Inflammatory Bowel Disease Candidate Research Articles

Myeloid Ptpn2 Deficiency in Mice Causes Functional Iron Deficiency

Background: Anemia is the most prevalent extraintestinal complication of inflammatory bowel disease (IBD) and is associated with the manifestation of Crohn’s disease. Anemia significantly reduces the quality of life, increases hospitalization visits, and increases medical care expenses of IBD patients. Iron deficiency is the leading cause of anemia in IBD; however, the physiological mechanisms involved are still poorly understood. Macrophages significantly contribute to iron homeostasis by mechanisms including iron recycling by phagocytosing senescent erythrocytes and damaged cells, and nurturing erythropoiesis in the bone marrow. In this study, we investigated the role of the IBD candidate gene, protein tyrosine phosphatase non-receptor type 2 ( PTPN2), in regulating macrophage iron homeostasis. Methods: Myeloid cell-specific knockout mice ( Ptpn2LysMCre) and control littermates ( Ptpn2fl/fl) were analyzed for serum iron content and liver and spleen non-heme iron. Duodenal epithelial cell (DEC) proteins were assayed by Western blotting. Localization of the brush border ferrous iron transporter, DMT1, and the coupled ferrous iron absorption driver, NHE3, in duodenal tissue was determined by immunohistochemistry (IHC). Gene expression profiles of macrophages were analyzed by Nanostring. Results: Ptpn2LysMCre mice had reduced serum iron ( p<0.0001; n=12) and reduced spleen non-heme iron (p=0.0017; n=6), whereas liver non-heme iron was unaltered (n=6), indicating iron deficiency in select tissues. DMT1 (n=8) and NHE3 (n=5) protein expression in DECs was unaltered and IHC confirmed the unaltered localization of DMT1 and NHE3 (n=6) in the duodenal epithelium of Ptpn2LysMCre mice, suggesting the reduction in serum iron was not due to an impairment in intestinal non-heme iron uptake. Nanostring analyses revealed that the gene expression of the iron transporter Dmt1, iron exporter Fpn, and iron importer Tfr1 were significantly downregulated (p<0.05; n=5) in macrophages from Ptpn2LysMCre mice, indicating an iron sequestering phenotype. Conclusions: Loss of myeloid Ptpn2 in mice causes functional iron deficiency. These findings indicate a major role of myeloid PTPN2 in regulating systemic iron homeostasis. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Chensinin-1b Alleviates DSS-Induced Inflammatory Bowel Disease by Inducing Macrophage Switching from the M1 to the M2 Phenotype.

Inflammatory bowel disease (IBD) is a chronic relapsing inflammatory disorder with an increasing prevalence worldwide. Macrophage polarization is involved in the pathogenesis of IBD. Repolarization of macrophage has thus emerged as a novel therapeutic approach for managing IBD. Chensinin-1b, derived from the skin of Rana chensinensis, is a derivative of a native antimicrobial peptide (AMP). It shows anti-inflammatory effects in sepsis models and can potentially modulate macrophage polarization. The objective of this research was to study the role of chensinin-1b in macrophage polarization and dextran sulfate sodium (DSS)-induced colitis. RAW264.7 macrophages were polarized to the M1 phenotype using lipopolysaccharide (LPS) and simultaneously administered chensinin-1b at various concentrations. The ability of chenisnin-1b to reorient macrophage polarization was assessed by ELISA, qRT-PCR, and flow cytometry analysis. The addition of chensinin-1b significantly restrained the expression of M1-associated proinflammatory cytokines and surface markers, including TNF-α, IL-6, NO, and CD86, and exaggerated the expression of M2-associated anti-inflammatory cytokines and surface markers, including IL-10, TGF-β1, Arg-1, Fizz1, Chil3, and CD206. Mechanistically, via Western Blotting, we revealed that chensinin-1b induces macrophage polarization from the M1 to the M2 phenotype by inhibiting the phosphorylation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK). In mouse models of colitis, intraperitoneal administration of chensinin-1b alleviated symptoms induced by DSS, including weight loss, elevated disease activity index (DAI) scores, colon shortening, colonic tissue damage, and splenomegaly. Consistent with our in vitro data, chensinin-1b induced significant decreases in the expression of M1 phenotype biomarkers and increases in the expression of M2 phenotype biomarkers in the mouse colitis model. Furthermore, chensinin-1b treatment repressesed NF-κB phosphorylation in vivo. Overall, our data showed that chensinin-1b attenuates IBD by repolarizing macrophages from the M1 to the M2 phenotype, suggesting its potential as a therapeutic candidate for IBD.

