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SARS-CoV-2 drives JAK1/2-dependent local complement hyperactivation

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Science Immunology  07 Apr 2021:
Vol. 6, Issue 58, eabg0833
DOI: 10.1126/sciimmunol.abg0833
  • Fig. 1 SARS-CoV-2 infection activated complement transcription in lung epithelial cells.

    (A and B) Significantly enriched pathways by GSEA comparing transcriptomes of lung samples from patients infected with SARS-CoV-2 (n = 2) versus uninfected controls (A) and similar GSEA analyses on NHBE cells infected in vitro, or not, with SARS-CoV-2 (n = 3) (B). (C) GSEA of A549 cells transduced with ACE2 (A549-ACE2) or not (A549), comparing cells infected with SARS-CoV-2 versus control cells (n = 3 or 4). Pathways in (A) to (C) were ranked by significance (FDR q values), with complement pathways highlighted in red. Only enriched pathways with FDR of <0.25 are shown. (D and E) Comparison of all pathways significantly induced (FDR q value of <0.25) by SARS-CoV-2 in patients (A), NHBE cells (B), and A549 and A549-ACE2 cells (C), indicating 14 shared enriched pathways (D) and their normalized enrichment score (NES) displayed as a heatmap, with complement pathways highlighted in red (E). (F and G) Representative GSEA plot for one of the complement pathways in (E) and expression of the leading-edge genes from this pathway, with C3, C1R, C1S, and CFB highlighted in red (G). (H) Expression of CFB (top) and C3 (bottom) mRNA in control (Ctrl.) versus SARS-CoV-2–infected cells. (I) Spearman correlation between C3 mRNA expression and SARS-CoV-2 viral load across virus-bearing samples in (A) to (H). ppm, parts per million mapped reads. Data have been sourced from GSE147507. *P < 0.05, **P < 0.01, and ***P < 0.001, by ANOVA.

  • Fig. 2 SARS-CoV-2 infection generated C3a protein in lung epithelial cells.

    (A to D) Confocal images (A and C) and quantification (B and D) from n = 2 independent experiments showing expression of C3a and SARS-CoV-2 N-protein in SARS-CoV-2–treated or mock-infected Calu-3 cells (A and B) or iAEC2s (C and D). Scale bars in (A) and (C), 100 μm. Cell numbers are indicated below each violin, and median values are denoted by dots in (B) and (D). (E and F) Correlation between SARS-CoV-2 N-protein intensity and C3a intensity on a per-cell basis in Calu-3 cells (E) and iAEC2s (F). Indicated are Pearson correlation coefficients and associated P values. Infected and uninfected cells in (B) to (D) have been distinguished by red and blue fills, respectively. ****P < 0.0001 by ANOVA. MOI, multiplicity of infection.

  • Fig. 3 SARS-CoV-2 infection invoked distinct complement signatures across lymphoid, myeloid, and epithelial cells in patients.

    (A) UMAP showing three major cell types and seven sub-cell types in uninfected subject lung biopsies (n = 8) and COVID-19 BAL specimens from patients with mild (n = 3) and severe (n = 3) COVID-19. (B) Expression of cell-defining features across all cell types. (C) Expression of C3, C3AR1, and CD46 in select cell types across uninfected, mild, and severe COVID-19 samples (see also fig. S4, A and B, for all cell types). (D and E) UMAP projection (D) and module (Mod) score (54) (E) of CD46-regulated genes (top), C3aR1-regulated genes (middle), and IFN-α/β–regulated genes (see table S2). In (E), selected cell types are shown. Single-cell data are from GSE145926 and GSE122960. ***P < 0.001 and ****P < 0.0001 by Wilcoxon test.

  • Fig. 4 STAT1 and RELA bound to complement genes induced by SARS-CoV-2.

    (A) Numbers of differentially expressed genes in NHBE cells and A549 alveolar cell lines infected with SARS-CoV-2 in comparison with mock infection. (B) The top 10 IPA-predicted TFs regulating the SARS-CoV-2–driven transcriptional response in NHBE cells and human alveolar basal epithelial cell lines (A549). Highlighted in red are TFs transducing IFN-mediated gene transcription and in blue NF-κB–mediated gene transcription. (C) H3K27Ac, STAT1, and RELA ChIP-seq binding profiles across SARS-CoV-2–induced and repressed genes. (D) STAT1, RELA, and H3K27Ac ChIP-seq tracks showing the IRF9, CFB, and C3 gene loci. Data in (A) are from GSE147507, and data in (C) and (D) have been sourced from ENCODE (H3K27Ac and STAT1) and from GSE132018 (RELA). RELA profiles in (C) are from LPS-treated cells. ***P < 0.001; ****P < 0.0001 by Fisher’s exact test.

  • Fig. 5 The JAKi ruxolitinib neutralized SARS-CoV-2–mediated complement transcription.

