留言板

尊敬的读者、作者、审稿人, 关于本刊的投稿、审稿、编辑和出版的任何问题, 您可以本页添加留言。我们将尽快给您答复。谢谢您的支持!

姓名
邮箱
手机号码
标题
留言内容
验证码

Role of neutrophil chemoattractant CXCL5 in SARS-CoV-2 infection-induced lung inflammatory innate immune response in an in vivo hACE2 transfection mouse model

Yan Liang Heng Li Jing Li Ze-Ning Yang Jia-Li Li Hui-Wen Zheng Yan-Li Chen Hai-Jing Shi Lei Guo Long-Ding Liu

Yan Liang, Heng Li, Jing Li, Ze-Ning Yang, Jia-Li Li, Hui-Wen Zheng, Yan-Li Chen, Hai-Jing Shi, Lei Guo, Long-Ding Liu. Role of neutrophil chemoattractant CXCL5 in SARS-CoV-2 infection-induced lung inflammatory innate immune response in an in vivo hACE2 transfection mouse model. Zoological Research, 2020, 41(6): 621-631. doi: 10.24272/j.issn.2095-8137.2020.118
Citation: Yan Liang, Heng Li, Jing Li, Ze-Ning Yang, Jia-Li Li, Hui-Wen Zheng, Yan-Li Chen, Hai-Jing Shi, Lei Guo, Long-Ding Liu. Role of neutrophil chemoattractant CXCL5 in SARS-CoV-2 infection-induced lung inflammatory innate immune response in an in vivo hACE2 transfection mouse model. Zoological Research, 2020, 41(6): 621-631. doi: 10.24272/j.issn.2095-8137.2020.118

中性粒细胞趋化因子CXCL5在人ACE2受体转染小鼠感染SARS-CoV-2病毒模型中诱导肺部炎症天然免疫反应的作用

doi: 10.24272/j.issn.2095-8137.2020.118

Role of neutrophil chemoattractant CXCL5 in SARS-CoV-2 infection-induced lung inflammatory innate immune response in an in vivo hACE2 transfection mouse model

Funds: This work was supported by the National Natural Science Foundation of China (82041017) and Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (2016-I2M-1-014)
More Information
  • 摘要: 了解严重急性呼吸综合征冠状病毒2型(SARS-CoV-2)的发病机制,阐明宿主的抗病毒免疫功能,是开发疫苗和抗病毒药物的关键。小鼠作为模式动物因其方便获取和操作并能够进行遗传编辑,广泛用于传染病动物模型。然而研究表明正常成年小鼠对SARS-CoV-2病毒并不敏感。因此,我们建立了一种肺部转染SARS-CoV-2病毒受体人血管紧张素转换酶2(human angiotensin-converting enzyme 2, hACE2)的小鼠模型,以期在正常小鼠上快速建立SARS-CoV-2的感染模型。基于该模型, hACE2受体转染小鼠在转染2天后感染SARS-CoV-2病毒。与对照小鼠相比,感染后在hACE2受体转染小鼠肺部可检测到病毒核酸和蛋白、肺部病理改变、天然免疫细胞浸润和炎症因子增高。进一步研究表明,中性粒细胞是病毒感染后最早也是数量最多浸润肺部的白细胞。此外,利用CXCL5基因敲除小鼠建立hACE2受体转染小鼠模型,该小鼠感染SARS-CoV-2病毒后嗜中性粒细胞募集入感染肺组织显著下调,同时降低了感染肺部的炎症反应。CXCL5的敲除并未影响到病毒感染后肺部的清除,提示了该因子作为潜在的控制SARS-CoV-2病毒感染肺炎的靶标。
    #Authors contributed equally to this work
  • Figure  1.  Characterization of in-vitro transfection of hACE2 and SARS-CoV-2 infection in MLE-12 cells

    A: MLE-12 cells were transfected with human ACE2 (hACE2) expression plasmid pCMV-ACE2-GFPSpark or pCMV3 plasmid as a control. B: hACE2 protein expression was detected by western blotting using anti-hACE2 antibody. C: hACE2 protein expression was detected by immunofluorescence using anti-hACE2 antibody. D: hACE2-transfected cells were infected with SARS-CoV-2 at an MOI of 5 at 48 h post-transfection, and immunofluorescence detection of viral spike protein and GFP protein was performed at 24 h post-infection by confocal microscopy. E: hACE2-transfected cells and control-transfected cells were infected with SARS-CoV-2 at an MOI of 1 at 48 h post-transfection, and viral titers at different days post-infection (dpi) were determined by a viral CCID50 assay (lower). Error bars indicate standard deviation of triplicate biological samples.