Bioadhesive Microcarriers Encapsulated with IL-27 High Expressive MSC Extracellular Vesicles for Inflammatory Bowel Disease Treatment.

Mesenchymal stem cell (MSC) therapy is a promising candidate for inflammatory bowel disease (IBD) treatment, while overcoming the limitations of naive seeding cells function and realizing efficient intestinal targeting remains a challenge. Here, a bioadhesive microparticle carrying interleukin-27 (IL-27) MSC-derived extracellular vesicles (MSCIL-27 EVs) is developed to treat IBD. The MSCIL-27 EVs prepared through lentivirus-mediated gene transfection technology show ideal anti-inflammatory and damage repair function. By encapsulating MSCIL-27 EVs into dopamine methacrylamide-modified hydrogel, a bioadhesive EVs microcarrier via microfluidic technology is fabricated. The resultant microcarriers exhibit ideal MSCIL-27 EVs sustained release effect and effective wet adhesion property. Furthermore, the therapeutic potential of MSCIL-27 EVs-loaded microcarriers in treating IBD is demonstrated. Through giving IBD rats a rectal administration, it is found that the microcarriers can firmly anchor to the surface of colon, reduce the inflammatory response, and repair the damaged barrier. Therefore, the bioadhesive MSCIL-27 EVs-loaded microcarriers provide a promising strategy for the biomedical application of MSC-derived EVs, and broaden the clinical potential of MSC therapy.

Foodomics-Based Approaches Shed Light on the Potential Protective Effects of Polyphenols in Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a chronic and progressive inflammatory disorder affecting the gastrointestinal tract (GT) caused by a wide range of genetic, microbial, and environmental factors. IBD is characterized by chronic inflammation and decreased gut microbial diversity, dysbiosis, with a lower number of beneficial bacteria and a concomitant increase in pathogenic species. It is well known that dysbiosis is closely related to the induction of inflammation and oxidative stress, the latter caused by an imbalance between reactive oxygen species (ROS) production and cellular antioxidant capacity, leading to cellular ROS accumulation. ROS are responsible for intestinal epithelium oxidative damage and the increased intestinal permeability found in IBD patients, and their reduction could represent a potential therapeutic strategy to limit IBD progression and alleviate its symptoms. Recent evidence has highlighted that dietary polyphenols, the natural antioxidants, can maintain redox equilibrium in the GT, preventing gut dysbiosis, intestinal epithelium damage, and radical inflammatory responses. Here, we suggest that the relatively new foodomics approaches, together with new technologies for promoting the antioxidative properties of dietary polyphenols, including novel delivery systems, chemical modifications, and combination strategies, may provide critical insights to determine the clinical value of polyphenols for IBD therapy and a comprehensive perspective for implementing natural antioxidants as potential IBD candidate treatment.

Molecular networking-guided isolation strategy of a new C-glycosyl flavone rotamer from Stellaria alsine and evaluation of anti-inflammatory and antioxidant activities.

Stellaria alsine has traditionally been used as both a famine relief food and an alternative medicine in East Asia. Modern pharmacological studies have revealed that S. alsine has various biological effects such as anticancer, anti-hepatoma, anti-inflammatory, and antioxidative effects. However, the anti-inflammatory properties of chemical constituents derived from this plant have not been studied well. To identify potential therapeutic candidate for treating inflammatory diseases such as inflammatory bowel disease (IBD). The distribution of chemical compounds was investigated by Global Natural Product Social (GNPS)-based molecular networking (MN) analysis using UPLC-Orbitrap tandem mass spectrometry. The anti-inflammatory and antioxidative effects of S. alsine extracts and fractions were evaluated by measuring interleukin (IL)-8 and reactive oxygen species (ROS) productions. The active EA layer of S. alsine showed the highest percentage of major compounds by feature-based molecular networking. The top candidate structures of EA fraction were rapidly annotated as flavone C- or O-glycosides via an advanced analysis tool, Network Annotation Propagation (NAP). With the GNPS molecular networking-guided isolation strategy, a new C-glycosyl flavone rotamer (1) was isolated. The structures of the major (1a) and minor (1b) rotational isomers were determined by extensive NMR analysis and MS/MS fragmentation. Finally, the anti-inflammatory activity of 1 was predicted by molecular docking simulations with IL-8 protein. These results suggested that the compound 1 is a potential therapeutic candidate for inflammatory bowel disease (IBD).

