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Short Report
Jie Na
School of Medicine, Tsinghua University
Corresponding Author
Qizhou Lian
Department of Medicine, Li Ka Shing Faculty of Medicine; the University of Hong Kong
Corresponding Author
Huanhuan Joyce Chen
The Pritzker School of Molecular Engineering, the University of Chicago
Corresponding Author
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Declarations
- This study involved human subjects.
- The author confirmed that all appropriate ethical guidelines for the use of human subjects have been followed, any necessary IRB and/or ethics committee review has been obtained, and information about the IRB/ethics committee is included in the manuscript.
- The author has confirmed that all necessary patient/participant consent or assent has been obtained and the appropriate institutional forms have been archived. If the IRB/ethics committee waived the requirement for patient/participant consent or assent, an explanation for the waiver is included in the text.
- The author has confirmed that a statement listing potential conflicts of interest or lack thereof is included in the text.
Dysfunctional immune responses contribute critically to the progression of Coronavirus Disease-2019 (COVID-19) from mild to severe stages including fatality, with pro-inflammatory macrophages as one of the main mediators of lung hyper-inflammation. Therefore, there is an urgent need to better understand the interactions among SARS-CoV-2 permissive cells, macrophage, and the SARS-CoV-2 virus, thereby offering important insights into new therapeutic strategies. Here, we used directed differentiation of human pluripotent stem cells (hPSCs) to establish a lung and macrophage co-culture system and model the host-pathogen interaction and immune response caused by SARS-CoV-2 infection. Among the hPSC-derived lung cells, alveolar type II and ciliated cells are the major cell populations expressing the viral receptor ACE2 and co-effector TMPRSS2, and both were highly permissive to viral infection. We found that alternatively polarized macrophages (M2) and classically polarized macrophages (M1) had similar inhibitory effects on SARS-CoV-2 infection. However, only M1 macrophages significantly up-regulated inflammatory factors including IL-6 and IL-18, inhibiting growth and enhancing apoptosis of lung cells. Inhibiting viral entry into target cells using an ACE2 blocking antibody enhanced the activity of M2 macrophages, resulting in nearly complete clearance of virus and protection of lung cells. These results suggest a potential therapeutic strategy, in that by blocking viral entrance to target cells while boosting anti-inflammatory action of macrophages at an early stage of infection, M2 macrophages can eliminate SARS-CoV-2, while sparing lung cells and suppressing the dysfunctional hyper-inflammatory response mediated by M1 macrophages.
Keywords
COVID-19, SARS-CoV-2, Human pluripotent stem cell, Macrophages, disease modeling
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
This is a list of supplementary files associated with this preprint. Click to download.
- FigureS1.jpg
Figure S1. Generation of lung cells by directed differentiation of hPSC (A) Scheme on distal lung structure and gene signatures of lung lineage cells. (B) Scheme of directed differentiation of hPSC to lung organoids. (C and D) Representative bright and IF images in the hPSC-derived cell cultures at day 3.5, 15 -25; the cells that are triple-positive for SOX2, FOXA2 and NKX2.1 at anterior foregut endoderm/lung progenitor stage or lung progenitor stage. Scale bars=100 µm.
- FigureS2.jpg
Figure S2. ACE2 expression in hPSC-derived lung cells (A) Representative bright images on the hPSC-derived lung cells at day 50 in the matrigel-coated 2-D culture. Scale bars=200 µm. (B) Expression of ACE2 was detected by immunostaining in hPSC-derived lung cells at day 50, in SP-B+ or Pro-SP-C+ AT2 cells, and FOXJ1+ ciliated cells. Scale bars= 100 µm.
- FigureS3.jpg
Figure S3. Characterization of hPSC-derived lung cells by scRNA seq (A) UMAP hPSC-derived lung cells at day 50, colored and annotated with cluster 0-5. AT1, Alveolar Epithelial Type 1 cells, AT2 cells, Alveolar Epithelial Type 2 cells. (B) Putative lung cell-fate related markers differentially expressed in each cluster in UMAPs. Relative expression of each marker gene range from low (light yellow) to high (pink) as indicated. Individual cell positive for lung cell markers are donated by red dots. (C) Heatmap examination of each cluster for top differential expression of genes. (D) Dot plot of gene signatures known for lung cell types in 5 clusters of hPSC-derived lung cell culture. Relative expression levels of each gene range from low (yellow) to high (red) as indicated. Percentages of cells with differential gene expression are indicated by the size of circles. (E&F) Expression of ACE2 and TMPRSS2, in each cluster in UMAPs. The violin plot shows the expression level (log2(UMI+1)) of indicated gene in each cluster. ACE2 and TMPRSS2 enriched in AT2, AT1, and ciliated cells.
