Spatiotemporal landscape of SARS-CoV-2 pulmonary infection reveals Slamf9 + Spp1 + macrophages promoting viral clearance and inflammation resolution

  1. Boyi Cong
  2. Xuan Dong
  3. Zongheng Yang
  4. Pin Yu
  5. Yangyang Chai
  6. Jiaqi Liu
  7. Meihan Zhang
  8. Yupeng Zang
  9. Jingmin Kang
  10. Yu Feng
  11. Yi Liu
  12. Weimin Feng
  13. Wei Deng
  14. Fengdi Li
  15. Qinyi Yu
  16. Yan Gu
  17. Zhiqing Li
  18. Shuxun Liu
  19. Xun Xu
  20. Nanshan Zhong
  21. Xianwen Ren
  22. Chuan Qin
  23. Longqi Liu
  24. Jian Wang
  25. Xuetao Cao

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Abstract

While SARS-CoV-2 pathogenesis has been intensively investigated, the host mechanisms of viral clearance and inflammation resolution are still elusive because of the ethical limitation of human studies based on COVID-19 convalescents. Here we infected Syrian hamsters by authentic SARS-CoV-2 and built an ideal model to simulate the natural recovery process of SARS-CoV-2 infection from severe pneumonia 1,2 . We developed and applied a spatial transcriptomic sequencing technique with subcellular resolution and tissue-scale extensibility, i.e. , Stereo-seq 3 , together with single-cell RNA sequencing (scRNA-seq), to the entire lung lobes of 45 hamsters and obtained an elaborate map of the pulmonary spatiotemporal changes from acute infection, severe pneumonia to the late viral clearance and inflammation resolution. While SARS-CoV-2 infection caused massive damages to the hamster lungs, including naïve T cell infection and deaths related to lymphopenia, we identified a group of monocyte-derived proliferating Slamf9 + Spp1 + macrophages, which were SARS-CoV-2 infection-inducible and cell death-resistant, recruiting neutrophils to clear viruses together. After viral clearance, the Slamf9 + Spp1 + macrophages differentiated into Trem2 + and Fbp1 + macrophages, both responsible for inflammation resolution and replenishment of alveolar macrophages. The existence of this specific macrophage subpopulation and its descendants were validated by RNAscope in hamsters, immunofluorescence in hACE2 mice, and public human autopsy scRNA-seq data of COVID-19 patients. The spatiotemporal landscape of SARS-CoV-2 infection in hamster lungs and the identification of Slamf9 + Spp1 + macrophages that is pivotal to viral clearance and inflammation resolution are important to better understand the critical molecular and cellular players of COVID-19 host defense and also develop potential interventions of COVID-19 immunopathology.