    Figure  2.  In vivo hACE2 pulmonary transfection in mice

    Mice underwent pulmonary transfection with hACE2 expression plasmid (35 μg each) or control pCMV3 plasmid via orotracheal intubation, after which lungs were harvested at different days post-transfection. A: Expression of hACE2 in transfected lung tissue via western blotting. B: Lung sections at 2 days post-transfection were stained with anti-hACE2 antibody and hematoxylin (original magnification, 40×). Red arrows indicate obvious expression of hACE2. C: Immunofluorescence detection of proSP-C and hACE2 proteins in lung tissue sections at 2 days post-transfection. D: Lung sections at 2 and 7 days post-transfection were stained with hematoxylin and eosin for histopathological analysis (original magnification, 20×).

    3.  Pulmonary SARS-CoV-2 infection in hACE2-transfected mice

    Two days (48 h) after in vivo transfection with hACE2 plasmid or control plasmid (mock transfection), mice were intranasally infected with SARS-CoV-2 (1×105 CCID50). A: Lungs were harvested from infected mice at 2 days post-infection (dpi), fixed in formalin, and embedded in paraffin. Lung sections were stained with anti-proSP-C and viral nucleoprotein for immunofluorescence detection. B, C: Lungs were harvested at different dpi, viral RNA was determined based on number of nucleocapsid (N) gene RNA copies detected by qRT-PCR (B), and viral titers were determined using a CCID50 assay (C). Error bars indicate standard deviation of triplicate biological samples. D: Total numbers of leukocytes in BALF at different dpi were determined. E: Percentages of neutrophils, monocytes and macrophages, and lymphocytes in BALF of infected hACE2 mice at 2 dpi were analyzed by flow cytometry using anti-Ly6G, anti-CD64, and anti-CD3 antibodies, respectively (left). Statistical analysis of BALF lymphocyte composition was carried out with data from triplicate biological samples (right). F: mRNA levels of cytokines from lung tissue homogenates of infected mice at 2 dpi were examined by qRT-PCR (normalized to β-actin). Error bars indicate standard deviation of triplicate biological samples. *: P<0.05 based on Student’s t-test. G: Lungs were harvested from infected mice at 2 dpi, fixed in formalin, and embedded in paraffin. Lung sections were stained with hematoxylin and eosin for histopathological analysis (original magnification, 20×).

    Figure  4.  Comparison of SARS-CoV-2 infection in hACE2-transfected WT mice and CXCL5-/- mice

    Two days (48 h) after in vivo transfection with hACE2 plasmid, WT and CXCL5-knockout mice (CXCL5-/-) were intranasally infected with SARS-CoV-2 (1×105 CCID50). A: Percentages of neutrophils (Ly6G antibody-positive) from two infected mouse strains at 2 dpi were analyzed by flow cytometry (left) and calculated from triplicate samples (right). Total numbers of leukocytes and neutrophils in BALF of infected mice at 2 dpi were counted (lower right). B: Lungs were harvested from infected mice at 2 dpi, fixed in formalin, and embedded in paraffin. Lung sections were stained with hematoxylin and eosin for histopathological analysis (original magnification, 20×). Lung histological scores were assessed by a veterinary pathologist blind to the study, as described in the Methods and Materials section. C: mRNAs levels of indicated cytokines in lung tissue homogenates of infected mice at 2 dpi were examined by qRT-PCR (normalized to β-actin). Mock-infected mice were transfected with control pCMV3 plasmid. D, E: Lungs were harvested at different dpi, viral RNA was determined based on number of nucleocapsid (n) gene RNA copies detected by qRT-PCR (D), and viral titers were determined using a CCID50 assay (E). Error bars indicate standard deviation of triplicate biological samples. *: P<0.05 based on Student’s t-test.

