Transcriptional analysis of immune-related gene expression in p53-deficient mice with increased susceptibility to influenza A virus infection

Abstract

Background: p53 is a tumor suppressor that contributes to the host immune response against viral infections in addition to its well-established protective role against cancer development. In response to influenza A virus (IAV) infection, p53 is activated and plays an essential role in inhibiting IAV replication. As a transcription factor, p53 regulates the expression of a range of downstream responsive genes either directly or indirectly in response to viral infection. We compared the expression profiles of immune-related genes between IAV-infected wild-type p53 (p53WT) and p53-deficient (p53KO) mice to gain an insight into the basis of p53-mediated antiviral response.

Methods: p53KO and p53WT mice were infected with influenza A/Puerto Rico/8/1934 (PR8) strain. Clinical symptoms and body weight changes were monitored daily. Lung specimens of IAV-infected mice were collected for analysis of virus titers and gene expression profiles. The difference in immune-related gene expression levels between IAV-infected p53KO and p53WT mice was comparatively determined using microarray analysis and confirmed by quantitative real-time reverse transcription polymerase chain reaction.

Results: p53KO mice showed an increased susceptibility to IAV infection compared to p53WT mice. Microarray analysis of gene expression profiles in the lungs of IAV-infected mice indicated that the increased susceptibility was associated with significantly changed expression levels in a range of immune-related genes in IAV-infected p53KO mice. A significantly attenuated expression of Ifng (encoding interferon (IFN)-gamma), Irf7 (encoding IFN regulator factor 7), and antiviral genes, such as Mx2 and Eif2ak2 (encoding PKR), were observed in IAV-infected p53KO mice, suggesting an impaired IFN-mediated immune response against IAV infection in the absence of p53. In addition, dysregulated expression levels of proinflammatory cytokines and chemokines, such as Ccl2 (encoding MCP-1), Cxcl9, Cxcl10 (encoding IP-10), and Tnf, were detected in IAV-infected p53KO mice during early IAV infection, reflecting an aberrant inflammatory response.

Conclusion: Lack of p53 resulted in the impaired expression of genes involved in IFN signaling and the dysregulated expression of cytokine and chemokine genes in IAV-infected mice, suggesting an essential role of p53 in the regulation of antiviral and inflammatory responses during IAV infection.

Fig. 1

Increased susceptibility of p53KO mice to PR8 infection. p53WT and p53KO mice (n = 10 per group) were intranasally inoculated with a sublethal dose of PR8 virus. Clinical signs and weight loss were assessed daily for 16 days. Lungs of infected mice were collected 3 and 6 dpi for analysis of viral loads. a Weight loss analysis in PR8-infected mice. Results are percentages of mean weight loss relative to initial weight. b The survival rates of PR8-infected mice. c Viral loads were determined by serial titration of lung homogenates in 10-day-old embryonated SPF chicken eggs. The EID₅₀ was calculated. d The expression of viral hemagglutinin (HA) in the lungs of infected mice was determined by qRT-PCR. Values are means ± SE of at least 4 mice. *, p < 0.05 as assessed by the Student’s t-test; dpi, days post-infection

Fig. 2

Detection of gene expression in PR8-infected mice by qRT-PCR. Lung samples were collected from PR8-infected mice 3 and 6 dpi and subjected to qRT-PCR for expression analysis of the indicated genes. FC, fold change. Results are means ± SE from 3 mice. *, p < 0.05 between PR8-infected p53WT and p53KO mice