朱武飛 羅亞?wèn)| 趙 博 王艷玲 楊 巍

(吉林大學(xué)基礎(chǔ)醫(yī)學(xué)院免疫學(xué)系，長(zhǎng)春130021)

·基礎(chǔ)免疫學(xué)·

Aire對(duì)巨噬細(xì)胞極化影響的研究①

朱武飛 羅亞?wèn)| 趙 博 王艷玲②楊 ?、?/p>

(吉林大學(xué)基礎(chǔ)醫(yī)學(xué)院免疫學(xué)系，長(zhǎng)春130021)

目的：研究自身免疫調(diào)節(jié)因子(Aire)對(duì)巨噬細(xì)胞極化的影響。方法：分別用LPS、IL-4以及LPS聯(lián)合免疫復(fù)合物刺激小鼠單核巨噬細(xì)胞系RAW264.7細(xì)胞、穩(wěn)定表達(dá)GFP-Aire的RAW264.7細(xì)胞(A33-3)細(xì)胞和穩(wěn)定表達(dá)GFP的RAW264.7細(xì)胞(C1-6)，使其向M1(LPS)、M2a(IL-4)和M2b(LPS聯(lián)合免疫復(fù)合物)型巨噬細(xì)胞極化。通過(guò)Real-time PCR檢測(cè)各組細(xì)胞中M1型巨噬細(xì)胞特征分子IL-1α、iNOS和IL-6，M2a型特征分子Arg-1和M2b型特征分子IL-10的表達(dá)水平，研究Aire對(duì)各種類型巨噬細(xì)胞極化的影響。結(jié)果：LPS在 0.5 μg/ml濃度時(shí)，RAW264.7細(xì)胞中M1型巨噬細(xì)胞產(chǎn)物IL-1α、iNOS和IL-6基因表達(dá)量最高；而IL-4以及LPS聯(lián)合免疫復(fù)合物的刺激作用有顯著的劑量依賴性，都在濃度最高時(shí)RAW264.7細(xì)胞中Arg1(M2a)和IL-10(M2b)基因表達(dá)量最高。LPS刺激后，A33-3細(xì)胞中IL-1α和iNOS表達(dá)水平明顯高于C1-6細(xì)胞，IL-6則相反；IL-4及LPS聯(lián)合免疫復(fù)合物刺激后，A33-3細(xì)胞中Arg1和IL-10的表達(dá)水平明顯低于C1-6細(xì)胞。結(jié)論：Aire可能促進(jìn)巨噬細(xì)胞向M1極化，同時(shí)抑制其向M2a和M2b極化。

自身免疫調(diào)節(jié)因子；巨噬細(xì)胞；極化

自身免疫調(diào)節(jié)因子(Aire)是一種轉(zhuǎn)錄因子，人類Aire基因的突變將導(dǎo)致由自身免疫應(yīng)答所介導(dǎo)的、以多器官損傷為特征的一種常染色體隱性遺傳病(Autoimmune polyendocrinopathy candidiasis ectoderm-al dystrophy，APECED)，也稱為自身免疫性多腺體綜合征 Ⅰ(Autoimmune polyendocrine syndrome type Ⅰ，APS-Ⅰ)。APECED其典型臨床表現(xiàn)為甲狀旁腺功能減退、Addison′s病和慢性皮膚黏膜念珠菌感染的三聯(lián)征[1]?，F(xiàn)有的研究證實(shí)Aire主要表達(dá)在胸腺上皮細(xì)胞，起到維持中樞耐受的作用。然而，對(duì)APECED病人易患慢性皮膚黏膜念珠菌感染仍然沒(méi)有合理的解釋。有研究發(fā)現(xiàn)Aire也表達(dá)于外周巨噬細(xì)胞，但其具體功能作用以及與念珠菌感染之間是否存在聯(lián)系尚不清楚。我們前期的研究發(fā)現(xiàn)TLR1、TLR3和TLR8在過(guò)表達(dá)Aire的小鼠的巨噬細(xì)胞系RAW264.7細(xì)胞中是上調(diào)的[2,3]，提示Aire可能在抗原提呈細(xì)胞針對(duì)病原體的應(yīng)答中發(fā)揮重要作用。

