CD73 is critical for the resolution of murine colonic inflammation.

CD73 is a glycosyl-phosphatidylinositol-(GPI-) linked membrane protein that catalyzes the extracellular dephosphorylation of adenosine monophosphate (AMP) to adenosine. Adenosine is a negative regulator of inflammation and prevents excessive cellular damage. We investigated the role of extracellular adenosine in the intestinal mucosa during the development of Dextran-Sulfate-Sodium-(DSS-)salt-induced colitis in mice that lack CD73 (CD73(-/-)) and are unable to synthesize extracellular adenosine. We have found that, compared to wild-type (WT) mice, CD73(-/-) mice are highly susceptible to DSS-induced colitis. CD73(-/-) mice exhibit pronounced weight loss, slower weight recovery, an increase in gut permeability, a decrease in expression of tight junctional adhesion molecules, as well as unresolved inflammation following the removal of DSS. Moreover, colonic epithelia in CD73(-/-) mice exhibited increased TLR9 expression, high levels of IL-1ß and TNF-α, and constitutive activation of NF-κB. We conclude that CD73 expression in the colon is critical for regulating the magnitude and the resolution of colonic immune responses.

CD73-generated adenosine facilitates Toxoplasma gondii differentiation to long-lived tissue cysts in the central nervous system.

Toxoplasma gondii is an obligate intracellular protozoan pathogen that traffics to the central nervous system (CNS) following invasion of its host. In the CNS, T. gondii undergoes transformation from a rapidly dividing tachyzoite to a long-lived, slow-dividing bradyzoite contained within cysts. The role of extracellular adenosine in T. gondii pathogenesis has not been previously investigated. T. gondii uses host purines such as adenosine for its energy needs, as it is unable to make its own. Here, we show that CD73(-/-) mice, which lack the ability to generate extracellular adenosine, are protected from T. gondii chronic infection, with significantly fewer cysts and reduced susceptibility to reactivation of infection in the CNS independent of host effector function. Parasite dissemination to the brain was unimpaired in CD73(-/-) hosts, suggesting that the reduced cyst number is due to impaired parasite differentiation in the CNS. Confirming this, T. gondii tachyzoites formed fewer cysts following alkaline pH stress in astrocytes isolated from CD73(-/-) mice compared with wild type, and in fibroblasts treated with a CD73 inhibitor. Cyst formation was rescued in CD73(-/-) astrocytes supplemented with adenosine, but not with adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine. Furthermore, mice lacking adenosine receptors had no defect in cyst formation. Based on these findings, we conclude that CD73 expression promotes Toxoplasma bradyzoite differentiation and cyst formation by a mechanism dependent on the generation of adenosine, but independent of adenosine receptor signaling. Overall, these findings suggest that modulators of extracellular adenosine may be used to develop therapies aimed at defending against human toxoplasmosis.

Extracellular adenosine signaling induces CX3CL1 expression in the brain to promote experimental autoimmune encephalomyelitis.

BACKGROUND: Multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE) are debilitating neuroinflammatory diseases mediated by lymphocyte entry into the central nervous system (CNS). While it is not known what triggers lymphocyte entry into the CNS during neuroinflammation, blockade of lymphocyte migration has been shown to be effective in controlling neuroinflammatory diseases. Since we have previously shown that extracellular adenosine is a key mediator of lymphocyte migration into the CNS during EAE progression, we wanted to determine which factors are regulated by adenosine to modulate EAE development. METHODS: We performed a genetic analysis of wild type and CD73-/- (that are unable to produce extracellular adenosine and are protected from EAE development) to identify factors that are both important for EAE development and controlled by extracellular adenosine signaling. RESULTS: We show that extracellular adenosine triggered lymphocyte migration into the CNS by inducing the expression of the specialized chemokine/adhesion molecule CX3CL1 at the choroid plexus. In wild type mice, CX3CL1 is upregulated in the brain on Day 10 post EAE induction, which corresponds with initial CNS lymphocyte infiltration and the acute stage of EAE. Conversely, mice that cannot synthesize extracellular adenosine (CD73-/- mice) do not upregulate CX3CL1 in the brain following EAE induction and are protected from EAE development and its associated lymphocyte infiltration. Additionally, blockade of the A2A adenosine receptor following EAE induction prevents disease development and the induction of brain CX3CL1 expression. The CX3CL1 induced during EAE is found on the choroid plexus, which is the barrier between the blood and cerebral spinal fluid in the brain and is a prime entry point into the CNS for immune cells. Furthermore, CX3CL1 expression can be induced in the brains of mice and in choroid plexus cell line following A2A adenosine receptor agonist administration. Most importantly, we show that CX3CL1 blockade protects against EAE development and inhibits lymphocyte entry into the CNS. CONCLUSIONS: We conclude that extracellular adenosine is an endogenous modulator of neuroinflammation during EAE that induces CX3CL1 at the choroid plexus to trigger lymphocyte entry into the brain.

