探討NLRX1在巨噬細胞中調控粒線體功能及參與先天免疫反應的角色

Abstract

NLRX1為先天免疫受體中NOD類受體（NLR）的一員，過去主要被報導位於粒線體，並涉及免疫細胞中的多種免疫反應，包括抑制RIG-I誘導的第一型干擾素產生，抑制RIG-I-MAVS和TLR4-TRAF6-NF-κB的訊息傳遞路徑，增強活性氧化物質(ROS)而促進腫瘤壞死因子(TNF-α)，志賀氏菌和雙鏈RNA誘導的NF-κB和JNK的活化。過去研究指出NLRX1會與TUFM產生交互作用，進一步活化小鼠胚胎成纖維細胞的自噬途徑，然而透過與Beclin 1-UVRAG複合物的相互作用而負調節Hela細胞的自噬功能。最近的研究表明NLRX1可藉由與LC3蛋白的作用而為粒線體自噬受體。另外，NLRX1也對粒線體功能扮演調控的角色，包含藉由Drp1的磷酸化來誘導神經元細胞的粒線體分裂，調控肝臟細胞的粒線體呼吸鏈等。迄今為止，NLRX1對調控先天免疫反應的兩項因素，即粒線體功能及細胞自噬的作用尚未完全清楚闡明。因此，在本研究中，我們使用盲腸結紮穿刺作為體內敗血症的小鼠模型及小鼠骨髓衍生的巨噬細胞，比較野生型和NLRX1基因剔除對於盲腸結紮穿刺及脂多醣誘導的發炎反應和自噬激活的作用。接著，我們也使用LPS處理的巨噬細胞以了解NLRX1調節粒線體功能的機制。首先我們在盲腸結紮穿刺誘導的敗血症模型中觀察小鼠的肝和肺組織，發現NLRX1基因剔除小鼠能產生更嚴重的發炎反應但未能誘導LC3II蛋白的積累，顯示NLRX1基因剔除使敗血症惡化可能與自噬功能的失調有關。此外，我們發現在巨噬細胞NLRX1基因剔除也會增強LPS誘導的發炎反應，然而LC3II的蛋白表現量因缺乏NLRX1而降低。後者的結果並非基於基因表現量降低或經增強溶酶體之分解而來。此外，我們發現NLRX1基因剔除會降低靜止狀態及LPS激活之細胞質的ROS產生，及改善LPS誘導粒線體膜電位的下降，但並沒有顯著改變粒線體的ROS水平。除此之外，NLRX1基因剔除會減少靜止狀態粒線體質量，及抑制LPS誘導的粒線體質量減少。共軛焦顯微鏡的結果顯示NLRX1基因剔除會增加巨噬細胞粒線體的融合。值得注意的是，NLRX1基因剔除會同時促進粒線體的呼吸作用及LPS誘導的糖解作用。總的來說，NLRX1在敗血症及LPS誘導的發炎反應中扮演負調節的作用，此作用除了源自NF-B抑制外，亦與誘導細胞自噬及影響能量代謝有關。我們的研究提供NLRX1藉由調節粒線體功能參與發炎反應的新見解，未來有必要進一步研究NLRX1調控粒線體合成、能量代謝和細胞自噬之間相互作用的分子機制。

NLRX1, which belongs to NOD-like receptors (NLR) family, is mostly localized at the mitochondria and involves in multiple responses in immune cells. These include inhibition of RIG-I-induced type I IFNs production, interference with the RIG-I-MAVS and TLR4-TRAF6-NF-κB signaling pathways, but enhancement of ROS-mediated NF-κB and JNK activation induced by TNF-, shigella infection and double-stranded RNA. Moreover, NLRX1 associates with the mitochondrial Tu translation elongation factor (TUFM) to activate autophagic pathway in MEFs, but interacts with the Beclin 1-UVRAG complex to negatively regulate autophagy in Hela cells. A recent study reveals that NLRX1 serves as a mitophagy receptor through interaction with LC3. In addition, NLRX1 can be a modulator of mitochondrial functions by inducing mitochondrial fission in neuronal cells via Drp1 phosphorylation and controlling mitochondrial respiration in hepatocytes. To date, the roles of NLRX1 in innate immune response and underlying mechanisms related to mitochondrial functions and autophagy have not been fully elucidated. Therefore, in this study we used cecal ligation and puncture (CLP) as a sepsis model in vivo and cultured bone marrow-derived macrophages (BMDMs) to determine the functional role of NLRX1 in innate response. We found that NLRX1 deficiency induced higher inflammatory responses but failed to induce LC3II accumulation in both liver and lung tissues in mice after CLP surgery. These findings suggest that NLRX1 deficiency worsens the pathological status of sepsis possibly owing to dysfunctional autophagy. In addition, LPS-induced inflammatory responses and LC3II protein expression at resting state were decreased in NLRX1-/- BMDMs. The latter event is not related to the gene expression nor lysosomal degradation of LC3. The NLRX1 deficiency also decreased basal and LPS-induced cytosolic ROS production as well as LPS-induced mitochondrial membrane potential loss, but it had no effect on mitochondrial ROS level. Furthermore, NLRX1 deficiency attenuated mitochondrial mass and prevented the inhibition of this event in response to LPS. The data from confocal microscopy revealed the higher mitochondrial fusion in NLRX1 deficienct macrophages. Of note, NLRX1-/- BMDMs displayed higher mitochondrial respiration before and after LPS stimulation, as well as higher glycolysis after LPS stimulation. Taken together, our data indicate that NLRX1 is a negative regulator of inflammation in septic mice and LPS-stimulated macrophages, and these effects might ascribe to the effects of NLRX1 on inhibition of NF-B pathway as previously observed, induction of autophagy and regulation of metabolism as observed in our work. This study expands the knowledge of NLRX1 in regulation of mitochondrial functions during inflammation. It is necessary to further investigate the molecular mechanisms and signaling interplay in mitochondrial biogenesis, metabolism and autophagy regulated by NLRX1.