Early upregulation of acute respiratory distress syndrome-associated cytokines promotes lethal disease in an aged-mouse model of severe acute respiratory syndrome coronavirus infection

Abstract

Several respiratory viruses, including influenza virus and severe acute respiratory syndrome coronavirus (SARS-CoV), produce more severe disease in the elderly, yet the molecular mechanisms governing age-related susceptibility remain poorly studied. Advanced age was significantly associated with increased SARS-related deaths, primarily due to the onset of early- and late-stage acute respiratory distress syndrome (ARDS) and pulmonary fibrosis. Infection of aged, but not young, mice with recombinant viruses bearing spike glycoproteins derived from early human or palm civet isolates resulted in death accompanied by pathological changes associated with ARDS. In aged mice, a greater number of differentially expressed genes were observed than in young mice, whose responses were significantly delayed. Differences between lethal and nonlethal virus phenotypes in aged mice could be attributed to differences in host response kinetics rather than virus kinetics. SARS-CoV infection induced a range of interferon, cytokine, and pulmonary wound-healing genes, as well as several genes associated with the onset of ARDS. Mice that died also showed unique transcriptional profiles of immune response, apoptosis, cell cycle control, and stress. Cytokines associated with ARDS were significantly upregulated in animals experiencing lung pathology and lethal disease, while the same animals experienced downregulation of the ACE2 receptor. These data suggest that the magnitude and kinetics of a disproportionately strong host innate immune response contributed to severe respiratory stress and lethality. Although the molecular mechanisms governing ARDS pathophysiology remain unknown in aged animals, these studies reveal a strategy for dissecting the genetic pathways by which SARS-CoV infection induces changes in the host response, leading to death.

Figures

FIG. 1.

Differences in clinical outcomes did not correlate with differences in viral replication efficiencies. Weight loss (A and B) and lung titer (C and D) results are shown for 8-week-old (young) (A and C) and 12-month-old (aged) (B and D) female BALB/c mice infected with icUrbani, icGZ02, and icHC/SZ/61/03. (A and B) Body weights of infected mice were measured on a daily basis (n = 5 per group). Weight changes are expressed as the mean percent changes for infected animals relative to the initial weights at day zero. Lung tissues were harvested from infected mice at 3, 12, 24, 48, and 72 h p.i. and assayed for infectious virus as described in Materials and Methods. Tissue samples from five mice were analyzed at each time point. The error bars represent standard deviations.

FIG. 2.

Tropism of lethal SARS-CoV is primarily for bronchial and bronchiolar cells at early stages of infection, while that of nonlethal SARS-CoV is primarily for alveolar epithelial cells. With SARS-CoV icUrbani (top row), alveolar epithelial cells of aged mice showed SARS-CoV N immunohistochemical staining, with greater numbers of alveolar epithelial cells being stained (arrows with closed arrowheads) as the infection progressed. With icGZ02 and icHC/SZ/61/03 (middle and bottom rows), SARS-CoV N staining was observed in many bronchial and bronchiolar cells (arrows with open arrowheads) and few alveolar epithelial cells at 24 h p.i. in aged mice. At 48 and 72 h p.i., the numbers of bronchial and bronchiolar cells positive for N decreased while the number of alveolar epithelial cells positive for N increased. In addition, positive staining was also observed in intraluminal debris (asterisks). Immunoperoxidase with hematoxylin counterstain was used. Magnification, ×20.

FIG. 3.

Expression of ACE2 is downregulated in lethal infection of aged mice. Quantitative RT-PCR using TaqMan chemistry was performed on individual lung samples from aged (A) and young (B) animals at 12, 24, 48, and 72 h p.i. The changes over aged and young mock controls, respectively, are plotted. The error bars represent standard deviations. *, P < 0.01 (analysis of variance; Bonferroni posttest compared to icUrbani).

FIG. 4.

Global gene expression profiles show a prominent kinetic difference in gene expression, with aged mice initiating an earlier gene transcription response to viral infection than young mice. (A) Global gene expression data are the results of a comparison of gene expression in the lungs of experimental mice versus gene expression in the lungs of mock-infected mice, and genes were included if they met the criteria of an absolute change of ≥2-fold (P ≤ 0.01) in at least two experiments. Each bar represents one microarray experiment (n = 4). Y, young; A, aged; U, icUrbani; G, icGZ02; H, icHC/SZ/61/03 (12, 24, and 72 h p.i.). (B) Genes were included in the heat map if they showed an absolute change of ≥2-fold (P ≤ 0.01) in at least one experiment. These data were plotted as a heat map in which each matrix entry represents a gene expression value. Red corresponds to gene expression higher than that of the reference; green corresponds to lower gene expression. Data are presented for young and aged mice infected with either icUrbani (U), icGZ02 (G), or icHC/SZ/61/03 (H) at the indicated time points.