巨噬細(xì)胞作為一類重要的抗原提呈細(xì)胞，廣泛分布于機(jī)體，最近研究表明巨噬細(xì)胞具有明顯的異質(zhì)性，表現(xiàn)為針對(duì)不同微環(huán)境應(yīng)答可使其功能狀態(tài)發(fā)生改變。目前認(rèn)為巨噬細(xì)胞可以功能性地極化為M1型(經(jīng)典激活的巨噬細(xì)胞)或M2型(非經(jīng)典激活的巨噬細(xì)胞)。M1型巨噬細(xì)胞由Ⅰ型干擾素(IFN-γ)和微生物代謝產(chǎn)物(LPS)激活，通過(guò)產(chǎn)生大量促炎細(xì)胞因子如白介素1(IL-1)、IL-6、IL-23、超氧化物陰離子和NO[4,5]，具有殺滅細(xì)菌、真菌等多種病原體的潛能和介導(dǎo)宿主防御的功能。根據(jù)表達(dá)的活化標(biāo)志不同M2型巨噬細(xì)胞又可分成三個(gè)亞群：M2a、M2b和M2c。Th2細(xì)胞產(chǎn)生的細(xì)胞因子IL-4/IL-13可促進(jìn)M2a巨噬細(xì)胞極化，通過(guò)產(chǎn)生精氨酸1(Arg1)和細(xì)胞外基質(zhì)(ECM)參與組織重構(gòu)。用免疫復(fù)合物或Toll樣受體(TLR)激動(dòng)劑可誘導(dǎo)M2b極化。M2c則被抗炎物質(zhì)所誘導(dǎo)，如糖皮質(zhì)激素或IL-10[6,7]。目前，大多數(shù)研究都集中在巨噬細(xì)胞的異質(zhì)性以及M1和M2巨噬細(xì)胞的產(chǎn)生條件。然而，調(diào)控M1和M2巨噬細(xì)胞極化的分子機(jī)制還沒(méi)有得到深入的研究。

鑒于Aire突變可導(dǎo)致機(jī)體易感染念珠菌，而M1型巨噬細(xì)胞又具有抵抗真菌的作用，那么Aire是否可通過(guò)參與不同類型巨噬細(xì)胞的極化過(guò)程，從而在抗真菌感染中發(fā)揮作用有待探究。因此本研究試圖通過(guò)觀察Aire對(duì)巨噬細(xì)胞極化類型的影響，探討其在外周免疫系統(tǒng)如抗真菌感染中的作用。

1 材料與方法

1.1 細(xì)胞系 RAW264.7細(xì)胞從上海細(xì)胞研究所獲得。課題組前期已完成穩(wěn)定轉(zhuǎn)染pEGFPC1/Aire 和pEGFPC1質(zhì)粒至RAW264.7細(xì)胞中，分別得到A33-3和C1-6細(xì)胞[6]，用含10%胎牛血清的RPMI-1640(Gibco，USA)進(jìn)行培養(yǎng)。

1.2 方法

1.2.1 巨噬細(xì)胞亞群的極化 RAW264.7細(xì)胞調(diào)濃度至1×106ml-1，吸取細(xì)胞懸液加至6孔細(xì)胞培養(yǎng)板,每孔2 ml含 10%NCS的RPMI1640，置于37℃、5% CO2培養(yǎng)箱中培養(yǎng)過(guò)夜。RPMI1640洗一遍細(xì)胞，然后用LPS(Sigma，USA)，IL-4(R&D Systems， minneapoils，MN)和LPS聯(lián)合免疫復(fù)合物(OVA和抗OVA IgG)(Sigma，USA)刺激24 h后收細(xì)胞。

1.2.2 RT-PCR 從細(xì)胞中提取總RNA(如上所述)用TRIzol(Invitrogen，USA)裂解，溶解于DEPC水中。用紫外分光光度計(jì)檢測(cè)總RNA的A260/A280的比值和濃度。取1 μg RNA為模板,加入隨機(jī)引物和AMV逆轉(zhuǎn)錄酶(TaKaRa，Japan)逆轉(zhuǎn)錄成cDNA，反應(yīng)條件：30℃ 10 min,45℃ 30 min，5℃ 5 min，1循環(huán)。cDNA用表1中的引物進(jìn)行PCR。應(yīng)用ImageMaster VDS(Pharmacia Biotech，China)圖像分析軟件分析光密度值，各種基因的mRNA表達(dá)水平均表示為目的基因/β-actin光密度值。