A2A adenosine receptor signaling in lymphocytes and the central nervous system regulates inflammation during experimental autoimmune encephalomyelitis.

Extracellular adenosine has an important role in regulating the severity of inflammation during an immune response. Although there are four adenosine receptor (AR) subtypes, the A2AAR is both highly expressed on lymphocytes and known as a prime mediator of adenosine's anti-inflammatory effects. To define the importance of A2AAR signaling during neuroinflammatory disease progression, we used the experimental autoimmune encephalomyelitis (EAE) animal model for multiple sclerosis. In EAE induction experiments, A2AAR antagonist treatment protected mice from disease development and its associated CNS lymphocyte infiltration. However, A2AAR(-/-) mice developed a more severe acute EAE phenotype characterized by more proinflammatory lymphocytes and activated microglia/macrophages. Interestingly, very high levels of A2AAR were expressed on the choroid plexus, a well-established CNS lymphocyte entry point. To determine the contribution of A2AAR signaling in lymphocytes and the CNS during EAE, we used bone marrow chimeric mice. Remarkably, A2AAR(-/-) donor hematopoietic cells potentiated severe EAE, whereas lack of A2AAR expression on nonhematopoietic cells protected against disease development. Although no defect in the suppressive ability of A2AAR(-/-) regulatory T cells was observed, A2AAR(-/-) lymphocytes were shown to proliferate more and produced more IFN-γ following stimulation. Despite this more proinflammatory phenotype, A2AAR antagonist treatment still protected against EAE when A2AAR(-/-) lymphocytes were adoptively transferred to T cell-deficient A2AAR(+/+) mice. These results indicate that A2AAR expression on nonimmune cells (likely in the CNS) is required for efficient EAE development, while A2AAR lymphocyte expression is essential for limiting the severity of the inflammatory response.

Adenosine receptor signaling modulates permeability of the blood-brain barrier.

The blood-brain barrier (BBB) is comprised of specialized endothelial cells that form the capillary microvasculature of the CNS and is essential for brain function. It also poses the greatest impediment in the treatment of many CNS diseases because it commonly blocks entry of therapeutic compounds. Here we report that adenosine receptor (AR) signaling modulates BBB permeability in vivo. A(1) and A(2A) AR activation facilitated the entry of intravenously administered macromolecules, including large dextrans and antibodies to ß-amyloid, into murine brains. Additionally, treatment with an FDA-approved selective A(2A) agonist, Lexiscan, also increased BBB permeability in murine models. These changes in BBB permeability are dose-dependent and temporally discrete. Transgenic mice lacking A(1) or A(2A) ARs showed diminished dextran entry into the brain after AR agonism. Following treatment with a broad-spectrum AR agonist, intravenously administered anti-ß-amyloid antibody was observed to enter the CNS and bind ß-amyloid plaques in a transgenic mouse model of Alzheimer's disease (AD). Selective AR activation resulted in cellular changes in vitro including decreased transendothelial electrical resistance, increased actinomyosin stress fiber formation, and alterations in tight junction molecules. These results suggest that AR signaling can be used to modulate BBB permeability in vivo to facilitate the entry of potentially therapeutic compounds into the CNS. AR signaling at brain endothelial cells represents a novel endogenous mechanism of modulating BBB permeability. We anticipate these results will aid in drug design, drug delivery and treatment options for neurological diseases such as AD, Parkinson's disease, multiple sclerosis and cancers of the CNS.

Human brain endothelial cells are responsive to adenosine receptor activation.

The blood-brain barrier (BBB) of the central nervous system (CNS) consists of a unique subset of endothelial cells that possess tight junctions which form a relatively impervious physical barrier to a large variety of blood components. Until recently, there have been no good in vitro models for studying the human BBB without the co-culture of feeder cells. The hCMEC/D3 cell line is the first stable, well-differentiated human brain endothelial cell line that grows independently in culture with characteristics that closely resemble those of resident human brain endothelial cells. As our previously published findings demonstrated the importance of adenosine receptor (AR) signaling for lymphocyte entry into the CNS, we wanted to determine if human brain endothelial cells possess the capacity to generate and respond to extracellular adenosine. Utilizing the hCMEC/D3 cell line, we determined that these cells express CD73, the cell surface enzyme that converts extracellular AMP to adenosine. When grown under normal conditions, these cells also express the A(1), A(2A), and A(2B) AR subtypes. Additionally, hCMEC/D3 cells are responsive to extracellular AR signaling, as cAMP levels increase following the addition of the broad spectrum AR agonist 5'-N-ethylcarboxamidoadenosine (NECA). Overall, these results indicate that human brain endothelial cells, and most likely the human BBB, have the capacity to synthesize and respond to extracellular adenosine.

