Complement Activation Contributes to Severe Acute Respiratory Syndrome Coronavirus Pathogenesis

Abstract

Acute respiratory distress syndrome (ARDS) is immune-driven pathologies that are observed in severe cases of severe acute respiratory syndrome coronavirus (SARS-CoV) infection. SARS-CoV emerged in 2002 to 2003 and led to a global outbreak of SARS. As with the outcome of human infection, intranasal infection of C57BL/6J mice with mouse-adapted SARS-CoV results in high-titer virus replication within the lung, induction of inflammatory cytokines and chemokines, and immune cell infiltration within the lung. Using this model, we investigated the role of the complement system during SARS-CoV infection. We observed activation of the complement cascade in the lung as early as day 1 following SARS-CoV infection. To test whether this activation contributed to protective or pathologic outcomes, we utilized mice deficient in C3 (C3^-/-), the central component of the complement system. Relative to C57BL/6J control mice, SARS-CoV-infected C3^-/- mice exhibited significantly less weight loss and less respiratory dysfunction despite equivalent viral loads in the lung. Significantly fewer neutrophils and inflammatory monocytes were present in the lungs of C3^-/- mice than in C56BL/6J controls, and subsequent studies revealed reduced lung pathology and lower cytokine and chemokine levels in both the lungs and the sera of C3^-/- mice than in controls. These studies identify the complement system as an important host mediator of SARS-CoV-induced disease and suggest that complement activation regulates a systemic proinflammatory response to SARS-CoV infection. Furthermore, these data suggest that SARS-CoV-mediated disease is largely immune driven and that inhibiting complement signaling after SARS-CoV infection might function as an effective immune therapeutic.IMPORTANCE The complement system is a critical part of host defense to many bacterial, viral, and fungal infections. It works alongside pattern recognition receptors to stimulate host defense systems in advance of activation of the adaptive immune response. In this study, we directly test the role of complement in SARS-CoV pathogenesis using a mouse model and show that respiratory disease is significantly reduced in the absence of complement even though viral load is unchanged. Complement-deficient mice have reduced neutrophilia in their lungs and reduced systemic inflammation, consistent with the observation that SARS-CoV pathogenesis is an immune-driven disease. These data suggest that inhibition of complement signaling might be an effective treatment option following coronavirus infection.

Keywords: SARS-CoV; animal models; complement; coronavirus; respiratory viruses.

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.