探討脾酪胺酸激酶調控類鐸受體訊息傳遞及NLRP3發炎小體活化的角色

Abstract

先天免疫對於宿主免疫反應是一個很重要的戰略，可以抵抗病原菌入侵。類鐸受體Toll-like receptors (TLRs) 和NOD-like receptors (NLRs) 是重要的模式辨識受體 (PRRs)，負責辨識入侵病原菌的特定病原體相關的分子模式 (PAMPs)後進一步啟動發炎反應活化的訊息路徑。脾酪胺酸激酶 (Syk) 是屬於非受體酪胺酸激酶家族(non-receptor tyrosine kinase)，會參與多種含有immunoreceptor tyrosine-based activation motifs (ITAMs) 的免疫相關受體所引起的訊息路徑，包含B細胞受體(BCR) 和各種Fc受體(FcR)，而在後天免疫反應中扮演著重要角色，同時也與很多免疫相關疾病的病程發展息息相關。近年來，已有報導指出Syk蛋白激酶也與類鐸受體所激發的訊息路徑和調控NLRP3發炎小體 (inflammasome)活化有關連性存在，不過在其中的詳細作用機制目前還不是十分明朗。在本篇論文中，我們提出Syk在調控類鐸受體和NLRP3發炎小體所誘導的發炎反應訊息路徑中有多種的調控角色存在。 首先，我們發現到Syk不僅與TLR4内噬作用有關係，同時也在TLR4媒介的訊息傳遞中扮演雙重的角色。當由其配位體 (LPS) 刺激而使TLR4活化時，與野生型 (wild type) 相比之下，在Syk缺失的巨噬細胞中會更增強TAK1激酶磷酸化及活化，及下游媒介的促發炎細胞激素(proinflammatory cytokines)產生。相反地，在Syk缺失的巨噬細胞中，顯示出會降低由TLR4依賴性的TBK1-IRF3活化路徑，這一個路徑對於產生第一型干擾素是被需要的。我們的結果同時也顯示，當LPS刺激後 Syk會出現在包含TRAF6和TRAF3的訊息複合體中，且由LPS引起的TRAF6和TRAF3的lysine-63–linked ubiquitination會受Syk相反性的調控。我們也確定Syk利用何種domains來與TRAF3、TRAF6、TAK1和TBK1結合在一起，因此這些結果顯示出，Syk在各種不同的TLR媒介反應中是一個重要的調控角色，扮演著對於TLR4媒介的TRAF6及TRAF3的訊息路徑有相反的角色。 除此之外，我們也證明Syk媒介由NLRP3刺激物引起的caspase-1活化。在HEK293T重組系統中，Syk的激酶活性 (kinase activity) 對於caspase-1的活化是必要的，且促進接合器蛋白ASC將NLRP3和其行動器蛋白procaspase-1連結在一起。在本篇研究中，我們發現Syk可以藉由它的激酶區域 (kinase domain) 直接與ASC和NLRP3結合在一起，不過會以間接方式與procaspase-1相互交互作用。Syk可以磷酸化ASC的Y146和Y187的胺基酸，且這兩個位置的磷酸化對於增強ASC寡聚合作用 (oligomerization) 和招募procaspase-1是非常重要的步驟。我們的結果顯示一個新的分子訊息傳遞路徑，發現Syk可以磷酸化ASC而促進NLRP3發炎小體的形成，及成熟發炎細胞激素IL-1b的產生。 總和以上結果，我們發現Syk可以調控先天免疫反應，因此控制Syk活性可能對於調節由TLRs媒介的發炎反應和NLRP3 發炎小體活化是一個有效的方式。在本篇研究中我們提供了一個藥理上發展調節Syk激酶活性的新視野，同時可以讓Syk成為一個重要的標靶蛋白，來治療如類風濕性關節炎和紅班性狼瘡這一類免疫相關的疾病。

The innate immune response triggered by pattern-recognition receptors (PRRs) is an important strategy for the host defense and resists a wide range of pathogens invasion. Toll-like receptors (TLRs) and NOD-like receptors (NLRs) are important PRRs which can recognize specific pathogen-associated molecular patterns (PAMPs) on invading pathogens and further trigger inflammatory signaling pathway activation. Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase that is involved in numerous immunoreceptors signaling pathways, including B cell receptor (BCR), T cell receptor (TCR) and FcgR that contain immunoreceptor tyrosine-based activation motifs (ITAMs). Thus, Syk plays crucial roles in adaptive immunity and is related with the development of several autoimmune diseases. Recent studies have reported that Syk is involved in the signaling mediated by TLRs and NLRP3 inflammasome; however, the detailed mechanisms linking Syk to TLRs and NLRP3 inflammasome remain unclear. In this thesis, we demonstrate that Syk has multiple roles in controlling TLRs- and NLRP3 inflammasome-mediated signaling pathway. First of all, we found that Syk was not only involved in the endocytosis of TLR4, but also played a dual role in TLR4-mediated signaling. LPS-dependent stimulation of TLR4 in Syk-deficient macrophages led to enhanced activation of TAK1 and increased production of proinflammatory cytokines, compared to that in wild-type macrophages. In contrast, Syk-deficient macrophages exhibited decreased TLR4-dependent activation of the TBK1 signaling and production of type I IFNs. We demonstrated that Syk was present in both TRAF6- and TRAF3-containing signaling complexes; however, the LPS-dependent, lysine-63–linked ubiquitination of TRAF6 and TRAF3 was oppositely regulated by Syk. We also identified the domains of Syk that interacted with TRAF3, TRAF6, TAK1, and TBK1, factors activated by multiple TLRs, thus suggesting the role of Syk as a common regulator of various TLR responses and played the opposing regulatory roles in TLR4-mediated TRAF6 and TRAF3 signaling pathways. In addition, we also showed Syk mediates NLRP3 stimuli-induced processing of procaspase-1 and the consequent activation of caspase-1. Moreover, the kinase activity of Syk is required to potentiate caspase-1 activation in reconstituted HEK293T cells system. The adaptor protein ASC bridges NLRP3 with the effector protein caspase-1. Herein, we find that Syk can directly associate with ASC and NLRP3 by its kinase domain, but indirectly interact with procaspase-1. Syk can phosphorylate ASC at Y146 and Y187 residues, and the phosphorylation of both residues is critical to enhance ASC oligomerization and the recruitment of procaspase-1. Collectively, our results reveal new molecular pathway through which Syk promotes NLRP3 inflammasome formation resulting from the phosphorylation of ASC. Taken together, we found that controlling Syk activity might be effective to modulating TLRs-mediated inflammation and NLRP3 inflammasome activation, which suggest that Syk may fine-tune the innate immune responses. These findings not only provide a new insight into the pharmacologic modulator of Syk kinase but also reveal Syk as a promising therapeutic target in immune-related diseases.