- FigureS4.jpg
Figure S4. Generation and characterization of macrophages by directed differentiation of hPSC (A) Scheme of directed differentiation of hPSC to macrophage. (B) Representative bright image on hPSC-derived macrophages in culture. Scale bars = 50μm. (C) Giemsa staining of hPSC-derived macrophages. Scale bars = 50μm.. (D) Flow cytometry characterized hPSC-derived macrophages expressed major macrophage /monocyte markers such as CD14, CD11b, and CD68.
- FigureS5.jpg
Figure S5. Characterization of iM1φ and iM2φ polarized from hPSC-derived macrophages (A) UMAP on iM1φ and iM2φ. (B) ACE2, TMPRSS2, putative cell-fate related markers, and inflammatory factors differentially expressed in the cluster of iM1φ and iM2φ in UMAPs. Relative expression of each marker gene range from low (light yellow) to high (red) as indicated. Individual cells positive for each marker are donated by red dots. (C) Dot plot presenting the cytokine and chemokine related genes in iM1φ and iM2φ. Relative expression of each gene ranges from low (light blue) to high (red) as indicated.
- FigureS6.jpg
Figure S6. Characterization of the co-culture of lung cells and macrophages derived from hPSCs (A) Heatmap examination of iM2φ, iM1φ, lung epithelial cells and stromal cells for top differential expression of genes. (B) Gene set enrichment analysis by GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) in iM1φ, versus iM2φ. (C) GO Gene set enrichment analysis in iM2φ, versus iM1φ. (D) Dot plot of cell-death related genes in iLung cells co-cultured with iM1φ or iM2φ. Relative expression levels of each gene range from low (black) to high (yellow) as indicated. Percentages of cells with differential gene expression are indicated by the size of circles.
- FigureS7.jpg
Figure S7 The effects of M1 or M2 macrophages on SARS-CoV-2-Pseudo virus infection (A) IF staining on the co-cultures of iLung cells and iM1φ, iM2φ, or 293T, at Mock or infected with virus at 24 hpi (MOI=0.01), using antibodies detecting luciferase protein expressing in SARS-CoV-2-Pseudo virus, CD80 or CD206. ILung cells expressed GFP. Scale bar = 100 µm. (B) Luciferase protein was co-localized with ACE2 in lung cells by IF staining. Scale bar = 100 µm. (C) Luciferase activity of the co-cultures of lung cells and M1, M2 macrophages (U937) or 293T cells (control) at Mock or infected with SARS-CoV-2 pseudo-entry virus at 24 hpi (MOI=0.01). P values were calculated by unpaired two-tailed Student’s t test. **P < 0.01, ****P < 0.0001.
- FigureS8.jpg
Figure S8. scRNA analysis of iM1φ, iM2φ or iLung upon viral infection (A) Volcano plot analysis of differential expression of iM1φ versus iM2φ co-cultured with ilung on viral infection. Individual genes denoted by gene name. (B) Volcano plot analysis of differential expression of ilung co-cultured with iM1φ versus iM2φ on viral infection. Individual genes denoted by gene name. (C) Dot plot examined differential expression genes related to cell death or mitochondria in iM1φ, or iM2φ. Relative expression levels of each gene range from low (blue) to high (red) as indicated. Percentages of cells with differential gene expression are indicated by the size of circles. (D) Dot plot examined differential expression genes related to IL-10 signaling in iM1φ, or iM2φ. (E) GO and KEGG enrichment analysis in iLung cells co-cultured with iM1φ, Important pathways related to immune response or viral reaction is highlighted in red (iM1φ).
- FigureS9.jpg
Figure S9. The effects of macrophages in combination with ACE2 blockage on SARS-CoV-2 Pseudo virus infection (A) The ACE2 blockage antibody was applied two hours prior to the virus presence, IF staining was performed on the co-cultures of iLung cells and iM1φ, iM2φ, or 293T, at Mock or infected with virus at 24 hpi (MOI=0.01), using antibodies detecting luciferase protein expressing in SARS-CoV-2-Pseudo virus, CD80 or CD206. ILung cells expressed GFP. Scale bar = 100 µm.
