靈芝免疫調節蛋白 LZ-8 和 GMI 對發炎體活化之作用

Abstract

IL-1β 是一個關鍵的促發炎反應細胞激素，同時可調控先天與後天免疫反應，而IL-1β 的產生受到發炎體的嚴密管控。發炎體是一類多蛋白複合體，主要負責 caspase-1 的活化，而使得 caspase-1 對 pro-IL-1β 進行酵素作用而成為具有功能性的 IL-1β ，才能分泌至胞外。本篇研究探討靈芝免疫調節蛋白(fungal immunomodulatory proteins, FIPs) LZ-8 和 GMI 刺激小鼠巨噬細胞後，對於發炎體的活化及 IL-1β 分泌的影響。結果發現 LZ-8 和 GMI 可使經 LPS 刺激的巨噬細胞之 caspase-1 活化，導致 IL-1β 的分泌量上升。進一步發現，於細胞培養中添加高濃度氯化鉀、使用P2X7 受體或 pannexin-1 的抑制劑皆能顯著降低 FIP 所引起的 IL-1β 分泌量，顯示 FIPs 所引發的發炎體活化需要透過鉀離子由離子通道外流的途徑。此外，抑制活性氧自由基 (ROS) 的產生也可以有效降低 IL-1β 的分泌量，代表 ROS 也可能參與其中的活化路徑。此外， LZ-8 以腹腔注射方式打入小鼠體內後，可引發嗜中性球聚集至腹腔中，而對於後續李斯特菌的感染具有保護的效果，上述的保護現象在 IL-1 受體缺陷 (IL-1R-/-) 小鼠中則顯著下降，顯示 IL-1β-IL-1R 訊息在 LZ-8 誘發的先天免疫反應中具有重要的角色。相反地，經初步實驗結果發現， IL-1β-IL-1R 訊息在 LZ-8 的佐劑活性中較不具影響力。總結而言，本研究證實 FIPs 是可活化發炎體的蛋白質，故在使用 FIPs 作為免疫調節物時，須同時考慮其活化發炎體的特性。

IL-1β is a key proinflammatory cytokine regulating both innate and adaptive immune responses, and its production is controlled by the inflammasome. Inflammasomes are multiprotein platforms responsible for caspase-1 activation and subsequent processing and secretion of mature IL-1β. Here we studied the stimulatory effect of fungal immunomodulatory proteins (FIPs) LZ-8 and GMI on inflammasome activation and IL-1β production in murine macrophages. FIP stimulation resulted in caspase-1 activation and robust IL-1β production in LPS-primed macrophages. FIP-induced IL-1β production was inhibited in the presence of high extracellular KCl or inhibitors targeting the membrane pores P2X7 and pannexin-1, indicating that FIP-induced inflammasome activation requires potassium efflux through ion channels. In addition, inhibition of reactive oxygen species (ROS) production also reduced FIP-induced IL-1β production, indicating that ROS generation is also involved in the process. Intraperitoneal administration of LZ-8 in mice elicited a robust influx of neutrophils and protected mice against subsequent L. monocytogenes infection, which were both attenuated in IL-1R-/- mice, indicating that the IL-1β-IL-1R signal plays a significant role in the innate immune response triggered by LZ-8. In contrast, our preliminary results indicate that the adjuvant function of LZ-8 is less dependent on the IL-1β-IL-1R signal. Overall, our findings provide evidence that FIPs are a new type of protein ligand for inflammasome activation, and this activity should be taken into consideration when using FIPs as an immunomodulatory agent.