Exact causes for autoimmune diseases remain unclear and no cures are available. Breakdown of immunotolerance could set the stage for unfettered immune responses that target self-antigens. Impaired regulatory immune mechanisms could have permissive roles in autoreactivity. Abnormal regulatory immune cell function, therefore, might be a major determinant of the pathogenesis of autoimmune disease. All current treatments are associated with some level of clinical toxicity. Treatment to specifically target dysregulated immunity in these diseases would be a great advance. Extracellular adenosine is a signaling mediator that suppresses inflammation through activation of P1 receptors, most active under pathological conditions. Mounting evidence has linked alterations in the generation of adenosine from extracellular nucleotides by ectonucleotidases, and associated perturbations in purinergic signaling, to the immunological disruption and loss of immunotolerance in autoimmunity. Targeted modulation of the purinergic signaling by either targeting ectonucleotidases or modulating P1 purinergic receptors could therefore restore the balance between autoreactive immune responses; and thereby allow reestablishment of immunotolerance. We review the roles of CD39 and CD73 ectoenzymes in inflammatory states and with the dysregulation of P1 receptor signaling in systemic and organ-specific autoimmunity. Correction of such perturbations could be exploited in potential therapeutic applications.
CD39 is an ectonucleotidase that initiates conversion of extracellular nucleotides into immunosuppressive adenosine. CD39 is expressed by regulatory T (Treg)-cells, where it mediates immunosuppression, and by a subset of T-helper (Th) 17-cells, where it limits pathogenicity. CD39 is regulated via single-nucleotide-polymorphisms and upon activation of aryl-hydrocarbon-receptor and oxygen-mediated pathways. Here we report a mechanism of CD39 regulation that relies on the presence of an endogenous antisense RNA, transcribed from the 3′-end of the human CD39/ENTPD1 gene. CD39-specific antisense is increased in Treg and Th17-cells of Crohn’s disease patients over controls. It largely localizes in the cell nucleus and regulates CD39 by interacting with nucleolin and heterogeneous-nuclear-ribonucleoprotein-A1. Antisense silencing results in CD39 upregulation in vitro and amelioration of disease activity in a trinitro-benzene-sulfonic-acid model of colitis in humanized NOD/scid/gamma mice. Inhibition/blockade of antisense might represent a therapeutic strategy to restore CD39 along with immunohomeostasis in Crohn’s disease.
CD39 is an ectonucleotidase that hydrolyzes extracellular ATP/ADP to generate immunosuppressive adenosine. CD39 is expressed by Tregs, where it contributes to immunosuppression; and by a subset of effector Th17 cells where it limits pathogenicity. CD39 is regulated at the genetic level via single nucleotide polymorphisms in non-coding regions of the gene. These are associated with decreased CD39 expression and with predisposition to Crohn’s disease. CD39 is also regulated at transcriptional level through the activation of pathways involving the aryl-hydrocarbon-receptor and O2. We have noted the presence of antisense RNA at the 3’ end of the human CD39/ENTPD1 gene. Hence, another form of regulation might be operational at post-transcriptional level and could relate to the presence of endogenous antisense RNAs. CD39-specific antisense levels were increased in Treg and Th17 cells obtained from the peripheral blood of Crohn’s patients over controls. CD39 antisense is largely localized in the cell nucleus. CD39 expression was at low levels in both cell types in Crohn’s. When testing lamina propria-derived cells, we noted higher CD39 expression and lower antisense levels in Th17 cells isolated from non-inflamed bioptic areas over inflamed areas. Functional analysis revealed that antisense silencing by FANA oligos (AumBiotec) resulted in consequent CD39 upregulation. In conclusion, our data indicate that regulation of CD39 antisense RNA impacts Treg suppression and Th17 cell modulation of pathogenic potential in Crohn’s disease. Inhibition/blockade of antisense should be considered as a novel therapeutic strategy to restore CD39 along with immune homeostasis in Crohn’s disease and other inflammatory conditions.
The ectoenzyme CD39 hydrolyzes ATP to generate immunosuppressive adenosine. We have shown that CD39 levels and activity are reduced in Treg and Th17-cells from Crohn’s patients. This impairment is linked to heightened levels of endogenous antisense (AS) present at the 3′ end of the human CD39 gene and regulating CD39 at mRNA and protein level. Here we note that CD39-specific AS is predominantly located in the nucleus of peripheral blood-derived Treg and Th17-cells of healthy subjects (HS) and Crohn’s disease patients. RNA pulldown assay followed by mass spectrometry shows that AS regulates CD39 upon interaction with nucleolin (NUCL) and heterogenous-nuclear-ribonucleoprotein-A1 (HNRNPA1). Silencing of NUCL and/or HNRNPA1 results in higher CD39 levels in both Treg and Th17-cells. Blockade of antisense in vitro using FANA oligonucleotides decreases AS while boosting CD39 in HS and Crohn’s Treg and Th17-cells. AS silencing in vivo ameliorates experimental colitis induced by trinitrobenzene-sulfonic-acid in NOD/scid/gamma mice reconstituted with antisense+ human CD4-cells and concomitantly administered FANA oligonucleotides. Compared to untreated controls, FANA-treated mice show decreased disease activity index, higher colon length, lower histology score and, phenotypically, increased proportions of circulating CD39+CD4+-cells and decreased frequencies of peripheral blood and splenic CD4+IL-17+-cells. In conclusion, CD39-specific AS dampens CD39 levels in Crohn’s Treg and Th17-cells by interacting with NUCL and HNRNPA1. Inhibition of AS controls disease activity in experimental colitis suggesting that AS blockade might serve as a novel therapeutic tool to restore CD39 and curb inflammation in Crohn’s disease.
CD39/ENTPD1 is an ectonucleotidase that scavenges pro-inflammatory nucleotides to ultimately generate adenosine. Single nucleotide polymorphisms within the promoter region of the ENTPD1 gene are linked to higher rates of inflammatory bowel disease (IBD) in humans. Furthermore, decreased levels of CD39 expression are noted in Th17 cells purified from patients with Crohn’s disease, because of aberrant signaling by aryl-hydrocarbon-receptor (AhR) in response to endogenous ligands e.g. unconjugated bilirubin (UCB). Deletion of CD39 increases susceptibility to disease in experimental colitis while overexpression of human CD39 (hCD39) in transgenic mice results in protection from ischemia reperfusion injury and thromboembolism. Here, we show that overexpression of hCD39 results in amelioration of dextran-sulfate-sodium (DSS)-induced experimental colitis and protects mice from the deleterious effects of systemic hypoxia, pharmacologically induced by deferoxamine. We further report that treatment with APT102, soluble recombinant apyrase, boosts Foxp3 expression in wild type Treg and renders Crohn’s-derived Treg and Tr1 cells more responsive to UCB-AhR stimulation in vitro. Further, APT102 enhances the beneficial regulatory effects of UCB in DSS colitic mice, in vivo. We conclude that transgenic overexpression of hCD39 is beneficial in experimental colitis and ameliorates systemic hypoxic injury in vivo. Exogenous administration of APT102 promotes AhR-mediated regulatory effects in vivo while boosting the regulatory T-cell pool in Crohn’s disease ex vivo. Boosting endogenous CD39 or augmenting ectonucleotidase activity, as with pharmacological treatment with APT102, might represent therapeutic options in IBD.
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