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. 2010 Aug;84(15):7613-24.
doi: 10.1128/JVI.00553-10. Epub 2010 May 26.

Lethal dissemination of H5N1 influenza virus is associated with dysregulation of inflammation and lipoxin signaling in a mouse model of infection

Cristian Cilloniz  1 , Mary J Pantin-Jackwood, Chester Ni, Alan G Goodman, Xinxia Peng, Sean C Proll, Victoria S Carter, Elizabeth R Rosenzweig, Kristy J Szretter, Jacqueline M Katz, Marcus J Korth, David E Swayne, Terrence M Tumpey, Michael G Katze
Affiliations

Lethal dissemination of H5N1 influenza virus is associated with dysregulation of inflammation and lipoxin signaling in a mouse model of infection

Cristian Cilloniz et al. J Virol. 2010 Aug.

Abstract

Periodic outbreaks of highly pathogenic avian H5N1 influenza viruses and the current H1N1 pandemic highlight the need for a more detailed understanding of influenza virus pathogenesis. To investigate the host transcriptional response induced by pathogenic influenza viruses, we used a functional-genomics approach to compare gene expression profiles in lungs from 129S6/SvEv mice infected with either the fully reconstructed H1N1 1918 pandemic virus (1918) or the highly pathogenic avian H5N1 virus Vietnam/1203/04 (VN/1203). Although the viruses reached similar titers in the lung and caused lethal infections, the mean time of death was 6 days for VN/1203-infected animals and 9 days for mice infected with the 1918 virus. VN/1203-infected animals also exhibited an earlier and more potent inflammatory response. This response included induction of genes encoding components of the inflammasome. VN/1203 was also able to disseminate to multiple organs, including the brain, which correlated with changes in the expression of genes associated with hematological functions and lipoxin biogenesis and signaling. Both viruses elicited expression of type I interferon (IFN)-regulated genes in wild-type mice and to a lesser extent in mice lacking the type I IFN receptor, suggesting alternative or redundant pathways for IFN signaling. Our findings suggest that VN/1203 is more pathogenic in mice as a consequence of several factors, including the early and sustained induction of the inflammatory response, the additive or synergistic effects of upregulated components of the immune response, and inhibition of lipoxin-mediated anti-inflammatory responses, which correlated with the ability of VN/1203 to disseminate to extrapulmonary organs.

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Figures

FIG. 1.
FIG. 1.
VN/1203 infection is more pathogenic than 1918 infection in a mouse model of infection. (A) Mortality data from a total of 12 mice (6 mice/virus). (B) Viral dissemination data from a total of 12 mice (3 mice/virus/time point). d, day(s). (C) Lung virus titer data from a total of 18 wild-type mice (3 mice/virus/time point) infected with 1918 or VN/1203 virus. The limit of detection in this assay is indicated by black and grey bars at 1.2 × 101 EID50/ml.
FIG. 2.
FIG. 2.
Global transcriptional responses to highly pathogenic 1918 and VN/1203 viruses. Shown is microarray analysis of whole lung tissue from 1918 and VN/1203 virus-infected wild-type mice at 1, 3, and 4 days p.i. The heat map illustrates the global transcriptional profile of 3,470 genes activated within cutoff values of ≥2-fold change and P ≤ 0.01. The genes shown in yellow were upregulated and those in blue were downregulated in infected relative to mock-infected animals. The box outlined in red indicates the attenuated transcriptional response in 1918 virus-infected animals.
FIG. 3.
FIG. 3.
VN/1203 virus differentially regulates the expression of inflammatory response genes. (A) Heat map illustrating ANOVA results for 142 inflammatory genes differentially transcribed within cutoff values of ≥2-fold change and ANOVA P ≤ 0.01 in wild-type mouse lungs infected with 1918 or VN/1203 virus. (B) Biological-network analysis of the top functional category inflammatory response (P = 4.22E−16) determined by Ingenuity Pathway Analysis. This analysis highlights a subset of genes that were upregulated during VN/1203 infection but not during 1918 infection (blue shading). Another subset of genes was anti-coregulated, that is, upregulated during VN/1203 infection but downregulated during 1918 infection (orange shading). A third subset of genes was upregulated by both viruses (green shading). The boxed genes highlight the differential regulation of the inflammasome components CASP1, IL-1β, and NLRP3.
FIG. 4.
FIG. 4.
The VN/1203 and 1918 viruses differentially regulate key cellular signaling pathways. Shown is ANOVA of microarray data from lung samples using cutoff values of ≥2-fold change and ANOVA P ≤ 0.01 comparing the infections of wild-type animals with 1918 and VN/1203 viruses. The results were uploaded into IPA for functional analysis. Selected canonical pathways were analyzed, and the results are presented as heat maps illustrating the expression of genes associated with viral sensing, neutrophil activation, NF-κB signaling, and chemokine signaling to 1918 and VN/1203 viruses. Yellow indicates upregulated genes; blue indicates downregulated genes.
FIG. 5.
FIG. 5.
Association of hematological function and lipoxin signaling with VN/1203 virus dissemination. (A) Differential regulation of hematological system development- and function-related genes. (B) Differential regulation of lipoxin signaling. Shown is ANOVA of lung gene expression data using cutoff values of ≥2-fold change and ANOVA P ≤ 0.01 comparing the infection of wild-type animals with VN/1203 or the 1918 virus. The results were uploaded into IPA for functional analysis. Selected biological functions were analyzed, and the results are presented as heat maps. Yellow indicates upregulated genes; blue indicates downregulated genes. WT, wild type.
FIG. 6.
FIG. 6.
Expression of interferon-regulated genes in response to interferon treatment or VN/1203 or 1918 infection. (A) Expression of type I interferon response genes in wild-type or IFNR1−/− mice infected with 1918 and VN/1203 viruses. Expression was determined by ANOVA of microarray data using cutoff values of ≥2-fold change and ANOVA P ≤ 0.01, respectively, from mock-infected wild-type mouse lung samples treated with interferon or untreated. This analysis generated a subset of 854 genes (right) that were used to cluster the mouse data. KO, knockout. (B) Expression of type I interferon response genes in MEFs infected with 1918 and VN/1203 viruses. Expression was determined by ANOVA analysis using cutoff values of ≥2-fold change and ANOVA P ≤ 0.01, respectively.
FIG. 7.
FIG. 7.
Histopathology analysis of 1918 and VN/1203 infections in IFNR1−/− mice. (A) Normal spleen of an IFNR1−/− mouse infected by 1918 virus at day 1 p.i. (B) Normal liver of an IFNR1−/− mouse infected by 1918 virus at day 4 p.i. (C) Apoptotic spleen of an IFNR1−/− mouse infected by VN/1203 virus at day 1 p.i. (D) Lymphocyte depletion in the periarteriolar lymphatic sheath with heterophilic splenitis of an IFNR1−/− mouse infected by VN/1203 virus at day 1 p.i. (E) Common viral staining in bronchus of an IFNR1−/− mouse infected by 1918 virus at day 3 p.i. (F) Intense antigen staining in the bronchus of an IFNR1−/− mouse infected by VN/1203 virus at day 3 p.i.
FIG. 8.
FIG. 8.
Model illustrating the consolidation of data obtained from macaques and mice infected with highly pathogenic 1918 and VN/1203 viruses. Provided is an illustration depicting our current understanding of influenza virus infections in macaques and mice. The network diagrams for macaques (8) and mice (Fig. 3B) emphasize the differential regulation of the inflammatory response and activation of inflammasome components that was found during 1918 and VN/1203 virus infections, respectively.
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