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Transcriptional analysis of immune-related gene expression in p53-deficient mice with increased susceptibility to influenza A virus infection
- Wenjun Yan†1,
- Jianchao Wei†1,
- Xufang Deng1,
- Zixue Shi1,
- Zixiang Zhu2,
- Donghua Shao1,
- Beibei Li1,
- Shaohui Wang1,
- Guangzhi Tong1 and
- Zhiyong Ma1Email author
© Yan et al. 2015
Received: 17 February 2015
Accepted: 6 August 2015
Published: 18 August 2015
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.
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.
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.
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.
Influenza A virus (IAV) is a member of the Orthomyxoviridae family of RNA viruses and a primary cause of respiratory tract infections that result in approximately 500,000 deaths per year worldwide . IAV evokes the host immune response to inhibit viral replication and clear viral infections. Meanwhile, an aberrant host immune response during IAV infection has been hypothesized to be the main cause of IAV-related pneumonia. The host immune response to IAV infection has been extensively studied for more than 70 years; however, many uncertainties still exist . For example, host gene involvement in both the host immune response and IAV pathogenesis remain unclear [3, 4].
The p53 protein is a major tumor suppressor that plays important roles in regulating various cellular activities, including cell cycle arrest, DNA repair, senescence, and apoptosis . The p53 protein primarily functions as a transcription factor that positively and negatively regulates the expression of a large and disparate group of responsive genes . In addition to its well-established role in protecting against cancer development, p53 has been recently shown to contribute to the host immune response against viral infections due to vesicular stomatitis virus, Newcastle disease virus, and hepatitis C virus [7–9]. The expression of p53 can be induced at the transcriptional level by type I interferon (IFN). The IFN-stimulated response elements have been identified in p53 gene . On the other hand, p53 upregulates the expression of several IFN-inducible proteins, including IFN regulatory factor (IRF) 9, IRF5, IFN-stimulated gene 15, and toll-like receptor 3, suggesting a crosstalk between the p53 and IFN pathways .
In response to IAV infection, p53 is significantly upregulated and activated in cultured cells [11–13] as well as in the lungs of IAV-infected mice . Previous studies indicated that p53 activation plays an essential role in inhibiting IAV replication and regulating apoptosis of IAV-infected cells [11, 15]. In this study, we observed increased mortality, severe weight loss, and increased viral loads in the lungs of p53-deficient mice after IAV infection, indicating an increased susceptibility to IAV. It is known that p53, as a transcription factor, upregulates or downregulates a series of immune-related genes either directly or indirectly in response to viral infection [10, 16]. To gain an insight into the basis of different susceptibilities to IAV infection, we compared the expression profiles of immune-related genes in the lung tissues of IAV-infected p53WT and p53 knockout (p53KO) mice and found that a range of immune-related genes involved in the regulation of host immune and inflammatory responses showed significantly altered expression levels in the absence of p53.
Virus and mice infection
Influenza A/Puerto Rico/8/1934 (PR8) (H1N1 subtype) virus was propagated in the allantoic cavities of 9-day-old embryonated specific-pathogen-free (SPF) chicken eggs. The lethal dose to 50 % (LD50) of the test animals due to the PR8 virus was measured by intranasally infecting p53WT C57BL/6 mice and calculated using the method of Reed and Muench . Homozygous p53+/+ (p53WT) and p53−/− (p53KO) mice on C57BL/6 backgrounds were obtained from a breeding colony at the SPF facility of the Shanghai Veterinary Research Institute (Shanghai, China) by mating heterozygous p53+/− mice originally obtained from the Jackson Laboratory (Bar Harbor, ME, USA). For viral infection, 8 to 10-week-old p53WT and p53KO mice (n = 10/group) were intranasally inoculated with a sublethal dose (0.75 LD50/mouse) of PR8 virus. Mock-infection of mice (n = 10/group) was performed in an identical fashion to the viral infection using inoculums of phosphate-buffered saline (PBS). Clinical symptoms and body weight changes in PR8- and mock-infected mice were monitored daily for 16 days. Mice were euthanized upon a decrease in body weight >25 % from the initial weight. The 50 % egg infectious dose (EID50) in lung homogenates from PR8-infected mice was determined in 10-day-old embryonated chicken eggs and calculated using the method of Reed and Muench . All animal experiments were performed in compliance with the Guidelines on the Humane Treatment of Laboratory Animals (Ministry of Science and Technology of the People’s Republic of China, Policy No. 2006 398) and were approved by the Institutional Animal Care and Use Committee at the Shanghai Veterinary Research Institute.
