CD14对生殖支原体LAMPs激活NF-κB的影响

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何军, 曾焱华, 游晓星, 伍绍坚, 田巍, 刘君, 吴移谋. CD14对生殖支原体LAMPs激活NF-κB的影响. 2014, 30(8): 797-799[HE Jun, ZENG Yan-hua, YOU Xiao-xing, WU Shao-jian, TIAN Wei, LIU Jun, WU Yi-mou. Effect of CD14 on lipid-associated membrane proteins ofMycoplasmagenitalium activate NF-kappaB . 中国人兽共患病学报, 2014, 30(8): 797-799] 复制到剪切板

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CD14对生殖支原体LAMPs激活NF-κB的影响

何军¹, 曾焱华², 游晓星², 伍绍坚¹, 田巍¹, 刘君¹, 吴移谋²

1.南华大学附属南华医院检验科,衡阳 421002

2.南华大学病原生物学研究所,衡阳 421001

基金:

摘要

目的

探讨CD14在生殖支原体(Mg)脂质相关膜蛋白(LAMPs)激活NF-κB中作用。

方法

THP-1细胞分别加入人血清和CD14抗体孵育,提取对数期Mg的LAMPs刺激,ELISA法检测其NF-κBp65水平;采用共转染技术和sCD14预孵育LAMPs,刺激Hela细胞,双荧光素酶报告基因分析CD14在LAMPs激活NF-κB中的作用。

结果

人血清上调LAMPs诱导THP-1细胞激活NF-κBp65;CD14中和抗体抑制LAMPs诱导THP-1细胞激活NF-κBp65;mCD14和sCD14上调LAMPs诱导Hela细胞激活NF-κB。

结论

CD14上调生殖支原体LAMPs激活NF-κB。

关键词: CD14; 生殖支原体; 脂质相关膜蛋白; 核转录因子κB; 人血清

中图分类号:R375 文献标志码:A 文章编号:1002-2694(2014)08-0797-03

Effect of CD14 on lipid-associated membrane proteins ofMycoplasmagenitalium activate NF-kappaB

HE Jun¹, ZENG Yan-hua², YOU Xiao-xing², WU Shao-jian¹, TIAN Wei¹, LIU Jun¹, WU Yi-mou²

1.Department of Clinical Laboratory, Nanhua Affiliated Hospital, University of South China, Hengyang 421002, China

2.Pathogenic Biology Institute, University of South China, Hengyang 421001, China

Fund:Supported by the National Natural Science Foundation of China (No.31100137), the National Science Foundation of Hunan Province (No.14JJ7044), and the 12th Five-year Technology Innovation Team in University of South China;

Abstract

To investigate the effects of CD14 on nuclear transcription factor κB (NF-κB) was activated by lipid-associated membrane proteins (LAMPs) ofMycoplasmagenitalium (Mg), THP-1 cells were pretreated with serum human or CD14 neutralizing antibody, and then were stimulated by LAMPs. The activation of NF-κBp65 was detected by ELISA. After LAMPs was pretreated with sCD14 stimulated Hela cells with the co-transfection, the activity of NF-κB luciferase were detected by the dual-luciferase reporter gene to analyze the role of CD14-mediated NF-κB activation by LAMPs. The activation of NF-κBp65 was significantly up-regulated in LAMPs activated THP-1 cells by human serum. It’s suggested that CD14 neutralizing antibody could inhibit the activation of NF-κBp65 in LAMPs stimulated THP-1. The activation of NF-κB was significantly up-regulated in LAMPs activated Hela cells by mCD14 or sCD14. CD14 could augment the activation of NF-κB by Mg LAMPs.

