Interleukin-1β and Interferon-γ increase GAD-67 levels and the immunosuppressive properties of murine Mesenchymal Stem Cells in vitro

Abstract

Mesenchymal Stem Cells (MSC) are multipotent cells widely used in regenerative medicine that possess strong immunosuppressive capabilities. Recent studies show that Interferon-γ (IFN-γ), in combination with other pro-inflammatory cytokines, stimulates MSC-mediated immunosuppression. It has been shown that immune system cells do produce and react to neurotransmitters, like γ-aminobutyric acid (GABA), indicating functional interaction between the immune and nervous systems. This neurotransmitter is synthesized from glutamate by glutamic acid decarboxylase (GAD). Two isoforms of different size, subcellular localization and biochemical properties, GAD-67 and GAD-65, do exist in mammals. GABA exerts potent immunosuppressive actions and pre-clinical studies demonstrate its effectiveness in preventing autoimmune diseases. However, it is not known whether GABA biosynthesis relates to the immunosuppressive capabilities of MSC. By using RT-qPCR and Western blot analysis, we determined mRNA and protein levels of GAD-67 and GAD-65 in MSC treated with combinations of IFN-γ and IL-1β, and the immunosuppressive activities of treated MSC were assessed in co-culture using a CD4+ T-cell proliferation assay. We demonstrate: (1) sufficiency of IL- 1β to increase GAD-67 mRNA levels; (2) requirement of IFN-γ for increased GAD-67 protein levels in IL-1β/IFN-γ co-treatment; (3) sufficiency of IL-1β priming to increase MSC-mediated immunosuppression. Our results reveal a selective increase in GAD-67 levels in MSC exposed to pro-inflammatory conditions that agrees with increased immunosuppressive capabilities of MSC. Our results suggest that GAD-67/GABA synthesis might be involved in the immunosuppressive machinery of MSC. Knockdown experiments will allow us to determine GAD-67/GABA role in MSC-mediated immunosuppression.