Dysregulated Type I Interferon and Inflammatory Monocyte-Macrophage Responses Cause Lethal Pneumonia in SARS-CoV-Infected Mice

Rudragouda Channappanavar, Anthony R Fehr, Rahul Vijay, Matthias Mack, Jincun Zhao, David K Meyerholz, Stanley Perlman, Rudragouda Channappanavar, Anthony R Fehr, Rahul Vijay, Matthias Mack, Jincun Zhao, David K Meyerholz, Stanley Perlman

Abstract

Highly pathogenic human respiratory coronaviruses cause acute lethal disease characterized by exuberant inflammatory responses and lung damage. However, the factors leading to lung pathology are not well understood. Using mice infected with SARS (severe acute respiratory syndrome)-CoV, we show that robust virus replication accompanied by delayed type I interferon (IFN-I) signaling orchestrates inflammatory responses and lung immunopathology with diminished survival. IFN-I remains detectable until after virus titers peak, but early IFN-I administration ameliorates immunopathology. This delayed IFN-I signaling promotes the accumulation of pathogenic inflammatory monocyte-macrophages (IMMs), resulting in elevated lung cytokine/chemokine levels, vascular leakage, and impaired virus-specific T cell responses. Genetic ablation of the IFN-αβ receptor (IFNAR) or IMM depletion protects mice from lethal infection, without affecting viral load. These results demonstrate that IFN-I and IMM promote lethal SARS-CoV infection and identify IFN-I and IMMs as potential therapeutic targets in patients infected with pathogenic coronavirus and perhaps other respiratory viruses.

Copyright © 2016 Elsevier Inc. All rights reserved.

Figures

Graphical abstract
Graphical abstract
Figure 1
Figure 1
Type I Interferon Signaling Promotes Lung Pathology following Lethal SARS-CoV Infection (A and B) Percentage of initial weight (A) and survival (B) of mice infected with SARS-CoV. (C) Clinical scores determined at days 3 and 5 p.i. (D) SARS-CoV titers in the lungs as determined by plaque assay (ND, not determined). (E) Gross lung pathology at days 3 and 5 p.i. (F) Microscopic changes in the lungs of naive and SARS-CoV-challenged mice at day 5 p.i. (G and H) Weight curves (G) and survival (H) of SARS-CoV infected anti-IFNAR or control (MOPC21) antibody treated mice. Data are representative of 2–3 independent experiments with 4–5 mice/group. Data in (A), (C), and (D) are represented as ±SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Gross and histopathology results are derived from 4–5 mice/group. See also Figures S1 and S2.
Figure 2
Figure 2
Characterization of Virus Replication and IFN-I Production in SARS-CoV-Infected Lungs (A) Lung virus titers determined at early times after SARS-CoV infection. (B) Immunohistochemical examination of SARS-CoV N protein at different times p.i. (C) BALF cytokine/chemokine levels from BALB/c and Ifnar−/− mice at different times p.i. (D) Weight curves and survival after treatment with IFN-β (2,000 U, i.n., single dose at 6, 12, or 24 hr p.i.). (E) Lung viral loads at days 1 and 3 p.i. in IFN-β (2,000 U, 6 hr p.i.) and PBS-treated mice. (F) Lung cells harvested from SARS-CoV-infected mice (24 hr p.i.) were MACS sorted into Siglec-H positive and negative and Siglec-F positive and negative cells. Sorted cells were analyzed for IFN-α4 and IFN-β mRNA transcript levels. (G) Immunohistochemical examination for IFN-β expression in the lung airway (top panel) and lung parenchyma (bottom panel) at different times p.i. (C). Data in (A) and (C)–(E) are derived from two independent experiments, 4–5 mice/group/experiment. Data in (A) and (C)–(F) are represented as ±SEM. ∗∗p < 0.01, ∗∗∗p < 0.001.
Figure 3
Figure 3
IFN-I Promotes Accumulation of Inflammatory Monocyte-Macrophages (A) FACS plots show kinetics of Ly6Chi CD11b+ inflammatory monocyte accumulation in the lungs. (B) Percentage and total number of Ly6Chi CD11b+ cells in the lungs. (C) Phenotypic marker expression on inflammatory monocytes from the lungs of BALB/c mice at day 3 p.i. (D) Cell surface levels of activation markers on lung IMMs at day 3 p.i. (E) Total number of cytokine positive IMMs in the lungs determined following 7 hr ex vivo incubation in the presence of Golgi plug. Data are derived from 2–3 independent experiments with 4 mice/group/experiment. Data in (B), (D), and (E) are represented as ±SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figures S3 and S4.
Figure 4
Figure 4
Depletion of Inflammatory Monocytes Ameliorates SARS-CoV-Induced Lethal Disease Survival and lung pathology were determined in young BALB/c mice treated with IMM-depleting and control antibodies. (A and C) Weight curves and survival of SARS-CoV-infected BALB/c mice after control and MC21 (A) or anti-BST-2 (C) antibody treatment. (B and D) Virus titers in the lungs at days 1 and 3 post control Ig and MC21 (B) or anti-BST-2 mAb (D) treatment. (E) Histological changes in the lungs of naive, isotype-antibody-treated and IMM-depleted SARS-CoV-challenged BALB/c mice at day 5 p.i. (F) Weight curves and survival of control and anti-TNF-α antibody-treated BALB/c mice. (G) Survival of SARS-CoV-infected BALB/c mice after neutrophil depletion. Data are representative of 2 independent experiments (4–5 mice/group/experiment). Data in (A)–(D) are represented as ±SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. See also Figures S2–S4.
Figure 5
Figure 5
IFN-I Signaling on Hematopoietic Cells Promotes SARS-CoV-Induced Morbidity and Mortality (A) PBMCs from uninfected bone-marrow chimera mice (5 weeks post BM transfer) were analyzed by flow cytometry to assess bone-marrow reconstitution. (B) BM-chimera mice were monitored for survival after SARS-CoV challenge (103 PFU, i.n., 4–5 mice/group, 2 independent experiments). (C) Lung cell suspensions from SARS-CoV-infected bone marrow chimeric mice were analyzed for IMM infiltration at day 3 p.i. (D) Percentage and total number of IMMs in chimeric mice at day 3 p.i. For (C) and (D), Data are representative of 2–3 independent experiments (2–3 mice/group/experiment). Data in (D) are represented as ±SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.
Figure 6
Figure 6
IFN-I Signaling Impairs T Cell Response Lung T cell responses were measured in SARS-CoV-challenged BALB/c, Ifnar−/−, and rat IgG and MC21 antibody-treated BALB/c mice at day 6 p.i. (A and B) FACS plots (A) and bar graphs (B) show percentage and total number of virus-specific CD4 and CD8 T cells in the lungs of BALB/c and Ifnar−/− mice. (C and D) FACS plots (C) and bar graphs (D) show percentage and total number of virus-specific CD4 and CD8 T cells in the lungs of rat Ig and MC21 antibody-treated BALB/c mice. (E and F) Mediastinal lymph node cell suspensions were prepared and analyzed at 18 hr p.i. for CFSE-labeled rDC migration. Representative FACS plots (E) and percentages and total numbers (F) are shown. (G and H) Percentage of apoptotic CD4 and CD8 T cells in SARS-CoV-infected lungs at day 5 p.i. Representative histograms (G) and percentage of apoptotic T cells (H) are shown. Data are representative of 2 independent experiments (4–5 mice/group/experiment). Data in (B), (D), (F), and (H) are represented as ±SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. See also Figure S5.

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