Differential effects of macrophage subtypes on SARS-CoV-2 infection in a human pluripotent stem cell-derived model

Abstract

Dysfunctional immune responses contribute critically to the progression of Coronavirus Disease-2019 (COVID-19), with macrophages as one of the main cell types involved. It is urgent to understand the interactions among permissive cells, macrophages, and the SARS-CoV-2 virus, thereby offering important insights into effective therapeutic strategies. Here, we establish a lung and macrophage co-culture system derived from human pluripotent stem cells (hPSCs), modeling the host-pathogen interaction in SARS-CoV-2 infection. We find that both classically polarized macrophages (M1) and alternatively polarized macrophages (M2) have inhibitory effects on SARS-CoV-2 infection. However, M1 and non-activated (M0) macrophages, but not M2 macrophages, significantly up-regulate inflammatory factors upon viral infection. Moreover, M1 macrophages suppress the growth and enhance apoptosis of lung cells. Inhibition of viral entry using an ACE2 blocking antibody substantially enhances the activity of M2 macrophages. Our studies indicate differential immune response patterns in distinct macrophage phenotypes, which could lead to a range of COVID-19 disease severity.

Fig. 1. Macrophages were highly involved at the severe stage of COVID-19.

a H&E (Hematoxylin and Eosin) staining of bronchial or alveolar region in Non-COVID-19 individuals and patients with severe COVID-19. Pulmonary hemorrhagic infarct regions were denoted by arrowheads. Images are representative of three independent experiments. b Immunohistochemistry (IHC) using the antibody against CD68 showed macrophages exhibit aggregated phenotype and enlarged nuclei in the lungs of COVID-19 patients, compared to the Non-COVID-19 samples. Images are representative of three independent experiments. c Immunofluorescence (IF) staining on Non-COVID-19 or COVID-19 distal lung tissues using antibodies against CD80 (M1 marker) or CD163 (M2 marker). CD163⁺ or CD80⁺ cells were denoted by arrows and arrowheads, respectively. d Quantification of CD80⁺ and CD163⁺ macrophages in Non-COVID-19 or COVID-19 distal lung tissues. Data are presented as mean values ± SD. Two-tailed unpaired Student’s t-test. e IF staining of non-COVID-19 or COVID-19 distal lung tissues using antibodies against CD68 and IL-6. f Quantification of CD68 + and IL-6+ macrophages in Non-COVID-19 or COVID-19 distal lung tissues. Data are presented as mean values ± SD. Two-tailed unpaired Student’s t-test. Scale bars indicate 50 μm in all images in Fig. 1. n  =  3 independent experiments.

Fig. 2. Activation of hPSC-derived macrophages and their co-culture with iLungs.

a–d Bulk RNA sequencing and analysis of the hPSC-derived macrophages, iM0φs, iM1φs, and iM2φs, respectively. a PCA plot showing the gene expression profiles of iM0φs, iM1φs, iM2φs. b Heatmap of differentially expressed genes (DEGs) in iM0φs, iM1φs, iM2φs. The highly expressed signature genes for each phenotype were highlighted in gray (iM0φ), red (iM1φ) and blue (iM2φ), respectively. c GO and KEGG analysis of the genes or pathways that were upregulated or enriched in iM1φs compared with iM2φs and iM2φs compared with iM1φs. d Gene Set Enrichment Analysis (GSEA) of KEGG pathways in iM1φs and iM2φs (P < 0.05, FDR < 0.25). e Schematic of the experimental flowchart of the co-culture systems. f Representative bright-field and fluorescence images of the co-culture of lung cells and macrophages derived from hPSC line RUES2. Lung cells are GFP positive. Scale bar = 50 µm. g Quantification of lung cells and macrophages (iMφs or THP-1 cells) in the co-cultures of lung cells and iM0φs, iM1φs, iM2φs, and 293T cells. n  =  3 independent experiments. Data are presented as mean values ± SD. p values were calculated by one-way ANOVA with Tukey’s multiple comparison test.