Oxidative Stress, Inflammation, Gut Dysbiosis: What Can Polyphenols Do in Inflammatory Bowel Disease?

Inflammatory bowel disease (IBD) is a long-term, progressive, and recurrent intestinal inflammatory disorder. The pathogenic mechanisms of IBD are multifaceted and associated with oxidative stress, unbalanced gut microbiota, and aberrant immune response. Indeed, oxidative stress can affect the progression and development of IBD by regulating the homeostasis of the gut microbiota and immune response. Therefore, redox-targeted therapy is a promising treatment option for IBD. Recent evidence has verified that Chinese herbal medicine (CHM)-derived polyphenols, natural antioxidants, are able to maintain redox equilibrium in the intestinal tract to prevent abnormal gut microbiota and radical inflammatory responses. Here, we provide a comprehensive perspective for implementing natural antioxidants as potential IBD candidate medications. In addition, we demonstrate novel technologies and stratagems for promoting the antioxidative properties of CHM-derived polyphenols, including novel delivery systems, chemical modifications, and combination strategies.

Deinococcus radiodurans R1 Lysate Induces Tolerogenic Maturation in Lipopolysaccharide-Stimulated Dendritic Cells and Protects Dextran Sulfate Sodium-Induced Colitis in Mice.

Deinococcus radiodurans is an extremophilic bacterium that can thrive in harsh environments. This property can be attributed to its unique metabolites that possess strong antioxidants and other pharmacological properties. To determine the potential of D. radiodurans R1 lysate (DeinoLys) as a pharmacological candidate for inflammatory bowel disease (IBD), we investigated the anti-inflammatory activity of DeinoLys in bone marrow-derived dendritic cells (BMDCs) and a colitis mice model. Lipopolysaccharide (LPS)-stimulated BMDCs treated with DeinoLys exhibited alterations in their phenotypic and functional properties by changing into tolerogenic DCs, including strongly inhibited proinflammatory cytokines (TNF-α and IL-12p70) and surface molecule expression and activated DC-induced T cell proliferation/activation with high IL-10 production. These phenotypic and functional changes in BMDCs induced by DeinoLys in the presence of LPS were abrogated by IL-10 neutralization. Furthermore, oral administration of DeinoLys significantly reduced clinical symptoms against dextran sulfate sodium-induced colitis, including body weight loss, disease activity index, histological severity in colon tissue, and lower myeloperoxidase level in mice. Our results establish DeinoLys as a potential anti-inflammatory candidate for IBD therapy.

Antibacterial and Immunosuppressive Effects of OPS-2071, a Candidate Therapy for Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract. Although many types of drug are used, clinical outcomes are still unsatisfactory. Previous studies have suggested that intestinal bacteria are involved in the pathogenesis of IBD. Accordingly, in an IBD model we evaluated the therapeutic effects of OPS-2071, a low-absorption quinolone antibacterial agent indicated for intestinal infection, and investigated its mechanism of action. The therapeutic effects of OPS-2071 and comparison therapies were evaluated using naive CD4 + T cell-transfer IBD model mice. In vitro inhibition of LPS-induced TNF-α production and inhibitory effects on T cell responses stimulated using anti-CD3/CD28 antibody-loaded beads were evaluated using mouse splenocytes and human peripheral blood mononuclear cells. In addition, in vitro activities against bacteria implicated in IBD pathogenesis were tested. OPS-2071 dose-dependently decreased both colonic weight/length ratio and the colitis histological score as compared with the vehicle group. The therapeutic effect of OPS-2071 was equivalent to that of anti-IL-12/23 (p40) antibody. In vitro, OPS-2071 suppressed TNF-α production induced by LPS stimulation and T cell responses in a dose-dependent manner. At high concentrations, these effects were comparable to those of existing immunosuppressive agents, such as prednisolone, in both mouse and human cells. OPS-2071 also showed antibacterial activity against IBD-related bacteria. Our results suggest that OPS-2071 had both immunosuppressive and antibacterial effects. This dual effect makes OPS-2071 a unique and promising candidate for IBD.