  • [1] Baggiolini M, Dewald B, Moser B. 1993. Lnterleukin-8 and related chemotactic cytokines–CXC and CC chemokines. Advances in Immunology, 55: 97−179. doi: 10.1016/S0065-2776(08)60509-X
    [2] Balamayooran G, Batra S, Cai SS, Mei JJ, Worthen GS, Penn AL, et al. 2012. Role of CXCL5 in leukocyte recruitment to the lungs during secondhand smoke exposure. American Journal of Respiratory Cell and Molecular Biology, 47(1): 104−111. doi: 10.1165/rcmb.2011-0260OC
    [3] Balamayooran G, Batra S, Fessler MB, Happel KI, Jeyaseelan S. 2010. Mechanisms of neutrophil accumulation in the lungs against bacteria. American Journal of Respiratory Cell and Molecular Biology, 43(1): 5−16. doi: 10.1165/rcmb.2009-0047TR
    [4] Bao LL, Deng W, Huang BY, Gao H, Liu JN, Ren LL, et al. 2020. The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice. Nature, 583(7818): 830−833. doi: 10.1038/s41586-020-2312-y
    [5] Chan JFW, Zhang AJ, Yuan SF, Poon VKM, Chan CCS, Lee ACY, et al. 2020. Simulation of the clinical and pathological manifestations of Coronavirus Disease 2019 (COVID-19) in a golden Syrian hamster model: implications for disease pathogenesis and transmissibility. Clinical Infectious Diseases: ciaa325.
    [6] Chen SC, Mehrad B, Deng JC, Vassileva G, Manfra DJ, Cook DN, et al. 2001. Impaired pulmonary host defense in mice lacking expression of the CXC chemokine lungkine. The Journal of Immunology, 166(5): 3362−3368. doi: 10.4049/jimmunol.166.5.3362
    [7] Das S, MacDonald K, Chang HYS, Mitzner W. 2013. A simple method of mouse lung intubation. Journal of Visualized Experiments, (73): e50318.
    [8] Deng W, Bao LL, Liu JN, Xiao C, Liu JY, Xue J, et al. 2020. Primary exposure to SARS-CoV-2 protects against reinfection in rhesus macaques. Science, 369(6505): 818−823. doi: 10.1126/science.abc5343
    [9] Driscoll KE, Hassenbein DG, Howard BW, Isfort RJ, Cody D, Tindal MH, et al. 1995. Cloning, expression, and functional characterization of rat MIP-2: a neutrophil chemoattractant and epithelial cell mitogen. Journal of Leukocyte Biology, 58(3): 359−364. doi: 10.1002/jlb.58.3.359
    [10] Frevert CW, Huang S, Danaee H, Paulauskis JD, Kobzik L. 1995. Functional characterization of the rat chemokine KC and its importance in neutrophil recruitment in a rat model of pulmonary inflammation. Journal of Immunology, 154(1): 335−344.
    [11] Guo L, Feng K, Wang YC, Mei JJ, Ning RT, Zheng HW, et al. 2017. Critical role of CXCL4 in the lung pathogenesis of influenza (H1N1) respiratory infection. Mucosal Immunology, 10(6): 1529−1541. doi: 10.1038/mi.2017.1
    [12] Huang CL, Wang YM, Li XW, Ren LL, Zhao JP, Hu Y, et al. 2020. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. The Lancet, 395(10223): 497−506. doi: 10.1016/S0140-6736(20)30183-5
    [13] Jiang RD, Liu MQ, Chen Y, Shan C, Zhou YW, Shen XR, et al. 2020. Pathogenesis of SARS-CoV-2 in transgenic mice expressing human angiotensin-converting enzyme 2. Cell, 182(1): 50−58.e8. doi: 10.1016/j.cell.2020.05.027
    [14] Li WH, Moore MJ, Vasilieva N, Sui JH, Wong SK, Berne MA, et al. 2003. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature, 426(6965): 450−454. doi: 10.1038/nature02145
    [15] Lu SY, Zhao Y, Yu WH, Yang Y, Gao JH, Wang JB, et al. 2020. Comparison of nonhuman primates identified the suitable model for COVID-19. Signal Transduction and Targeted Therapy, 5(1): 157. doi: 10.1038/s41392-020-00269-6
    [16] Lukacs NW, Hogabaom C, Campbell E, Kunkel SL. 1999. Chemokines: function, regulation and alteration of inflammatory responses. Chemical Immunology, 72: 102−120. doi: 10.1159/000058729
    [17] McCray PB Jr, Pewe L, Wohlford-Lenane C, Hickey M, Manzel L, Shi L, et al. 2007. Lethal infection of K18-hACE2 mice infected with wevere acute respiratory sundrome coronavirus. Journal of Virology, 81(2): 813−821. doi: 10.1128/JVI.02012-06
    [18] Mei JJ, Liu YH, Dai N, Favara M, Greene T, Jeyaseelan S, et al. 2010. CXCL5 regulates chemokine scavenging and pulmonary host defense to bacterial infection. Immunity, 33(1): 106−117. doi: 10.1016/j.immuni.2010.07.009
