Previously we had reported that astrocytes physiologically express high levels of CD73 in their membrane, converting extracellular AMP to immune suppressive adenosine, mediates an anti-inflammatory effect. Following an interaction with effector T cells (CD4+CD25- ), astrocytes lost most of their membrane expressed CD73, which rendered astrocytes’ immune suppressive function and accelerated neural inflammation such as EAE. Here, we investigated the mechanism leading to the loss of membrane CD73 in astrocytes. Our results revealed that there was no significant difference in Cd73 mRNA expressions between CD73high and CD73low astrocytes. Membrane shedding of CD73 by matrix metalloproteinase-9 (MMP-9) accounted for its membrane loss in astrocytes; meanwhile, C terminal truncated CD73 could be found in the medium of induced CD73low astrocytes. With an MMP-9 inhibitor in existence, the shedding of CD73 in wt-astrocytes, when interacted with CD73-/- effector CD4 cells, was almost completely blocked, and the production of pro-inflammatory cytokines, such as IL-17 and IFNγ, from interacted CD73-/- effectors, were significantly decreased. However, when a CD73 inhibitor was added together with MMP-9 inhibitor, decreased production of pro-inflammatory cytokines were completely restored. As conclusion, our findings suggested that under active inflammatory condition, MMP-9 releases CD73 from astrocytes. The block of CD73 shedding in astrocytes by the addition of MMP-9 inhibitor could significantly decrease the activation of interacted effector T cells.

CD73, an ectonucleotidase, participates in the regulation of immune responses by controlling the conversion of extracellular AMP to adenosine. In this study, we investigated whether any type of brain cells, especially neuroglia cells, exhibit altered CD73 expression, localization or activity upon experimental autoimmune uveitis (EAU) induction and whether altered CD73 manipulates the activation of effector T cells that interact with such cell types. First, the amount of cell membrane-exposed CD73 was detected by flow cytometry in various types of brain cells collected from either naïve or EAE mice. Compared to that in astrocytes from naïve control mice, the amount of membrane-bound CD73 was significantly decreased in astrocytes from EAE mice, while no significant differences were detected in other cell types. Thereafter, wild-type and CD73-/- astrocytes were used to study whether CD73 influences the function of inflammatory astrocytes, such as the production of cytokines/chemokines and the activation of effector T cells that interact with astrocytes. The results indicated that the addition of exogenous AMP significantly inhibited cytokine/chemokine production by wild type astrocytes but had no effect on CD73-/- astrocytes and that the effect of AMP was almost completely blocked by the addition of either a CD73 inhibitor (APCP) or an adenosine receptor A1 subtype (ARA1) antagonist (DPCPX). Although the addition of AMP did not affect CD73-/- astrocytes, the addition of adenosine successfully inhibited their cytokine/chemokine production. The antigen-specific interaction of astrocytes with invading CD4 cells caused CD73 downregulation in astrocytes from mice that underwent EAE induction. Collectively, our findings support the conclusion that, upon EAE induction, likely due to an interaction with invading CD4+ cells, astrocytes lose most of their membrane-localized CD73; this inhibits the generation of adenosine in the local microenvironment. As adenosine has anti-inflammatory effects on astrocytes and CNS-infiltrating effector T cells in EAE, the downregulation of CD73 in astrocytes may be considered a pro-inflammatory process for facilitating the pathogenesis of EAE.

The four kinds of adenosine receptor subtypes (ARs), named as ARA1, ARA2A, ARA2B and ARA3, have multiple biological functions. ARs are differently distributed across the body and have distinguished ability of binding adenosine. We try to figure out how these ARs were expressed in astrocytes and which one has the first priority of utilizing adenosine. Firstly, mRNA expressions and membrane localization of all ARs were evaluated by qPCR and western blot. After the membrane localization of all ARs in astrocytes was being confirmed their individual adenosine binding ability was determined by radio-active ligand binding assay respectively. It was revealed that ARA1 had much superior adenosine binding ability than other AR subtypes. Functional study demonstrated that ARA1 potentially mediated an immune suppressive effect in astrocytes. The activation of ARA1 signaling lead to decreased IL-12 and IL-23 production, and decreased chemokine production, including CCL2, CXCL8 and IP-10. When interacted with CD4 cells ARA1 agonist pre-treated astrocytes showed hindered ability of stimulating CD4 cells to secret IL-17 and IFN-γ and inducing CD4 cells' chemo taxi. Finally, in vivo experiment confirmed that local administration of ARA1agonist ameliorated EAE in wild type B6 recipients, but not Ara1-/- recipients. As a conclusion, this paper suggested that adenosine receptor A1 subtype predominantly binds adenosine in astrocytes and mediates an immunosuppressive effect.

