apyrase and Colitis

ATP is both an important mediator of physiological gut functions such as motility and epithelial function, and a key danger signal that mediates cell death and tissue damage. The actions of extracellular ATP are regulated through the catalytic functions extracellular nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), -2, -3, and -8, which ultimately generate nucleosides. Ectonucleotidases have distinct cellular associations, but the specific locations and functional roles of individual NTPDases in the intestine are still poorly understood. Here, we tested the hypothesis that differential and cell-selective regulation of purine hydrolysis by NTPDase1 and -2 plays important roles in gut physiology and disease. We studied

Topics: Adenosine Triphosphatases; Animals; Antigens, CD; Apyrase; Colitis; Colon; Dextran Sulfate; Female; Male; Mice; Mice, Knockout

In Crohn's disease, pathogenic Th17-cells express low levels of CD39 ectonucleotidase and are refractory to the immunosuppressive effects of unconjugated bilirubin (UCB), an endogenous ligand for aryl-hydrocarbon-receptor (AhR). This resistance to AhR ligation might be associated with alterations in responses to hypoxia. Limited exposure to hypoxia appears beneficial in acute tissue injury. However, in protracted inflammation, hypoxemia may paradoxically result in Th17-cell activation. We report here that in vitro exposure of Th17-cells from Crohn's disease patients to hypoxia limits responsiveness to AhR stimulation by UCB, as reflected by lower CD39 levels. Blockade of hypoxia-inducible-factor-1alpha (HIF-1α) upregulates CD39 and favors Th17-cell regulatory responses. Resistance of Th17-cells to AhR signaling results, in part, from HIF-1α-dependent induction of ATP-binding cassette (ABC) transporters: multidrug-resistance-protein-1 (MDR1) and multidrug-resistance-associated-protein-4 (MRP4). Increased ABC transporters promote efflux of suppressive AhR ligands, such as UCB, from Th17-cells. Inhibition of MDR1, MRP4 and/or HIF-1α with ritonavir (RTV) reconstitutes AhR function in Th17-cells, enhancing therapeutic effects of UCB in dextran-sulfate-sodium-induced experimental colitis. Deleterious effects of hypoxia on Th17-cells in Crohn's disease can be ameliorated either by inhibiting HIF-1α or by suppressing ABC transporters to increase UCB availability as an AhR substrate. Targeting HIF-1α-ABC transporters could provide innovative therapeutic pathways for IBD.

Topics: Animals; Anti-Inflammatory Agents; Apyrase; ATP Binding Cassette Transporter, Subfamily B; Basic Helix-Loop-Helix Transcription Factors; Bilirubin; Cell Hypoxia; Colitis; Crohn Disease; Dextran Sulfate; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Knockout; Mucous Membrane; Multidrug Resistance-Associated Proteins; Primary Cell Culture; Protein Binding; Receptors, Aryl Hydrocarbon; Ritonavir; RNA, Small Interfering; Th17 Cells

Recent studies have suggested that the enteric nervous system can modulate gut immunity. Ecto-nucleoside triphosphate diphosphohydrolases [E-NTPDases] regulate purinergic signalling by sequential phosphohydrolysis of pro-inflammatory extracellular adenosine 5'-triphosphate [ATP]. Herein, we test the hypothesis that E-NTPDases modulate gut inflammation via neuro-immune crosstalk.. We determined expression patterns of NTPDase2 and NTPDase3 in murine and human colon. Experimental colitis was induced by dextran sodium sulphate [DSS] in genetically engineered mice deficient in NTPDase2 or NTPDase3. We compared plasma adenosine diphosphatase [ADPase] activity from Crohn's patients and healthy controls, and linked the enzyme activity to Crohn's disease activity.. NTPDase2 and -3 were chiefly expressed in cells of the enteric nervous system in both murine and human colon. When compared with wild type, DSS-induced colitis was exacerbated in Entpd2, and to a lesser extent, Entpd3 null mice as measured by disease activity score and histology, and marked anaemia was seen in both. Colonic macrophages isolated from Entpd2 null mice displayed a pro-inflammatory phenotype compared with wild type. In human plasma, Crohn's patients had decreases in ADPase activity when compared with healthy controls. The drop in ADPase activity was likely associated with changes in NTPDase2 and -3, as suggested by inhibitor studies, and were correlated with Crohn's disease activity.. NTPDase2 and -3 are ecto-enzymes expressed in the enteric nervous system. Both enzymes confer protection against gut inflammation in experimental colitis and exhibit alterations in Crohn's disease. These observations suggest that purinergic signalling modulated by E-NTPDases governs neuro-immune interactions that are relevant in Crohn's disease.

