Mx1+/+ mice partially control 1918 virus infection.
Previous studies by our group and others (6
) have shown that knock-in mice carrying a fully functional Mx1 gene are resistant to lethal influenza virus infection; however, those studies did not investigate the host response elicited under such experimental conditions. In the present study, our main goal was to understand the contribution of the elicited host response to survival. To achieve this goal, we performed global gene expression analyses on lung tissue from mice infected with the fully reconstructed 1918 virus.
Our initial experiments showed that Mx1+/+
mice were able to reduce the growth of the 1918 virus by 1 log unit at 12 and 24 h postinfection (p.i.) with a reduction of 2 log units by 72 h p.i. compared to the virus growth observed in Mx1−
mice (A, left). Administration of interferon to the animals prior to infection resulted in an even greater decrease of 1918 virus growth in Mx1+/+
mice, with 2.5-, 3.5-, and 5-log-unit reductions at 12, 24, and 72 h p.i., respectively. However, there was no significant change in virus growth in BALB/c (Mx1−
) mice (A, right); these observations were in agreement with previous studies (33
). Although there was a large amount of virus detected in the lungs of infected animals, especially in BALB/c mice, we did not detect viral dissemination to the brain or spleen (data not shown). However, morbidity data showed dramatic differences between the two mouse strains. While BALB/c mice were highly susceptible to 1918 virus infection, as shown by the rapid and steady decrease in body weight, interferon-treated Mx1+/+
mice were insensitive to 1918 infection (as measured by weight loss) throughout the experiment. In the absence of interferon treatment, an initial slight decline followed by a recovery in body weight in Mx1+/+
mice may be observed as a characteristic of interferon treatment (B).
Fig 1 Mx1 competent mice control lethal influenza virus infection. (A) Lung virus titer data from a total of 36 mice (3 animals per time point/treatment/mouse strain). The limit of detection of the assay was 100.95 PFU/ml. (B) Morbidity data from a total of (more ...) 1918 virus infection generates less tissue damage in Mx1+/+ mice.
In the absence of interferon treatment, infection with the 1918 virus resulted in lung and airway lesions typical of influenza A virus infection in both Mx1+/+ and BALB/c mice. The lesions varied depending on the individual animals and the area of the lung examined, but in general, the lesions in BALB/c mice were more severe than the lesions observed in the Mx1+/+ mice (). At 24 h p.i., BALB/c mice exhibited moderate to severe necrotizing bronchiolitis and bronchitis. The bronchiolar walls showed vacuolation and loss of epithelium and were thickened by the presence of edema fluid, fibrin, neutrophils, eosinophils, and mononuclear cells. There was perivascular and peribronchiolar infiltration of macrophages, lymphocytes, and plasma cells. At this time point, Mx1+/+ mice exhibited only mild diffuse histiocytic alveolitis with congestion and edema, whereas BALB/c mice had moderate interstitial pneumonia. At 72 h p.i., loss of the bronchial and bronchiolar epithelium was evident, accompanied by severe perivascular and peribronchiolar inflammation, mostly composed of macrophages, but also lymphocytes, plasma cells, and neutrophils. Mild to severe diffuse interstitial pneumonia was present, with thickening of the alveolar walls; presence of inflammatory cells; congestion; and areas of alveolar necrosis, atelectasia, hemorrhage, or consolidation.
Fig 2 Histopathological changes in tissues from 1918 virus-infected mice. Shown are photomicrographs of lung tissue sections stained with hematoxylin and eosin. (A) Severe necrotizing bronchiolitis and associated peribronchiolar lymphocytic inflammation and (more ...)
The character of the lesions varied in both the virus-infected Mx1+/+ mice and BALB/c mice treated with interferon compared to the mice not treated with interferon. At 24 h p.i., lesions to the bronchiolar and bronchial epithelium were moderate in all mice with, in some cases, loss of the respiratory epithelium, but with minimal or no peribronchial or peribronchiolar inflammation. Alveolar lesions were moderate to severe at this time point, with diffuse alveolar necrosis, hemorrhage, and edema. At 72 h p.i., Mx1+/+ mice had mild to moderate diffuse interstitial pneumonia with alveolar necrosis, and the inflammatory cells were mostly macrophages. In contrast, the BALB/c mice exhibited more severe pathology, with mild to severe necrotizing bronchiolitis and moderate to severe alveolitis with alveolar necrosis, congestion, and edema.