20 kDa PEGylated Adrenomedullin as a New Therapeutic Candidate for Inflammatory Bowel Disease

Human adrenomedullin (AM), a hypotensive peptide, also has anti-colitis activity. We prepared a polyethylene glycol (PEG) ylated form of AM through the conjugation of PEG-AM (1–15) and AM (15–52). Highly pure monomeric 20 kDa PEG-AM (20kPEG-AM) stimulated cyclic adenosine monophosphate production in HEK-293 cells stably expressing the type 1 AM receptor in a dose-dependent manner. The half-life of 20kPEG-AM was 7.4 h following subcutaneous administration in mice. We assessed the anti-colitis effect of subcutaneous 20kPEG-AM administration in the dextran sodium sulfate murine colitis model. Single and double subcutaneous injection of 20kPEG-AM significantly reduced total inflammation scores. These results suggest that 20kPEG-AM is a promising therapeutic candidate for the treatment of human inflammatory bowel diseases.

The IBD candidate gene, PTPN2, Restricts Epithelial and Macrophage Invasion of a Novel Reporter Adherent‐Invasive Escherichia coli

BackgroundDysbiosis of the gut microbiota and expansion of pathobionts such as adherent‐invasive Escherichia coli (AIEC) are associated with the pathogenesis of Inflammatory Bowel Disease (IBD). Mice deficient in expression of the IBD gene candidate, Ptpn2, exhibit pronounced expansion of a novel murine adherent‐invasive Escherichia coli (mAIEC). The aim of this study was to generate a fluorescent mAIEC to study host susceptibility to mAIEC invasion, and to identify how loss of PTPN2 expression in host cells modifies mAIEC invasiveness.MethodsmAIEC was isolated from the distal colon of a Ptpn2 knockout (KO) mouse. mAIEC was made chemically competent and then transformed with a plasmid expressing mCherry fluorescent protein and a chloramphenicol resistance cassette. For bacterial adherence and invasion studies, parental Caco‐2bbe cells were infected with mAIEC‐mCherry at multiplicity of infection (MOI) of 10 for 3 hours. Caco‐2bbe intestinal epithelial cells (IEC), stably expressing either scrambled shRNA or shRNA against Ptpn2 were infected with mAIEC‐mCherry (MOI=10) for 5 hours. Peritoneal macrophages from Ptpn2 wildtype, heterozygous and KO mice were infected with mAIEC‐mCherry for 5 hours.ResultsWe previously demonstrated that mAIEC can adhere to and invade IECs. We next validated that mAIEC‐mCherry adhered to (0.94 ±0.61 bacteria/cell compared to control E. coli K12; 0.14±0.03 bacteria/cell, n=4, P=0.0039) IECs. mAIEC‐mCherry had comparable adherence (n=4, P=0.35) to IECs as control (unlabeled) mAIEC. Bacterial invasion of mAIEC‐mCherry invasion of IECs was visually confirmed by fluorescence microscopy. To better understand if the loss of expression of PTPN2, which drives increased luminal abundance of mAIEC, also affected mAIEC pathogenicity, we next tested if Ptpn2 deficiency in host cells altered mAIEC‐mCherry invasion. We observed by immunofluorescence that Ptpn2 knockdown (KD) IECs were more susceptible to mAIEC‐mCherry invasion compared to control IECs (n=3). We also confirmed that macrophages from Ptpn2‐KO mice were more susceptible to mAIEC‐mCherry invasion compared to wildtype and Ptpn2‐heterozygous mouse macrophages (n=5, wildtype vs. KO, P=0.0044; KO vs. heterozygous, P=−0.8665). Ptpn2‐heterozygous macrophages were also more susceptible to invasion than wildtype, indicating a gene‐dosing effect of macrophage PTPN2 loss on bacterial invasion (wildtype vs. heterozygous, P=0.0117). These data identify that loss of PTPN2 expression increases susceptibility to mAIEC invasion.ConclusionHere, we validated a newly generated fluorescently tagged mAIEC to study interactions between this novel mouse AIEC with host cells. We further demonstrated that Ptpn2 deficiency promotes mAIEC of intestinal epithelial cells and macrophages. These data suggest that IBD patients with loss‐of‐function mutations in PTPN2 may have increased susceptibility to colonization by disease‐relevant AIEC.Support or Funding InformationNIH 2R01DK091281 (DFM)

18 A HUMAN-DERIVED 5-MER PEPTIDE (MTADV), WHICH RESTRICTIVELY ALLEVIATES THE PRO-INFLAMMATORY ACTIVITY OF SERUM AMYLOID A (SAA), SUBSTANTIALLY AMELIORATES IBD PATHOLOGY: NEW POTENTIAL DRUG (MTADV) AND THERAPEUTIC TARGET CANDIDATE (SAA) FOR IBD

