Human Inflammatory Bowel Disease Patients Research Articles

Longitudinal single-cell data informs deterministic modelling of inflammatory bowel disease

Single-cell-based methods such as flow cytometry or single-cell mRNA sequencing (scRNA-seq) allow deep molecular and cellular profiling of immunological processes. Despite their high throughput, however, these measurements represent only a snapshot in time. Here, we explore how longitudinal single-cell-based datasets can be used for deterministic ordinary differential equation (ODE)-based modelling to mechanistically describe immune dynamics. We derived longitudinal changes in cell numbers of colonic cell types during inflammatory bowel disease (IBD) from flow cytometry and scRNA-seq data of murine colitis using ODE-based models. Our mathematical model generalised well across different protocols and experimental techniques, and we hypothesised that the estimated model parameters reflect biological processes. We validated this prediction of cellular turnover rates with KI-67 staining and with gene expression information from the scRNA-seq data not used for model fitting. Finally, we tested the translational relevance of the mathematical model by deconvolution of longitudinal bulk mRNA-sequencing data from a cohort of human IBD patients treated with olamkicept. We found that neutrophil depletion may contribute to IBD patients entering remission. The predictive power of IBD deterministic modelling highlights its potential to advance our understanding of immune dynamics in health and disease.

Antioxidant and Anti-Inflammatory Phytochemicals for the Treatment of Inflammatory Bowel Disease: A Systematic Review

Inflammatory bowel disease (IBD) remains a burden for patients with increasing prevalence in industrialized countries. Phytochemicals are non-nutrient plant derived bioactive substances with antioxidant and anti-inflammatory effects that may prove beneficial to IBD patients. This review aims to overview current evidence on the application and impact of isolated phytochemicals or phytochemicals contained in plant extracts and essential oils on patients suffering from IBD. A systematic literature search was conducted for studies relating to the use of phytochemicals for the treatment of IBD. Ultimately, 37 human clinical trials and 3 systematic reviews providing human IBD patient data relevant to phytochemicals as therapeutic agents were included. Phytochemicals in the form of curcumin, Plantago ovata seeds, polyphenon E, silymarin, resveratrol supplements or an herbal preparation of myrrh, chamomile and coffee charcoal have evidence from human clinical trials supporting their safety and beneficial effects. Cannabinoids improve quality of life but not IBD outcomes. The addition of probiotics like B. longum to fructo-oligosaccharides promote healthy composition of the gut microbiome. Phytochemicals like mastiha, anthocyanins, berberine, tormentil, T2, ecabet sodium and Pycnogenol need more well-designed trials. Systematic research on phytochemicals can lead to the discovery of useful therapeutics. These secondary metabolites can be incorporated in current IBD treatment strategies to limit side effects, promote mucosal healing and provide higher quality of life to patients.

B cell-mediated CD4 T-cell costimulation via CD86 exacerbates pro-inflammatory cytokine production during autoimmune intestinal inflammation

Dysregulated B cell responses have been described in inflammatory-bowel disease (IBD) patients; however, the role of B cells in IBD pathology remained incompletely understood. We here provide evidence for a detrimental role of activated B cells during the onset of autoimmune intestinal inflammation. Using Wiskott-Aldrich Syndrome interacting protein deficient (Wipf1-/-) mice as a mouse model of chronic colitis, we identified CD86 expression on activated B cells as a crucial factor exacerbating pro-inflammatory cytokine production of intestinal CD4 T cells. Depleting B cells through anti-CD20 antibody treatment or blocking co-stimulatory signals mediated by CD86 through CTLA-4-Ig diminished intestinal inflammation in our mouse model of chronic IBD at the onset of disease. This was due to a reduction in aberrant humoral immune responses and reduced CD4 T cell pro-inflammatory cytokine production, especially IFN-γ and GM-CSF. Interestingly, in addition to B cells isolated from the inflamed colon of Wipf1-/- mice, we also found CD86 mRNA and protein expression up-regulated on activated B cells isolated from inflamed tissue of human IBD patients. B cell activation and CD86 expression was boosted by soluble CD40L in vitro, which we found in the serum of mice and human IBD patients. In summary, our data provides detailed insight into the contribution of B cells to intestinal inflammation, with implications for the treatment of IBD.

Epithelial SMYD5 Exaggerates IBD by Down-regulating Mitochondrial Functions via Post-Translational Control ofPGC-1α Stability.