Topics: Adenosine Triphosphatases; Adolescent; Adult; Animals; Apyrase; Biomarkers; Case-Control Studies; Colitis; Colon; Crohn Disease; Dextran Sulfate; Enteric Nervous System; Female; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Young Adult

T helper type 17 (Th17) lymphocytes, characterized by the production of interleukin-17 and other pro-inflammatory cytokines, are present in intestinal lamina propria and have been described as important players driving intestinal inflammation. Recent evidence, supporting the notion of a functional and phenotypic instability of Th17 cells, has shown that Th17 differentiate into type 1 regulatory (Tr1) T cells during the resolution of intestinal inflammation. Moreover, it has been suggested that the expression of CD39 ectonucleotidase endows Th17 cells with immunosuppressive properties. However, the exact role of CD39 ectonucleotidase in Th17 cells has not been studied in the context of intestinal inflammation. Here we show that Th17 cells expressing CD39 ectonucleotidase can hydrolyze ATP and survive to ATP-induced cell death. Moreover, in vitro-generated Th17 cells expressing the CD39 ectonucleotidase produce IL-10 and are less pathogenic than CD39 negative Th17 cells in a model of experimental colitis in Rag-/- mice. Remarkably, we show that CD39 activity regulates the conversion of Th17 cells to IL-10-producing cells in vitro, which is abrogated in the presence of ATP and the CD39-specific inhibitor ARL67156. All these data suggest that CD39 expression by Th17 cells allows the depletion of ATP and is crucial for IL-10 production and survival during the resolution of intestinal inflammation.

Topics: 5'-Nucleotidase; Adenosine Triphosphate; Animals; Antigens, CD; Apyrase; Cell Death; Cell Survival; Colitis; Hydrolysis; Inflammation; Interferon-gamma; Interleukin-10; Interleukin-23; Intestines; Mice, Inbred C57BL; Phenotype; Receptors, Purinergic; Signal Transduction; Th17 Cells; Transforming Growth Factor beta1

Immune suppression by regulatory T cells and regulatory B cells is a critical mechanism to limit excess inflammation and autoimmunity. IL-10 is considered the major mediator of B cell-induced immune suppression. We report a novel mechanism for immune suppression through adenosine generation by B cells. We identified a novel population of B cells that expresses CD73 as well as CD39, two ectoenzymes that together catalyze the extracellular dephosphorylation of adenine nucleotides to adenosine. Whereas CD39 expression is common among B cells, CD73 expression is not. Approximately 30-50% of B-1 cells (B220(+)CD23(-)) and IL-10-producing B (B10) cells (B220(+)CD5(+)CD1d(hi)) are CD73(hi), depending on mouse strain, whereas few conventional B-2 cells (B220(+)CD23(+)AA4.1(-)) express CD73. In keeping with expression of both CD73 and CD39, we found that CD73(+) B cells produce adenosine in the presence of substrate, whereas B-2 cells do not. CD73(-/-) mice were more susceptible to dextran sulfate sodium salt (DSS)-induced colitis than wild type (WT) mice were, and transfer of CD73(+) B cells ameliorated the severity of colitis, suggesting that B cell CD73/CD39/adenosine can modulate DSS-induced colitis. IL-10 production by B cells is not affected by CD73 deficiency. Interestingly, adenosine generation by IL-10(-/-) B cells is impaired because of reduced expression of CD73, indicating an unexpected connection between IL-10 and adenosine and suggesting caution in interpreting the results of studies with IL-10(-/-) cells. Our findings demonstrate a novel regulatory role of B cells on colitis through adenosine generation in an IL-10-independent manner.

