Mechanisms of severe acute respiratory syndrome coronavirus-induced acute lung injury

Abstract

Systems biology offers considerable promise in uncovering novel pathways by which viruses and other microbial pathogens interact with host signaling and expression networks to mediate disease severity. In this study, we have developed an unbiased modeling approach to identify new pathways and network connections mediating acute lung injury, using severe acute respiratory syndrome coronavirus (SARS-CoV) as a model pathogen. We utilized a time course of matched virologic, pathological, and transcriptomic data within a novel methodological framework that can detect pathway enrichment among key highly connected network genes. This unbiased approach produced a high-priority list of 4 genes in one pathway out of over 3,500 genes that were differentially expressed following SARS-CoV infection. With these data, we predicted that the urokinase and other wound repair pathways would regulate lethal versus sublethal disease following SARS-CoV infection in mice. We validated the importance of the urokinase pathway for SARS-CoV disease severity using genetically defined knockout mice, proteomic correlates of pathway activation, and pathological disease severity. The results of these studies demonstrate that a fine balance exists between host coagulation and fibrinolysin pathways regulating pathological disease outcomes, including diffuse alveolar damage and acute lung injury, following infection with highly pathogenic respiratory viruses, such as SARS-CoV.

Importance: Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002 and 2003, and infected patients developed an atypical pneumonia, acute lung injury (ALI), and acute respiratory distress syndrome (ARDS) leading to pulmonary fibrosis and death. We identified sets of differentially expressed genes that contribute to ALI and ARDS using lethal and sublethal SARS-CoV infection models. Mathematical prioritization of our gene sets identified the urokinase and extracellular matrix remodeling pathways as the most enriched pathways. By infecting Serpine1-knockout mice, we showed that the urokinase pathway had a significant effect on both lung pathology and overall SARS-CoV pathogenesis. These results demonstrate the effective use of unbiased modeling techniques for identification of high-priority host targets that regulate disease outcomes. Similar transcriptional signatures were noted in 1918 and 2009 H1N1 influenza virus-infected mice, suggesting a common, potentially treatable mechanism in development of virus-induced ALI.

FIG 1

SARS MA15 dose response. (A) Weight loss is shown as percent starting weight over the course of a 7-day infection in 20-week-old B6 mice. Mice infected with 10² to 10⁴ PFU of SARS-CoV MA15 had low levels of transient weight loss, while mice infected with 10⁵ PFU showed increasing weight loss over time. (B) Virus titer in the lung was quantitated by plaque assay. The mean value of all samples with detectable virus in each group is shown (three mice at 10² PFU and two each at 10³, 10⁴, and 10⁵ PFU had detectable virus by plaque assay at day 7; BLD, below the limit of detection of 100 PFU per lung).

FIG 2

Dose-response differential gene expression. (A) Differential expression (DE) of transcripts for each dose is shown at each day postinfection. The number of DE transcripts was greatest for sublethal (10⁴-PFU) and lethal (10⁵-PFU) infections at day 2, with 2,091 and 2,251, respectively. In total across all 4 days, there were 3,138 unique DE transcripts for the 10⁴-PFU infections and 3,683 for the 10⁵-PFU infections. (B) The heat map shows the number of overlapping transcripts for each time point in both sublethal- and lethal-dose MA15 infections. Coloring represents the odds ratio or the effect size of each overlap, and gray numbers within the cells are the numbers of common differentially expressed (DE) transcripts. Analogous to differences in phenotype between infection doses, the overlap is strongest at day 2 and weakest at day 7 postinfection.

FIG 3

Eigengene analysis. The consensus network is represented as a dendrogram (A), and modules are shown as colors below. The blue module (circled in red) displayed distinct behavior for each dose (10⁴ and 10⁵ PFU), indicating potential mediators of MA15 infection pathogenesis (B). The arrow in panel A indicates the approximate location of Serpine1 and PLAT within the blue module.

FIG 4

(A) Identification of urokinase and tissue remodeling pathway members. (A) Peptide levels from total lung homogenates were analyzed to determine expression of select ECM and urokinase pathway proteins. Mock-infection values are shown by dashed lines, sublethal infection values are shown by gray lines, and lethal infection values are shown by black lines. Significance values: *, P < 0.05; **, P < 0.01; ***, P < 0.001; #, lethal dose significant at P < 0.05. VWF, von Willebrand factor. (B) Lung sections from 7-dpi lethally or sublethally infected mice or mock-infected mice were stained for the presence of fibrin using MSB (Martius scarlet blue). Yellow staining indicates red blood cells, blue staining indicates connective tissue, and red staining indicates fibrin. Arrows point to positive fibrin staining.

FIG 5

Serpine1^−/− mice are susceptible to SARS-CoV infection. (A) Serpine1^−/− mice lost more weight than did B6 control mice when infected with 10⁴ PFU of SARS-CoV MA15 (P value of <0.05 for Serpine1 versus B6 at days 5, 6, and 7 postinfection). (B) Serpine1^−/− mice succumbed to infection more rapidly than did B6 controls when infected with 10⁵ PFU of MA15 (** = P value of <0.01). (C) Lung mean virus load was quantitated by plaque assay. There was no statistical difference in viral titers at 4 dpi; at 7 dpi, most mice had lung titers below the limit of detection (BLD, <100 PFU; two Serpine1^−/− mice and one B6 control mouse with detectable virus). Independent replicate experiments confirmed significant differences in weight loss but no difference in lung titer between Serpine1^−/− and B6 controls at both 4 and 7 dpi (data not shown). (D) Representative histology images from Serpine1^−/− or B6 mouse lungs at 7 days postinfection show that infected knockout mice had extensive hemorrhage after infection with MA15. Exudates are indicated by open arrows with dashed lines; hemorrhage is shown by filled arrows with solid lines. (E) Log₂ fold change ratio of ARDS-related gene expression from the lungs of SARS-CoV-infected Serpine1^−/− and B6 mice at 4 and 7 dpi (log₂ fold change = mean log₂ FC [WT] − mean log₂ FC [knockout]). Green indicates that expression in Serpine1-knockout mice is lower than that in B6 mice, and red indicates that expression in Serpine1-knockout mice is higher than that in B6 mice.

FIG 6

Urokinase pathway model. (A) Representation of the unperturbed urokinase pathway signaling pathway. (B) Without the presence of Serpine1, an inhibitor of both PLAU/urokinase and PLAT/tPA, there is increased cleavage of plasminogen into the active plasmin and thus increased breakdown of fibrin clots and hemorrhage compared to an unperturbed system. Red T shapes indicate inhibition, and blue arrows indicate activation.