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A new preprint design is coming!
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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Short Report
Jie Na
School of Medicine, Tsinghua University
Corresponding Author
Qizhou Lian
Department of Medicine, Li Ka Shing Faculty of Medicine; the University of Hong Kong
Corresponding Author
Huanhuan Joyce Chen
The Pritzker School of Molecular Engineering, the University of Chicago
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 2
Posted 15 Sep, 2020
No community comments so far
No comments provided
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Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Declarations:
Declarations
- This study involved human subjects.
- The author confirmed that all appropriate ethical guidelines for the use of human subjects have been followed, any necessary IRB and/or ethics committee review has been obtained, and information about the IRB/ethics committee is included in the manuscript.
- The author has confirmed that all necessary patient/participant consent or assent has been obtained and the appropriate institutional forms have been archived. If the IRB/ethics committee waived the requirement for patient/participant consent or assent, an explanation for the waiver is included in the text.
- The author has confirmed that a statement listing potential conflicts of interest or lack thereof is included in the text.
Dysfunctional immune responses contribute critically to the progression of Coronavirus Disease-2019 (COVID-19) from mild to severe stages including fatality, with pro-inflammatory macrophages as one of the main mediators of lung hyper-inflammation. Therefore, there is an urgent need to better understand the interactions among SARS-CoV-2 permissive cells, macrophage, and the SARS-CoV-2 virus, thereby offering important insights into new therapeutic strategies. Here, we used directed differentiation of human pluripotent stem cells (hPSCs) to establish a lung and macrophage co-culture system and model the host-pathogen interaction and immune response caused by SARS-CoV-2 infection. Among the hPSC-derived lung cells, alveolar type II and ciliated cells are the major cell populations expressing the viral receptor ACE2 and co-effector TMPRSS2, and both were highly permissive to viral infection. We found that alternatively polarized macrophages (M2) and classically polarized macrophages (M1) had similar inhibitory effects on SARS-CoV-2 infection. However, only M1 macrophages significantly up-regulated inflammatory factors including IL-6 and IL-18, inhibiting growth and enhancing apoptosis of lung cells. Inhibiting viral entry into target cells using an ACE2 blocking antibody enhanced the activity of M2 macrophages, resulting in nearly complete clearance of virus and protection of lung cells. These results suggest a potential therapeutic strategy, in that by blocking viral entrance to target cells while boosting anti-inflammatory action of macrophages at an early stage of infection, M2 macrophages can eliminate SARS-CoV-2, while sparing lung cells and suppressing the dysfunctional hyper-inflammatory response mediated by M1 macrophages.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
This is a list of supplementary files associated with this preprint. Click to download.
- FigureS1.jpg
Figure S1. Generation of lung cells by directed differentiation of hPSC (A) Scheme on distal lung structure and gene signatures of lung lineage cells. (B) Scheme of directed differentiation of hPSC to lung organoids. (C and D) Representative bright and IF images in the hPSC-derived cell cultures at day 3.5, 15 -25; the cells that are triple-positive for SOX2, FOXA2 and NKX2.1 at anterior foregut endoderm/lung progenitor stage or lung progenitor stage. Scale bars=100 µm.
- FigureS2.jpg
Figure S2. ACE2 expression in hPSC-derived lung cells (A) Representative bright images on the hPSC-derived lung cells at day 50 in the matrigel-coated 2-D culture. Scale bars=200 µm. (B) Expression of ACE2 was detected by immunostaining in hPSC-derived lung cells at day 50, in SP-B+ or Pro-SP-C+ AT2 cells, and FOXJ1+ ciliated cells. Scale bars= 100 µm.
- FigureS3.jpg
Figure S3. Characterization of hPSC-derived lung cells by scRNA seq (A) UMAP hPSC-derived lung cells at day 50, colored and annotated with cluster 0-5. AT1, Alveolar Epithelial Type 1 cells, AT2 cells, Alveolar Epithelial Type 2 cells. (B) Putative lung cell-fate related markers differentially expressed in each cluster in UMAPs. Relative expression of each marker gene range from low (light yellow) to high (pink) as indicated. Individual cell positive for lung cell markers are donated by red dots. (C) Heatmap examination of each cluster for top differential expression of genes. (D) Dot plot of gene signatures known for lung cell types in 5 clusters of hPSC-derived lung cell culture. Relative expression levels of each gene range from low (yellow) to high (red) as indicated. Percentages of cells with differential gene expression are indicated by the size of circles. (E&F) Expression of ACE2 and TMPRSS2, in each cluster in UMAPs. The violin plot shows the expression level (log2(UMI+1)) of indicated gene in each cluster. ACE2 and TMPRSS2 enriched in AT2, AT1, and ciliated cells.
