在先前的研究中我們已經證明在病原相關分子(PAMPs)活化巨噬細胞的路徑當中，iNOS/Src/FAK是一個通用的路徑。而且Eps8參與在TLR4所誘導的通用路徑當中，促進吞噬作用以及殺菌作用。在第一篇研究中我們發現在聚肌胞苷酸 (polyI:C)刺激巨噬細胞下TLR3 的Y759位置會有早期和晚期兩個階段的磷酸化。 除了短時間的第一階段TLR3 Y759位置的磷酸化，在雙股RNA的刺激後期，我們還發現了第二階段的TLR3 Y759位置的磷酸化，而且與Src的表現量還有IFN-β的產生有關。有趣的是，不論在in vitro或是in vivo實驗中皆發現Src可以磷酸化TLR3 的Y759位置。然而，Src的抑制會破壞晚期TLR3 Y759位置的磷酸化，而且降低IRF3和IRF7的入核量以及IFN-β的產生。重新表現Src則會回復所有因Src被抑制而改變的分子機制。值得注意是，在iNOS缺乏的巨噬細胞中我們也觀察到透過抑制Src的表現，polyI:C所誘導TLR3 的Y759位置磷酸化、IRF3和IRF7的入核以及IFN-β的產生都受到抑制。在巨噬細胞受到LPS的刺激下(LPS，一種TLR4的配體，已知會誘導Src與IFN-β的產生)，抑制TLR3的表現會造成Src蛋白的不穩定，降低IRF3和IRF7在核中的表現量以及減少IFN-β的產生。異位表達原始的TLR3會回復Src的活性和ifn-β轉錄能力，但異位表達Y759位置磷酸突變的TLR3則無法回復Src的活性和ifn-β轉錄能力。總括來說，這些結果可以推測在巨噬細胞產生IFN-β的路徑中，iNOS/Src/TLR3 路徑軸是不可或缺的。在第二個研究中，我們觀察到Eps8的表達是受到PAMP誘導並且需要iNOS / Src。當Eps8減少同時會降低Src的活性，並且抑制巨噬細胞的移動能力。值得注意的是，在病原相關分子刺激Src受抑制的巨噬細胞中，異位表達Eps8可以回復部分Src的活性以及細胞移行能力。這些結果表明Eps8會調控TLR4誘導的訊息傳遞並且參與在TLRs刺激巨噬細胞由Src所調控的細胞移行能力中。在第三個研究中，我們發現降低Src的表現量會去抑制NO的產生和細胞激素的分泌；如果將Src 的表達量回復的話，NO的產生和激素的分泌則會有回復的現象。抑制Eps8也會降低LPS所誘導的iNOS的表現以及Src的活性。的確，Eps8會透過NF-κB訊息的活化來調控NO的產生以及TNF-α、IL-1β和IL-6的分泌。總括來說，我們的實驗證據指出Eps8和Src在LPS誘導NF-κB的活性中是不可或缺的，而且對於巨噬細胞所調控的先天免疫系統有很大的貢獻。

We earlier demonstrated iNOS/Src/FAK axis as a general mechanism of macrophage motility in response to various pathogen-associated molecular patterns (PAMPs) and Eps8 took part in TLR4-mediated signal transduction, enhancing phagocytosis and bacterial killing effect. In the first study, we found that dsRNA stimulation induces biphasic TLR3 Tyr-759 phosphorylation in macrophages. In addition to the immediate TLR3 Tyr-759 phosphorylation, we identified a second wave of Tyr-759 phosphorylation accompanied by an increase of both Src and ifn-β transcription in the later phase of dsRNA stimulation. Interestingly, Src phosphorylated TLR3 Tyr-759 in vitro and in vivo. However, knockdown of Src abolished the late phase of TLR3 Tyr-759 phosphorylation and decreased the nuclear accumulation of interferon regulatory factors 3 and 7 (IRF3 and -7) and IFN-β production. Reintroduction of Src restored all of these molecular changes. Notably, via down-regulation of Src, dsRNA-elicited TLR3 Tyr-759 phosphorylation, the nuclear accumulation of IRF3/IRF7, and IFN-β generation were inhibited in inducible nitric-oxide synthase (iNOS)-null macrophages. TLR3 knockdown destabilized Src and reduced the nuclear level of IRF3/IRF7 and IFN-β production in macrophages exposed to LPS (a TLR4 ligand known to induce Src and IFN-β expression). Ectopic expression of wild type TLR3, but not its 759-phenylalanine mutant, restored Src activity and ifn-β transcription. Taken together, these results suggested an essential role of the iNOS/Src/ TLR3 axis in IFN-β production in macrophages. In the second study, we observed that expression of Eps8 was PAMP-inducible and iNOS/Src-dependent. Attenuation of Eps8 simultaneously impaired Src activity and suppressed macrophage mobility. Notably, ectopic Eps8 partly restored motility and Src activity in Src-attenuated macrophages exposed to PAMPs. These findings indicated Eps8 modulating TLR4-mediated signal transduction and taking part in Src-mediated cell migration in TLRs-stimulated macrophages. In the third study, we found that Src knockdown impaired LPS-induced NO production and cytokines secretion, which was reverted by ectopically expressed avian Src. Attenuation of Eps8 also reduced LPS-mediated iNOS expression and Src activation. Indeed, via activation of NF-κB signaling, Eps8 affected NO production and the secretion of TNF-α, IL-1β, and IL-6. Taken together, our data indicates that Eps8 and Src are necessary for LPS mediated NF-κB activation and contributes to macrophage-mediated innate immunity.

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