Identification of stage-specific gene modulation during early thymocyte development by whole-genome profiling analysis after aryl hydrocarbon receptor activation.

The aryl hydrocarbon receptor (AHR) is a basic helix-loop-helix transcription factor, implicated as an important modulator of the immune system and of early thymocyte development. We have shown previously that AHR activation by the environmental contaminant and potent AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to a significant decline in the percentage of S-phase cells in the CD3(-)CD4(-)CD8(-) triple-negative stage (TN) 3 and TN4 T-cell committed thymocytes 9 to 12 h after exposure. In the more immature TN1- or TN2-stage cells, no effect on cell cycle was observed. To identify early molecular targets, which could provide insight into how the AHR acts as a modulator of thymocyte development and cell cycle regulation, we performed gene-profiling experiments using RNA isolated from four intrathymic progenitor populations in which the AHR was activated for 6 or 12 h. This microarray analysis of AHR activation identified 108 distinct gene probes that were significantly modulated in the TN1-4 thymocyte progenitor stages. Although most of the genes identified have specific AHR recognition sequences, only seven genes were altered exclusively in the two T-cell committed stages of early thymocyte development (TN3 and TN4) in which the decline of S-phase cells is seen. Moreover, all seven of these genes were reduced in expression, and five of the seven are associated with cell cycle regulatory processes. These seven genes are novel targets for modulation by the TCDD-activated AHR and may be involved in the observed cell-cycle arrest and suppression of early thymocyte development.

SAP97 regulates Kir2.3 channels by multiple mechanisms.

We examined the impact of coexpressing the inwardly rectifying potassium channel, Kir2.3, with the scaffolding protein, synapse-associated protein (SAP) 97, and determined that coexpression of these proteins caused an approximately twofold increase in current density. A combination of techniques was used to determine if the SAP97-induced increase in Kir2.3 whole cell currents resulted from changes in the number of channels in the cell membrane, unitary channel conductance, or channel open probability. In the absence of SAP97, Kir2.3 was found predominantly in a cytoplasmic, vesicular compartment with relatively little Kir2.3 localized to the plasma membrane. The introduction of SAP97 caused a redistribution of Kir2.3, leading to prominent colocalization of Kir2.3 and SAP97 and a modest increase in cell surface Kir2.3. The median Kir2.3 single channel conductance in the absence of SAP97 was approximately 13 pS, whereas coexpression of SAP97 led to a wide distribution of channel events with three distinct peaks centered at 16, 29, and 42 pS. These changes occurred without altering channel open probability, current rectification properties, or pH sensitivity. Thus association of Kir2.3 with SAP97 in HEK293 cells increased channel cell surface expression and unitary channel conductance. However, changes in single channel conductance play the major role in determining whole cell currents in this model system. We further suggest that the SAP97 effect results from SAP97 binding to the Kir2.3 COOH-terminal domain and altering channel conformation.

CD73 is required for efficient entry of lymphocytes into the central nervous system during experimental autoimmune encephalomyelitis.

CD73 is a cell surface enzyme of the purine catabolic pathway that catalyzes the breakdown of AMP to adenosine. Because of the strong immunosuppressive and antiinflammatory properties of adenosine, we predicted that cd73(-/-) mice would develop severe experimental autoimmune encephalomyelitis (EAE), an animal model for the central nervous system (CNS) inflammatory disease, multiple sclerosis. Surprisingly, cd73(-/-) mice were resistant to EAE. However, CD4 T cells from cd73(-/-) mice secreted more proinflammatory cytokines than wild-type (WT) mice and were able to induce EAE when transferred into naïve cd73(+/+) T cell-deficient recipients. Therefore, the protection from EAE observed in cd73(-/-) mice was not caused by a deficiency in T cell responsiveness. Immunohistochemistry showed that cd73(-/-) mice had fewer infiltrating lymphocytes in their CNS compared with WT mice. Importantly, susceptibility to EAE could be induced in cd73(-/-) mice after the transfer of WT CD73(+)CD4(+) T cells, suggesting that CD73 must be expressed either on T cells or in the CNS for disease induction. In the search for the source of CD73 in the CNS that might facilitate lymphocyte migration, immunohistochemistry revealed a lack of CD73 expression on brain endothelial cells and high expression in the choroid plexus epithelium which regulates lymphocyte immunosurveillance between the blood and cerebrospinal fluid. Because blockade of adenosine receptor signaling with the A(2a) adenosine receptor-specific antagonist SCH58261 protected WT mice from EAE induction, we conclude that CD73 expression and adenosine receptor signaling are required for the efficient entry of lymphocytes into the CNS during EAE development.