[1]李鹏,崔佳,王丽清,等.ASAP1调节巨噬细胞对结核分枝杆菌吞噬作用的研究[J].南京农业大学学报,2018,41(3):481-488.[doi:10.7685/jnau.201801042]
 LI Peng,CUI Jia,WANG Liqing,et al.ASAP1 involvement of the phagocytosis of Mycobacterium tuberculosis in macrophages[J].Journal of Nanjing Agricultural University,2018,41(3):481-488.[doi:10.7685/jnau.201801042]
点击复制

ASAP1调节巨噬细胞对结核分枝杆菌吞噬作用的研究()
分享到:

《南京农业大学学报》[ISSN:1000-2030/CN:32-1148/S]

卷:
41卷
期数:
2018年3期
页码:
481-488
栏目:
出版日期:
2018-05-15

文章信息/Info

Title:
ASAP1 involvement of the phagocytosis of Mycobacterium tuberculosis in macrophages
作者:
李鹏 崔佳 王丽清 吴长新
山西大学生物医学研究院, 山西 太原 030006
Author(s):
LI Peng CUI Jia WANG Liqing WU Changxin
Institutes of Biomedical Sciences, Shanxi University, Taiyuan 030006, China
关键词:
ASAP1巨噬细胞结核分枝杆菌吞噬
Keywords:
ASAP1macrophagesMycobacterium tuberculosis(Mtb)phagocytosis
分类号:
R392.12
DOI:
10.7685/jnau.201801042
摘要:
[目的]结核病是全球最重要的人畜共患传染病之一,Arf核糖激化因子GTP酶活化蛋白(ASAP1)与结核病易感性相关。本文旨在明确在细胞水平ASAP1表达与结核分枝杆菌感染的关系,探究ASAP1是否参与巨噬细胞对Mycobacterium tuberculosisMtb)的吞噬以及了解该蛋白表达对巨噬细胞吞噬结核分枝杆菌的影响。[方法]以人源单核细胞建立的不同ASAP1表达水平的巨噬细胞作为模型,通过感染结核分枝杆菌开展了一系列体外试验,使用BCG和Mtb H37Ra菌株感染THP-1分化的巨噬细胞,Western blot以及荧光定量PCR检测感染后ASAP1的表达;建立不同ASAP1表达水平的巨噬细胞,进一步研究ASAP1对巨噬细胞吞噬Mtb H37Ra的调节作用;利用Mtb H37Ra感染不同表达ASAP1的巨噬细胞,通过细菌菌落计数及细胞免疫荧光试验,检测ASAP1表达水平与巨噬细胞吞噬Mtb H37Ra的关系。[结果]Mtb H37Ra感染诱导ASAP1表达上调,BCG感染不能诱导ASAP1表达上调,说明ASAP1参与了巨噬细胞对Mtb H37Ra的吞噬作用;利用Mtb H37Ra感染不同ASAP1表达水平的巨噬细胞,证实ASAP1低水平表达的巨噬细胞对Mtb H37Ra吞噬能力减弱,高水平表达的巨噬细胞对Mtb H37Ra的吞噬能力增强,ASAP1表达水平与巨噬细胞对Mtb H37Ra的吞噬能力呈正相关。[结论]Mtb感染能诱导ASAP1高水平表达,ASAP1参与巨噬细胞对Mtb吞噬,并通过该蛋白表达对巨噬细胞吞噬结核分枝杆菌进行调控,ASAP1的超高水平表达可诱导巨噬细胞对Mtb过多地吞噬。
Abstract:
[Objectives] Tuberculosis(TB)is one of the most important zoonosis all over the world. ASAP1 is associated with the susceptibility to TB in humans. The purpose of this research is to investigate the relationship between ASAP1 expression and Mtb infection, prove whether ASAP1 involves the phagocytosis of Mycobacterium tuberculosis (Mtb)in macrophages, and understand how ASAP1 affects the phagocytosis of Mtb in macrophages. [Methods] By using cell model, a THP-1 derived macrophage model expressing different levels of ASAP1 was generated, then a serials experiments were performed for analysis of affection of ASAP1 on phagocytosis of Mtb after infection of Mtb or BCG in vitro. BCG and Mtb H37Ra were used to infect macrophages, and ASAP1 expression was tested by using Western blotting and Real-time qPCR. ASAP1 specific siRNA was used to generate ASAP1 knockdown macrophages, ASAP1 expression lentiviral system was constructed to transduce THP-1 cell, and then it was made to differentiate into ASAP1 overexpression macrophages. Mtb H37Ra was used to infect macrophages expressing three different ASAP1 expression levels including knockdown, overexpression and normal expression of macrophages which were directly derived from THP-1 cell line, and then the intracellular Mtb number post-infection was counted by using colony forming unit(CFU)counting assays and image acquisition technique. [Results] This research first demonstrated that Mtb H37Ra infection significantly provoked higher ASAP1 expression in macrophages derived from THP-1 compared with BCG infection and non-infection control. ASAP1 overexpression macrophages(P<0.01)and knockdown macrophages(P<0.01)were generated to establish a macrophage cellular model for investigation into the affection of ASAP1 on phagocytosis of Mtb in macrophages. The CFU assays showed that ASAP1 overexpression macrophages loaded more bacteria(P<0.001), and ASAP1 knockdown macrophages loaded less bacteria compared with macrophages directly derived from wild type THP1 as a control(P<0.001). [Conclusions] Mtb infection provokes higher ASAP1 expression in macrophages, and a macrophage cellular model has been established for investigation into the mechanisms underling ASAP1 expression affecting on phagocytosis of Mtb in macrophages. Results also have shown ASAP1 expression has positive correlation with phagocytosis of Mtb, and overexpression of ASAP1 may introduce redundant phagocytosis of Mtb in macrophages, which may affect humans’ susceptibility to TB.