    (A) GSEA showing enrichment of genes normalized by pharmaceutical agents in the transcriptomes of control (Ctrl.) or SARS-CoV-2–infected NHBE (left) or A549 (right) cells. Drugs have been ranked by significance (FDR q values), with ruxolitinib, baricitinib, and atovaquone highlighted in red. (B) Representative GSEA plot showing enrichment (higher expression) of ruxolitinib–down-regulated genes in SARS-CoV-2–treated cells. (C) Heatmap showing expression of genes induced/repressed by SARS-CoV-2 in A549 cells transduced with ACE2 (A549-ACE2) and then infected with SARS-CoV-2 in the presence of ruxolitinib or vehicle. Genes are clustered according to their response to SARS-CoV-2 and ruxolitinib. (D) Scatterplot comparing the expression of all genes between STAT1 wild-type (STAT1+/+) and STAT1 knockout (STAT1−/−) HepG2 cells after IFN-α treatment. Differentially expressed genes (FC > 2) are highlighted in blue (down-regulated in knockout) and red (up-regulated in knockout), and selected key complement and IFN pathway genes are highlighted in orange. IL6 is also marked but not significantly expressed or changed. Transcriptomes are sourced from GSE147507 (A to C) (13) and GSE98372 (D) (25).

  • Fig. 6 Pharmacological inhibition of key targets inhibited C3a output from SARS-CoV-2–infected respiratory epithelial cells.

    (A) Chemoproteomic profiling of CFBi identified complement factor as the only target. Shown is the dose-dependent reduction of bead binding of CFB from protein extracts of cells. Shown are means and SD from three independent experiments. (B) C3a enzyme-linked immunosorbent assay in plasma treated with zymosan (an alternative complement pathway activator) in the presence of increasing concentrations of EDTA (a chelator of divalent cations, which stops convertase activity), a CFB blocking antibody (Ab) or isotype control, the chemical CFBi, or its carrier, DMSO. Bars show means + SEM; dots represent individual experiments. (C) Confocal images (left) and quantifications (right) showing generation of C3a in mock-infected or SARS-CoV-2–infected iAEC2s treated with CFBi, ruxolitinib, or a combination of ruxolitinib and remdesivir. Scale bar, 100 μm. Data are from n = 2 independent experiments; 18,191 + 660 (means + SD) cells per condition. Bars indicate means + SD (A) or SEM (B and C). *P < 0.05, ***P < 0.001, and ****P < 0.0001 by ANOVA. AU, arbitrary units.

  • Fig. 7 Schematic model of SARS-CoV-2 induction of complement in respiratory epithelial cells.

    SARS-CoV-2 infects respiratory epithelial cells and induces an IFN response. IFNs signal via the IFN receptor to activate STAT1 via JAK1/2. STAT1 cooperates with RELA to induce transcription of IL-6 and complement genes including C3, CFB, C1R, and C1S. CFB acts as an alternative pathway C3 convertase to cleave C3 intracellularly to C3a and C3b. C3a engages C3aR and C3b engages CD46 on leukocyte subsets in the lungs to drive inflammation. These events can be pharmacologically targeted with antivirals (e.g., remdesivir), JAK-STAT inhibitors (e.g., ruxolitinib), and/or cell-permeable complement inhibitors, including CFBi.

  • The PDF file includes:

    • Fig. S1. SARS-CoV-2 infection activates complement transcription in lung epithelial cells.
    • Fig. S2. SARS-CoV-2 infection activates complement transcription in human lung organoids and primary human bronchial epithelial cells.
    • Fig. S3. C3 protein is induced by SARS-CoV-2 infection of respiratory epithelial cells.
    • Fig. S4. Complement components are expressed in lymphoid, myeloid and epithelial cells.
    • Fig. S5. SARS-CoV2 infection activates complement transcription in bronchoalveolar lavage fluid (BALF) cells.
    • Fig. S6. SARS-CoV2 viral load correlates with C3 mRNA expression in lung tissues of patients with COVID-19.
    • Fig. S7. BALF cells from COVID-19 patients have a CD46 activated signature.
    • Fig. S8. SARS-CoV2 infection minimally affects complement pathways in PBMC across different cell types.
    • Fig. S9. STAT1 and RELA bind SARS-CoV2-induced genes.
    • Fig. S10. STAT1 dependence of SARS-CoV2-induced genes.
    • Fig. S11. Data showing activity of cell-permeable complement factor B inhibitor (CFBi).
    • ig. S12. Percentage N-protein+ and syncytial cells in SARs-CoV2 infected iAECs treated with chemical agents.
    • Table S1. Normalized expression of all transcripts in GSE147507 and SRP257667 and GSEA outputs used in Figure 1.
    • Table S2. List of CD46, C3aR and Interferon alpha/beta target genes curated experimentally for CD46 and from literature for the rest.
    • Table S3. . Differentially expressed genes in SARS-CoV-2-infected cells.
    • Table S4. GSEA-based drug prediction.
    • Table S5. Mass spectrometry data for profiling of CFB inhibitor.
    • Table S6. Data sources used in this study.
    • Table S7. Table of raw data.
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    Other Supplementary Material for this manuscript includes the following:

    • Table S1. Normalized expression of all transcripts in GSE147507 and SRP257667 and GSEA outputs used in Figure 1.
    • Table S2. List of CD46, C3aR and Interferon alpha/beta target genes curated experimentally for CD46 and from literature for the rest.
    • Table S3. . Differentially expressed genes in SARS-CoV-2-infected cells.
    • Table S4. GSEA-based drug prediction.
    • Table S5. Mass spectrometry data for profiling of CFB inhibitor.
    • Table S6. Data sources used in this study.
    • Table S7. Table of raw data.
  • The PDF file includes:

    • Figs. S1 to S12
    • Legends for tables S1 to S7

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    Other Supplementary Material for this manuscript includes the following:

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