Keyword: CD14; Mycoplasma genitalium; lipid-associated membrane protein; nuclear transcription factor κB; human serum

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1 材料与方法

1.1 材料

1.1.1 菌株和细胞

Mg-37标准株由南华大学病原生物学研究所保存,人单核细胞株THP-1细胞和宫颈癌细胞株HeLa细胞购自武汉大学中国典型培养物保藏中心。

1.1.2 试剂和试剂盒

青霉素、多粘菌素B购自Solarbio;无支原体牛血清和超级新生胎牛血清购自四季青生物工程公司;RPMI-1640和DMEM培养基购自Hyclone;PPLO购自BD公司;NF-κBp65 (Total) ELISA KIT购自Life Technologies公司;细胞核蛋白与细胞浆蛋白抽提试剂盒购自碧云天生物;Lipofectamine 2000购自Invitrogen公司;1%人血清来自正常体检者并经其同意使用。

1.2 方法

1.2.1 Mg培养及LAMPs的提取

将Mg菌液按1∶40比例接种于PPLO改良培养基中于95%CO₂、37℃恒温下培养3~4 d,培养基颜色由玫瑰红色变为清亮的橙色时,表示Mg生长已进入对数期。Mg LAMPs的提取按照参考文献进行^{[ 5]},BCA法测定蛋白浓度后置于-80℃备用。Mg LAMPs在刺激前用100 μg/mL多粘菌素B处理2 h,去除可能存在的内毒素污染。

1.2.2 THP-1细胞培养及LAMPs刺激

THP-1细胞在含10%胎牛血清的1640培养基中,于37 ℃、5%CO₂的培养箱中培养。①在THP-1细胞加入1%正常人血清37℃孵育2 h,再用4.0 μg/mL LAMPs刺激8 h;②在THP-1细胞与10 μg/mL CD14抗体37 ℃孵育30 min,再用4.0 μg/mL LAMPs刺激8 h。

1.2.3 细胞核蛋白的提取

离心收集THP-1细胞,PBS洗涤一遍,离心后弃上清,加入含PMSF的细胞浆蛋白抽提试剂A 200 μL,剧烈振荡完全悬浮,冰浴15 min后加入细胞浆蛋白抽提试剂B 10 μL,剧烈振荡后冰浴60 s,再振荡悬浮,4℃、14 000 r/min离心5 min后弃上清,再加入含PMSF的细胞核蛋白抽提试剂50 μL,剧烈振荡30 s重悬后冰浴,每隔120 s剧烈振荡30 s,共30 min,4℃、14 000 r/min离心10 min所提取的为细胞核蛋白保存至-80℃待测。

1.2.4 NF-κBp65水平的测定

提取细胞核蛋白,采用ELISA法根据NF-κBp65 ELISA试剂盒操作说明书进行,其结果以每mg核蛋白提取物中含NF-κBp65的 A₄₅₀值表示,即 A₄₅₀/mg。

1.2.5 共转染及NF-kB活性的测定

①以pcDNA-CD14,pFLAG-TLR2, pNF-kB-luc及pRL-TK质粒共转染Hela细胞24 h后,再用LAMPs刺激8 h;②LAMPs用10 μg/mL sCD14 37℃共孵育30 min后,再刺激0.1 μg/mL pFLAG-TLR2,0.05 μg/mL pNF-kB-luc,0.005 μg/mL pRL-TK共转染的Hela细胞8 h。分别消化收集HeLa细胞,加入细胞裂解液200 μL,再加入100 μL萤火虫荧光素酶检测试剂,测定pNF-kB-luc的RLU值,再加入Renilla荧光素酶检测工作液100 μL,测定pRL-TK的RLU值,根据pNF-kB-luc/pRL-TK比值计算NF-kB的活化程度。

1.3 统计学方法

计量资料以均数±标准差( ±s)表示,组间比较采用 t检验,统计分析运用SPSS 16.0软件, P<0.05表示其差异具有统计学意义。

2 结、果

2.1 人血清上调LAMPs激活NF-κBp65

前期研究证实Mg LAMPs能诱导THP-1细胞激活NF-κBp65^{[ 6]}。在与1%人血清孵育2 h后,再以4.0 μg/mL Mg LAMPs刺激THP-1细胞8 h,ELISA结果显示人血清组NF-κBp65水平明显高于同浓度未加人血清组,并且随LAMPs浓度增加而增加,结果见图1:

	Figure Option View Download New Window
	图1 人血清上调LAMPs激活NF-κBp65Fig.1 Activation of NF-κBp65 were significantly up-regulated in LAMPs by human serum Compared with no serum group

2.2 CD14中和抗体对NF-κBp65的影响

用10 μg/mL CD14中和抗体与THP-1细胞共孵育30 min,再用4.0 μg/mL LAMPs刺激8 h后,ELISA结果显示CD14中和抗体能明显降低激活的NF-κBp65水平,结果见图2:

	Figure Option View Download New Window
	图2 CD14中和抗体抑制LAMPs激活NF-κBp65Fig.2 CD14 neutralizing antibody inhibited the activation of NF-κBp65 in LAMPs stimulated THP-1 cells Compared with IgG group, P<0.05.