Background & AimsThe expression and role of methyltransferase SET and MYND domain-containing protein 5 (SMYD5) in inflammatory bowel disease (IBD) is completely unknown. Here, we investigated the role and underlying mechanism of epithelial SMYD5 in IBD pathogenesis and progression.MethodsThe expression levels of SMYD5 and the mitochondrial transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) were examined by Western blot, immunofluorescence staining, and immunohistochemistry in intestinal epithelial cells (IECs) and in colon tissues from human IBD patients and colitic mice. Mice with Smyd5 conditional knockout in IECs and littermate controls were subjected to dextran sulfate sodium–induced colitis and the disease severity was assessed. SMYD5-regulated mitochondrial biogenesis was examined by quantitative reverse-transcription polymerase chain reaction and transmission electron microscopy, and the mitochondrial oxygen consumption rate was measured in a Seahorse Analyzer system (Agilent, Santa Clara, CA). SMYD5 and PGC-1α interaction was determined by co-immunoprecipitation assay. PGC-1α degradation and turnover (half-life) were analyzed by cycloheximide chase assay. SMYD5-mediated PGC-1α methylation was assessed via in vitro methylation assay followed by mass spectrometry for identification of methylated lysine residues.ResultsUp-regulated SMYD5 and down-regulated PGC-1α were observed in intestinal epithelia from IBD patients and colitic mice. Smyd5 depletion in IECs protected mice from dextran sulfate sodium–induced colitis. SMYD5 was critically involved in regulating mitochondrial biology such as mitochondrial biogenesis, respiration, and apoptosis. Mechanistically, SMYD5 regulates mitochondrial functions in a PGC-1α–dependent manner. Furthermore, SMYD5 mediates lysine methylation of PGC-1α and subsequently facilitates its ubiquitination and degradation.ConclusionsSMYD5 attenuates mitochondrial functions in IECs and promotes IBD progression by enhancing PGC-1α degradation in a methylation-dependent manner. Strategies to decrease SMYD5 expression and/or increase PGC-1α expression in IECs might be a promising therapeutic approach to treat IBD patients.

Spatiotemporal regulation of galectin-1-induced T-cell death in lamina propria from Crohn's disease and ulcerative colitis patients.

Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is characterized by chronic, relapsing intestinal inflammation. Galectin-1 (Gal-1) is an endogenous lectin with key pro-resolving roles, including induction of T-cell apoptosis and secretion of immunosuppressive cytokines. Despite considerable progress, the relevance of Gal-1-induced T-cell death in inflamed tissue from human IBD patients has not been ascertained. Intestinal biopsies and surgical specimens from control patients (n = 52) and patients with active or inactive IBD (n = 97) were studied. Gal-1 expression was studied by RT-qPCR, immunoblotting, ELISA and immunohistochemistry. Gal-1-specific ligands and Gal-1-induced apoptosis of lamina propria (LP) T-cells were determined by TUNEL and flow cytometry. We found a transient expression of asialo core 1-O-glycans in LP T-cells from inflamed areas (p < 0.05) as revealed by flow cytometry using peanut agglutinin (PNA) binding and assessing dysregulation of the core-2 β 1-6-N-acetylglucosaminyltransferase 1 (C2GNT1), an enzyme responsible for elongation of core 2 O-glycans. Consequently, Gal-1 binding was attenuated in CD3+CD4+ and CD3+CD8+ LP T-cells isolated from inflamed sites (p < 0.05). Incubation with recombinant Gal-1 induced apoptosis of LP CD3+ T-cells isolated from control subjects and non-inflamed areas of IBD patients (p < 0.05), but not from inflamed areas. In conclusion, our findings showed that transient regulation of the O-glycan profile during inflammation modulates Gal-1 binding and LP T-cell survival in IBD patients.