Mice groups assigned for microarray analysis
Sampling 3 dpi
Sampling 6 dpi
Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)
Total RNA was extracted from lung tissue using the RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. The complementary DNA (cDNA) was synthesized using avian myeloblastosis virus reverse transcriptase (TaKaRa, Otsu, Japan). qRT-PCR analysis was performed using SYBR Premix Ex Taq™ (TaKaRa) according to the manufacturer’s protocol. Briefly, total reaction volumes of 20 μl were prepared containing 1 μl of cDNA, 10 μl of SYBR Premix Ex Taq™ (2×), and 0.2 μM of specific primers. The amplification parameters were an initial denaturation step at 95 °C for 2 min followed by 40 cycles of 15 s at 95 °C and 60 s at 60 °C. The primer sequences are shown in Additional file 1: Table S1. Relative quantification of gene expression was calculated using the 2-∆∆Ct method . Data are presented as the fold change (FC) in gene expression normalized to endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and relative to the mock-infected mice.
All measured values are expressed as the mean ± standard error (SE). The significance of the results was analyzed using the Student’s two-tailed t-test or the Gehan-Breslow-Wilcoxon test. A p value < 0.05 was considered statistically significant.
p53KO mice shows an increased susceptibility to IAV infection
Global analysis of immune-related gene expression between PR8-infected p53WT and p53KO mice
The p53 protein functions as a transcription factor that regulates expression of a series of immune-related genes in response to viral infection [10, 15, 16]. To explore the basis of the susceptibility differences to IAV infection between p53WT and p53KO mice, we compared expression profiles of immune-related genes in the lungs of PR8-infected p53WT and p53KO mice. To this end, we isolated RNA from lung homogenates of PR8- and mock-infected mice 3 and 6 dpi and performed microarray analysis. The genes that showed significantly changed expression levels between PR8-infected p53WT and p53KO mice are shown in Additional files 2: Table S2 and Additional file 3: Table S3. The possible functions of these significantly changed genes were analyzed by Gene Ontology (GO) analysis (Additional file 4: Figure S1).
Number of significantly changed genes
p53WT vs p53KOb
GO analysis of genes with attenuated expression in PR8-infected p53KO mice
Number of genes
Number of genes
Positive regulation of immune system process
Ifng, Il2, Lag3, Stat6, Trat1
Blm, Lag3, Nfkbia, Il6, Fcer1g, Il15, Ifng, etc.
Regulation of immune system process
Il2, Lag3, Orm1, Ifng, Trat1, Hmgb1, Jag1, Stat6
Blm, Orm1, Tnf, Nfkbia, Ifng, Tnfsf11, etc.
Tnf, Ifng, Fcer1g, Il1b, Selp, Tlr2
Fyn, Ifng, Il2, Lag3, Stat6, Exo1, Mink1
Lag3, Blm, Fcgr2b, Was, Nfkbid, etc.
Antigen processing and presentation
Ifng, Fcgr2b, Fcer1g, Slc11a1, Ctse
Ifng, Fcer1g, Gpam, Ikbkb, Il6, Pik3cd
Immune effecter process
Ifng, Il2, Lag3, Stat6, Exo1, Irf7
Irf7, Tnf, Lag3, Fcgr2b, Fcer1g, Msh2, etc.
Activation of immune response
Nfkbia, Fcer1g, C1qb, Lat2, Plcg2, etc.
Somatic diversification of immune receptors
Ifng, Il2, Stat6, Exo1
Ifng, Msh2, Cd40, Pms2, Exo1
Immune system development
Hmgb1, Ifng, Il2, Jag1, Exo1, Tlx1, Six4
Blm, Timp1, Tnf, Tnfsf11, Cxcl13, Msh2, etc.
Ctla4, Cxcl1, Ifng, Mx2, Irf7, Polr3c, etc.
Irf7, Cxcl9, Cxcl10, Ccl2, Ccl7, etc.