2.3 mCD14上调LAMPs激活NF-κB

以0.1~2.0 μg/mL pcDNA-CD14与0.1 μg/mL pFLAG-TLR2,0.05 μg/mL pNF-kB-luc,0.005 μg/mL pRL-TK共转染Hela细胞24 h后,再用4.0 μg/mL LAMPs刺激8 h。当pcDNA-CD14质粒浓度为1.0 μg/mL时,结果显示pNF-kB-luc/pRL-TK相对荧光比值最高,结果见图3:

	Figure Option View Download New Window
	图3 mCD14上调LAMPs激活NF-κBFig.3 Activation of NF-κB were significantly up-regulated in LAMPs activated Hela cells by mCD14 Compared with 0.5 group, P<0.05.

2.4 sCD14上调LAMPs激活NF-κB

预先用10 μg/mL sCD14与4.0 μg/mL LAMPs 37℃共孵育30 min,再刺激0.1 μg/mL pFLAG-TLR2,0.05 μg/mL pNF-kB-luc,0.005 μg/mL pRL-TK共转染的Hela细胞,8 h后pNF-kB-luc/pRL-TK相对荧光比值显示sCD14也能上调LAMPs激活NF-κB,结果见图4:

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	图4 sCD14上调LAMPs激活NF-κBFig.4 Activation of NF-κB were significantly up-regulated in LAMPs activated Hela cells by sCD14 Compared with Mg LAMPs group, P<0.05.

3 讨论

支原体广泛存在于自然界,是一类缺乏细胞壁、呈高度多形性、能通过滤菌器、可在无细胞的培养中繁殖的最小原核细胞型微生物。支原体的细胞膜是与宿主细胞相互作用的主要成分,而膜上丰富的LAMPs是影响机体炎症反应的主要蛋白。目前已证实LAMPs能诱导多种细胞分泌一系列炎症因子和诱导细胞凋亡、坏死^{[ 7]}。由于其生物活性的发挥可能要通过LAMPs中多种蛋白的相互作用,因些对LAMPs的整体研究相对单个膜蛋白来说更有意义。我们前期研究发现Mg LAMPs能激活NF-κB,并且与TLR2有关^{[ 6]}。NF-κB广泛存在于各种类型的真核细胞中,当机体在遭受外界病原感染时能通过识别相关分子模式引发机体相应的免疫应答反应,NF-κB信号通路诱导活化的重要作用就是通过诱导激活下游的免疫及炎性相关因子的转录表达^{[ 8]}。我们结果发现,人血清能明显上调LAMPs激活NF-κB,我们推测人血清中sCD14可能在上调中发挥了一定的作用。随后用CD14中和抗体处理THP-1细胞后发现能显著抑制LAMPs激活NF-κB。这些实验表明,CD14在LAMPs激活NF-κB过程中起着重要作用。

CD14是LPS的高亲和性受体,可分为膜CD14(mCD14)和可溶性CD14(sCD14)两种,两者均在细菌性感染过程中具有重要的免疫介导作用^{[ 9]}。mCD14通过糖基磷脂酰肌醇(GPI)锚定于细胞膜上,主要存在于单核细胞、巨噬细胞、树突细胞等细胞表面。sCD14结构与mCD14相似,但无GPI结构,主要存在于血清、尿液和培养mCD14阳性细胞的上清中。sCD14在血清中正常浓度为2~5 mg/mL,其来源可能是单核细胞上mCD14发生内源性酶促反应脱落或转录合成mCD14时不能PI化直接分泌入血^{[ 10]}。sCD14的生物学活性与mCD14相似,也能直接与LPS结合,介导LPS所致的NF-κB^{[ 11]}。Hela缺乏TLRs,但拥有NF-κB信号传导途径,有利于胞外各种刺激诱导的信号通路研究。我们通过共转染和sCD14预处理,结果发现,mCD14和sCD14均上调LAMPs激活NF-κB。

