases, p21WAF1 and γ-H2AX were identified as JNK-regulated proteins. Up-regulation of caspases was linked to cell survival and not, as expected, to apoptosis. Inhibition using the pan-caspase inhibitor Z-VAD-FMK caused up-regulation of γ-H2AX, a DNA-damage sensor, indicating its negative regulation via caspases. Cell cycle analysis revealed an accumulation of HCEC in the G1-phase as first response to oxidative stress and increased S-phase population and then apoptosis as second response following caspase inhibition. Thus, caspases execute a non-apoptotic function by promoting cells through G1- and S-phase by overriding the G1/S- and intra-S checkpoints despite DNA-damage. This led to the accumulation of cells in the G2/M-phase and decreased apoptosis. Caspases mediate survival of oxidatively damaged HCEC via γ-H2AX suppression, although its direct proteolytic inactivation was excluded. Conversely, we found that oxidative stress led to caspase-dependent proteolytic degradation of the DNA-damage checkpoint protein ATM that is upstream of γ-H2AX. As a consequence, undetected DNA-damage and increased proliferation were found in repeatedly H2O2-exposed HCEC. Such features have been associated with neoplastic transformation and appear here to be mediated by a non-apoptotic function of caspases. Overexpression of upstream p-JNK in active ulcerative colitis also suggests a potential importance of this pathway in vivo.

AMP-18 facilitates assembly and stabilization of tight junctions to protect the colonic mucosal barrier

Inflammatory bowel disease (IBD) is characterized by an injured epithelium. Development of agents that could enhance mucosal healing is a major goal in IBD therapeutics. The 18-kDa antrum mucosal protein (AMP-18) and a 21-mer peptide derived from AMP-18 stimulate accumulation of tight junction (TJ) proteins in cultured epithelial cells and mouse colonic mucosa to protect the mucosal barrier, suggesting it might be a useful agent to treat IBD. We searched for molecular mechanisms by which AMP peptide or recombinant AMP-18 act on TJs in intact cell monolayers, or those disrupted by low-calcium medium. Roles of the p38 mitogen-activated protein kinase (MAPK) / heat shock protein (hsp)25 pathway and PKCζ were investigated by immunoblotting and confocal microscopy. AMP peptide activated p38 MAPK, which subsequently phosphorylated hsp25. Accumulated phospho-hsp25 was associated with perijunctional actin. AMP-18 also induced rapid phosphorylation of PKCζ and its colocalization with perijunctional actin in Caco2/bbe cells, which was accompanied by translocation and formation of complexes of "polarity proteins" in the TJ-containing detergent-insoluble fraction. Treatment with AMP-18 also stimulated accumulation of ZO-1, ZO-2, and JAM-A in nascent TJs known to associate with the multimeric p-PKCζ/Par6/ Cdc42/ECT2·GTP/Par3 polarity protein complex. AMP-18 facilitates translocation and assembly of multiple proteins into TJs and their association with and subsequent stabilization of the actin filament network. We speculate that improved barrier function induced by AMP-18 is mediated by enhanced TJ assembly. Thus, AMP-18 may provide a promising lead to develop agents effective in healing injured colonic epithelium in IBD.

Beneficial Effect of Glatiramer Acetate Treatment on Syndecan-1 Expression in Dextran Sodium Sulfate Colitis

Syndecan-1, the most abundant heparan sulfate proteoglycan in the gastrointestinal tract, is reduced in the regenerative epithelium in inflammatory bowel disease (IBD). This study explored the effects of the immunomodulator glatiramer acetate (GA; Copaxone) treatment on syndecan-1 expression in dextran sodium sulfate (DSS)-induced colitis. Acute and chronic colitis was induced in C57BL/6 mice by 2 and 1.5% DSS in tap water, respectively. GA was applied subcutaneously, 2 mg per mouse per day, starting on the day of DSS induction until the mice were sacrificed. Syndecan-1 expression was assessed by immunohistochemistry. The effect of adoptive transfer of GA-specific T cells as an organ-specific therapy also was evaluated. Syndecan-1 expression was significantly lower in both colitis groups compared with that in naive mice (p < 0.0001). GA attenuated clinical scores and pathological manifestations of colitis and led to the reinstatement of normal levels of syndecan-1. After adoptive transfer, GA-specific cells homed to the surface epithelium of the distal colon, accompanied by the augmentation of syndecan-1 staining in their vicinity. We concluded that syndecan-1 expression is reduced in DSS-induced colitis and could be a potential prognostic factor in IBD. Treatment with GA exerts not only an anti-inflammatory effect but also a possible beneficial effect in stabilizing the intestinal epithelium barrier and tissue repair in DSS colitis. GA may be applied as a novel drug for IBD, shifting treatment from immunosuppression toward immunomodulation.