参考文献/References:

[1] Wang X,Wang X,Ma A,et al. ASAP1 gene polymorphisms are associated with susceptibility to tuberculosis in a Chinese Xinjiang Muslim population[J]. Experimental & Therapeutic Medicine,2018,15(4):3392-3398.
[2] 秦波,柳桐,徐聪,等. CARD15基因StyⅠ酶切多态性与奶牛结核病易感性的相关性分析[J]. 中国畜牧兽医,2015,42(6):1553-1558. Qin B,Liu T,Xu C,et al. Correlation study of CARD15 gene StyⅠ polymorphism and susceptibility of tuberculosis in dairy cows[J]. China Animal Husbandry and Veterinary Medicine,2015,42(6):1553-1558(in Chinese with English abstract).
[3] Huang Z,Luo Q,Guo Y,et al. Mycobacterium tuberculosis-induced polarization of human macrophage orchestrates the formation and development of tuberculous granulomas in vitro[J]. PLoS One,2015,10(6):e0129744.
[4] Podinovskaia M,Lee W,Caldwell S,et al. Infection of macrophages with Mycobacterium tuberculosis induces global modifications to phagosomal function[J]. Cellular Microbiology,2013,15(6):843-859.
[5] Guirado E,Schlesinger L S,Kaplan G. Macrophages in tuberculosis:friend or foe[J]. Seminars in Immunopathology,2013,35(5):563-583.
[6] Haines J L,Hauser M A,Schmidt S,et al. Complement factor H variant increases the risk of age-related macular degeneration[J]. Science,2005,308(5720):419-421.
[7] Fridkis-Hareli M,Storek M,Mazsaroff I,et al. Design and development of TT30,a novel C3d-targeted C3/C5 convertase inhibitor for treatment of human complement alternative pathway-mediated diseases[J]. Blood,2011,118(17):4705-4713.
[8] Curtis J,Luo Y,Zenner H L,et al. Susceptibility to tuberculosis is associated with variants in the ASAP1 gene encoding a regulator of dendritic cell migration[J]. Nature Genetics,2015,47(5):523-527.
[9] Kahn R A,Lambright D G. A pH domain with dual phospholipid binding sites regulates the ArfGAP,ASAP1[J]. Structure,2015,23(11):1971.
[10] Bharti S,Inoue H,Bharti K,et al. Src-dependent phosphorylation of ASAP1 regulates podosomes[J]. Molecular and Cellular Biology,2007,27(23):8271.
[11] Lin D,Watahiki A,Bayani J,et al. ASAP1,a gene at 8q24,is associated with prostate cancer metastasis[J]. Cancer Research,2008,68(11):4352-4359.
[12] Onodera Y,Hashimoto S,Hashimoto A,et al. Expression of AMAP1,an ArfGAP,provides novel targets to inhibit breast cancer invasive activities[J]. EMBO Journal,2005,24(5):963-973.
[13] Ehlers J P,Worley L,Onken M D,et al. DDEF1 is located in an amplified region of chromosome 8q and is overexpressed in uveal melanoma[J]. Clin Cancer Res,2005,11(10):3609-3613.