Abstract A human anti-inflammatory 5-MER peptide MTADV (methionine-threonine-alanine-aspartic acid-valine) derived from a sequence of a pro-inflammatory CD44 variant was first described by us in Nedvetzki et al., J. Clin. Invest. 111,1211-1220, 2003. A NCBI BLAST RefSeq protein sequence database, that contains at present 81,347 human protein sequences, reveals that only one additional protein contains this unique MTADV sequence of alternating hydrophobic and hydrophilic amino acids. This human peptide displays an efficient anti-inflammatory effect in Inflammatory Bowel Disease (IBD), Rheumatoid Arthritis and Multiple sclerosis mouse models, all share amyloid proteins in the relevant target organs. We show here that the 5-MER peptide, administered after the onset of disease,either by SC injections or oral delivery, inhibited the pathological activity of two IBD models (TNBS and DSS), as indicated by histopathology analysis (Ameho’s score) or Bioluminescence Imaging, sensing the release of Reactive Oxdative Species (ROS), which reflect colon inflammation (Fig 1). The 5-MER MTADV peptide, while inhibiting chronic inflammation, neither interferes with normal immune responses nor induce neutralizing antibodies. A pull-down experiment with mass spectrometric analysis revealed that the 5-MER peptide specifically binds to serum amyloid A (SAA), a stress pro-inflammatory protein, which generates aggregated amyloid deposits in the IBD colon (de Villiers at al Cytokine 12:1337-1347,2000). Our studies further provide in vitro evidence, strengthened by in vivo experiments, that SAA is a significant target of this pentamer. To this end, the 5-MER peptide (but not the corresponding scrambled peptide) inhibits the release of the pro-inflammatory cytokines IL-6 and IL-1β from SAA-activated fibroblasts. Furthermore, the 5-MER peptide was found to retard the early stages of amyloid-type aggregation of SAA in solution (Fig 2). Adopting the β-sheet conformation, MTADV would display opposing hydrophobic and hydrophilic faces that could interact with the β-sheet-forming amyloidogenic sequences in SAA. This suggests that the mechanism of action for the 5-MER peptide in vivo may depend on its ability to slow the aggregation of SAA, thus reduce its contribution to chronic inflammation. Finally, using bioinformatics and qRT-PCR, we have found the pentamer up-regulates set of genes involved in resistance to chronic inflammations. Hence, our study provides both a new potential drug (MTADV) and a new therapeutic target candidate (SAA) for IBD.

A hydroxyethyl derivative of chrysin exhibits anti-inflammatory activity in dendritic cells and protective effects against dextran sodium salt-induced colitis in mice

Inflammatory bowel disease (IBD) is a chronic disease that occurs in the intestinal tract. Phyto-ingredients have been evaluated for their ability to protect against IBD because of their anti-inflammatory activities. In our previous study, we identified a novel derivative of chrysin (HE-chrysin) using irradiation technology, which exhibited stronger anti-cancer activity in human colorectal cancer cells than the original chrysin. Here, to determine whether HE-chrysin is a new therapeutic candidate for IBD, we investigated the anti-inflammatory effects of HE-chrysin on bone marrow-derived dendritic cells (BMDCs) and dextran sodium salt (DSS)-induced colitis in mice. HE-chrysin more effectively inhibited BMDC maturation compared to chrysin, as demonstrated by the decreased levels of pro-inflammatory cytokines, surface molecules, antigen-presenting ability, and T cell proliferation/activation in lipopolysaccharide-stimulated BMDCs. These anti-inflammatory effects of HE-chrysin were regulated by mitogen-activated protein kinases and nuclear factor-κB. Furthermore, oral administration of HE-chrysin attenuated DSS-induced colitis symptoms and clinical signs in the mouse model. The protective effects of HE-chrysin treatment against colitis were mediated by decreasing Th1- and Th17-type cytokine levels. These results indicate that HE-chrysin is attractive candidate for IBD therapy.

Robustaflavone Isolated from Nandina domestica Using Bioactivity-Guided Fractionation Downregulates Inflammatory Mediators.