FIG. 5.

Viruses show more similar profiles of differentially expressed genes as time progresses. The Venn diagram shows the overlap of genes with ≥2-fold change (P ≤ 0.01) between icUrbani, icGZ02, and icHC/SZ/61/03 in young and aged mice over time. Data are presented for young (Y) and aged (A) mice infected with either icUrbani (U), icGZ02 (GZ), or icHC/SZ/61/03 (HC) at the indicated time points p.i. The numbers in the centers were shared by all three viruses.

FIG. 6.

Functional enrichment of disease-related pathways reveals the importance of kinetics in the host response. (A) GSEA (described in Materials and Methods) was performed on the change data for all experiments using GO classes. GO categories 6954 (inflammatory response), 6915 (apoptosis), and 2449 (lymphocyte-mediated immunity) were chosen for further investigation based on their high P values. The P values for these categories were plotted as a function of time p.i. for infections of young (Y) and aged (A) animals infected with icUrbani (U), icGZ02 (G), or icHC/SZ/61/03 (H). (B) Expression data are shown for young and aged mice infected with icUrbani (U), icGZ02 (G), or icHC/SZ/61/03 (H) for 24 and 72 h p.i. Genes were selected by GSEA within the GO categories inflammatory response (pink bar), apoptosis (blue bar), and lymphocyte-mediated immunity (green bar), with the additional condition that at least one experiment met the cutoff change of >1.5-fold (P < 0.01). The experiments are clustered using the unweighted-pair group method using average linkages, with similarity determined by Euclidean distance and ordering by average value. The column dendrogram, showing the relative similarity of the experiments, is at the top. The data for aged icUrbani-infected mice at 24 h p.i. clusters more distantly with the data for young mice at 24 h p.i. (bold gray line) than the data for aged mice at 24 h p.i. Bold black line, icGZ02 and icHC/SZ/61/03 data.

FIG. 7.

Confirmation of select gene expression results using RT-PCR. Quantitative RT-PCR for Cxcl10 (A), Ccl2 (B), IFN-β (C), IL-1β (D), IL-6 (E), and TNF (F) was performed on all separate lung samples from aged animals at 12, 24, 48, and 72 h p.i., with the exception of that for Cxcl10, which was not performed at 48 h. The data are shown as changes over mock-infected controls. The error bars represent standard deviations.

FIG. 8.

Canonical pathway regulation correlates with pathogenesis and lethality. At 24 h p.i., the top canonical pathways associated with lethal infections of aged mice (the significance criteria were >1.5-fold change with a P value of <0.01) are interferon signaling (P = 2.75 × 10−14 for HC/SZ/61/03 and P = 4.75 × 10−14 for GZ02) and acute-phase response (P = 1.62 × 10−9 for HC/SZ/61/03 and P = 5.2 × 10−9 for GZ02). These pathways are not as significantly upregulated by pathogenic but nonlethal infection (aged animals infected with Urbani, interferon signaling, P = 5.26 × 10−5, and acute-phase response, P = 1.88 × 10−2). Expression data for genes in these pathways are shown as a heat map for young and aged mice infected with icUrbani (U), icGZ02 (G), or icHC/SZ/61/03 (H) for 24 and 72 h p.i. The “NM” numbers on the right are GenBank accession numbers.

FIG. 9.

A network of genes suggests lethality in aged mice. The network was created by importing genes meeting the selection criteria (≥1.5-fold change; P ≤ 0.01 at 24 h p.i. in either the icHC/SZ/61/03-infected aged-mouse or icGZ02-infected aged-mouse experiments, but not the icUrbani-infected aged-mouse experiment) in ingenuity pathways analysis (IPA). The top two networks built by IPA were chosen and merged, keeping only “direct” IPA-curated interactions. Genes were separated into functional classes based on ingenuity functional annotations. The circles represent individual genes or protein complexes in the network and are colored according to the expression pattern observed in aged animals infected with icGZ02 spike variant virus at 24 h p.i. (A) or icHC/SZ/61/03 spike variant virus at 24 h p.i (B). The arrows represent direct biological interactions. The circles colored in shades of red represent genes upregulated in lethal infection versus mock infection. Pink indicates 1.5- to 2.0-fold change, red indicates 2.0- to 5.0-fold change, and dark red represents a >5-fold change. Likewise, light-green circles represent downregulation and indicate a 1.5- to 2.0-fold change. Gene names colored blue are differentially regulated by icHC/SZ/61/03 infection, but not icGZ02 infection, and are therefore associated with increased lethality.