靈芝免疫蛋白 LZ-8 與 GMI 活化發炎體之機制

Abstract

IL-1β 為調控先天與後天免疫反應的重要促發炎細胞激素，並且其生成受到發炎體嚴密的機制調控。本篇研究以探討真菌免疫調節蛋白 (fungal immunomodulatory proteins, FIPs) 如何調控發炎體活化及IL-1β的分泌為主軸，先前研究發現給予LZ-8 及 GMI 於經LPS刺激小鼠巨噬細胞，能導致 IL-1β 分泌量上升。此外，添加高濃度氯化鉀於細胞培養液中能顯著降低FIPs所引起的IL-1β分泌量，顯示鉀離子外流對於FIPs 所誘導發炎體活化之重要性，而以dihydrorhodamine 123 (DHR) 螢光 probe 偵測到在FIPs刺激下能產生活性氧自由基 (ROS) ，代表FIPs造成巨噬細胞的發炎體活化需透過鉀離子外流的方式，並且ROS生成亦可能參與活化發炎體的路徑；進一步以 NLRP3-/- 及 ASC-/- 小鼠巨噬細胞進行實驗，結果證實 LZ-8與 GMI 主要調控 IL-1β 生成的路徑是藉由活化 NLRP3 inflammasome 此種發炎體以達成免疫調節，然而以caspase-1 -/- 小鼠進行實驗也發現FIPs的免疫調節作用需藉由 caspase-1活化但並未全然經由caspase-1，其他蛋白酶也可能參與其中。此外以 qRT-PCR 偵測 FIPs 是否具有活化發炎體相關基因表現之第二訊號作用，結果證實 LZ-8 刺激能些微提升巨噬細胞內 IL-1β、NLRP3、caspase-1、caspase-11及type I IFN之基因表現量，並且此作用可能是經由 TLR4 來進行訊號之傳遞。因此本研究證實靈芝免疫調節蛋白 FIPs 同時具有促進發炎體相關基因表現之能力及發炎體組裝之功能，進而引起促發炎細胞激素 IL-1β 的分泌，達到免疫調節的效果。

IL-1β is a key proinflammatory cytokine that regulates innate and adaptive immune responses, and the production of IL-1β is precisely controlled by the inflammasome. We previously found that fungal immunomodulatory proteins (FIPs) LZ-8 and GMI could induce IL-1β production in LPS-primed murine macrophages. In this study, we further investigated the mechanisms regulating FIP-induced inflammasome activation and IL-1β secretion. High concentration of KCl in the culture medium significantly suppressed FIP-induced IL-1β production, indicating that potassium efflux plays a vital role in FIP-induced inflammasome activation. Using the fluorescent probe dihydrorhodamine 123 (DHR), ROS production was detected in FIP-treated macrophages, suggesting that ROS generation also plays a role in activation inflammasome. Furthermore, FIP-induced inflammasome activation was strongly attenuated in NLRP3-/- and ASC-/- macrophages, suggesting that LZ-8 and GMI activate the NLRP3 inflammasome in LPS-primed macrophages. However, FIP-induced IL-1β production was only partially reduced in casp-1-/- macrophages, indicating that besides caspase-1, other protease(s) may also contribute to the processing of pro-IL-1β. In addition to providing signal 2 for inflammasome activation, LZ-8 also stimulated the expression of genes encoding IL-1β, NLRP3, caspase-1, caspase-11 and type I IFNs, and TLR4 might be involved in transducing signal. Taken together, our findings provide evidence that FIPs may stimulate both the expression and assembly of inflammasome components, resulting in the production of IL-1β that mediates FIPs immunomodulatory activities.