Mx1+/+ mice exhibit increased survival of 1918 virus infection.
Our results indicated that when infected with the 1918 virus, Mx1+/+ mice had lower viral titers and less severe lung lesions than BALB/c mice. In addition, whereas the 1918 virus was uniformly lethal in BALB/c mice, 50% of the Mx1+/+ mice survived infection (A). In order to assess the effects of both genetic background (MX1+/+ and BALB/c) and time points (12, 24, and 72 h) on gene expression, two-way ANOVA was performed using genetic backgrounds and time points as two independent variables. We identified 547 genes that were differentially expressed (B). A large proportion of these genes were downregulated in the BALB/c animals, whereas the same genes were strongly upregulated in the Mx1+/+ mice. The top 10 biological categories in which these genes are involved are shown in C.
We used IPA to generate a network of genes functionally associated with survival. As annotated in IPA, the resulting network included genes associated with apoptosis, cell migration, connective tissue disorders, and the production of reactive oxygen species (). All of these genes, with the exception of spp1 and Ppp2r5a, were upregulated in the Mx1+/+ mice but were downregulated in BALB/c mice. These results suggest that increased survival of Mx1+/+ mice is associated with the upregulation of these pathways.
Fig 4 Molecular signature associated with partial protection of Mx1+/+ mice. The biological network shows the direct functional relationship of genes associated with partial protection determined by IPA identified from the 547 differentially regulated genes. (more ...) Interferon-dependent host response associated with complete protection.
Previous studies by our group and others (6
) showed that pretreatment of Mx1+/+
mice with interferon results in increased resistance to influenza virus infection. In the present study, we observed similar results, with all Mx1+/+
mice pretreated with interferon surviving a challenge with a high dose of 1918 virus (A). In this context, we wanted to gain insights into the specific host response required for complete protection. Using a two-way ANOVA approach, we identified 2,071 differentially expressed genes between the Mx1+/+
and BALB/c interferon-treated mice (B). There was a clear anticorrelation in gene expression between the two mouse strains; however, the Mx1+/+
mice showed a strong upregulation of these genes by 12 h p.i. that continued, although to a lesser extent, at later time points. Functional analysis of these genes using IPA showed that the majority of the genes were associated with host physiological functions, such as cell-to-cell signaling and interaction or cellular movement (C). Network analysis of these genes allowed us to identify a set of physically interacting genes that might be responsible for survival (). The most striking feature of this molecular signature was that the Mx1+/+
mice were able to downregulate the expression of inflammatory cytokine and chemokine genes, as well as acute-phase genes. These results suggest that complete survival of mice is linked to the downregulation of key modulator molecules. We also note that these changes in gene expression were not due to differences in the response to interferon, since in the absence of infection, the gene expression signatures of interferon-treated Mx1+/+
and BALB/c mice were similar (see Table S1 in the supplemental material for a complete list of differentially expressed genes).
Fig 6 Molecular signature associated with complete protection of Mx1+/+ mice. The biological network shows the direct functional relationship of genes associated with complete protection determined by IPA identified from the 2,071 differentially regulated genes. (more ...) Specific contribution of interferon to survival.
Because treatment of Mx1+/+ mice with interferon prior to 1918 virus infection increased survival from 50% to 100% (A), we next investigated the contribution of interferon to differences in the host transcriptional response to infection. Using two-way ANOVA, we identified 234 differentially expressed genes between these two experimental conditions (B), and the top 10 functional categories assigned to these genes are indicated in C.
Network analysis identified functionally related genes associated with survival. All of the genes depicted in this specific network were related to cell cycle regulation, including cyclins (CCNA1, CCNA2, CCNB1, and CCNB2), cell cycle DNA damage check point regulation (BRCA1, CDK1, CKS2, TOP2A), and cell division cycle-associated genes (CDCA5, CDCA8) (). A large proportion of these genes were upregulated in the interferon-treated animals but were downregulated in the absence of interferon, suggesting that tight control of this specific set of genes involved in cell cycle regulation is important to elicit a response capable of providing complete protection. Although during our previous studies we found that the expression of some cell cycle-related genes was differentially regulated upon influenza virus infection, the extent and functional network of cell cycle genes associated with IFN pretreatment of Mx1+/+ mice challenged with the 1918 virus is a novel finding.
Fig 8 Molecular signature of the contribution of interferon to complete protection. The biological network shows the direct functional relationship of cell cycle regulation genes associated with the specific contribution of interferon to survival determined (more ...)