Background and Objectives The conversion of ATP to adenosine is an important mechanism of immune suppression by Tregs, and this is done by expression of CD39 and CD73. CD39 is a membrane bound ATPase that converts extracellular ATP and ADP to AMP, whereas CD73 is a membrane bound ecto-nucleotidase that converts AMP to adenosine. We have previously found evidence that the balance between pro-inflammatory ATP and anti-inflammatory adenosine is skewed in the synovial compartment of rheumatoid arthritis (RA) patients. We have developed two novel systems for the delivery of CD39 and CD73. The first is a Recombinant Anti-Inflammatory fusioN protein (RAIN) that consists of soluble CD73 fused to soluble CD39 by a flexible linker. The second approach uses exosomes containing CD39 and CD73. Here we present data characterising these CD39-CD73 delivery methods and investigate their efficacy in vitro.
Materials and Methods CD39-CD73 samples were produced by transfecting 293T cells with CD39-CD73 expressing plasmids. Exosomes were isolated using ExoQuick TC (System Biosciences) and soluble proteins were concentrated using a spin column. Antigen levels were assayed by quantitative western blot (Odyssey). CD39 and CD73 activity was measured by Malachite Green Phosphate (R&D Systems) assay using ATP or AMP as a substrate. In vitro inflammation assay was performed by incubating LPS activated THP-1 cells with CD39-CD73 containing samples in the presence of ATP. Whole blood inflammasome activation assay was performed by incubating whole blood with CD39-CD73 samples prior to 2 hr LPS stimulation followed by 1 hr ATP stimulation. Pro-inflammatory cytokine/chemokine levels were measured by ELISA.
Results RAIN and exosomes demonstrated high specific activity (RAIN: CD39 = 6660 U/pmol, CD73 = 16100 U/pmol; Exo: CD39 = 10900 U/pmol, CD73 = 2820 U/pmol). Both were potent in reducing pro-inflammatory cytokine and/or chemokine (IL-6, CCL2) expression in a THP-1 based in vitro inflammation assay (CCL2 EC50: RAIN 44 pM ± 1.13, exosomes 12.4 pM ± 1.27; IL-6 EC50: RAIN 29.3 pM ± 1.1, exosomes 5.9 pM ± 1.2). RAIN and CD39-CD73 exosomes were also effective in reducing IL-1β secretion in a whole blood inflammasome activation assay (IL-1β EC50: RAIN ~4000 pM, exosome ~100 pM).
Conclusions We have developed two novel biological therapeutics, RAIN and exosomes expressing CD39 and CD73 that are able to simultaneously decrease a potent pro-inflammatory molecule while increasing an anti-inflammatory molecule. The use of CD39 and CD73 to modulate inflammation should be applicable to broad spectrum of acute and chronic inflammatory disorders.
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