[14] Müller T,Stein U,Poletti A,et al. ASAP1 promotes tumor cell motility and invasiveness,stimulates metastasis formation in vivo,and correlates with poor survival in colorectal cancer patients[J]. Oncogene,2010,29(16):2393-2403.
[15] Davidson A C,Humphreys D,Brooks A B E,et al. The Arf GTPase-activating protein family is exploited by Salmonella enterica serovar Typhimurium to invade nonphagocytic host cells[J]. mBio,2015,6(1):e02253-14.
[16] Ramakrishnan L. Revisiting the role of the granuloma in tuberculosis[J]. Nature Reviews Immunology,2012,12(5):352-366.
[17] Russell D G,Cardona P J,Kim M J,et al. Foamy macrophages and the progression of the human TB granuloma[J]. Nature Immunology,2009,10(9):943.
[18] Nunesalves C,Booty M G,Carpenter S M,et al. In search of a new paradigm for protective immunity to TB[J]. Nature Reviews Microbiology,2014,12(4):289-299.
[19] Kleinnijenhuis J,Oosting M,Joosten L A,et al. Innate immune recognition of Mycobacterium tuberculosis[J]. Clinical and Developmental Immunology,2011. DOI:10.1155/2011/4050310.
[20] Liu Y,Yerushalmi G M,Grigera P R,et al. Mislocalization or reduced expression of Arf GTPase-activating protein ASAP1 inhibits cell spreading and migration by influencing Arf1 GTPase cycling[J]. Journal of Biological Chemistry,2005,280(10):8884-8892.
[21] Randazzo P A,Andrade J,Miura K,et al. The Arf GTPase-activating protein ASAP1 regulates the actin cytoskeleton[J]. Proc Natl Acad Sci USA,2000,97(8):4011-4016.
[22] Shiba Y,Randazzo P A. GEFH1 binds ASAP1 and regulates podosome formation[J]. Biochemical and Biophysical Research Communications,2011,406(4):574.
[23] Kruljac-Letunic A,Moelleken J,Kallin A,et al. The tyrosine kinase Pyk2 regulates Arf1 activity by phosphorylation and inhibition of the Arf-GTPase-activating protein ASAP1[J]. Journal of Biological Chemistry,2003,278(32):29560-29570.
[24] Kam J L,Miura K,Jackson T R,et al. Phosphoinositide-dependent activation of the ADP-ribosylation factor GTPase-activating protein ASAP1[J]. Journal of Biological Chemistry,2000,275(13):9653-9663.
[25] Song O R,Queval C J,Iantomasi R,et al. ArfGAP1 restricts Mycobacterium tuberculosis entry by controlling the actin cytoskeleton[J]. EMBO Reports,2018,19:29-42.

备注/Memo

备注/Memo:
收稿日期:2018-01-25。
基金项目:国家重点研发计划子课题(2017YFD0500303)
作者简介:李鹏,硕士研究生。
通信作者:吴长新,教授,研究方向为感染与免疫,E-mail:cxw20@sxu.edu.cn。
更新日期/Last Update: 1900-01-01