最近研究证实LAMPs的生物活性主要与其脂质成分,脂肽的、脂肪酸链的饱和程度、数量及酰化程度有关^{[ 12]}。最新研究发现,CD14能结合三酰化脂肽介导免疫反应,但LAMPs中是否存在三酰化蛋白仍存在争议^{[ 13]}。因此CD14上调LAMPs的具体机制还有待于更深一步研究。

The authors have declared that no competing interests exist.

参考文献

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[1]	Tully JG, Taylor-Robinson D, Cole RM, et al. A newly discovered Mycoplasma in the human urogenital tract[J]. Lancet, 1981, 1(8233): 1288-1291. [本文引用:1] [JCR: 39.06]
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1981

39.06

0.0

2014

4.87

0.0

2013

2.594

0.0

2011

4.961

0.0

. 2011, 81(2):327-339

Transcriptional regulation of MG_149, an osmoinducible lipoprotein gene from Mycoplasma genitalium

Wenbo Zhang andJoel B. Baseman *

Department of Microbiology and Immunology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229-3900, USA. * E-mail baseman@uthscsa.edu ; Tel. (+1) 210 567 3939; Fax (+1) 210 567 6491.

> Transcriptional regulation remains poorly understood in Mycoplasma genitalium , the smallest self-replicating cell and the causative agent of a spectrum of urogenital diseases. Previously, we reported that MG_149, a lipoprotein-encoding gene, was highly induced under physiological hyperosmolarity conditions. In this study we further analysed MG_149 transcription with a focus on the identification of promoter elements and regulatory mechanisms. We established MG_149 as a genuine osmoinducible gene that exhibited the highest transcript abundance compared with other lipoprotein genes. Using genetic approaches, we demonstrated that the −10 region of the MG_149 promoter was essential for osmoinduction. Moreover, we showed that MG_149 osmoinduction was regulated by DNA supercoiling, as the presence of novobiocin decreased MG_149 expression in a dose-dependent manner. Taken together, these results indicate that DNA supercoiling participates in controlling MG_149 expression during in vivo -like conditions.

2007

1.743

0.0

. 2007, 121(4):473-483

Triacylated lipoproteins derived from Mycoplasma pneumoniae activate nuclear factor-κB through toll-like receptors 1 and 2

Takashi Shimizu,Yutaka Kida andKoichi Kuwano

Department of Bacteriology, Kurume University School of Medicine, Kurume, Japan * Dr K. Kuwano, Department of Bacteriology, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830–0011, Japan. Email: kuwano@med.kurume-u.ac.jp Senior author: Dr K. Kuwano

> Summary Pathogenesis of Mycoplasma pneumoniae infection is considered to be in part attributed to excessive immune responses. Recently, a mycoplasma lipoprotein has been shown to induce nuclear factor κB (NF-κB) activation through toll-like receptor 1 (TLR1), TLR2 and TLR6. In this study, we examined the ability of lipoproteins from M. pneumoniae to activate NF-κB through TLR1- and TLR2-dependent, but TLR6-independent, pathways, and the active components responsible for the NF-κB activation through the TLR6-independent pathway were identified. The active lipoproteins were found to be MPN611 and MPN162 of M. pneumoniae (designated N-ALP1 and N-ALP2, respectively). Purified N-ALP1 and N-ALP2 from M. pneumoniae and triacylated partial synthetic lipopeptides of N-ALP1 and N-ALP2 augmented the levels of NF-κB induction through TLR1- and TLR2-dependent pathways, whereas diacylated partial synthetic lipopeptides of N-ALP1 and N-ALP2 activated NF-κB through TLR1-, TLR2- and TLR6-dependent pathways. These data suggest that N-ALP1 and N-ALP2 would be triacylated lipoproteins. The activity of N-ALP1 and N-ALP2 was decreased with a pretreatment of lipoprotein lipase, and partially decreased by protease treatment, indicating that the lipid moiety of N-ALP1 and N-ALP2 is critical for the NF-κB activation. Thus, triacylated lipoproteins derived from M. pneumoniae might activate NF-κB through TLR1 and TLR2, but not TLR6.

