Role of neutrophil chemoattractant CXCL5 in SARS-CoV-2 infection-induced lung inflammatory innate immune response in an in vivo hACE2 transfection mouse model
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。Abstract: Understanding the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and clarifying antiviral immunity in hosts are critical aspects for the development of vaccines and antivirals. Mice are frequently used to generate animal models of infectious diseases due to their convenience and ability to undergo genetic manipulation. However, normal adult mice are not susceptible to SARS-CoV-2. Here, we developed a viral receptor (human angiotensin-converting enzyme 2, hACE2) pulmonary transfection mouse model to establish SARS-CoV-2 infection rapidly in the mouse lung. Based on the model, the virus successfully infected the mouse lung 2 days after transfection. Viral RNA/protein, innate immune cell infiltration, inflammatory cytokine expression, and pathological changes in the infected lungs were observed after infection. Further studies indicated that neutrophils were the first and most abundant leukocytes to infiltrate the infected lungs after viral infection. In addition, using infected CXCL5-knockout mice, chemokine CXCL5 was responsible for neutrophil recruitment. CXCL5 knockout decreased lung inflammation without diminishing viral clearance, suggesting a potential target for controlling pneumonia.
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.
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ZR-2020-118 Supplementary Material.pdf