Välimäki Elina, Cypryk Wojciech, Virkanen Juhani, Nurmi Katariina, Turunen Pauli M, Eklund Kari K, Åkerman Karl E, Nyman Tuula A, Matikainen Sampsa
Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland.
Finnish Institute of Occupational Health, 00250 Helsinki, Finland.
J Immunol. 2016 Oct 15;197(8):3315-3325. doi: 10.4049/jimmunol.1501840. Epub 2016 Sep 16.
Extracellular ATP is an endogenous danger signal that is known to activate inflammatory responses in innate immune cells, including macrophages. Activated macrophages start to secrete proteins to induce an immune response, as well as to recruit other immune cells to the site of infection and tissue damage. In this study, we characterized the secretome (i.e., the global pattern of secreted proteins) of ATP-stimulated human macrophages. We show that ATP stimulation activates robust vesicle-mediated unconventional protein secretion, including exosome release and membrane shedding, from human macrophages. Pathway analysis of the identified secreted proteins showed that calpain-related pathways were overrepresented in the secretome of ATP-stimulated cells. In accordance with this, calpains, which are calcium-dependent nonlysosomal cysteine proteases, were activated upon ATP stimulation through a P2X purinoceptor 7 receptor-dependent pathway. Functional studies demonstrated that calpain activity is essential for the P2X purinoceptor 7 receptor-mediated activation of unconventional protein secretion. Unconventional protein secretion was followed by cell necrosis and NLRP3 inflammasome-mediated secretion of the mature form of the proinflammatory cytokine IL-1β. Furthermore, ATP-driven NLRP3 inflammasome activation was also dependent on calpain activity. Interestingly, pro-IL-1β and inflammasome components ASC and caspase-1 were released by ATP-activated macrophages through a vesicle-mediated secretion pathway. In conclusion, to our knowledge, we provide the first global characterization of proteins secreted by ATP-activated human macrophages and show a pivotal role for calpains in the activation of the inflammatory response during ATP exposure.
细胞外ATP是一种内源性危险信号，已知其可激活包括巨噬细胞在内的先天免疫细胞中的炎症反应。活化的巨噬细胞开始分泌蛋白质以诱导免疫反应，并招募其他免疫细胞至感染和组织损伤部位。在本研究中，我们对ATP刺激的人巨噬细胞的分泌蛋白质组（即分泌蛋白的整体模式）进行了表征。我们发现，ATP刺激可激活人巨噬细胞中强大的囊泡介导的非常规蛋白质分泌，包括外泌体释放和膜脱落。对鉴定出的分泌蛋白进行的通路分析表明，钙蛋白酶相关通路在ATP刺激细胞的分泌蛋白质组中过度富集。与此一致的是，钙蛋白酶作为钙依赖性非溶酶体半胱氨酸蛋白酶，在ATP刺激下通过P2X嘌呤受体7受体依赖性途径被激活。功能研究表明，钙蛋白酶活性对于P2X嘌呤受体7受体介导的非常规蛋白质分泌激活至关重要。非常规蛋白质分泌之后是细胞坏死以及NLRP3炎性小体介导的促炎细胞因子IL-1β成熟形式的分泌。此外，ATP驱动的NLRP3炎性小体激活也依赖于钙蛋白酶活性。有趣的是，前体IL-1β以及炎性小体组分ASC和半胱天冬酶-1通过囊泡介导的分泌途径由ATP活化的巨噬细胞释放。总之，据我们所知，我们首次对ATP活化的人巨噬细胞分泌的蛋白质进行了全面表征，并表明钙蛋白酶在ATP暴露期间炎症反应激活中起关键作用。
