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. 2010 Nov;84(21):11297-309.
doi: 10.1128/JVI.01130-10. Epub 2010 Aug 11.

Transcriptomic analysis reveals a mechanism for a prefibrotic phenotype in STAT1 knockout mice during severe acute respiratory syndrome coronavirus infection

Gregory A Zornetzer  1 , Matthew B Frieman, Elizabeth Rosenzweig, Marcus J Korth, Carly Page, Ralph S Baric, Michael G Katze
Affiliations

Transcriptomic analysis reveals a mechanism for a prefibrotic phenotype in STAT1 knockout mice during severe acute respiratory syndrome coronavirus infection

Gregory A Zornetzer et al. J Virol. 2010 Nov.

Abstract

Severe acute respiratory syndrome coronavirus (SARS-CoV) infection can cause the development of severe end-stage lung disease characterized by acute respiratory distress syndrome (ARDS) and pulmonary fibrosis. The mechanisms by which pulmonary lesions and fibrosis are generated during SARS-CoV infection are not known. Using high-throughput mRNA profiling, we examined the transcriptional response of wild-type (WT), type I interferon receptor knockout (IFNAR1-/-), and STAT1 knockout (STAT1-/-) mice infected with a recombinant mouse-adapted SARS-CoV (rMA15) to better understand the contribution of specific gene expression changes to disease progression. Despite a deletion of the type I interferon receptor, strong expression of interferon-stimulated genes was observed in the lungs of IFNAR1-/- mice, contributing to clearance of the virus. In contrast, STAT1-/- mice exhibited a defect in the expression of interferon-stimulated genes and were unable to clear the infection, resulting in a lethal outcome. STAT1-/- mice exhibited dysregulation of T-cell and macrophage differentiation, leading to a TH2-biased immune response and the development of alternatively activated macrophages that mediate a profibrotic environment within the lung. We propose that a combination of impaired viral clearance and T-cell/macrophage dysregulation causes the formation of prefibrotic lesions in the lungs of rMA15-infected STAT1-/- mice.