Fig. 3. Differential responses of the iM0φ, iM1φ and iM2φ to SARS-CoV-2 infection.

a Schematic of the experimental flowchart of SARS-CoV-2 infection of the co-culture systems. b–e IF staining of the co-cultures of iLungs with 293 T cells (b), iM0φs (c), iM1φs (d), and iM2φs (e) infected with SARS-CoV-2 at 24 hpi (MOI = 0.1) and mock samples, using antibodies detecting SARS-CoV-2 nucleocapsid protein (SARS-CoV-2), CD68 (M0φ), CD80 (M1φ), and CD206 (M2φ). iLung cells expressed GFP. Scale bar = 100 µm. f Quantification of infected lung cells in the co-cultures at 24 hpi by FACS using antibodies against SARS-2 N and S protein. n  =  3 independent experiments. Data are presented as mean values ± SD. p values were calculated by one-way ANOVA with Tukey’s multiple comparison test.

Fig. 4. Immune reaction of macrophages following SARS-CoV-2 infection.

a Schematic of the experimental flowchart of SARS-CoV-2 infection of macrophages. b Heatmap analysis of cytokines, chemokines, receptors, phagocytosis and cell death related genes in iM0φ, iM1φs and iM2φ cells at 24hpi or 48 hpi. c Venn plot comparing the overlap of upregulated and downregulated genes upon SARS-CoV-2 infection in iM1φ and iM0φ cells at 24hpi. Bar graph showing the GO enrichments of the overlap genes. d GSEA diagrams comparing the cytokine–cytokine receptor interaction following SARS-CoV-2 infection in iM0φ, iM1φ, and iM2φ at 24hpi. e Schematic of the experimental design of the macrophages on SARS-CoV-2 infection for plaque assay and RT-qPCR. f Comparative graph of plaque assay results in A549(negative control), Calu-3(positive control), H1-ESC-derived iMACs (H1-iM0φs/iM1φs/iM2φs) and iPSC-derived iMACs (iPSC-iM0φs/ iM1φs/iM2φs) on SARS-CoV-2 infection at 6,24 and 48 hpi, respectively. Virus was only detected in the supernatant of Calu-3 cells. n  =  3 independent experiments. Data are presented as mean values ± SD. Statistically significant differences are calculated using an unpaired two-tailed unpaired Student’s t test. g Bar graph depicts RT-qPCR analysis of mRNA expression of SARS-CoV-2 nucleocapsid in control cells and iMACs on SARS-CoV-2 infection at 6,24 and 48 hpi, respectively. Increased mRNA of virus was only detected in Calu-3 cells. n  =  3 independent experiments. Data are presented as mean values ± SD. Statistically significant differences are calculated using an unpaired two-tailed unpaired Student’s t-test.

Fig. 5. Blockage of ACE2 enhanced the elimination of SARS-CoV-2 by macrophages.

a Schematic of the experimental flowchart showing the infection of co-cultures with SARS-CoV-2. The Immunofluorescence analysis of the SARS-CoV-2 infected co-culture systems in the presence or absence of ACE2 blocking antibody. ACE2 blockage antibody was applied two hours prior to the virus addition and IF staining was performed on the co-cultures of lung cells and 293 T (b), iM0φs (c), iM1φs (d), and iM2φs (e) that were infected with SARS-CoV-2 and mock samples at 24 hpi (MOI = 0.1), using antibodies against SARS-CoV-2 nucleocapsid protein (SARS-CoV-2), CD68 (M0φ), CD80 (M1φ), and CD206 (M2φ). Lung cells expressed GFP. Scale bar = 100 µm. f Comparison of the percentages of infected lung cells with or without ACE2 blocking at 24 hpi using FACS. n  =  3 independent experiments. Data are presented as mean values ± SD. p values were calculated by two-tailed unpaired Student’s t test.