陰電性低密度脂蛋白對巨噬細胞極化的影響：LOX-1 扮演的關鍵角色

肥胖問題隨著經濟繁榮與飲食型態改變而日益嚴重，甚至演變成全球性的健康問題──肥胖患者屬於代謝症候群的高危險族群，容易罹患如動脈粥狀硬化與非酒精性脂肪肝炎等代謝性疾病，並併發心血管疾病的發生，對健康的風險極大，一般認為巨噬細胞在這些慢性疾病扮演關鍵角色。巨噬細胞其功能具可塑性，稱為極化(polarization)，會隨著免疫環境的不同而變為典型(M1)或是選擇型(M2)。一般認為，在許多慢性的發炎疾病中巨噬細胞會極化為促發炎的 M1 型並引起發炎；相反的，M2 型巨噬細胞則是抵消 M1 型引起之發炎反應，有減緩發炎與修復組織的作用。過去有文獻指出肥胖患者中巨噬細胞極化為 M1 型的較多，並引起胰島素抗性的產生。此外，在動脈粥狀硬化病患的斑塊切片中，病情較輕微且損傷較小的區域主要是 M2 分布，M1 則是分布在病情嚴重且主要損傷的區域。而在肝損傷較輕微的肥胖病患的肝組織中，相較於嚴重者其組織 mRNA 表現是以 M2 為主，同時在組織切片中觀察到較多的 M1 細胞自噬的情形。然而是何種因子在這些疾病中誘導了巨噬細胞之極化，目前則是尚不清楚。在先前實驗室學長的研究結果指出自 STEMI 病人分離之陰電性低密度脂蛋白(LDL(-))會刺激巨噬細胞分泌 IL-1β；此外，從餵食 high fat-high cholesterol 飼料的黃金倉鼠分離之 LDL(-)亦會刺激大鼠肝臟 Kupffer cell 分泌 TNF-α 引起發炎反應，顯示 LDL(-)引起發炎因子生成，可能與巨噬細胞極化有關。我的實驗目的即是要探討 LDL(-)是否引起巨噬細胞之極化與其可能透過的途徑為何。以高脂高膽固醇飼料餵食紐西蘭白兔，由其血漿中分離得到 LDL(-)。再將LDL(-)加到 THP-1 細胞培養液中，再分析 M1 與 M2 細胞的標記基因的表現。實驗結果發現，LDL(-)會顯著促進巨噬細胞中發炎相關基因 mRNA 表現和蛋白質分泌，並且表面標記分析亦可見明顯的 M1 訊號，但是在 nLDL 卻只有增加些微的表現，甚至表面標記分析結果與對照的細胞無異；而 M2 相關的基因表現、蛋白質分泌與表面標記訊號則是沒有因 nLDL 或 LDL(-)誘導而有顯著的提升。由於 LDL(-)會誘導清道夫受體 LOX-1 顯著表現並較其他受體的表現突出，我們利用 shRNA knockdown 的方法抑制 LOX-1 表現並觀察 LDL(-)對其極化的誘導情形。研究結果顯示，LDL(-)誘導的發炎激素分泌會在 LOX-1 knockdown 的細胞中顯著減少，M1標記的訊號也是降低至與未處理的細胞相同；而 LOX-1 knockdown 之後巨噬細胞M2 標記 CD206 則是沒有明顯的變化。綜合以上，我們的實驗結果顯示 LDL(-)在人類單核球細胞分化而成的巨噬細胞中主要是透過清道夫受體 LOX-1 誘導巨噬細胞極化為促發炎之 M1 型，而過程可能是透過 NF-κB 等訊息傳遞途徑。對於 LDL(-)誘導的巨噬細胞極化，LOX-1 在其中扮演了關鍵角色，也提供了 LDL(-)、LOX-1 與代謝性疾病的發展的另一層關聯。