Nandina domestica (Berberidaceae) has been used in traditional medicine for the treatment of cough. This plant is distributed in Korea, Japan, China, and India This study aimed to investigate the anti-inflammatory phytochemicals obtained from the N. domestica fruits. We isolated a biflavonoid-type phytochemical, robustaflavone (R), from N. domestica fruits through bioactivity-guided fractionation based on its capacity to inhibit inflammation. The anti-inflammatory mechanism of R isolated from N. domestica has not yet been studied. In the present study, we evaluated the anti-inflammatory activities of R using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. We have shown that R reduces the production of nitric oxide (NO), pro-inflammatory cytokine interleukin-1 beta (IL-1β), and IL-6. Western blot analysis showed that R suppresses the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and downregulates the expression of LPS-induced nuclear factor-kappa B (NF-κB) and the phosphorylation of extracellular-regulated kinases (pERK 1/2). Moreover, R inhibited IL-8 release in LPS-induced human colonic epithelial cells (HT-29). These results suggest that R could be a potential therapeutic candidate for inflammatory bowel disease (IBD).

Computational drug repurposing for inflammatory bowel disease using genetic information

As knowledge of the genetics behind inflammatory bowel disease (IBD) has continually improved, there has been a demand for methods that can use this data in a clinically significant way. Genome-wide association analyses for IBD have identified 232 risk genetic loci for the disorder. While identification of these risk loci enriches our understanding of the underlying biology of the disorder, their identification does not serve a clinical purpose. A potential use of this genetic information is to look for potential IBD drugs that target these loci in a procedure known as drug repurposing. The demand for new drug treatments for IBD is high due to the side effects and high costs of current treatments. We hypothesize that IBD genetic variants obtained from GWAS and the candidate genes prioritized from the variants have a causal relationship with IBD drug targets. A computational drug repositioning study was done due to its efficiency and inexpensiveness compared to traditional in vitro or biochemical approaches. Our approach for drug repurposing was multi-layered; it not only focused on the interactions between drugs and risk IBD genes, but also the interactions between drugs and all of the biological pathways the risk genes are involved in. We prioritized IBD candidate genes using identified genetic variants and identified potential drug targets and drugs that can be potentially repositioned or developed for IBD using the identified candidate genes. Our analysis strategy can be applied to repurpose drugs for other complex diseases using their risk genes identified from genetic analysis.

The Probiotic, ID-JPL934, Attenuates Dextran Sulfate Sodium-Induced Colitis in Mice Through Inhibition of Proinflammatory Cytokines Expression.

Despite the increasing prevalence of inflammatory bowel disease (IBD), classified as immune-mediated disorders, the exact biological mechanisms leading to its development are undetermined, and treatment strategies remain elusive. Probiotics have been proposed as potential alternatives for treating IBD. The purpose of this research was to find therapeutic candidates of probiotics for colitis. We adopted dextran sulfate sodium (DSS)-induced colitis model to demonstrate the therapeutic effects of ID-JPL934, a mixture of three live bacterial strains at a 1:1:1 ratio: Lactobacillus johnsonii IDCC9203, Lactobacillus plantarum IDCC3501, and Bifidobacterium animalis subspecies lactis IDCC4301, on IBD. The severity was scored according to the disease activity index (DAI) for colitis by observing body weight (BW) and stool status of each mouse once a day. BALB/c mice given 3.5% DSS in drinking water suffered from symptoms of colitis such as weight loss, diarrhea, and bloody excrement. In our study, administration of ID-JPL934 reduced the DAI scores in a dose-dependent manner, and treatments with ID-JPL934 108 and 109 colony-forming unit per mouse per day showed similar inhibition compared with those of sulfasalazine 500 mg per kg BW per day. Moreover, the contraction of colon length improved. ID-JPL934 also suppressed inflammatory lesions such as infiltration of immune cells in mucosa and submucosa, severe crypt damage, and loss of goblet and epithelial cells on the histological analysis. These results might be due to downregulation of the expression of proinflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-1β, and IL-6. From these results, ID-JPL934 might be an effective therapeutic candidate for IBD.

Systematic analysis of chromatin interactions at disease associated loci links novel candidate genes to inflammatory bowel disease.