表1 RT-PCR和Real-time PCR所用引物及其擴(kuò)增片段大小

Tab.1 Primers for RT-PCR and quantitative Real-time PCR and their amplification size

GenePrimer(5′→3′)Amplificationsize(bp)GAPDH5′?GACTTCAACAGCAACTCCCACTC?3′1075′?TAGCCGTATTCATTGTCATACCAG?3′IL?1α5′?ACAGTTCTGCCATTGACCATCTC?3′1945′?CTTCAGAATCTTCCCGTTGCTTG?3′IL?65′?GAAACCGCTATGAAGTTCCTCTCTG?3′1255′?GTATCCTCTGTGAAGTCTCCTCTCC?3′iNOS5′?TGGAGCGAGTTGTGGATTGTC?3′1465′?GGTCGTAATGTCCAGGAAGTAG?3′IL?105′?GCTCTTACTGACTGGCATGAGGA?3′1795′?AGGAGTCGGTTAGCAGTATGTTG?3′LIGHT5′?GAGAAGCTGATACAAGATCAACGA?3′1035′?CCCATAACAGAGGTCCACCAATA?3′Arg15′?GCATATCTGCCAAAGACATCGT?3′1235′?CAATCCCCAGCTTGTCTACTTCA?3′

1.2.3 Real-time PCR 取上述cDNA為模板，以表1中的引物，采用SYBR Premix Ex Taq試劑盒(TaKaRa，Japan)和ABI7300儀器(Life Technol-ogies,USA)進(jìn)行實(shí)時(shí)定量PCR反應(yīng)，條件為：95℃ 30 s,95℃ 5 s和60℃ 30 s，40循環(huán)。結(jié)果用公式 2-ΔΔCt計(jì)算。

2 結(jié)果

2.1 不同的刺激物刺激巨噬細(xì)胞的極化 為了確定RAW264.7細(xì)胞是否能夠極化為不同類型的巨噬細(xì)胞，并明確激活巨噬細(xì)胞亞群的各種刺激物的有效濃度，分別采用不同濃度LPS、IL-4和LPS聯(lián)合免疫復(fù)合物來(lái)刺激RAW264.7細(xì)胞。結(jié)果顯示，LPS濃度在 0.5 μg/ml時(shí)，M1的產(chǎn)物IL-1α、IL-6和誘導(dǎo)的一氧化氮合成酶(iNOS)的水平在RAW264.7細(xì)胞中明顯地上調(diào)(圖1)。經(jīng)IL-4和LPS聯(lián)合免疫復(fù)合物刺激后，RAW264.7細(xì)胞中Arg1和IL-10基因表達(dá)呈劑量依賴的方式增加(圖2)。因此確定IL-4在200 pg/ml和0.5 μg/ml LPS聯(lián)合IC3(150 μg/ml OVA和15 μl anti-OVA IgG)分別為M2a和M2b極化的最佳濃度，用于后續(xù)實(shí)驗(yàn)。結(jié)果表明不同刺激物可激活RAW264.7細(xì)胞分化為M1或M2型巨噬細(xì)胞。

2.2 Aire促進(jìn)M1型巨噬細(xì)胞極化 為了研究Aire是否影響M1型巨噬細(xì)胞的極化，采用0.5 μg/ml LPS刺激C1- 6和A33-3細(xì)胞， Real-time PCR檢測(cè)特異的M1型巨噬細(xì)胞相關(guān)標(biāo)志的表達(dá)。結(jié)果顯示LPS刺激后，與C1-6細(xì)胞相比，A33-3細(xì)胞中iNOS和IL-1α表達(dá)增加，而IL-6表達(dá)水平降低(圖3)。這些數(shù)據(jù)表明Aire可能促進(jìn)M1的極化。

圖1 不同濃度的LPS刺激下RAW264.7細(xì)胞產(chǎn)生的 IL-1α、IL-6 和 iNOS的表達(dá)量Fig.1 Dose effects of IL-1α,IL-6 and iNOS produced by RAW264.7 cells in response to LPSNote:A.The expression of different genes in RAW264.7 cells stimulated with 0,0.5,5.0 or 50 μg/ml LPS (Lanes 1-4);B.The gene/β-actin optical density values from the top electrophoregram were calculated and plotted. All data were obtained from three independent experiments and expressed as the ±s.