Topics: 5'-Nucleotidase; Adenosine; Animals; Antigens, CD; Apyrase; B-Lymphocyte Subsets; Colitis; Disease Models, Animal; Gene Expression; Immunomodulation; Immunophenotyping; Interleukin-10; Male; Mice; Mice, Knockout; Models, Biological; Peritoneal Cavity; Phenotype

Although the etiology of two major forms of inflammatory bowel disease (IBD), Crohn's disease (CD) and ulcerative colitis (UC) are unknown and evidence suggests that chronic intestinal inflammation is caused by an excessive immune response to mucosal antigens. Previous studies support the role for TGF-β1 through 3 in the initiation and maintenance of tolerance via the induction of regulatory T cells (Tregs) to control intestinal inflammation. Leptin, a satiety hormone produced primarily by adipose tissue, has been shown to increase during colitis progression and is believed to contribute to disease genesis and/or progression.. We investigated the ability of a pegylated leptin antagonist (PG-MLA) to ameliorate the development of chronic experimental colitis.. Compared to vehicle control animals, PG-MLA treatment of mice resulted in an (1) attenuated clinical score; (2) reversed colitis-associated pathogenesis including a decrease in body weight; (3) reduced systemic and mucosal inflammatory cytokine expression; (4) increased insulin levels and (5) enhanced systemic and mucosal Tregs and CD39⁺ Tregs in mice with chronic colitis. The percentage of systemic and mucosal TGF-β1, -β2 and -β3 expressing CD4⁺ T cells were augmented after PG-MLA treatment. The activation of STAT1 and STAT3 and the expression of Smad7 were also reduced after PG-MLA treatment in the colitic mice. These findings clearly suggest that PG-MLA treatment reduces intestinal Smad7 expression, restores TGF-β1-3 signaling and reduces STAT1/STAT3 activation that may increase the number of Tregs to ameliorate chronic colitis.. This study clearly links inflammation with the metabolic hormone leptin suggesting that nutritional status influences immune tolerance through the induction of functional Tregs. Inhibiting leptin activity through PG-MLA might provide a new and novel therapeutic strategy for the treatment of IBD.

Topics: Animals; Antigens, CD; Apyrase; Body Weight; Chronic Disease; Colitis; Down-Regulation; Female; Insulin; Interleukin-10; Intestinal Mucosa; Leptin; Mice; Mice, Inbred C57BL; Mice, Knockout; Polyethylene Glycols; Recombinant Proteins; Signal Transduction; Smad7 Protein; STAT1 Transcription Factor; STAT3 Transcription Factor; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Dysregulation of immune responses in inflammatory bowel diseases (IBD) results in intestinal inflammation and vascular injury while exacerbating systemic disease. CD39 is an ectonucleotidase, expressed by T regulatory cells and dendritic cells, that hydrolyzes extracellular nucleotides to modify those cellular immune responses implicated in IBD. Genetic polymorphisms of CD39 have been linked to Crohn's disease while gene deletion in mice exacerbates dextran sodium sulphate-induced colitis.. The aim of this study was to test how global deletion of CD39 in mice impacts other models of experimental colitis.. Colitis was induced in CD39-null and -wt mice, using trinitrobenzene sulfonic acid (TNBS, 125 mg/kg) administered intrarectally. Oxazolone colitis (1.5% oxazolone in 50% alcohol) was induced in comparable groups. Morphology, clinical and molecular parameters, and FACS analyses of lamina propria mononuclear cells (LPMC) were examined in CD39-null mice. CD39 expression was analyzed in human IBD biopsies.. Paradoxically, TNBS colitis in CD39-null mice was characterized by improved survival, favorable clinical scores, and decreased MPO activity, when compared to wt mice (P < 0.05). LPMC from TNBS colitis contained significantly increased amounts of T-cells (CD3(+) and CD4(+)) and TNF-α mRNA expression were increased over those in CD39 null mice (P < 0.05). In contrast, oxazolone treated CD39-null and wt mice had comparable outcomes. In both ulcerative colitis and Crohn's disease, CD39 is present at high levels in intestinal tissue biopsies.. TNBS colitis was attenuated in CD39-null mice whereas oxazolone-induced colitis was not impacted. Impaired adaptive cellular immune reactivity in the CD39-null environment appears protective in hapten-mediated Th1-type colitis. CD39 is expressed at high levels in clinical IBD tissues.