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  1. ScreenIT

    SciScore for 10.1101/2022.05.03.490381: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    EthicsIACUC: The Institutional Animal Care and Use Committee (IACUC) of the ILAS, Peking Union Medical College & Chinese Academy of Medical Sciences, evaluated and gave permission to all the protocols in these studies, including animal experiments (Approval number QC21003).
    Sex as a biological variableEthics Statement: At the Institute of Laboratory Animal Science (ILAS) of Chinese Academy of Medical Sciences, the animal biosafety level 3 (ABSL-3) facility was used to accomplish all the experiments with Syrian hamsters (male and female, aged 8-10 weeks) and hACE2 mice (male and female, aged 6-11 months).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Different primary antibodies were sequentially applied to examine specific cell markers, including anti-CD68 Polyclonal antibody (28058-1-AP, 1:800; proteintech) and TREM2 Polyclonal antibody (13483-1-AP, 1:200; proteintech), or FBP1 Polyclonal antibody (12842-1-AP, 1:100, proteintech), or Osteopontin Polyclonal antibody (22952-1-AP, 1:200; proteintech) and SLAMF9 polyclonal antibody (LM-2205R, 1:200; LMAI BIO), followed by HRP-conjugated secondary antibody incubation and tyramide signal amplification (TSA).
    anti-CD68
    suggested: (Proteintech Cat# 28058-1-AP, RRID:AB_2881049)
    28058-1-AP
    suggested: (Proteintech Cat# 28058-1-AP, RRID:AB_2881049)
    TREM2
    suggested: (Proteintech Cat# 13483-1-AP, RRID:AB_2877955)
    13483-1-AP
    suggested: (Proteintech Cat# 13483-1-AP, RRID:AB_2877955)
    FBP1
    suggested: (Proteintech Cat# 12842-1-AP, RRID:AB_2103572)
    12842-1-AP
    suggested: (Proteintech Cat# 12842-1-AP, RRID:AB_2103572)
    Osteopontin
    suggested: (Proteintech Cat# 22952-1-AP, RRID:AB_2783651)
    22952-1-AP
    suggested: (Proteintech Cat# 22952-1-AP, RRID:AB_2783651)
    SLAMF9
    suggested: None
    LM-2205R
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Viruses: The SARS-CoV-2 virus (accession number is MT093631.2, SARS CoV-2/WH-09/human/2020/CHN) was put into use in this study and propagated in Vero E6 cells incubated in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, United States) supplemented with 10% fetal bovine serum (Gibco, Grand Island, United States) and incubated at 37 ◦C and 5% carbon dioxide.
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Experimental hACE2 mice: Specific-pathogen-free, 6-11-month-old male and female hACE2 mice were obtained from the Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences as described in our previous study15.
    hACE2
    suggested: None
    For multiplex staining the following probes were used: Fbp1 (Mau-Fbp1 1153521-C3), Cd4 (Mau-Cd4 1153531-C1), Il10 (Mau-Il10 1153551-C3), Mki67 (Mau-Mki67 1153561-C2), Trem2 (Mau-Trem2 1153571-C2), Sftpc (Mau-Sftpc 1058161-C3), Cd68 (Mau-Cd68 899591-C1), S (V-nCoV2019-S 848561-C2).
    Fbp1 (Mau-Fbp1 1153521-C3)
    suggested: None
    Software and Algorithms
    SentencesResources
    UMI counting, cell identification, and generation of expression matrices were accomplished by PISA.
    PISA
    suggested: (PISA, RRID:SCR_015749)
    Unsupervised cell clustering and annotation: Clustering analysis of the scRNA-seq dataset was performed using the Seurat package (v4.0.5) and the R program (v4.1.0).
    Seurat
    suggested: (SEURAT, RRID:SCR_007322)
    Differential gene expression and Gene Ontology enrichment analysis: To investigate the effects of viral RNA in one subpopulation, we identified differentially expressed genes between viral RNA positive and negative cells by performing two-sided unpaired Wilcoxon tests based on the tl.rank_genes_groups function of the python package Scanpy (v1.8.1) (method=’wilcoxon’, corr_method = ‘benjamini-hochberg’).
    python
    suggested: (IPython, RRID:SCR_001658)
    Mm.eg.db, pAdjustMethod=“BH”) function of the Cluster Profiler R package (v4.0.5)8.
    Cluster Profiler
    suggested: (clusterProfiler, RRID:SCR_016884)
    We processed the pseudo-time analysis followed tutorial described in http://cole-trapnell-lab.github.io/monocle-release/docs/ with default parameters.
    http://cole-trapnell-lab.github.io/monocle-release/docs/
    suggested: (Monocle2, RRID:SCR_016339)
    Mesocricetus auratus genome (BCM_Maur_2.0) and SARS-CoV-2 genome were integrated as one reference for read mapping by STAR.
    STAR
    suggested: (STAR, RRID:SCR_004463)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    However, because of the complexity of in vivo infection and the related host immune responses, traditional technologies cannot address such challenges due to the limitations of spatial, temporal, and cellular resolution, limiting the development of anti-infectious measurements and therapies. Although scRNA-seq provides sufficient resolution at the single-cell level, the loss of spatiotemporal information and the “survivor biases” introduced during scRNA-seq library preparation as we illustrated in this study prohibit its power to directly identify critical host factors important for pathogen clearance and inflammation resolution. Our study demonstrated the power of spatial omics technologies in illustrating the molecular and cellular details of in vivo biological events, specifically those locally related to cell damages and cell deaths. Therefore, single-cell spatiotemporal studies of in vivo biological phenomena will be a powerful paradigm to revolutionize studies including inflammatory damages, infectious diseases, tumours, and any other questions related to cell deaths. As single-cell spatiotemporal studies are inherently unbiased and observational, the generated hypotheses will be more reliable than guesses based on limited indices and more deserving to be validated and deepened by experimental studies. Our study revealed that Slamf9+Spp1+ macrophages were specifically induced by SARS-CoV-2 infection. However, the conditions sufficient or necessary for the induction of S...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  2. Version published to 10.1101/2022.05.03.490381v1 on bioRxiv