... Mg LAMPs的提取按照参考文献进行^[5],BCA法测定蛋白浓度后置于-80℃备用 ...

2010

0.0

0.629

... Bp65^[6] ...

... B,并且与TLR2有关^[6] ...

2008

3.882

0.0

... 目前已证实LAMPs能诱导多种细胞分泌一系列炎症因子和诱导细胞凋亡、坏死^[7] ...

2004

3.419

1.1301

... B信号通路诱导活化的重要作用就是通过诱导激活下游的免疫及炎性相关因子的转录表达^[8] ...

2013

0.0

... CD14是LPS的高亲和性受体,可分为膜CD14(mCD14)和可溶性CD14(sCD14)两种,两者均在细菌性感染过程中具有重要的免疫介导作用^[9] ...

2003

5.52

0.0

... sCD14在血清中正常浓度为2~5 mg/mL,其来源可能是单核细胞上mCD14发生内源性酶促反应脱落或转录合成mCD14时不能PI化直接分泌入血^[10] ...

2005

3.057

0.0

... B^[11] ...

2008

4.074

0.0

... 最近研究证实LAMPs的生物活性主要与其脂质成分,脂肽的、脂肪酸链的饱和程度、数量及酰化程度有关^[12] ...

2006

4.811

0.0

. 2006, 8(12):1899-1909

CD14 directly binds to triacylated lipopeptides and facilitates recognition of the lipopeptides by the receptor complex of Toll-like receptors 2 and 1 without binding to the complex

Takashi Nakata 1,2 ,Motoaki Yasuda 1 ,Mari Fujita 3 ,Hideo Kataoka 1 ,Kazuto Kiura 1 ,Hidehiko Sano 2 andKenichiro Shibata 1,*

1 Departments of Oral Pathobiological Science and 2 Oral Health Science, Hokkaido University Graduate School of Dental Medicine, Nishi 7, Kita 13, Kita-ku, Sapporo 060-8586, Japan. 3 Department of Oral Microbiology, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Tobetsu, Hokkaido 061-0293, Japan. * *E-mail shibaken@den.hokudai.ac.jp ; Tel. (+81) 11 706 4240; Fax (+81) 11 706 4901.

> It has demonstrated that the recognition of triacylated lipopeptides by Toll-like receptor (TLR) 2 requires TLR1 as a coreceptor. In the NF-κB reporter assay system in which human embryonic kidney 293 cells were transfected with TLR2 and TLR1 together with an NF-κB luciferase reporter gene, S-(2,3-bispalmitoyloxypropyl)-N-palmitoyl-Cys-Lys-Lys-Lys-Lys (Pam 3 CSK 4 ) and Pam 3 CSSNA were recognized by TLR2/TLR1, but the recognition level was unexpectedly very low. However, cotransfection of CD14 drastically enhanced the recognition of triacylated lipopeptides by TLR2/TLR1. The CD14-induced enhancement did not occur without cotransfection of TLR1. Both CD14 dS39-A48 , a mutant with deletion of the part of possible N-terminal ligand-binding pocket, and anti-CD14 monoclonal antibody reduced the CD14-induced enhancement. Transfection of a TIR domain-deficient mutant of TLR2 (TLR2 dE772-S784 ) or TLR1 (TLR1 dQ636-K779 ) completely abrogated the CD14-induced enhancement. Soluble recombinant CD14 added extracellularly enhanced the recognition of Pam 3 CSSNA by TLR2/TLR1. Immunoprecipitation analysis demonstrated that CD14 was not associated with TLR2 but that TLR1 was associated with TLR2. In addition, surface plasmon resonance-based assay demonstrated that CD14 binds to Pam 3 CSK 4 at a dissociation constant of 5.7 µM. This study suggests that CD14 directly binds to triacylated lipopeptides and facilitates recognition of the lipopeptides by the TLR2/TLR1 complex without binding to the receptor complex.

... 最新研究发现,CD14能结合三酰化脂肽介导免疫反应,但LAMPs中是否存在三酰化蛋白仍存在争议^[13] ...