- FigureS4.jpg
Figure S4. Generation and characterization of macrophages by directed differentiation of hPSC (A) Scheme of directed differentiation of hPSC to macrophage. (B) Representative bright image on hPSC-derived macrophages in culture. Scale bars = 50μm. (C) Giemsa staining of hPSC-derived macrophages. Scale bars = 50μm.. (D) Flow cytometry characterized hPSC-derived macrophages expressed major macrophage /monocyte markers such as CD14, CD11b, and CD68.
- FigureS5.jpg
Figure S5. Characterization of iM1φ and iM2φ polarized from hPSC-derived macrophages (A) UMAP on iM1φ and iM2φ. (B) ACE2, TMPRSS2, putative cell-fate related markers, and inflammatory factors differentially expressed in the cluster of iM1φ and iM2φ in UMAPs. Relative expression of each marker gene range from low (light yellow) to high (red) as indicated. Individual cells positive for each marker are donated by red dots. (C) Dot plot presenting the cytokine and chemokine related genes in iM1φ and iM2φ. Relative expression of each gene ranges from low (light blue) to high (red) as indicated.
- FigureS6.jpg
Figure S6. Characterization of the co-culture of lung cells and macrophages derived from hPSCs (A) Heatmap examination of iM2φ, iM1φ, lung epithelial cells and stromal cells for top differential expression of genes. (B) Gene set enrichment analysis by GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) in iM1φ, versus iM2φ. (C) GO Gene set enrichment analysis in iM2φ, versus iM1φ. (D) Dot plot of cell-death related genes in iLung cells co-cultured with iM1φ or iM2φ. Relative expression levels of each gene range from low (black) to high (yellow) as indicated. Percentages of cells with differential gene expression are indicated by the size of circles.
- FigureS7.jpg
Figure S7 The effects of M1 or M2 macrophages on SARS-CoV-2-Pseudo virus infection (A) IF staining on the co-cultures of iLung cells and iM1φ, iM2φ, or 293T, at Mock or infected with virus at 24 hpi (MOI=0.01), using antibodies detecting luciferase protein expressing in SARS-CoV-2-Pseudo virus, CD80 or CD206. ILung cells expressed GFP. Scale bar = 100 µm. (B) Luciferase protein was co-localized with ACE2 in lung cells by IF staining. Scale bar = 100 µm. (C) Luciferase activity of the co-cultures of lung cells and M1, M2 macrophages (U937) or 293T cells (control) at Mock or infected with SARS-CoV-2 pseudo-entry virus at 24 hpi (MOI=0.01). P values were calculated by unpaired two-tailed Student’s t test. **P < 0.01, ****P < 0.0001.
- FigureS8.jpg
Figure S8. scRNA analysis of iM1φ, iM2φ or iLung upon viral infection (A) Volcano plot analysis of differential expression of iM1φ versus iM2φ co-cultured with ilung on viral infection. Individual genes denoted by gene name. (B) Volcano plot analysis of differential expression of ilung co-cultured with iM1φ versus iM2φ on viral infection. Individual genes denoted by gene name. (C) Dot plot examined differential expression genes related to cell death or mitochondria in iM1φ, or iM2φ. Relative expression levels of each gene range from low (blue) to high (red) as indicated. Percentages of cells with differential gene expression are indicated by the size of circles. (D) Dot plot examined differential expression genes related to IL-10 signaling in iM1φ, or iM2φ. (E) GO and KEGG enrichment analysis in iLung cells co-cultured with iM1φ, Important pathways related to immune response or viral reaction is highlighted in red (iM1φ).
- FigureS9.jpg
Figure S9. The effects of macrophages in combination with ACE2 blockage on SARS-CoV-2 Pseudo virus infection (A) The ACE2 blockage antibody was applied two hours prior to the virus presence, IF staining was performed on the co-cultures of iLung cells and iM1φ, iM2φ, or 293T, at Mock or infected with virus at 24 hpi (MOI=0.01), using antibodies detecting luciferase protein expressing in SARS-CoV-2-Pseudo virus, CD80 or CD206. ILung cells expressed GFP. Scale bar = 100 µm.
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