圖2 RAW264.7 細(xì)胞中Arg-1和IL-10各自對(duì)不同刺激物的劑量反應(yīng)Fig.2 Dose effects of Arg-1 and IL-10 produced by RAW264.7 cells in response to different stimuliNote: A. RAW264.7 cells were stimulated with different doses of IL-4:0,10,50,100 or 200 pg/ml (Lanes 1-5)；B.RAW264.7 cells were stimulated with 0.5 μg/ml LPS with different doses of immune complexes. Lanes 1-6 represent normal saline,150 μg/ml OVA,LPS+150 μg/ml OVA,LPS+IC.1 (150 μg/ml OVA+2.5 μl anti-OVA IgG),LPS+IC.2 (150 μg/ml OVA+5 μl anti-OVA IgG),LPS+IC.3 (150 μg/ml OVA+15 μl anti-OVA IgG).The bottom graphs were plotted with the gene/β-actin optical density values from the top electropherogram. All data were obtained from three independent experiments and expressed as the ±s.

圖3 LPS刺激對(duì)C1-6和A33-3細(xì)胞中IL-1α、IL-6和iNOS表達(dá)水平的影響Fig.3 Stimulation with LPS affects expression levels of iNOS,IL-1α and IL-6 in C1-6 and A33-3 cellsNote: A. The expression levels of iNOS and IL-1α in C1-6 and A33-3 stimulated with LPS;B. The expression levels of IL-6 in C1-6 and A33-3 stimulated with LPS. All data were obtained from three independent experiments and expressed as the ±s. a,b and c represent comparisons of stimulated C1-6,unstimulated A33-3 and stimulated A33-3 cells,respectively,with unstimulated C1-6 cells.**.P< 0.01.

圖4 IL-4刺激對(duì)C1-6和A33-3細(xì)胞中Arg1表達(dá)水平的影響Fig.4 Stimulation with IL-4 affects expression levels of Arg1 in C1-6 and A33-3 cellsNote: All data were obtained from three independent experiments and expressed as the ±s. a and c represent comparisons of stimulated C1-6 and stimulated A33-3 cells,respectively,with unstimulated C1-6 cells. **.P<0.01.

圖5 LPS聯(lián)合免疫復(fù)合物刺激對(duì)C1-6和A33-3細(xì)胞中IL-10和LIGHT表達(dá)水平的影響Fig.5 Stimulation of LPS along with IC effects on expression levels of IL-10 and LIGHT in C1-6 and A33-3 cellsNote: All data were obtained from three independent experiments and expressed as the ±s. a,b and c represent comparisons of stimulated C1-6,unstimulated A33-3 and stimulated A33-3 cells,respectively,with unstimulated C1-6 cells. *.P< 0.05,**.P< 0.01.

2.3 Aire抑制M2a型巨噬細(xì)胞極化 為進(jìn)一步研究Aire對(duì)M2a巨噬細(xì)胞極化的影響。采用200 pg/ml IL-4刺激后，Real-time PCR檢測(cè)結(jié)果發(fā)現(xiàn)相比于C1-6細(xì)胞，A33-3細(xì)胞中Arg-1基因表達(dá)較低(圖4)。說(shuō)明Aire可能抑制M2a極化。

2.4 Aire抑制M2b型巨噬細(xì)胞極化 為明確Aire是否也影響另一種M2型巨噬細(xì)胞——M2b的極化。采用0.5 μg/ml LPS聯(lián)合IC3分別刺激A33-3和C1-6細(xì)胞，Real-time PCR檢測(cè)M2b相關(guān)分子的表達(dá)。結(jié)果顯示在有或沒(méi)有刺激物時(shí)，相比于C1-6細(xì)胞A33-3細(xì)胞中IL-10的表達(dá)均顯著降低。然而，在刺激后A33-3細(xì)胞中LIGHT基因的表達(dá)并未發(fā)生變化(圖5)。表明Aire可能抑制M2b極化。

