Good Biomarker Sciences, Leiden, the Netherlands.
Centre for Human Drug Research, Leiden, the Netherlands.
PLoS One. 2019 Apr 8;14(4):e0214999. doi: 10.1371/journal.pone.0214999. eCollection 2019.
Processing of pro-interleukin (IL)-1β and IL-18 is regulated by multiprotein complexes, known as inflammasomes. Inflammasome activation results in generation of bioactive IL-1β and IL-18, which can exert potent pro-inflammatory effects. Our aim was to develop a whole blood-based assay to study the inflammasome in vitro and that also can be used as an assay in clinical studies. We show whole blood is a suitable milieu to study inflammasome activation in primary human monocytes. We demonstrated that unprocessed human blood cells can be stimulated to activate the inflammasome by the addition of adenosine 5'-triphosphate (ATP) within a narrow timeframe following lipopolysaccharide (LPS) priming. Stimulation with LPS resulted in IL-1β release; however, addition of ATP is necessary for "full-blown" inflammasome stimulation resulting in high IL-1β and IL-18 release. Intracellular cytokine staining demonstrated monocytes are the major producers of IL-1β in human whole blood cultures, and this was associated with activation of caspase-1/4/5, as detected by a fluorescently labelled caspase-1/4/5 probe. By applying caspase inhibitors, we show that both the canonical inflammasome pathway (via caspase-1) as well as the non-canonical inflammasome pathway (via caspases-4 and 5) can be studied using this whole blood-based model.
白细胞介素 (IL)-1β 和 IL-18 的加工受多蛋白复合物（称为炎性体）调节。炎性体的激活导致生物活性的 IL-1β 和 IL-18 的产生，这可以发挥强大的促炎作用。我们的目的是开发一种基于全血的体外测定法来研究炎性体，该方法也可用于临床研究。我们表明全血是研究原代人单核细胞中炎性体激活的合适环境。我们证明，未经处理的人血细胞可以通过在脂多糖 (LPS) 引发后很短的时间内添加三磷酸腺苷 (ATP) 来刺激激活炎性体。用 LPS 刺激导致 IL-1β 的释放；然而，ATP 的添加对于导致高 IL-1β 和 IL-18 释放的“完全”炎性体刺激是必需的。细胞内细胞因子染色表明单核细胞是人类全血培养物中 IL-1β 的主要产生者，这与通过荧光标记的 caspase-1/4/5 探针检测到的 caspase-1/4/5 的激活相关。通过应用半胱天冬酶抑制剂，我们表明可以使用这种基于全血的模型研究经典炎性体途径（通过半胱天冬酶-1）和非经典炎性体途径（通过半胱天冬酶-4 和 5）。
