Background and objectives The conversion of extracellular ATP (eATP) to adenosine is an important mechanism of immune suppression through the coordinated activity of two enzymes, CD39 and CD73. CD39 is a membrane bound ATPase, and CD73 is a membrane bound ectonucleotidase that converts AMP to adenosine. We have previously presented evidence that the balance between pro-inflammatory eATP and anti-inflammatory adenosine is skewed in the synovial compartment of rheumatoid arthritis patients. Here we developed strategies for the use of CD39 and CD73 in inflammatory disease, including a soluble Recombinant Anti-Inflammatory fusioN (RAIN) CD39-CD73 fusion protein and transgenic exosomes containing CD39 and CD73.
Materials and methods Whole blood inflammasome activation assay was performed by incubating whole blood with CD39-CD73 samples prior to 2 h LPS stimulation followed by a 1 h ATP stimulation. Pro-inflammatory cytokine/chemokine levels were measured by ELISA. Cell factories seeded with stably expressing 293 cells were used for large scale production of RAIN or CD39-CD73 exosomes. Conditioned serum-free media was collected every 3–5 days until a total of ˜10 L was collected. Conditioned media was concentrated (100 fold) by tangential-flow filtration (TFF) and RAIN was purified by anion-exchange chromatography, whereas exosomes were purified using Total Exosome Isolation reagent. Large scale batches of RAIN and exosomes were characterised by western blot and CD39-CD73 activities were measured by malachite green assay.
Results Small scale batches of CD39-CD73 containing exosomes or RAIN were potent in reducing IL-1β secretion in a whole blood inflammasome activation assay (IL-1β EC50: RAIN ˜4000 pM, exosome ˜100 pM). Large scale production of RAIN was successful and yielded ˜2.5 mgs with ˜90% purity. The protein had a high specific activity (˜16000 U/μg CD39 activity, 24400 U/μg CD73 activity). Exosome purification yielded ˜25 mls of CD39-CD73 containing exosomes with ˜1316,4000 U/mL (36,5000 U/μg) CD39 activity and ˜253,9000 U/mL (13,5300 U/μg) of CD73 activity. These yields are sufficient for future in vivostudies. Interestingly, the exosomal enzymes had significantly higher specific activity (5–20 fold) when compared with RAIN.
Conclusion These data demonstrate that exosome or fusion protein delivery of CD39 and CD73 is effective in reducing pro-inflammatory cytokine production in vitro, and that large scale production of CD39 and CD73 containing fusion protein or exosomes is feasible.
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