BackgroundGenome-wide association studies (GWAS) have revealed many susceptibility loci for complex genetic diseases. For most loci, the causal genes have not been identified. Currently, the identification of candidate genes is predominantly based on genes that localize close to or within identified loci. We have recently shown that 92 of the 163 inflammatory bowel disease (IBD)-loci co-localize with non-coding DNA regulatory elements (DREs). Mutations in DREs can contribute to IBD pathogenesis through dysregulation of gene expression. Consequently, genes that are regulated by these 92 DREs are to be considered as candidate genes. This study uses circular chromosome conformation capture-sequencing (4C-seq) to systematically analyze chromatin-interactions at IBD susceptibility loci that localize to regulatory DNA.ResultsUsing 4C-seq, we identify genomic regions that physically interact with the 92 DRE that were found at IBD susceptibility loci. Since the activity of regulatory elements is cell-type specific, 4C-seq was performed in monocytes, lymphocytes, and intestinal epithelial cells. Altogether, we identified 902 novel IBD candidate genes. These include genes specific for IBD-subtypes and many noteworthy genes including ATG9A and IL10RA. We show that expression of many novel candidate genes is genotype-dependent and that these genes are upregulated during intestinal inflammation in IBD. Furthermore, we identify HNF4α as a potential key upstream regulator of IBD candidate genes.ConclusionsWe reveal many novel and relevant IBD candidate genes, pathways, and regulators. Our approach complements classical candidate gene identification, links novel genes to IBD and can be applied to any existing GWAS data.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-1100-3) contains supplementary material, which is available to authorized users.

Drug Repositioning in Inflammatory Bowel Disease Based on Genetic Information

Currently, 200 genetic risk loci have been identified for inflammatory bowel disease (IBD). Although these findings have significantly advanced our insight into IBD biology, there has been little progress in translating this knowledge toward clinical practice, like more cost-efficient drug development. Our aim was to use genetic knowledge to identify drugs that warrant further investigation in IBD treatment. We hypothesized that proteins encoded by IBD candidate genes are potential IBD drug targets because genetic information can increase successful drug identification. We identified drugs that target the proteins encoded by IBD candidate genes using the DrugBank. We included proteins that are in direct protein-protein interaction with proteins encoded by IBD risk genes. Promising potential IBD drugs were selected based on a manual literature search of all identified drugs (PubMed, ClinicalTrials.gov). We have identified 113 drugs that could potentially be used in IBD treatment. Fourteen are known IBD drugs, 48 drugs have been, or are being investigated in IBD, 19 are being used or being investigated in other inflammatory disorders treatment, and 32 are investigational new drugs that have not yet been registered for clinical use. We confirm that proteins encoded by IBD candidate genes are targeted by approved IBD therapies. Furthermore, we show that Food and Drug Administration-approved drugs could possibly be repositioned for IBD treatment. We also identify investigational new drugs that warrant further investigation for IBD treatment. Incorporating this process in IBD drug development will improve the utilization of genetic data and could lead to the improvement of IBD treatment.

Loss of PTPN2 in Intestinal Epithelial Cells Upregulates a Novel STAT‐Dependent Mechanism of Claudin‐2 Expression

Inflammatory bowel disease (IBD) is a multi‐factorial condition characterized by elevated levels of pro‐inflammatory cytokines such as TNF‐α and IFN‐γ. Genome‐wide‐association‐studies (GWAS) revealed that individuals with single nucleotide polymorphic mutations in the locus of the protein tyrosine phosphatase non‐receptor type 2 (PTPN2) gene that encodes T‐cell protein tyrosine phosphatase (TCPTP), have an increased susceptibility to IBD. We have reported that reduced TCPTP expression increased effects of IFN‐γ on Signal Transducer and Activator of Transcription (STAT) signaling and led to a greater reduction in barrier function in intestinal epithelial cells (IEC). The protein composition of tight junctions (TJ) determines the tightness of the barrier. Claudin‐2 is a TJ protein that is upregulated in IBD and increases paracellular permeability to cations thus reducing transepithelial electrical resistance (TER). We have previously shown that transient knockdown of TCPTP permits increased expression of claudin‐2 through Janus Kinase (JAK)‐STAT signaling. The aim of the present study was to identify the molecular mechanisms underlying STAT regulation of claudin‐2 expression in PTPN2 (TCPTP)‐knockdown (PTPN2‐KD) HT‐29 IEC. Western blot and immunofluorescence studies revealed that basal STAT1 phosphorylation (Y701) and claudin‐2 expression were increased in unchallenged PTPN2‐KD cells compared to control shRNA transfected cells, and this effect was greatly enhanced by IFN‐γ treatment. Protein analysis of soluble and insoluble fractions showed increased claudin‐2 in the insoluble fraction in KD cells compared to control cells. siRNA silencing of STAT1 mRNA levels significantly decreased claudin‐2 protein and mRNA expression. To confirm STAT binding to the claudin‐2 promoter region, we used Chromatin immunoprecipitation (ChIP) assay to immunoprecipitate STAT1 from IFN‐γ‐treated PTPN2‐KD and control IEC with anti‐STAT1 antibody, and probed the associated DNA with primers against the claudin‐2 promoter. ChIP analysis showed increased STAT1 binding to the claudin‐2 promoter region in KD cells compared to control cells. Next we determined if STAT1 activates claudin‐2 promoter activity through the STAT binding site. We performed a luciferase reporter assay using a human claudin‐2 promoter‐luciferase construct expressed in PTPN2‐KD or Con‐shRNA IEC. Luciferase activity was significantly increased (214 ± 9%, p<0.0001; n=3) in PTPN2‐KD cells over control IEC. In further studies using two different constructs of the claudin‐2 promoter region with mutations of the STAT binding site, there was a significant decrease in promoter activity (43±15%, p<0.0001; n=3) when compared to full length claudin‐2 promoter activity in KD cells, but no significant difference with control cells. In summary, we have demonstrated that STAT1 regulates claudin‐2 expression through binding to its promoter region under conditions of decreased activity of the IBD candidate gene, PTPN2.

