一個核糖核酸解旋酶於NLRP3發炎體活化的調控角色

Abstract

NLRP3發炎小體是由發炎體蛋白NLRP3、銜接蛋白ASC以及第一型半胱天冬酶(Caspase-1)所組裝而成的蛋白質複合體，於先天性免疫系統扮演重要的角色。當細胞受到感染或刺激而活化NLRP3發炎體，會促使第一型半胱天冬酶會產生蛋白酶的活性，進而催化第一型介白素β (IL-1β)以及第十八型介白素(IL-18)的成熟並啟動後續的發炎反應。同時第一型半胱天冬酶也具有引發細胞焦亡(pyroptosis)的能力。NLRP3發炎體的重要性在於幫助宿主抵禦外來物的侵犯，同時也可以維持組織的恆定性，然而當NLRP3發炎小體失去嚴謹的調控時，則導致自體發炎或自體免疫相關的疾病。因此，NLRP3發炎小體的調控相當重要，然而對於NLRP3發炎小體的分子調控機制至今尚未完全清楚。本實驗室先前的研究發現一個負向調控的蛋白質名為核糖核酸解旋酶27(DDX27)，而其在先天性免疫中扮演的角色目前尚未有任何報導。我們研究發現，在人類巨噬細胞株THP-1以及小鼠巨噬細胞株J774a.1抑制DDX27蛋白表現並處理第二型或第四型類鐸受體的活化劑時，可以偵測到第一型半胱天冬酶和第一型介白素β的釋放有大量增加的情形，同時也可以觀察到DDX27蛋白出核的現象，並與發炎小體蛋白NLRP3做結合。除此之外，已知鉀離子的流動、粒線體產生的活性氧化物質以及溶小體的損傷等事件是活化NLRP3發炎小體的重要條件，而我們的結果顯示DDX27並不會影響上述現象來達到調控NLRP3發炎小體的活化。另外，為了研究DDX27在生理功能上的意義，我們欲利用DDX27剔除小鼠，但過程中我們發現無法取得DDX27完全剔除的小鼠，而在小鼠骨髓分化的巨噬細胞中，DDX27的缺陷也可以看到NLRP3發炎小體過度活化的情形。這些研究結果證實DDX27是NLRP3發炎小體活化的負向調控者，但並不是透過影響上述活化條件來抑制第一型半胱天冬酶和第一型介白素β的釋放。

The NLRP3 inflammasome, an important component of innate immunity, is a multiple protein complex composed of NLRP3, ASC, and pro-caspase-1. Activation of the NLRP3 inflammasome induces caspase-1 activity, which results in the cleavage of pro-inflammatory cytokines, IL-1β and IL-18 into their bioactive forms, and induction of a type of inflammatory cell death, named pyroptosis. The NLRP3 inflammasome plays a crucial role in the clearance of microbial pathogens and tissue homeostasis. However, dysregulation of NLRP3 inflammasome has been associated with many autoimmune and auto-inflammatory diseases. Activation of the NLRP3 inflammasome, therefore, needs to be tightly regulated, but the molecular mechanism remains incompletely understood. We previously identified DEAD-box protein 27 (DDX27) from a yeast two-hybrid screening using PAI-2, a negative regulator of the NLRP3 inflammasome, as the bait, yet, the exact function of DDX27 in innate immunity remains unclear. We found that caspase-1 activation and IL-1β production increased in DDX27-depleted THP-1 macrophages treated with LPS. In this study, we further investigate the underlying mechanism by which DDX27 regulates NLRP3 inflammasome activation. We found that besides LPS, TLR2 ligand, Pam3CSK4 also enhanced caspase-1 activation and IL-1β production increased in DDX27-depleted THP-1 and J774a.1 macrophages. In addition, LPS induces DDX27 translocation from the nucleus to the cytoplasm, where it interacts with NLRP3 to suppress NLRP3 inflammasome activation. We further revealed that DDX27 was not involved in LPS-induced K+ efflux, mitochondrial ROS, and lysosomal destabilization, all of which have been reported to be the mediators of NLRP3 inflammasome activation. We also generated mice with DDX27 deletion and found out Ddx27-/- mice are embryonic lethal. DDX27-deficient bone marrow macrophages produced increased caspase-1 activation and IL-1β production upon NLRP3 activator challenge. Our results together suggest that DDX27 suppresses the formation of the NLRP3 inflammasome leading to negative regulation of caspase-1 activation and IL-1β production.