3 討論

本研究中，我們發(fā)現(xiàn)不同的誘導(dǎo)劑刺激可使RAW264.7細(xì)胞分別極化為M1、M2a和M2b型巨噬細(xì)胞。并且相比于C1-6細(xì)胞，A33-3細(xì)胞中M1型巨噬細(xì)胞特異的下游分子IL-1α和iNOS的表達(dá)增加，而M2a和M2b型巨噬細(xì)胞特異的下游分子表達(dá)降低。說(shuō)明AIRE可能促進(jìn)M1極化，抑制M2極化。

M1型巨噬細(xì)胞的極化發(fā)生在Th1細(xì)胞介導(dǎo)的免疫應(yīng)答過(guò)程中[8]，在清除胞內(nèi)病原體中發(fā)揮重要作用。在感染早期，激活的NK細(xì)胞和Th1細(xì)胞分泌大量的IFN-γ，為促進(jìn)M1極化的重要物質(zhì)。其他的細(xì)菌產(chǎn)物如LPS或某些細(xì)胞因子(TNF和GM-CSF)也可以誘導(dǎo)M1極化。活化的M1型巨噬細(xì)胞分泌大量促炎細(xì)胞因子(IL-1、IL-6和IL-23)和大量的NO和超氧陰離子，從而增強(qiáng)細(xì)胞的殺傷能力和抗感染能力。本研究表明在LPS刺激M1巨噬細(xì)胞極化過(guò)程中，A33-3細(xì)胞中IL-1α和iNOS的mRNA的表達(dá)水平顯著高于C1-6細(xì)胞，而IL-6的表達(dá)水平降低。iNOS可以促進(jìn)瓜氨酸形成NO，增加巨噬細(xì)胞的殺傷和抗感染能力。IL-1在免疫防御中也具有重要作用。另外，過(guò)多分泌的IL-6和IL-23也可以引起組織損傷。IL-6和IL-23與Th17細(xì)胞的分化和增殖有關(guān)[9-11]。Th17分泌的IL-17可以引起組織中大量多形核白細(xì)胞的募集，導(dǎo)致一種炎癥性自身反應(yīng)，因此Aire可能通過(guò)促進(jìn)巨噬細(xì)胞極化為M1發(fā)揮抗感染作用，同時(shí)抑制IL-6分泌所誘導(dǎo)的自身反應(yīng)性炎癥。

M2a巨噬細(xì)胞的極化可以被Th2型細(xì)胞分泌的IL-4和IL-13所誘導(dǎo)[8]。這些巨噬細(xì)胞有較弱的殺傷病原體的能力且不具有提呈外來(lái)抗原的能力[12]。然而，它可以產(chǎn)生大量的Arg-1，催化精氨酸變?yōu)轼B(niǎo)氨酸，鳥(niǎo)氨酸是一種多胺和膠原的前體，有助于形成細(xì)胞外基質(zhì)(ECM)[13]，這些復(fù)合物都具有促進(jìn)傷口愈合的功能。我們研究發(fā)現(xiàn)IL-4刺激后，A33-3細(xì)胞中Arg1的上調(diào)水平較C1-6細(xì)胞低，證明Aire可抑制M2a的極化。M2b和M2c都具有免疫調(diào)節(jié)作用，經(jīng)常被稱為調(diào)節(jié)型巨噬細(xì)胞。在體外試驗(yàn)中，IgG免疫復(fù)合物或TLR激動(dòng)劑刺激下，會(huì)產(chǎn)生M2b[14]，M2b巨噬細(xì)胞高表達(dá)免疫抑制性細(xì)胞因子IL-10。本研究結(jié)果表明，LPS聯(lián)合OVA和anti-OVA IgG免疫復(fù)合物可以促進(jìn)巨噬細(xì)胞的極化，并且相比于未刺激的細(xì)胞，刺激后A33-3和C1-6細(xì)胞中IL-10的表達(dá)水平明顯增加，但A33-3細(xì)胞中IL-10的表達(dá)明顯低于C1-6細(xì)胞(無(wú)論刺激前或刺激后)。表明Aire可能通過(guò)降低IL-10的表達(dá)抑制M2b巨噬細(xì)胞極化。