DEVELOPMENT OF SMALL MOLECULE EPIGENETIC THERAPEUTICS FOR INFLAMMATORY BOWEL DISEASE

Abstract Long-term persistence of chronic inflammation in inflammatory bowel disease (IBD) including ulcerative colitis (UC) and Crohn’s disease (CD) is among the major factors contributing to neoplastic transformation and the development of colitis-associated colorectal cancer. There exists a lack of efficient medications for IBD, primarily due to emergence of resistance or side effects. Antibodies against tumor necrosis factor-α (anti-TNFα) are effective therapies in the armamentarium; however, up to 40% of patients become resistant to this therapy. Thus, target-based improved therapy with higher efficacy and enhanced safety profile is urgently needed to fill these gaps. We have demonstrated that bromodomain-containing protein 4 (BRD4), an epigenetic regulator, is required for stabilization of NFkB binding on the promoters of inflammatory genes, activation of RNA polymerase II, and histone H3 Lys122 acetylation (H3K122ac) to permit high levels of inflammatory gene expressions in sentinel immune cells, epithelial cells, and fibroblasts. Our compelling data using human IBD patient samples suggest that BRD4 activation is coincident with the initiation of colonic inflammation. Inhibition of the BRD4 activation is an attractive target for the development of superior therapeutics for IBD, especially for anti-TNFα-resistant patients. We have successfully identified proprietary highly potent and specific BRD4 inhibitors (patent WO 2018/112037 A1) with direct binding modes validated by the solved co-complex crystal structures. We observed that our lead compounds exhibit low toxicity both in vitro and in vivo. Therapeutic administration of our lead inhibitors ZL0516 and ZL0590 significantly reduces mucosal inflammation in several animal models of IBD and restores tissue architecture. Our data show that inhibition of BRD4 results in a decrease of key inflammatory cytokines associated with pathological responses in IBD such as TNFα, IL-6, IL-17A, and IL-1β expression in human colonic epithelial cells and peripheral blood mononuclear cells as well as several animal models of IBD colitis. Collectively, our data suggest that BRD4 activation is critical to the initiation of human colonic inflammation during IBD. BRD4 inhibition disrupts its protein-protein interactions with acetylated histone lysine residues, thereby blocking the pathological activation of the BRD4-NFκB signaling and suppressing colonic inflammation. Thus, BRD4 represents a unique drug target, and novel BRD4 inhibitors ZL0516 and ZL0590 have been identified as promising drug candidates towards the development of small molecule epigenetic therapeutics for the treatment of IBD and its chronic sequelae. Funding support: Crohn’s &amp; Colitis Foundation Entrepreneurial Investing (EI) Initiative award, Litwin IBD Pioneers Program, and a research fellowship award from the Crohn’s &amp; Colitis Foundation of America.

Hypoxia Reduces the Transcription of Fibrotic Markers in the Intestinal Mucosa

Introduction: Intestinal fibrosis, characterized by excessive deposition of extracellular matrix proteins, is a common and severe clinical complication of inflammatory bowel disease (IBD). However, the mechanisms underlying fibrosis remain elusive, and currently, there are limited effective pharmacologic treatments that target the development of fibrosis. Hypoxia is one of the key microenvironmental factors influencing intestinal inflammation and has been linked to fibrosis. Objective: In the present study, we sought to elucidate the impact of hypoxia on fibrotic gene expression in the intestinal mucosa. Methods: Human volunteers, IBD patients, and dextran sulphate sodium-treated mice were exposed to hypoxia, and colonic biopsies were collected. The human intestinal epithelial cell line Caco-2, human THP-1 macrophages, and primary human gut fibroblasts were subjected to hypoxia, and changes in fibrotic gene expression were assessed. Results: Human volunteers subjected to hypoxia presented reduced transcriptional levels of fibrotic and epithelial-mesenchymal transition markers in the intestinal mucosa. IBD patients showed a trend towards a decrease in tissue inhibitor of metalloproteinase 1 protein expression. In mice, hypoxic conditions reduced the colonic expression of several collagens and matrix metalloproteinases. Hypoxic Caco-2 cells, THP-1 cells, and primary gut fibroblasts showed a significant downregulation in the expression of fibrotic and tissue remodelling factors. Conclusions: Stabilization of hypoxia-inducible factors might represent a novel therapeutic approach for the treatment of IBD-associated fibrosis.

6 NOVEL BRD4 INHIBITORS BLOCK THE PATHOLOGICAL ACTIVATION OF BRD4-NFκB SIGNALING AND SUPPRESS COLONIC INFLAMMATION IN IBD MOUSE MODELS