    [19] Moore JB, June CH. 2020. Cytokine release syndrome in severe COVID-19. Science, 368(6490): 473−474. doi: 10.1126/science.abb8925
    [20] Puneet P, Moochhala S, Bhatia M. 2005. Chemokines in acute respiratory distress syndrome. American Journal of Physiology - Lung Cellular and Molecular Physiology, 288(1): L3−L15. doi: 10.1152/ajplung.00405.2003
    [21] Qin C, Zhou LQ, Hu ZW, Zhang SQ, Yang S, Tao Y, et al. 2020. Dysregulation of immune response in patients with Coronavirus 2019 (COVID-19) in Wuhan, China. Clinical Infectious Diseases, 71(15): 762−768. doi: 10.1093/cid/ciaa248
    [22] Read LJ, Muench H. 1938. A simple method of estimating fifty percent endpoints. American Journal of Epidemiology, 27: 493−497. doi: 10.1093/oxfordjournals.aje.a118408
    [23] Rockx B, Kuiken T, Herfst S, Bestebroer T, Lamers MM, Oude Munnink BB, et al. 2020. Comparative pathogenesis of COVID-19, MERS, and SARS in a nonhuman primate model. Science, 368(6949): 1012−1015.
    [24] Shan C, Yao YF, Yang XL, Zhou YW, Gao G, Peng Y, et al. 2020. Infection with novel coronavirus (SARS-CoV-2) causes pneumonia in Rhesus macaques. Cell Research, 30(8): 670−677. doi: 10.1038/s41422-020-0364-z
    [25] Shi JZ, Wen ZY, Zhong GX, Yang HL, Wang C, Huang BY, et al. 2020. Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARS-coronavirus 2. Science, 368(6494): 1016−1020. doi: 10.1126/science.abb7015
    [26] Song TZ, Zheng HY, Han JB, Jin L, Yang X, Liu FL, et al. 2020. Delayed severe cytokine storm and immune cell infiltration in SARS-CoV-2-infected aged Chinese rhesus macaques. Zoological Research, 41(5): 503−516. doi: 10.24272/j.issn.2095-8137.2020.202
    [27] Sun J, Zhuang Z, Zheng J, Li K, Wong RLY, Liu DL, et al. 2020. Generation of a broadly useful model for COVID-19 pathogenesis, vaccination, and treatment. Cell, 182(3): 734−743. doi: 10.1016/j.cell.2020.06.010
    [28] Tate MD, Deng YM, Jones JE, Anderson GP, Brooks AG, Reading PC. 2009. Neutrophils ameliorate lung injury and the development of severe disease during influenza infection. The Journal of Immunology, 183(11): 7441−7450. doi: 10.4049/jimmunol.0902497
    [29] Tseng CTK, Huang C, Newman P, Wang N, Narayanan K, Watts DM, et al. 2007. Severe acute respiratory syndrome coronavirus infection of mice transgenic for the human Angiotensin-converting enzyme 2 virus receptor. Journal of Virology, 81(3): 1162−1173. doi: 10.1128/JVI.01702-06
    [30] Xu L, Yu DD, Ma YH, Yao YL, Luo RH, Feng XL, et al. 2020a. COVID-19-like symptoms observed in Chinese tree shrews infected with SARS-CoV-2. Zoological Research, 41(5): 517−526. doi: 10.24272/j.issn.2095-8137.2020.053
    [31] Xu Z, Shi L, Wang YJ, Zhang JY, Huang L, Zhang C, et al. 2020b. Pathological findings of COVID-19 associated with acute respiratory distress syndrome. The Lancet Respiratory Medicine, 8(4): 420−422. doi: 10.1016/S2213-2600(20)30076-X
    [32] Yang XH, Deng W, Tong Z, Liu YX, Zhang LF, Zhu H, et al. 2007. Mice transgenic for human angiotensin-converting enzyme 2 provide a model for SARS coronavirus infection. Comparative Medicine, 57(5): 450−459.
    [33] Zhang YN, Li XD, Zhang ZR, Zhang HQ, Li N, Liu J, et al. 2020. A mouse model for SARS-CoV-2 infection by exogenous delivery of hACE2 using alphavirus replicon particles. Cell Research. doi: 10.1038/s41422-020-00405-5.
    [34] Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, et al. 2020. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature, 579(7798): 270−273. doi: 10.1038/s41586-020-2012-7
    [35] Ziegler CGK, Allon SJ, Nyquist SK, Mbano IM, Miao VN, Tzouanas CN, et al. 2020. SARS-CoV-2 receptor ACE2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues. Cell, 181(5): 1016−1035. doi: 10.1016/j.cell.2020.04.035
  • ZR-2020-118 Supplementary Material.pdf
  • 加载中
图(5)
计量
  • 文章访问数:  602
  • HTML全文浏览量:  307
  • PDF下载量:  160
  • 被引次数: 0
出版历程
  • 收稿日期:  2020-05-14
  • 网络出版日期:  2020-10-12
  • 刊出日期:  2020-11-18

目录

    /

    返回文章
    返回