關鍵字

代謝性疾病；巨噬細胞；發炎反應；極化；陰電性低密度脂蛋白；類凝集素氧化低密度脂蛋白受器-1

並列摘要

Obesity has been a health problem worldwide because of economic prosperity and changing of diet preference, and has become a serious epidemic nowadays. Morbidly obese people usually suffer from metabolic syndromes, which are linked to diabetes mellitus, atherosclerosis and non-alcoholic steatohepatitis (NASH), and complicates with cardiovascular diseases, such as myocardial infarction. It is generally believed that macrophages play a central role in the formation of these metabolic diseases. Macrophages possess a functional plasticity known as polarization which is driven by their immunological microenvironment. The spectrum of macrophages activation and polarization can be classified into two extreme states: classically activated (M1) phenotype and alternatively activated (M2) phenotype. In general, M1 macrophages promote inflammation in a variety of chronic inflammatory diseases; and M2 macrophages counterbalance the effect, promoting inflammation resolution and tissue repair. Results of several studies indicate that insulin resistance in morbidly obesity patients is associated with M1 macrophages, and in human atherosclerotic lesions, immunohistochemical analysis shows M2 macrophages are localized to more stable locations within asymptomatic plaques, while M1 macrophages accumulate in developed lipid core and diseased portion of the symptomatic plaques. Furthermore, hepatic M2 marker mRNA expression is higher and M1 Kupffer cell apoptosis is promoted by M2 counterparts in liver biopsies of morbidly obese patients with limited hepatic injury. These results suggest that polarization plays an important role in the progression of the metabolic diseases. However, what factor induces the polarization is unknown. In this study, we investigated if electronegative LDL (LDL(-)) is able to induce macrophage polarization and the underlying mechanism. Based on our previous data, LDL(-) from STEMI patients can induce IL-1β secretion in THP-1 macrophages and LDL(-) from Mesocricetus auratus fed with an high fat- high cholesterol diet induced TNF-α secretion in rat Kupffer cell. The results suggest LDL(-) may modulate polarization of macrophages. In this study, LDL(-) is isolated from plasma of high-fat/high-cholesterol diet fed rabbit. And then added to the THP-1 cells which were treated with PMA for 2 days. The results show that LDL(-) significantly provoke mRNA expressions and cytokine secretions of pro-inflammatory genes, and M1 surface marker, CD86; while nLDL exert much lower effects. However, levels of mRNA and protein of M2-related genes, and the levels of M2 surface markers were not significantly induced by nLDL or LDL(-). Besides M1 markers, LDL(-) also induced higher expression level of LOX-1 than other receptors. Using LOX-1 knockdown THP-1 cells, our results demonstrated that LDL(-)-induced secretions of pro-inflammatory cytokines were remarkably decreased, and LDL(-)-induced CD86 up-regulation was almost abolished demonstrated by flow cytometry and immunofluorescence assays. In parallel, M2 marker CD206 was not induced in LOX-1 knockdown cells. Taken together, our results show that LDL(-) induces macrophages polarize into pro-inflammatory M1 type in THP-1 cells and LOX-1 serves a crucial role in LDL(-)-induced polarization of macrophages. These results suggest that pharmacological interventions targeting LOX-1 may be a relevant strategy to polarization-related chronic inflammatory diseases.

並列關鍵字

Metabolic disease ； Macrophages ； Inflammation ； Polarization ； LDL(-) ； LOX-1

參考文獻

Anstee, Q.M., Targher, G., and Day, C.P. (2013). Progression of NAFLD to diabetes mellitus, cardiovascular disease or cirrhosis. Nat Rev Gastroenterol Hepatol 10, 330-344.

Bancells, C., Canals, F., Benitez, S., Colome, N., Julve, J., Ordonez-Llanos, J., and Sanchez-Quesada, J.L. (2010). Proteomic analysis of electronegative low-density lipoprotein. J Lipid Res 51, 3508-3515.

Benitez, S., Bancells, C., Ordonez-Llanos, J., and Sanchez-Quesada, J.L. (2007). Pro-inflammatory action of LDL(-) on mononuclear cells is counteracted by increased IL10 production. Biochim Biophys Acta 1771, 613-622.

Trapping of oxidized LDL in lysosomes of Kupffer cells is a trigger for hepatic inflammation. Liver Int 33, 1056-1061.

Browning, J.D., and Horton, J.D. (2004). Molecular mediators of hepatic steatosis and liver injury. J Clin Invest 114, 147-152.

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陰電性低密度脂蛋白對巨噬細胞極化的影響：LOX-1 扮演的關鍵角色

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