Abstract Ulcerative Colitis (UC) and Crohn’s Disease (CD) are two major types of inflammatory bowel disease (IBD), with recurrent symptoms and significant morbidity. Long-term persistence of chronic inflammation in IBD is among the major factors contributing to neoplastic transformation and development of colitis-associated colorectal cancer. There is a lack of efficient medications for IBD, due to either limited efficacy or side effects. Antibodies against TNFα are effective therapies; however, they are expensive, and up to 40% of patients are non-responders. Thus, improved therapy with higher efficacy, enhanced safety and better patient affordability is urgently needed. The master regulator of innate immune response NFκB plays a critical role in the initiation and chronicity of IBD mucosal inflammation. We demonstrated that bromodomain-containing protein 4 (BRD4), an epigenetic reader, is required for stabilization of NFκB binding on the promoters of inflammatory genes, activation of RNA polymerase II, and histone H3 Lys122 acetylation (H3K122ac) to permit high levels of inflammatory gene expressions by sentinel epithelial cells and stromal fibroblasts. Our data using human IBD patient samples suggest that BRD4 activation is critical to the initiation of colonic inflammation. We have successfully identified highly potent and specific BRD4 inhibitors, demonstrating that inhibition of BRD4 suppresses expression of inflammatory cytokines TNFα, IL-6, IL-17A, and IL-8. Administration of our lead inhibitors (ZL0454 &amp; ZL0420) significantly reduces initiation of the mucosal inflammation in both dextran sulfate sodium (DSS)-induced and Cbir1 T cell transfer colitis models in vivo, but with low toxicity and much safer than the generic NFκB/IKK inhibitor BMS345541. The levels of NFkB and BRD4 activation were also compared using immunofluorescence staining of NFkB/RelA translocation, phospho-Ser276 RelA formation, and induction of the BRD4 marker H3K122Ac. The BRD4 inhibitors reduced DSS-induced NFkB-BRD4 activation in colon tissue, demonstrating the target specificity in vivo. Our data suggest that BRD4 activation is critical to the initiation of the colonic inflammation during IBD. BRD4 inhibition disrupts its interactions with acetylated histone lysine residues, thereby blocking the pathological activation of the BRD4-NFκB signaling and suppressing colonic inflammation. Therefore, inhibition of the BRD4 activation is an attractive target for the development of novel IBD therapy and in prevention of CAC. Using our novel BRD4 inhibitors, we will further seek to evaluate the effect of long-term BRD4 inhibition on UC-induced neoplastic transformation and development of CAC. Funding support: Crohn’s &amp; Colitis Foundation Entrepreneurial Investing (EI) Initiative award and a research fellowship award from the Crohn’s &amp; Colitis Foundation of America.

Pharmacological inhibition of NPY receptors illustrates dissociable features of experimental colitis in the mouse DSS model: Implications for preclinical evaluation of efficacy in an inflammatory bowel disease model.

Administration of dextran sodium sulfate (DSS) to rodents at varying concentrations and exposure times is commonly used to model human inflammatory bowel disease (IBD). Currently, the criteria used to assess IBD-like pathology seldom include surrogate measures of visceral pain. Thus, we sought to standardize the model and then identify surrogate measures to assess effects on visceral pain. We used various 4% DSS protocols and evaluated effects on weight loss, colon pathology, biochemistry, RNA signature, and open field behavior. We then tested the therapeutic potential of NPY Y1 and/or Y2 receptor inhibition for the treatment of IBD pathology using this expanded panel of outcome measures. DSS caused weight loss and colon shrinkage, increased colon NPY and inflammatory cytokine expression, altered behaviors in the open field and induced a distinct gene metasignature that significantly overlapped with that of human IBD patients. Inhibition of Y1 and/or Y2 receptors failed to improve gross colon pathology. Y1 antagonism significantly attenuated colon inflammatory cytokine expression without altering pain-associated behaviors while Y2 antagonism significantly inhibited pain-associated behaviors in spite of a limited effect on inflammatory markers. A protocol using 7 days of 4% DSS most closely modeled human IBD pathology. In this model, rearing behavior potentially represents a tool for evaluating visceral pain/discomfort that may be pharmacologically dissociable from other features of pathology. The finding that two different NPY receptor antagonists exhibited different efficacy profiles highlights the benefit of including a variety of outcome measures in IBD efficacy studies to most fully evaluate the therapeutic potential of experimental treatments.

Persistent Salmonella enterica Serovar Typhimurium Infection Induces Protease Expression During Intestinal Fibrosis.

BackgroundIntestinal fibrosis is a common and serious complication of Crohn’s disease characterized by the accumulation of fibroblasts, deposition of extracellular matrix, and formation of scar tissue. Although many factors including cytokines and proteases contribute to the development of intestinal fibrosis, the initiating mechanisms and the complex interplay between these factors remain unclear.MethodsChronic infection of mice with Salmonella enterica serovar Typhimurium was used to induce intestinal fibrosis. A murine protease-specific CLIP-CHIP microarray analysis was employed to assess regulation of proteases and protease inhibitors. To confirm up- or downregulation during fibrosis, we performed quantitative real-time polymerase chain reaction (PCR) and immunohistochemical stainings in mouse tissue and tissue from patients with inflammatory bowel disease. In vitro infections were used to demonstrate a direct effect of bacterial infection in the regulation of proteases.ResultsMice develop severe and persistent intestinal fibrosis upon chronic infection with Salmonella enterica serovar Typhimurium, mimicking the pathology of human disease. Microarray analyses revealed 56 up- and 40 downregulated proteases and protease inhibitors in fibrotic cecal tissue. Various matrix metalloproteases, serine proteases, cysteine proteases, and protease inhibitors were regulated in the fibrotic tissue, 22 of which were confirmed by quantitative real-time PCR. Proteases demonstrated site-specific staining patterns in intestinal fibrotic tissue from mice and in tissue from human inflammatory bowel disease patients. Finally, we show in vitro that Salmonella infection directly induces protease expression in macrophages and epithelial cells but not in fibroblasts.ConclusionsIn summary, we show that chronic Salmonella infection regulates proteases and protease inhibitors during tissue fibrosis in vivo and in vitro, and therefore this model is well suited to investigating the role of proteases in intestinal fibrosis.

