Article Figures & Data
Figures
- FIG 1
Omics characterization of complement pathway expression and activation. (A) Protein abundance at 1, 2, 4, and 7 days postinfection relative to that in mock-treated samples. Samples were taken from total lung homogenates, and error bars indicate standard errors of the means (SEM). Each point indicates the mean of results for 5 mice at a given time. (B) C3 protein cleavage is observed in the lung by Western blotting as early as 24 h following SARS-CoV MA15 infection of C57BL/6J mice. Numbers at the left are molecular weights (in thousands).
- FIG 2
Characterization of C3 knockout mice. (A) Weight loss of SARS-CoV MA15-infected C57BL/6J mice, C3–/– mice, or mock-infected mice were measured over time. (B) Lung titers of SARS-CoV MA15-infected C57BL/6J or C3–/– mice at 2, 4, and 7 days postinfection. nd, not determined. (A and B) Six to 8 mice were used through day 4, and 3 to 4 mice were used for days 5 to 7. The respiratory function of SARS-CoV MA15-infected C57BL/6J and C3–/– mice and mock-infected mice was measured using a Buxco whole-body plethysmography system for Penh, a measure of calculated airway resistance (C), EF50, midbreath expiratory flow (D), and RPEF, the rate of peak expiratory flow (E). *, P < 0.05 between mock-infected mice and a given condition. (C to E) Three mice were used for each infection group, and two mock-infected mice were used.
- FIG 3
Histological analysis of C57BL/6J and C3−/− lungs at 2 and 4 days postinfection. Representative images show 400× magnifications of the large airways (top row), vasculature (middle row), and parenchyma (bottom row) of the lung after SARS-CoV MA15 or mock infection of C57BL/6J or C3−/− mice.
- FIG 4
Complement deposition staining. Complement deposition on lung tissue of C57BL/6J mice (top three rows) was assessed by immunohistochemistry. Mice were examined at 2 and 4 dpi and mock infected or infected with SARS-CoV MA15. C3–/– mice (bottom row) showed no positive staining.
- FIG 5
Inflammatory cells of C57BL/6J mice and C3 knockout mice. Flow cytometric analysis of inflammatory cells present in the lungs of SARS-CoV MA15-infected or mock-infected C57BL/6J or C3–/– mice at 4 days postinfection. (A) Lymphocytes; (B) myeloid-cell-derived cells (as defined by Misharin et al. [68]); (C) CD4 T cell activation markers; (D) CD8 T cell activation markers; (E) CD11c– neutrophil activation markers. *, P < 0.05. Error bars indicate SEM. Eight mice were used for all infection groups, and 4 mice were used for all mock-infected groups. Neuts, neutrophils; DC, dendritic cells; MHCIIhi, a high fluorescence intensity for MHCII staining; MonoMac (inf), inflammatory monocyte-macrophages; Macs, macrophages.
- FIG 6
Cytokine and chemokine abundance levels. Protein abundance in the lung was measured by Bioplex multiplex magnetic bead assay at days 2, 4, and 7 postinfection or in mock-infected mice. MIP-1a, MIP-1b, and monocyte chemoattractant protein (MCP) had similar concentrations in the lungs of C57BL/6J and C3–/– mice, with peak abundance at 2 days postinfection (A), while G-CSF, IL-6, TNF, and IL-1a expression was highest in C57BL/6J mice at 2 dpi (B). *, P < 0.05; ‡, P < 0.1. Error bars indicate SEM, and 3 to 4 mice were tested for each condition.
- FIG 7
Systemic response to SARS-CoV MA15 infection. (A) C3 protein cleavage products are observed by Western blotting in the serum following infection. Molecular weights are noted at the left (in thousands). rMA15, recombinant MA15. (B) MCP-1 and RANTES levels are similarly elevated following infection in C57BL/6J and C3–/– mice. (C) G-CSF, KC, and IL-5 all have significantly higher expression in the sera of C57BL/6J mice than in those of C3–/– mice. IL-6 expression was suggestive of differences. (D) Complement deposition staining in the kidneys of C57BL/6J mice. *, P < 0.05; ‡, P < 0.06. Error bars indicate SEM, and 3 to 4 mice were used for each condition.
Supplemental Material
FIG S1
Complement pathway knockouts. C4 and fB complement pathway-specific knockout mice were intranasally infected with 105 PFU of MA15; both show an intermediate phenotype, with significantly less weight loss than that of C57BL/6J mice but more weight loss than that of C3 knockout mice at day 3 postinfection. *, P < 0.05 between C57BL/6J and pathway-specific knockout mice; †, P < 0.05 between C3–/– and pathway-specific knockout mice. Error bars indicate SEM, with 8 C4–/– mice, 14 C57BL/6J mice, 7 C3–/– mice, and 3 fB–/– mice. Download FIG S1, TIF file, 0.45 MB.
Copyright © 2018 Gralinski et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
TABLE S1
Histology scoring summary of C57BL/6J and C3–/– mice at 2 and 4 days postinfection. Download Table S1, DOCX file, 0.01 MB.
Copyright © 2018 Gralinski et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S2
Bronchoalveolar lavage. Platelet counts in BAL fluid were assessed using a VetscanHM5 at both day 2 and day 4 post infection. (Four mice were used for all infected groups, and 2 mice were used for all mock-infected groups.) Download FIG S2, TIF file, 0.96 MB.
Copyright © 2018 Gralinski et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
