環狀核苷酸磷酸二酯酶4對LPS/TLR4訊息傳導誘導小鼠巨噬細胞表現IFN-β的影響;Cyclic nucleotide phosphodiesterase 4 regulates LPS/TLR4 signaling-induced IFN-β expression in mouse macrophages

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Title:	環狀核苷酸磷酸二酯酶4對LPS/TLR4訊息傳導誘導小鼠巨噬細胞表現IFN-β的影響;Cyclic nucleotide phosphodiesterase 4 regulates LPS/TLR4 signaling-induced IFN-β expression in mouse macrophages
Authors:	陳慶戎;Chen,Ching-jung
Contributors:	生命科學系
Keywords:	環狀核苷酸磷酸二酯酶4;巨噬細胞;干擾素-β;環狀核苷酸;Phosphodiesterase4;Macrophage;IFN-β;cAMP
Date:	2014-08-28
Issue Date:	2014-10-15 14:39:19 (UTC+8)
Publisher:	國立中央大學
Abstract:	干擾素-β(IFN-β)可促進多種干擾素刺激基因(IFN stimulated genes)表現，提高宿主的先天免疫能力。文獻指出，多種抗原結構如細菌表面結構lipopolysaccharide (LPS)會藉由活化細胞表面Toll-like receptor (TLR)的訊息傳導使IFN-β產生，而這些免疫反應多會受到cAMP的訊息傳導調控。環狀核苷酸磷酸二酯酶4 (phosphodiesterase 4；PDE4)是免疫細胞中主要水解cAMP的酵素，因此可有效調控細胞內cAMP的濃度。本研究主要是探討在巨噬細胞中PDE4是否參與IFN-β的產生及其作用機制。我們使用LPS處理Raw264.7巨噬細胞發現IFN-β的表現會隨著LPS處理濃度增加而上升，且在處理三小時達到最高。此IFN-β的表現會被PDE4抑制劑rolipram顯著抑制，其IC50約為0.115 µM。此rolipram的抑制作用是經由活化cAMP訊息傳導所致，因為以cAMP類似物dibutyryl cAMP處理Raw264.7細胞也有相同的抑制作用。進一步使用exchange-protein activated by cAMP (Epac)抑制劑ESI-09或protein kinase A (PKA)抑制劑Rp-8-CPT-cAMP處理細胞，結果顯示抑制Epac不會使rolipram對IFN-β的抑制作用回復，而抑制PKA可回復約57 %，同時，PKA活化劑6-Bnz-cAMP也可顯著抑制IFN-β表現，其抑制程度與rolipram相當，這表示rolipram可藉由活化cAMP/PKA訊息傳導來抑制IFN-β表現。為進一步了解PDE4亞型對IFN-β的調控，我們使用PDE4基因剔除小鼠之腹腔巨噬細胞進行實驗，結果顯示，去除PDE4B的細胞其LPS誘導IFN-β的表現呈現顯著下降，且rolipram不再進一步抑制此反應。相反的，PDE4A-/-與PDE4D-/-細胞其IFN-β表現與野生型巨噬細胞相當，且均會被rolipram顯著抑制。這些結果表示rolipram抑制IFN-β表現主要是藉由抑制PDE4B所致。此外，本研究進一步探討抑制PDE4是否會影響TRIF-dependent訊息傳導路徑，西方點墨法分析轉錄因子IRF3活化的結果顯示，在Raw264.7細胞內IRF3磷酸化作用會隨著LPS處理時間增加而上升，至三小時後有下降的趨勢，然而在三小時前rolipram對IRF3鄰酸化無顯著影響，僅至五小時才有顯著下降。再者，LPS可使IRF3的上游分子TBK1活化，但此TBK1磷酸化作用也不受rolipram調控。綜合上述結果證實，在巨噬細胞內抑制PDE4B可活化cAMP/PKA訊息傳導，進而抑制LPS誘導IFN-β mRNA表現，但這反應並非經由調控TLR4/TRIF/TBK1/IRF3訊息傳導路徑所致。;Interferon-β (IFN-β) is a crucial component of innate immune system which enhances IFN-stimulated genes (ISGs) expression to promote the host’s immunity. Numerous data indicated that various pathogenic structures, such as lipopolysaccharide (LPS), can up-regulate IFN-β production by activating toll like receptor (TLR) signal pathways. Many of these responses can be modulated by cAMP signaling. Type 4 cAMP-specific phosphodiesterases (PDE4s) are the predominant cAMP-hydrolyzing enzymes in most immune cells and, thereby important in modulating cAMP concentration in these cells. In this study, we aimed to determine whether PDE4 is involved in regulation of IFN-β production in mouse macrophages, and its underlying mechanisms. By stimulation of Raw264.7 macrophages with LPS, we found a dose-dependent increase in IFN-β mRNA expression and the IFN-β mRNA level reached maximum at 3 h after LPS treatment. The PDE4 inhibitor rolipram effectively suppressed the IFN-β expression with the IC50 of approximately 0.115 µM. Such inhibiting effect of rolipram was mediated by activating cAMP signaling because the cAMP analog dibutyryl-cAMP produced the same inhibitory effect in Raw264.7 cells. In addition, the Epac inhibitor ESI-09 did not reverse the inhibitory effect of rolipram, while the PKA inhibitor Rp-8-CPT-cAMP reversed the inhibition by approximately 57 %, suggesting that the inhibitory effect of rolipram on the IFN-β expression was mediated by activation of the cAMP/PKA, but not cAMP/Epac signal pathway. Moreover, the PKA activator 6-Bnz-cAMP significantly decreased the LPS-induced IFN-β expression at the level similar to that of rolipram. Using the peritoneal macrophages isolated from PDE4 null mice and the corresponding wild-type mice, we further found that the LPS-induced IFN-β expression in PDE4B-/- macrophages, but not PDE4A-/- or PDE4D-/- macrophages, was significantly decreased, and the level of expression was similar to that of PDE4B+/+ macrophages treated with rolipram. Additionally, rolipram did not further decrease the IFN- expression in PDE4B-/- macrophages. Contrarily, PDE4A-/- and PDE4D-/- macrophages, like their wild-type macrophages, exhibited significant reduction in IFN-β expression in the presence of rolipram. These results demonstrated that the rolipram effect on the IFN-β expression was mediated by inhibition of PDE4B. To explore whether inhibition of IFN-β expression by rolipram acts through regulation of TRIF-dependent signal pathway, the activation of the transcription factor IRF3 and its upstream factor TBK1 was monitored by western blotting. The results showed that LPS-induced IRF3 phosphorylation in Raw264.7 cells was elevated with time until 3 h, but was not altered by rolipram until 5 h after LPS stimulation. LPS-induced TBK1 phosphorylation was unchanged by rolipram up to 5 h of LPS stimulateion. Collectively, our data demonstrated that inhibition of PDE4B is sufficient to block LPS-induced IFN-β expression via activation of cAMP/PKA signal pathway in mouse macrophages but not by regulation of TLR4/TRIF/TBK1/IRF3 signal pathway.
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