Topics: Animals; Antigens, CD; Apyrase; Colitis; Colon; Cytokines; Humans; Leukocytes, Mononuclear; Mice; Mice, Knockout; Oxazolone; Specific Pathogen-Free Organisms; Trinitrobenzenesulfonic Acid

Evidence from patients with inflammatory bowel disease (IBD) and animal models suggests that inflammation alters blood flow to the mucosa, which precipitates mucosal barrier dysfunction. Impaired purinergic sympathetic regulation of submucosal arterioles, the resistance vessels of the splanchnic vasculature, is one of the defects identified during IBD and in mouse models of IBD. We hypothesized that this may be a consequence of upregulated catabolism of ATP during colitis. In vivo and in vitro video microscopy techniques were employed to measure the effects of purinergic agonists and inhibitors of CD39, an enzyme responsible for extracellular ATP catabolism, on the diameter of colonic submucosal arterioles from control mice and mice with dextran sodium sulfate [DSS, 5% (wt/vol)] colitis. Using a luciferase-based ATP assay, we examined the degradation of ATP and utilized real-time PCR, Western blotting, and immunohistochemistry to examine the expression and localization of CD39 during colitis. Arterioles from mice with DSS colitis did not constrict in response to ATP (10 microM) but did constrict in the presence of its nonhydrolyzable analog alpha,beta-methylene ATP (1 microM). alpha,beta-Methylene ADP (100 microM), an inhibitor of CD39, restored ATP-induced vasoconstriction in arterioles from mice with DSS-induced colitis. CD39 protein and mRNA expression was markedly increased during colitis. Immunohistochemical analysis demonstrated that, in addition to vascular CD39, F4/80-immunoreactive macrophages accounted for a large proportion of submucosal CD39 staining during colitis. These data implicate upregulation of CD39 in impaired sympathetic regulation of gastrointestinal blood flow during colitis.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Antigens, CD; Antigens, Differentiation; Apyrase; Arterioles; Colitis; Colon; Dextran Sulfate; Disease Models, Animal; Electric Stimulation; Enzyme Inhibitors; Macrophages; Male; Mice; RNA, Messenger; Splanchnic Circulation; Submucous Plexus; Sympathetic Nervous System; Up-Regulation; Vasoconstriction

CD39/ENTPD1 hydrolyzes proinflammatory nucleotides to generate adenosine. As purinergic mediators have been implicated in intestinal inflammation, we hypothesized that CD39 might protect against inflammatory bowel disease. We studied these possibilities in a mouse model of colitis using mice with global CD39 deletion. We then tested whether human genetic polymorphisms in the CD39 gene might influence susceptibility to Crohn's disease. We induced colitis in mice using Dextran Sodium Sulfate (DSS). Readouts included disease activity scores, histological evidence of injury, and markers of inflammatory activity. We used HapMap cell lines to find SNPs that tag for CD39 expression, and then compared the frequency of subjects with high vs. low CD39-expression genotypes in a case-control cohort for Crohn's disease. Mice null for CD39 were highly susceptible to DSS injury, with heterozygote mice showing an intermediate phenotype compared to wild type (WT). We identified a common SNP that tags CD39 mRNA expression levels in man. The SNP tagging low levels of CD39 expression was associated with increased susceptibility to Crohn's disease in a case-control cohort comprised of 1,748 Crohn's patients and 2,936 controls (P = 0.005-0.0006). Our data indicate that CD39 deficiency exacerbates murine colitis and suggest that CD39 polymorphisms are associated with inflammatory bowel disease in humans.

Topics: Animals; Antigens, CD; Apyrase; Colitis; Crohn Disease; Disease Models, Animal; Genetic Predisposition to Disease; Genotype; Humans; Inflammatory Bowel Diseases; Mice; Mice, Inbred C57BL; Polymorphism, Genetic; RNA, Messenger; Sequence Deletion