Intestinal glucocorticoid synthesis enzymes in pediatric inflammatory bowel disease patients.

Inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis are devastating chronic immunopathologies of the intestinal mucosa, which are frequently treated by immunosuppressive glucocorticoids. Endogenous glucocorticoids are not only produced by the adrenal glands, but also by the intestinal epithelium. Local glucocorticoid synthesis critically contributes to the immune homeostasis of the intestinal mucosa. As defective intestinal glucocorticoid synthesis has been associated with the development of IBD, we investigated the expression of steroidogenic enzymes and the key transcriptional regulator Liver Receptor Homolog-1 (LRH-1/NR5A2) in ileal and colonic biopsies human pediatric IBD and control patients. Furthermore, the induction of steroidogenic enzymes and their transcriptional regulation by LRH-1 was investigated in a mouse model of experimental colitis. These analyses revealed that colitis-induced expression of steroidogenic enzymes in the murine colon is dependent on the presence of LRH-1, as intestinal deletion of LRH-1 strongly reduced their colitis-induced expression. Similarly, a strong correlation between the expression of LRH-1 and different steroidogenic enzymes was seen in intestinal biopsies of human pediatric patients. Importantly, reduced expression of hydroxysteroid dehydrogenase 11B1 (HSD11B1) was observed in IBD patients compared to control patients, suggesting that defective local reactivation of glucocorticoids could contribute to the pathogenesis of IBD.

Microbiota of Inflammatory Bowel Disease Models.

Gut microbiome plays an important role in inflammatory bowel disease (IBD), a group of intestinal chronic inflammation conditions that affect a large population. The animal models of IBD have long been established on basis of pathological features, but their ability to recapitulate patient gut microbiota is unknown. We investigated and compared the composition and biodiversity of bacterial population in the fecal samples from rat models of the two IBD subtypes, and compared them with patient samples. Our analyses revealed that inflammation reduces overall microbiome diversity and increased variation between individuals. We identified specific microbial signatures associated with the two IBD subtypes that were consistent between the animal models and human IBD patients, suggesting that the animal models can partially recapitulate the microbiota in human diseases. Furthermore, metagenome prediction analysis suggested microbial functions that were likely altered by host-microbiota interactions in IBD models.

Low dose Naltrexone for induction of remission in inflammatory bowel disease patients

BackgroundAround 30% of patients with inflammatory bowel disease (IBD) are refractory to current IBD drugs or relapse over time. Novel treatments are called for, and low dose Naltrexone (LDN) may provide a safe, easily accessible alternative treatment option for these patients. We investigated the potential of LDN to induce clinical response in therapy refractory IBD patients, and investigated its direct effects on epithelial barrier function.MethodsPatients not in remission and not responding to conventional therapy were offered to initiate LDN as a concomitant treatment. In total 47 IBD patients prescribed LDN were followed prospectively for 12 weeks. Where available, endoscopic remission data, serum and biopsies were collected. Further the effect of Naltrexone on wound healing (scratch assay), cytokine production and endoplasmic reticulum (ER) stress (GRP78 and CHOP western blot analysis, immunohistochemistry) were investigated in HCT116 and CACO2 intestinal epithelial cells, human IBD intestinal organoids and patient samples.ResultsLow dose Naltrexone induced clinical improvement in 74.5%, and remission in 25.5% of patients. Naltrexone improved wound healing and reduced ER stress induced by Tunicamycin, lipopolysaccharide or bacteria in epithelial barriers. Inflamed mucosa from IBD patients showed high ER stress levels, which was reduced in patients treated with LDN. Cytokine levels in neither epithelial cells nor serum from IBD patients were affected.ConclusionsNaltrexone directly improves epithelial barrier function by improving wound healing and reducing mucosal ER stress levels. Low dose Naltrexone treatment is effective and safe, and could be considered for the treatment of therapy refractory IBD patients.

