Overexpression of Glutamate Decarboxylase in Mesenchymal Stem Cells Enhances Their Immunosuppressive Properties and Increases GABA and Nitric Oxide Levels

The neurotransmitter GABA has been recently identified as a potent immunosuppressive agent that targets both innate and adaptive immune systems and prevents disease progression of several autoimmunity models. Mesenchymal stem cells (MSCs) are self-renewing progenitor cells that differentiate into various cell types under specific conditions, including neurons. In addition, MSC possess strong immunosuppressive capabilities. Upon cytokine priming, undifferentiated MSC suppress T-cell proliferation via cell-to-cell contact mechanisms and the secretion of soluble factors like nitric oxide, prostaglandin E2 and IDO. Although MSC and MSC-derived neuron-like cells express some GABAergic markers in vitro, the role for GABAergic signaling in MSC-mediated immunosuppression remains completely unexplored. Here, we demonstrate that pro-inflammatory cytokines selectively regulate GAD-67 expression in murine bone marrow-MSC. However, expression of GAD-65 is required for maximal GABA release by MSC. Gain of function experiments using GAD-67 and GAD-65 co-expression demonstrates that GAD increases immunosuppressive function in the absence of pro-inflammatory licensing. Moreover, GAD expression in MSC evokes an increase in both GABA and NO levels in the supernatants of co-cultured MSC with activated splenocytes. Notably, the increase in NO levels by GAD expression was not observed in cultures of isolated MSC expressing GAD, suggesting crosstalk between these two pathways in the setting of immunosuppression. These results indicate that GAD expression increases MSC-mediated immunosuppression via secretion of immunosuppressive agents. Our findings may help reconsider GABAergic activation in MSC for immunological disorders.近期研究证实，神经递质γ-氨基丁酸（GABA）是一种强效免疫抑制剂，可靶向先天免疫与适应性免疫系统，延缓多种自身免疫模型的疾病进展。间充质干细胞（MSCs）是一类具备自我更新能力的祖细胞，可在特定条件下分化为包括神经元在内的多种细胞类型。此外，间充质干细胞具备强大的免疫抑制能力：在经细胞因子预刺激后，未分化的间充质干细胞可通过细胞间接触机制，以及分泌一氧化氮（NO）、前列腺素E2、吲哚胺2,3-双加氧酶（IDO）等可溶性因子，抑制T细胞增殖。尽管间充质干细胞及其诱导的神经元样细胞在体外可表达部分GABA能标志物，但GABA能信号通路在间充质干细胞介导的免疫抑制中的作用仍完全未被探明。本研究证实，促炎细胞因子可选择性调控小鼠骨髓间充质干细胞中谷氨酸脱羧酶67（GAD-67）的表达；而间充质干细胞要实现最大程度的γ-氨基丁酸释放，则需要谷氨酸脱羧酶65（GAD-65）的参与。采用共表达GAD-67与GAD-65的功能获得性实验证实，在未经过促炎预激活的情况下，谷氨酸脱羧酶可增强间充质干细胞的免疫抑制功能。此外，间充质干细胞中谷氨酸脱羧酶的表达，可在与活化脾细胞共培养的上清液中同时提升γ-氨基丁酸与一氧化氮的水平。值得注意的是，仅表达谷氨酸脱羧酶的分离培养间充质干细胞中，并未观测到一氧化氮水平的升高，这提示在免疫抑制过程中，这两条通路存在交叉对话。上述结果表明，谷氨酸脱羧酶的表达可通过分泌免疫抑制因子，增强间充质干细胞介导的免疫抑制作用。本研究结果或有助于重新审视针对免疫紊乱性疾病，采用GABA能通路激活间充质干细胞的治疗思路。