探討巨噬細胞NOD2在Toll-Like Receptor 4的訊息傳遞及誘發的發炎基因表現中所扮演的角色

Roles of NOD2 in TLR4-mediated signal transduction and induced inflammatory genes in macrophages

蔡文翔(Wen-Hsiang Tsai)

指導教授 ： 林琬琬

國立臺灣大學/醫學院/藥理學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.02392

摘要

NLR家族中的NOD2蛋白是屬於細胞內的受體，可辨識細菌的peptidoglycan所分離的組成份，並且在先天免疫反應裡扮演重要角色。最近一些報導指出，在克隆氏症(Crohn’s disease)的病人中，發現NOD2蛋白的LRR功能區段受到突變，但是NOD2蛋白的功能及調控其訊號傳遞的機制還尚未清楚。在先天免疫系統中，Toll-like receptors (TLRs)是胞漿內模式識別受體家族成員中的一個主要份子，而它的活化反應對於免疫扮演著重要的角色。最近的一些報導提到，TLR4受體在潰瘍性結腸炎(ulcerative colitis)和克隆氏症的病人中有大量表現的情形，也有探討TLR4受體的基因多型性(Asp299Gly and Thr399Ile)與克隆氏症的關係，但是TLR4受體與NOD2蛋白所引起的訊息傳遞之間的分子交互作用關係為何還是相當具有爭議。在此，我們利用研究小鼠的巨噬細胞，在TLR主導的訊息傳導與基因調控的過程，探討NOD2在其中扮演的角色。在小鼠巨噬細胞株(RAW 264.7)，TLR4的專一辨識配體(LPS)可以活化TNFα、IL-1β、iNOS及COX-2的表現，且這些表現會受到NOD2的專一辨識配體(MDP)所加強。另外，我們發現TLR4受體活化會誘發NOD2表現的能力，在RAW巨噬細胞中，我們發現由TLR4受體所主導而活化的MAPK和IKK或IκBα降解過程則不受MDP作用影響，而卻發現MDP作用會對TLR4受體所主導而活化的NF-κB有正向調控。這些結果顯示，NOD2蛋白受到MDP的活化之下，對於TLR4受體所引導的訊息傳遞路徑為一正調控因子。有趣的是，在NOD2蛋白壓抑(NOD2 silencing)的細胞中，LPS所主導活化的發炎反應會更增加並且在LPS耐受機制(LPS tolerance)下也可以誘發發炎反應。因此，我們提出一個論點，在不受MDP刺激之下，NOD2蛋白扮演著一個負向調控TLR4受體所引導的訊息傳遞的角色。此外，我們也證明此負向調控機制是同時需要NOD2蛋白中的CARD和LRR功能區段。在另外的實驗中，我們也發現NOD2蛋白能夠負向調控巨噬細胞的殺菌能力以及TLR4所引發的LC3蛋白的活化(是一個細胞自噬的標誌蛋白)。總括結果，NOD2蛋白對於TLR4受體所引導的訊息傳遞路徑有雙面的調控機制，於是我們提出一個論點﹕在不受配體刺激之下，細胞中持續性表現NOD2蛋白或因細胞受感染而誘發NOD2表現，此情形對於TLR4所引導的訊息傳遞扮演負向調控角色並且正調控TLR4耐受機制以及減少巨噬細胞殺菌能力。儘管NOD2蛋白受到配體而活化能夠增強TLR4所誘發的發炎反應，但如何透過NOD2所引導的雙面調控角色去解釋NOD2參與在克隆氏症的病理機制及過度發炎情形，例如，在克隆氏症的病人中發現NOD2有序列上的突變，但此突變如何導致疾病的形成，仍需要更多的研究。

關鍵字

NOD2蛋白 ； TLR4受體

並列摘要

NOD2 of NLR family has an important role in innate immunity as the cytosolic sensor of microbial components derived from bacterial peptidoglycan. Recent reports indicated that mutations in the leucine-rich repeat (LRR) domain of NOD2 have been implicated in the pathogenesis of Crohn’s disease (CD), yet the function of NOD2 and regulation of the NOD2 pathway remain unclear. Moreover, Toll-like receptor (TLR) is a major family of pattern recognition receptors (PRRs) and plays a crucial role in innate immune system. Recently, TLR4 is strongly up-regulated in both ulcerative colitis and CD. TLR4 polymorphisms (Asp299Gly and Thr399Ile) have been described in CD. Molecular interaction between TLR- and NOD2-signaling pathways is still controversial. Herein, we investigated the role of NOD2 in TLR-mediated signaling and gene regulation in murine macrophages. In murine RAW 264.7 macrophages, MDP (NOD2 ligand) can further increase LPS-induced pro-inflammatory cytokines and mediators, including TNFα, IL-1β, iNOS and COX-2. Accordingly we found the ability of TLR4 to induce NOD2 mRNA expression. We also found that MDP treatment could not affect TLR4-mediated activation of MAPK and IKK or IκBα degradation in RAW 264.7 macrophages, but it could up-regulate NF-κB activation. It is suggested that NOD2 is a positive regulator of TLR4-mediated signaling upon MDP treatment. Interestingly, NOD2 silencing cells lead to a further increase of LPS-induced inflammatory responses, but a decrease of LPS tolerance. We proposed that NOD2 could play an important role in negative regulation of TLR4-mediated signaling pathway in the absence of MDP treatment. Further, we demonstrated that both CARD and LRR domains of NOD2 protein were responsible for negative regulating function of TLR4. Besides, we also found that NOD2 could participate in down-regulation of macrophage bactericidal activity and TLR4-induced LC3 activation (an autophagy marker protein). In summary, NOD2 could have dual effects on TLR4 signaling. We proposed that the constitutively expressed NOD2 or infection up-regulated NOD2 without ligand stimulation plays roles to inhibit TLR4-mediated signaling, increase TLR4 tolerance and decrease bactericidal activity of macrophages. Nevertheless, activated NOD2 upon binding to pathogen-derived ligands can integrate with TLR4 signaling to enhance inflammatory response. How to interpret these two distinct roles together which possibly concomitantly happen in pathogenesis of infectious diseases and control the outcomes of infectious severity, e.g. the development of CD in patients whose NOD2 is functional mutant, still needs more investigation.

並列關鍵字

NOD2 ； TLR4

參考文獻

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