Increased Expression of Hypoxia-Inducible Factor 1α in Coeliac Disease

Previously, it has been suggested that hypoxia-inducible factor (HIF) 1 signaling may play determinative role in the maintenance of the barrier function of the intestinal epithelium in inflammatory bowel disease. Our aim was to depict the alteration of HIF-1alpha and related genes in celiac disease (CD) where the importance of the barrier function is well known. Duodenal biopsy specimens were collected from 16 children with untreated CD, 9 children with treated CD and 10 controls. HIF-1alpha, trefoil factor 1 (TFF1), ecto-5-prime nucleotidase (CD73), and multi drug resistance gene 1 (MDR1) mRNA and HIF-1alpha protein expression were determined by real-time PCR and Western blot, respectively. Localization of HIF-1alpha was determined by immunofluorescent staining. We found increased HIF-1alpha and TFF1 mRNA and HIF-1alpha protein expression in the duodenal mucosa of children with untreated CD compared with controls or children with treated CD (p < 0.05). In untreated CD children, HIF-1alpha staining was present in cytoplasmic and nuclear region of the villous enterocytes. In treated CD mRNA expression of CD73 and MDR1 were increased compared with controls (p < 0.01 and 0.05, respectively). Our results of increased mucosal HIF-1alpha expression in CD children suggest the contribution of this signaling pathway in the pathomechanism of CD.

Differential expression of the TRAIL/TRAIL-receptor system in patients with inflammatory bowel disease

TNF-related apoptosis inducing-ligand (TRAIL) is a potent inducer of apoptosis and plays an important role in immune regulation. To explore the role of TRAIL in inflammatory bowel disease (IBD), we examined the expression of the TRAIL/TRAIL-receptor system in colonic resections from patients with ulcerative colitis and Crohn's disease in comparison to normal colon and appendicitis. TRAIL and TRAIL-receptor (TRAIL-R) expression was assessed in resections of normal colon, colon of IBD patients, and appendicitis by immunohistochemistry. TRAIL was downregulated in enterocytes of patients with IBD, but was upregulated in mononuclear cells in areas of active mucosal inflammation. For TRAIL-R1, we detected a strong downregulation in the surface epithelium in IBD but not in appendicitis. TRAIL-R2 and TRAIL-R4 were strongly downregulated in the surface epithelium in any kind of mucosal inflammation. TRAIL and TRAIL-R1 are downregulated in enterocytes, and TRAIL is upregulated in mononuclear cells only in IBD but not in normal colon or appendicitis. This may point to a pathophysiologic role of the TRAIL system in inflammatory bowel disease.

Heparanase upregulation by colonic epithelium in inflammatory bowel disease

AbstractHeparanase is an endo-β-D-glucuronidase capable of cleaving heparan sulfate (HS) side chains at a limited number of sites, yielding HS fragments of still appreciable size (∼5–7 kDa). Heparanase activity has long been detected in a number of cell types and tissues. Importantly, heparanase activity correlated with the metastatic potential of tumor-derived cells, attributed to enhanced cell dissemination as a consequence of HS cleavage and remodeling of the extracellular matrix barrier. Similarly, heparanase activity was implicated in neovascularization, inflammation and autoimmunity, involving migration of vascular endothelial cells and activated cells of the immune system. The involvement of heparanase in inflammatory processes of the gastrointestinal tract has not been examined. Here, we utilized immunohistochemical analysis to investigate heparanase expression in acute and chronic inflammatory conditions. Heparanase expression was not detected in specimens derived from normal colon tissue. In contrast, strong heparanase staining was observed in Crohn’s disease and ulcerative colitis, but not in infectious colitis. Interestingly, heparanase staining was primarily observed in epithelial rather than immune cells. Importantly, un-fractionated as well as low molecular weight heparin (enoxaparin), which exhibit a strong inhibitory activity towards heparanase, have proven efficacious in ulcerative colitis and Crohn’s disease patients, suggesting that heparanase is actively involved in these pathologies and thus may be considered as a target for the development of anti-inflammatory therapies.