MicroRNA-132 and microRNA-223 control positive feedback circuit by regulating FOXO3a in inflammatory bowel disease.

Although many progresses have been achieved for inflammatory bowel disease (IBD), it is still remained as idiopathic disease to be completely controlled. MicroRNAs (miRNAs) have been identified as key players in many human diseases through degradation or translational inhibition of target genes. Because role of miRNAs in IBD is not completely understood yet, we need to identify miRNAs as novel targets for treatment of IBD. Microarray analysis for miRNAs was performed using dextran sulfate sodium-induced colitis samples and selected differentially regulated miRNAs. Candidate genes were validated using in vitro system and IBD patient samples. Molecular mechanism for regulation of inflammatory signaling was identified using gene modulation system of miRNAs. We selected 14 upregulated and 15 downregulated miRNAs through microarray analysis. Among candidate miRNAs, significant upregulation of miR-132 and miR-223 was confirmed in inflamed mouse tissues as well as human IBD patient tissues. Through bioinformatics analysis, we identified FOXO3a as direct target of miRNAs and confirmed regulatory mechanism using luciferase assay. Expression of miRNAs clearly suppressed the level of IκBα through downregulation of FOXO3a, leading to enhanced NF-κB signaling to promote the production of pro-inflammatory cytokines. The downregulation of FOXO3a concurrent with upregulation of cytokines was significantly reversed by sequestration of miRNAs with miRNA sponges. Our findings provided the evidences that miR-132 and 223 are critical mediators in positive circuit for pathogenesis of IBD by negatively regulating FOXO3a to enhance the expression of inflammatory cytokines and can be a good therapeutic target for IBD treatment.

MD-1 deficiency attenuates dextran sodium sulfate (DSS)-induced colitis through modulating the function of colonic lamina propria dendritic cells.

Available evidence suggests that both dysregulated innate and adaptive immune pathways contribute to the aberrant intestinal inflammatory response in patients with inflammatory bowel disease (IBD). Myeloid Differentiation 1 (MD-1), also known as Lymphocyte Antigen 86 (Ly86), a secreted protein interacting with radioprotective 105 (RP105), plays an important role in Toll-like receptor 4 (TLR4) signaling pathway. Previous studies showed that MD-1 may be involved in the (patho) physiological regulation of the innate immune system and inflammation. In this study, we reported for the first time that MD-1 mRNA expression was up-regulated in both human IBD patients and DSS-treated WT mice. We showed that MD-1(-/-) mice were less susceptible to the development of colitis than WT controls as demonstrated by significantly reduced weight loss, disease activity index, colon histological scores, cellular infiltration and expression of inflammatory mediators. In addition, mucosal barrier function seemed to be intact in response to the loss of MD-1. Finally, lamina propria dendritic cells (LPDCs) from the colon of MD-1(-/-) mice after DSS exposure not only decreased in number but also significantly down-regulated the expression of surface maturation co-stimulatory molecules MHC-II, CD40 and CD86 compared with those from WT mice. Taken together, our results reveal that MD-1 deficiency is of critical importance in down-regulating induction and progression of DSS colitis, thereby suggesting that MD-1 might be a target for future interventional therapies of IBD.

Loss of PHLPP protects against colitis by inhibiting intestinal epithelial cell apoptosis

A common feature of inflammatory bowel disease (IBD) is the loss of intestinal epithelial barrier function due to excessive apoptosis of intestinal epithelial cells (IECs). However, the molecular mechanism underlying increased IEC apoptosis remains unclear. Here, we investigated the role of PHLPP, a novel family of protein phosphatases, in regulating inflammation-induced IEC apoptosis in mouse models of colitis. Both Phlpp1 and Phlpp2 genes were deleted in mice. Compared with wild-type mice, PHLPP double knockout (DKO) mice were protected from colitis induced by DSS as demonstrated by lower histopathological scores, and this reduced susceptibility to colitis was associated with decreased apoptosis and increased Akt activity in IECs in vivo. In addition, epithelial organoids derived from PHLPP DKO mice were more resistant to inflammation-induced apoptosis while inhibition of Akt activity abolished the protective effect of PHLPP-loss. Furthermore, we found that PHLPP expression was significantly reduced in IECs following the induction of colitis by DSS and in human IBD patient samples. This inflammation-induced downregulation of PHLPP was partially blocked by treating cells with a proteasome inhibitor. Taken together, our results indicated that proteasome-mediated degradation of PHLPP at the onset of inflammation plays an important role in protecting IEC injury by inhibiting apoptosis.