Physiologically, retinal pigment epithelium (RPE) expresses high levels of CD73 in their membrane, converting AMP to immune suppressive adenosine, mediates an anti-inflammatory effect. However, after being exposed to inflammatory factors, RPE rapidly becomes CD73-negative cells, which render RPE’s immune suppressive function and accelerate local inflammation. Here, we investigated the mechanism leading to the loss of membrane CD73 in RPE. We found the controversy that when membrane CD73 was significantly diminished in inflammatory RPE, Cd73 mRNA levels were not changed at all. It was further verified that, matrix metalloproteinase-9 (MMP-9) mediated the shedding of CD73 from the cell membrane of inflammatory RPE by catalyzing its K547/F548 site. However, MMP-9 could not catalyze uncomplexed CD73, the interaction of CD73 with adenosine receptor A1 subtype (ARA1) is necessary for being catalyzed by MMP-9. After being treated by LPS and TNF-α, the formation of CD73/ARA1 complex in RPE was verified by co-immunoprecipitation and FRET-based assays. It was also revealed that CD73 need to be localized in lipid rafts to be capable of interacting with ARA1, since CD73/ARA1 interaction and CD73 shedding were completely blocked by the addition of lipid raft synthesis inhibitor. As a conclusion, multiple steps are involved in CD73 shedding in RPE, including up-regulation of MMP-9 activity, localization of CD73 in lipid rafts, and the formation of CD73/ARA1 complex. Lipid rafts committed CD73 with high mobility, shuttled CD73 to ARA1 to form a complex, which was capable of being recognized and catalyzed by MMP-9.

PURPOSE:Mechanism study of why astrocytes isolated from experimental autoimmune encephalomyelitis (EAE)-induced B6 mice or after being exposed to inflammatory factors had the highest transfection efficiency to larger-sized, but not compacted, pspCS/pDNA particles.METHODS:Phosphorylatable short peptide conjugated chitosan (pspCS) was compounded with plasmid DNA (pDNA) at different N:P ratios to form pspCS/pDNA particles of different size and zeta potentials. These pspCS/pDNA particles were used for the transfection of astrocytes isolated from either EAE induced or healthy B6 mice. Transfection efficiency and cell permeability of the particles were determined by the internalization of radio [H3]-labeled plasmid and the expression of a luciferase reporter gene respectively. Phagocytosis of EAE-astrocytes was determined by the internalization of FITC labeled dextran beads. By comparing the transfection efficiency of differently-sized pspCS/pDNA particles to normal and phagocytic astrocytes, with or without cytochalasin D, a phagocytosis inhibitor, in the presence, the contribution of phagocytosis to cell permeability and transfection efficiency was evaluated.RESULTS:In vivo EAE-induction or in vitro inflammatory factors treatment transferred normal astrocytes to be phagocytic astrocytes which underwent phagocytosis, had the highest cell permeability and transfection efficiency to larger-sized pspCS/pDNA particles formed at lower N:P ratios. When phagocytosis was inhibited by cytochalasin D, both cell permeability and transfection efficiency of phagocytic astrocytes to larger were significantly decreased. Thereafter, particle size, not zeta potential, was verified as the key factor for determining whether the particles could be phagocytosed. In addition phagocytosis was successfully induced in ARPE-19 cells as well, which also improved the transfection efficiency of larger pspCS/pDNA particles.CONCLUSION:A generally accepted concept is that the internalization of cationic polymer/pDNA particles, chitosan-DNA complex for instance, is mainly through the procedure of endocytosis of the transfected cells. More compacted particles with higher zeta potential were used to be considered had higher cell permeability and transfection efficiency. However, here we reported that phagocytosis is another important procedure for determining internalization and transfection efficiency of cationic polymer/pDNA nanoparticles, especially for advanced transfection efficiency of large pspCS/pDNA particles. Thus, for gene delivery applications, the environmental condition of the target cells should be seriously considered for selecting an appropriate gene transfer strategies.