VSL#3 Probiotic Stimulates T‐Cell Protein Tyrosine Phosphatase‐Mediated Protection of Intestinal Epithelial Barrier Function

VSL#3 is a probiotic cocktail that has shown efficacy in treating murine colitis and patients with ulcerative colitis. VSL#3 can also exert a beneficial effect on epithelial barrier function which is dysregulated in inflammatory bowel disease (IBD). We have previously shown that the enzyme T-cell protein tyrosine phosphatase (TCPTP), encoded by the IBD candidate gene PTPN2, protects intestinal epithelial barrier function. The aim of this study was to identify if VSL#3 activates TCPTP and protects intestinal epithelial cells (IEC) from the effects of the IBD-associated pro-inflammatory cytokine interferon-gamma (IFN-γ). Administration of VSL#3 to T84 or HT-29 IEC polarized monolayers increased expression and enzymatic activity of TCPTP in a dose-dependent and time-dependent manner. VSL#3 reduced IFN-γ signaling, as reflected by decreased phosphorylation of STAT-1, in a TCPTP-dependent manner. In addition, VSL#3 alone increased IEC barrier integrity and promoted recovery of barrier function in IFN-γ pre-treated IEC monolayers. VSL#3 conditioned medium also promoted partial recovery from IFN-γ-induced barrier defects. Using a stable TCPTP-deficient HT-29 cell line, we observed that the recovery of TER induced by VSL#3 in IFN-γ treated cells was TCPTP-dependent. In conclusion, the probiotic VSL#3 increased activity and expression of TCPTP to reduce inflammatory signaling and correct epithelial barrier defects.

PTPN2 Gene Deficiency Leads to Increased Epithelial Permeability and Promotes STAT‐1‐dependent Claudin‐2 Expression

Interferon gamma (IFN‐γ) is an inflammatory cytokine involved in inflammatory bowel disease (IBD), and is known to compromise the intestinal barrier. siRNA silencing of the IBD candidate gene protein tyrosine phosphatase non‐receptor type‐2 (PTPN2) in human intestinal epithelial cells (IEC) elevates expression of claudin‐2 causing increased cation permeability of tight junctions. The aim of this study was to identify the cellular and molecular mechanisms underlying claudin‐2 expression in the absence of PTPN2. We developed a HT‐29 IEC line stably expressing short hairpin RNA for PTPN2 using a lentiviral system to knockdown PTPN2 expression. RT‐PCR and densitometry analysis indicated that PTPN2 expression was significantly reduced (60 ± 4%, p<0.0001) in PTPN2‐depleted cells, paralleled with an increased expression in claudin‐2 protein (180 ± 4%, p<0.0005). Western blot and immunofluorescence studies revealed that basal phosphorylation of the PTPN2 substrate, STAT‐1, as well as claudin‐2 protein expression, were significantly increased in PTPN2 KD cells. This effect was potentiated following IFN‐γ treatment. siRNA silencing of STAT‐1 decreased claudin‐2 protein expression in PTPN2 KD cells. There was also a significant increase in in vivo intestinal permeability to FITC‐Dextran (4 kD) in 3‐week old Ptpn2 knockout (298 ± 5%, p<0.0001) and heterozygote mice (206 ± 12%, p<0.001) compared to wild‐type mice. STAT‐1 phosphorylation and claudin‐2 protein expression were increased in Ptpn2 KO mouse cecum vs. wild type. In summary, PTPN2 deficiency leads to increased intestinal permeability and elevated claudin‐2 expression through a STAT‐1‐dependent pathway.