STAT3-Activating Cytokines: A Therapeutic Opportunity for Inflammatory Bowel Disease?

The gastrointestinal tract is lined by a single layer of epithelial cells that secrete mucus toward the lumen, which collectively separates the immune sentinels in the underlying lamina propria from the intestinal microflora to prevent aberrant immune responses. Inflammatory bowel disease (IBD) describes a group of autoimmune diseases that arise from defects in epithelial barrier function and, as a consequence, aberrant production of inflammatory cytokines. Among these, interleukin (IL)-6, IL-11, and IL-22 are elevated in human IBD patients and corresponding mouse models and, through activation of the JAK/STAT3 pathway, can both propagate and ameliorate disease. In particular, cytokine-mediated activation of STAT3 in the epithelial lining cells affords cellular protection, survival, and proliferation, thereby affording therapeutic opportunities for the prevention and treatment of colitis. In this review, we focus on recent insights gained from therapeutic modulation of the activities of IL-6, IL-11, and IL-22 in models of IBD and advocate a cautionary approach with these cytokines to minimize their tumor-promoting activities on neoplastic epithelium.

Systemic levels of the anti-inflammatory decoy receptor soluble RAGE (receptor for advanced glycation end products) are decreased in dogs with inflammatory bowel disease

Inflammatory bowel disease (IBD) is a common condition in dogs, and a dysregulated innate immunity is believed to play a major role in its pathogenesis. S100A12 is an endogenous damage-associated molecular pattern molecule, which is involved in phagocyte activation and is increased in serum/fecal samples from dogs with IBD. S100A12 binds to the receptor of advanced glycation end products (RAGE), a pattern-recognition receptor, and results of studies in human patients with IBD and other conditions suggest a role of RAGE in chronic inflammation. Soluble RAGE (sRAGE), a decoy receptor for inflammatory proteins (e.g., S100A12) that appears to function as an anti-inflammatory molecule, was shown to be decreased in human IBD patients. This study aimed to evaluate serum sRAGE and serum/fecal S100A12 concentrations in dogs with IBD.Serum and fecal samples were collected from 20 dogs with IBD before and after initiation of medical treatment and from 15 healthy control dogs. Serum sRAGE and serum and fecal S100A12 concentrations were measured by ELISA, and were compared between dogs with IBD and healthy controls, and between dogs with a positive outcome (i.e., clinical remission, n=13) and those that were euthanized (n=6). The relationship of serum sRAGE concentrations with clinical disease activity (using the CIBDAI scoring system), serum and fecal S100A12 concentrations, and histologic disease severity (using a 4-point semi-quantitative grading system) was tested.Serum sRAGE concentrations were significantly lower in dogs with IBD than in healthy controls (p=0.0003), but were not correlated with the severity of histologic lesions (p=0.4241), the CIBDAI score before (p=0.0967) or after treatment (p=0.1067), the serum S100A12 concentration before (p=0.9214) and after treatment (p=0.4411), or with the individual outcome (p=0.4066). Clinical remission and the change in serum sRAGE concentration after treatment were not significantly associated (p=0.5727); however, serum sRAGE concentrations increased only in IBD dogs with complete clinical remission. Also, dogs that were euthanized had significantly higher fecal S100A12 concentrations than dogs that were alive at the end of the study (p=0.0124).This study showed that serum sRAGE concentrations are decreased in dogs diagnosed with IBD compared to healthy dogs, suggesting that sRAGE/RAGE may be involved in the pathogenesis of canine IBD. Lack of correlation between sRAGE and S100A12 concentrations is consistent with sRAGE functioning as a non-specific decoy receptor. Further studies need to evaluate the gastrointestinal mucosal expression of RAGE in healthy and diseased dogs, and also the formation of S100A12-RAGE complexes.

Tu1712 Analysis of the IL-17C-IL-17RE Cytokine Ligand-Receptor Pair in Human Inflammatory Bowel Disease Reveals Chromogranin a-Positive Epithelial Cells As Major Producers of IL-17C and IL-17RE-Positive Lamina Propria Mononuclear Cells As Main Colonic IL-17C Targets