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Figures

FIG. 1.
FIG. 1.
Viral replication in the lungs of wild-type and knockout mice. Lung viral titer data from 5 mice infected with rMA15 per strain per time point are shown. The limit of detection for this assay is 5 × 102 PFU.
FIG. 2.
FIG. 2.
Sustained inflammation in the lungs of STAT1−/− mice infected with rMA15. Representative tissue samples from WT, IFNAR−/−, and STAT1−/− mice infected with rMA15 or mock-infected (2, 5, and 9 dpi) were stained with hematoxylin and an eosin counterstain. Images at lower magnification are available in Fig. S1 in the supplemental material. Magnification, ×40.
FIG. 3.
FIG. 3.
Abnormal protein deposition in the lungs of STAT1−/− mice infected with rMA15. Representative tissue samples from STAT1−/− mice infected with rMA15 or subjected to mock infection (9 dpi) were stained with Masson's Trichrome. The presence of blue-purple staining indicates protein deposition in the alveolar spaces. Images at lower magnification are available in Fig. S2 in the supplemental material.
FIG. 4.
FIG. 4.
Kinetics of the transcriptional response to infection correlate with pathological findings. Expression profiles of 261 immune-related genes (top) and 204 cell cycle-related genes (bottom) selected by ANOVA are shown. Each column represents averages of data from the results of 3 replicate animal experiments performed using WT, IFNAR1−/−, or STAT1−/− mice at 2, 5, or 9 dpi compared to averages of data from experiments performed using mock-infected mice of the same strain. Genes shown in red were upregulated, genes shown in green were downregulated, and genes shown in black indicate no change in expression relative to mock-infected mice. Genes in the top panel were identified as part of the IPA “immunological disease” functional category in at least one ANOVA comparison between mock-infected animals and animals sacrificed at 2 dpi. A yellow box highlights 81 genes similarly upregulated by the 3 strains at 2 dpi. The identities of these genes are listed in Table S1 in the supplemental material. Genes in the lower panel were identified as part of the IPA “cell stage” functional category in at least one ANOVA comparison between mice sacrificed at 2 and at 5 dpi. A cyan box highlights 80 genes commonly upregulated by all strains of mice by 5 dpi. The identities of these genes are listed in Table S2 in the supplemental material.
FIG. 5.
FIG. 5.
Deviant interferon response in STAT1−/− mice. Expression profiles are shown for interferon-stimulated genes (identified by intranasal administration of 10,000 units of interferon alpha for 24 h) (see details in Materials and Methods and reference 14) that were significantly different between STAT1−/− mice and WT or IFNAR1−/− mice as judged by ANOVA. Each column represents gene expression data from an average of 3 animal replicate experiments compared to mock-infected animal results. Genes shown in red were upregulated, genes shown in green were downregulated, and genes shown in black indicate no change in expression relative to mock-infected animal results. Genes are marked according to whether greater differential regulation was observed in STAT1−/− mice (blue and cyan) or in WT and IFNAR1−/− mice (orange and yellow). Genes marked with a cyan bar were functionally annotated as involved with “cell movement of leukocytes” by IPA (14 genes; P = 8.61 × 10−11). Genes marked with a yellow bar were functionally annotated as “antiviral response” genes by IPA (18 genes; P = 8.58 × 10−12). Genes in these functionally annotated categories are listed by Entrez Gene name. Genes comprising this heat map are listed in Table S3 in the supplemental material.
FIG. 6.
FIG. 6.
Increased fibrotic gene expression in STAT1−/− mice. Expression profiles are shown for 48 genes associated with the IPA “hepatic fibrosis” canonical pathway. The heat map is colored according to the ratio of gene expression at 5 dpi to that seen with mock-infected mice. Red indicates upregulation, green indicates downregulation, and black indicates no change in expression upon infection.
FIG. 7.
FIG. 7.
Increased TH2- and M2-associated gene expression in STAT1−/− mice. (A and B) Expression profiles are shown for genes associated with TH1 and TH2 responses (A) and for M1 classical macrophage activation and M2 alternative macrophage activation (45) (B). Individual genes are identified by the mouse Entrez Gene name. TH2 responses and M2 responses were upregulated only in STAT1−/− mice and are highlighted by yellow and cyan boxes, respectively.
FIG. 8.
FIG. 8.
Detection of AAM markers in lung tissue of infected STAT1−/− mice by the use of immunohistochemistry. Lung sections were stained for the AAM markers arginase I, Ym1, and Mac3 with a hematoxylin counterstain. At 9 dpi, prominent staining is observed in STAT1−/− mice infected with rMA15 compared with uninfected animals and WT animals infected with rMA15. Magnification, ×40.
FIG. 9.
FIG. 9.
Enrichment of alternatively activated macrophages in STAT1−/− mice infected with SARS-CoV. Ten-week-old strain 129 WT and STAT1−/− mice were infected with MA15, and their induction of AAMs was analyzed at 8 days postinfection. A total of 50,000 lung cells were sorted for the total macrophage population according to the presence of CD11b and CD14 double positives. Of these cells, AAMs were identified by the presence of Mac3 in the cells by intracellular staining. (A) The numbers of CD11b+, CD14+, and MAC3+ cells are graphed. (B) The numbers of AAMs are graphed as percentages of the total number of macrophages (CD11b+/CD14+ cells).
FIG. 10.
FIG. 10.
Dysregulation of T-helper and macrophage differentiation in STAT1−/− mice. A functional network that depicts the effects of T-helper differentiation on macrophage phenotype and disease resolution is shown. The regulation of genes associated with TH1, TH2, M1, and M2 is represented with red arrows to indicate upregulation or with a gray bar to indicate no significant change from mock-infected animal results. A heat map is shown for select macrophage-associated genes that contribute to fibrosis and eosinophilia. Genes are colored according to differential regulation at 9 dpi.
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