然而，Aire調(diào)節(jié)巨噬細(xì)胞極化的機(jī)制尚不明確。最近一項(xiàng)研究證明Micro RNA 155(miR-155)是調(diào)控M1型巨噬細(xì)胞極化的關(guān)鍵分子[15]。過(guò)表達(dá)miR-155可以將抗炎的M2型巨噬細(xì)胞轉(zhuǎn)變?yōu)榇傺椎腗1型巨噬細(xì)胞。相反，沉默M1中miR-155可使M1型巨噬細(xì)胞重新極化為M2型[15]。此外，Macedo等[16]發(fā)現(xiàn)胸腺髓質(zhì)上皮細(xì)胞中的Aire可調(diào)控miR-155的表達(dá)。因此，我們推測(cè)miR-155可能在Aire誘導(dǎo)M1極化過(guò)程中發(fā)揮重要作用，但還需要進(jìn)一步研究證實(shí)。

綜上，在巨噬細(xì)胞極化為不同亞群的過(guò)程中，Aire可促進(jìn)M1極化，同時(shí)抑制M2的極化，有助于機(jī)體形成抗感染能力，參與某些病原體(包括真菌)的清除，這也為APECED病人出現(xiàn)的真菌易感提供了一個(gè)新的解釋。

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[收稿2016-08-01]

(編輯 張曉舟)

Effects of Aire on macrophage polarization

ZHUWu-Fei,LUOYa-Dong,ZHAOBo,WANGYan-Ling,YANGWei.

DepartmentofImmunology,CollegeofBasicMedicalSciences,JilinUniversity，Changchun130021,China

Objective:To study whether autoimmune regulator (Aire) affects macrophage polarization.Methods: The mouse mononuclear macrophage cell line RAW264.7 cells,stable expressing GFP-Aire protein RAW264.7 cells (A33-3) and stable expressing GFP protein RAW264.7 cells (C1-6) were stimulated with LPS,IL-4 and LPS combined with immune complex respectively to make the macrophage polarize to M1 (LPS),M2a (IL-4) and M2b (LPS with immune complex).To investigate the effects of Aire on the various types of macrophages polarization,the M1 related molecules (IL-1α,iNOS and IL-6),M2a related molecules (Arg-1) and M2b related molecule (IL-10) were detected by Real-time PCR.Results: The expression of IL-1α，IL-6 and inducible nitric oxide synthase (iNOS),the products of M1 macrophages,were significantly upregulated in RAW264.7 cells treated with LPS at 0.5 μg/ml.The expression of Arg-1 and IL-10 mRNA in RAW264.7 cells increased in a dose-dependent manner after stimulation with IL-4 or LPS combined with immune complexes,respectively.M1 macrophage-related marker iNOS and IL-1 increased,whereas IL-6 levels decreased in A33-3 cells compared with C1-6 cells after treatment with LPS.The expression of Arg1 and IL-10 were downregulated in A33-3 cells compared with C1-6 cells after IL-4 and LPS combined with immune complexes stimulation,respectively.Conclusion: Aire may promote M1 macrophage polarization while inhibit M2a and M2b macrophage polarization.

Autoimmune regulator;Macrophage;Polarization

10.3969/j.issn.1000-484X.2017.01.002

①本文受國(guó)家自然科學(xué)基金項(xiàng)目(81373127)和吉林省科技廳項(xiàng)目(20160414038GH)資助。

朱武飛(1981年- )，男，博士，主要從事免疫耐受方面的研究。

R392

A

1000-484X(2017)01-0011-05

②吉林大學(xué)基礎(chǔ)醫(yī)學(xué)院病原免疫細(xì)胞遺傳學(xué)實(shí)驗(yàn)中心，長(zhǎng)春130021。

③通訊作者，E-mail：ywei@jlu.edu.cn。