Background: To date, the role of IL-17C and its specific receptor subunit IL-17RE in inflammatory bowel disease (IBD) settings is entirely based on murine models. Therefore, the aim of this study was to analyze the role of the novel IL-17 family member IL-17C and its receptor IL-17RE in human IBD. Methods: IL-17C expression in intestinal epithelial cells (IEC) was analyzed by qPCR, immunoblotting, immunohistochemistry, EMSA, RNA interference and pharmacological or antibody-mediated inhibition. IL-17C protein and mRNA expression in IBD patient samples was assessed by ELISA, qPCR and immunohistochemistry. IL-17C serum levels were correlated to IL23R genotype status. Results: IL-17A amplifies IL-17C expression in human IEC upon TNF-α stimulation; this depends on TNFα-activated NF-κB, ERK-1/2 and p38 MAP kinases, and IL-17A-activated Akt kinase, MCPIP and C/EBP-δ pathways. IL-17C serum levels are elevated in patients with ulcerative colitis (UC) (P=0.008). IL-17C mRNA is increased in inflamed colonic sections of IBD patients (P=0.005) and correlates significantly with the mRNA expression of Th1 cytokines (e.g., IFN-γ; P<0.01) and Th17 cytokines (e.g., IL-17A, IL-23, CCL20; P<0.01). Genotyping revealed that UC patients carrying a minor allele of the IL23R SNP rs134351 have decreased IL-17C serum levels (P=0.037). Immunohistochemistry displayed increased IL-17C and IL17RE staining in IBD patients with active disease, compared to IBD patients in remission and healthy controls. Chromogranin A-positive IEC are major producers of IL-17C in the colon, whereas the IL-17RE subunit was only present on lamina propria mononuclear cells but not in IEC. In vitro experiments suggested that IEC lines are not responsive to IL-17C stimulation, even when combined with other cytokines, such as IL-22. Conclusions: This is the first comprehensive study involving human IBD patients which demonstrates an upregulation of the novel IL-17C-IL-17RE cytokine ligand-receptor pair during active human IBD. In contrast to murine models, IL-17RE expression is confined to lamina propria leukocytes and not epithelial cells; specialized chromogranin A-positive cells are among the major producers of IL-17C in the epithelium. Our data indicate that serum IL-17C is modulated by IL23R genotype status. In addition, our data reveal potential novel targets for the pharmacological modulation of epithelium-derived IL-17C.

Tu1713 Role of miR-19b-Targeting SOCS3 in Modulating Production of Chemokine in Colonic Epithelial Cells and Is Associated With the Pathogenesis of Crohn's Disease

Background: To date, the role of IL-17C and its specific receptor subunit IL-17RE in inflammatory bowel disease (IBD) settings is entirely based on murine models. Therefore, the aim of this study was to analyze the role of the novel IL-17 family member IL-17C and its receptor IL-17RE in human IBD. Methods: IL-17C expression in intestinal epithelial cells (IEC) was analyzed by qPCR, immunoblotting, immunohistochemistry, EMSA, RNA interference and pharmacological or antibody-mediated inhibition. IL-17C protein and mRNA expression in IBD patient samples was assessed by ELISA, qPCR and immunohistochemistry. IL-17C serum levels were correlated to IL23R genotype status. Results: IL-17A amplifies IL-17C expression in human IEC upon TNF-α stimulation; this depends on TNFα-activated NF-κB, ERK-1/2 and p38 MAP kinases, and IL-17A-activated Akt kinase, MCPIP and C/EBP-δ pathways. IL-17C serum levels are elevated in patients with ulcerative colitis (UC) (P=0.008). IL-17C mRNA is increased in inflamed colonic sections of IBD patients (P=0.005) and correlates significantly with the mRNA expression of Th1 cytokines (e.g., IFN-γ; P<0.01) and Th17 cytokines (e.g., IL-17A, IL-23, CCL20; P<0.01). Genotyping revealed that UC patients carrying a minor allele of the IL23R SNP rs134351 have decreased IL-17C serum levels (P=0.037). Immunohistochemistry displayed increased IL-17C and IL17RE staining in IBD patients with active disease, compared to IBD patients in remission and healthy controls. Chromogranin A-positive IEC are major producers of IL-17C in the colon, whereas the IL-17RE subunit was only present on lamina propria mononuclear cells but not in IEC. In vitro experiments suggested that IEC lines are not responsive to IL-17C stimulation, even when combined with other cytokines, such as IL-22. Conclusions: This is the first comprehensive study involving human IBD patients which demonstrates an upregulation of the novel IL-17C-IL-17RE cytokine ligand-receptor pair during active human IBD. In contrast to murine models, IL-17RE expression is confined to lamina propria leukocytes and not epithelial cells; specialized chromogranin A-positive cells are among the major producers of IL-17C in the epithelium. Our data indicate that serum IL-17C is modulated by IL23R genotype status. In addition, our data reveal potential novel targets for the pharmacological modulation of epithelium-derived IL-17C.