GO analysis of genes significantly expressed in PR8-p53KO mice
Number of genes
Number of genes
Negative regulation of immune system process
Regulation of immune system process
Msh2, Ndrg1, Il20rb
Ms4a1, Cd80, Il7
Positive regulation of immune system process
Immune effector process
Il20rb, Msh2, Ung
Somatic diversification of immune receptors
Immune system development
Chuk, Nfkb2, Pbx1, Six1, Msh2, Ung
Cebpa, Il7, Mb, Mlf1, Med1, Six1, Tgfb2
Spon2, Bmi1, Il1rap, Msh2, Nfkb2, Ccl25, Il20rb, Ung
Impaired expression of immune-related genes involved in IFN signaling pathways in the absence of p53
The expression of selected genes involved in interferon signaling pathway
myxovirus (influenza virus) resistance 2
2'-5' oligoadenylate synthetase 2
2'-5' oligoadenylate synthetase 3
eukaryotic translation initiation factor 2-alpha kinase 2 (PKR)
guanylate binding protein 1
interferon induced transmembrane protein 1
bone marrow stromal cell antigen 2 (Tetherin)
interferon-induced protein 44
receptor transporter protein 4
three prime repair exonuclease 1
Fas death domain-associated protein
interferon alpha B
signal transducer and activator of transcription 4
signal transducer and activator of transcription 6
interferon regulatory factor 5
interferon regulatory factor 7
The expression of ISGs is mainly regulated by IFN at the transcriptional level. The attenuated expression of antiviral ISGs in the absence of p53 may result from altered IFN expression. Next, we compared the expression of IFN and IFN receptor genes between PR8-infected p53WT and p53KO mice. Among IFN and IFN receptor genes analyzed, only Ifng and Ifnab were found to show a significantly attenuated expression in PR8-infected p53KO mice (Table 5). In addition, the Jak-Stat signaling pathway, which is activated by IFN, plays an essential role in the expression and activation of ISGs . In the absence of p53, the expression levels of Stat4 and Stat6 were attenuated significantly (Table 5). The difference in the expression levels of Ifng, Stat4, and Stat6 between PR8-infected p53WT and p53KO mice were confirmed by qRT-PCR (Fig. 2).
The IFN regulatory factors (Irf) are essential for expression and regulation of IFN and ISGs . Among 9 Irf genes analyzed, Irf1, Irf5, Irf7, and Irf9 were significantly upregulated in PR8-infected p53WT mice compared to mock-infected mice (data are available upon request), especially, Irf7, which is the master regulator of type I IFN-dependent immune responses  and significantly upregulated in IAV-infected mice  with a FC > 93 (Table 5). However, in PR8-infected p53KO mice, although Irf7 was expressed at a significant level compared to mock-infected p53KO mice, the FC was remarkably less than that in PR8-infected p53WT mice, showing significantly attenuated expression. Significantly attenuated expression of Irf5 was also found 6 dpi in the absence of p53 (Table 5). The difference in the expression levels of Irf5 and Irf7 between PR8-infected p53WT and p53KO mice was confirmed by qRT-PCR (Fig. 2).
Taken together, a number of genes essential for regulating IFN-mediated immune responses against viral infection were expressed at significantly attenuated levels in the absence of p53 during IAV infection, suggesting that the IFN-mediated immune response against IAV infection was impaired in the absence of p53.
Dysregulated expression of cytokine and chemokine genes in the absence of p53
The expression of selected cytokine and chemokine genes
interleukin 1 beta
interleukin 3 receptor, alpha chain
Interleukin 10 receptor, alpha (Il10ra), mRNA
interleukin 17 receptor D
interleukin 17 receptor E
interleukin 20 receptor beta
chemokine (C-C motif) ligand 2 (MCP-1)
chemokine (C-C motif) ligand 3 (MIP-1α)
chemokine (C-C motif) ligand 4 (MIP-1β)
chemokine (C-C motif) ligand 7
chemokine (C-C motif) ligand 11
chemokine (C-C motif) ligand 19
chemokine (C-C motif) ligand 25
chemokine (C-X-C motif) ligand 1
chemokine (C-X-C motif) ligand 9
chemokine (C-X-C motif) ligand 10 (IP-10)
chemokine (C-X-C motif) ligand 13
chemokine (C-X-C motif) ligand 14
chemokine (C-C motif) receptor 6
chemokine (C-C motif) receptor-like 2
tumor necrosis factor
tumor necrosis factor receptor superfamily, member 10b
tumor necrosis factor (ligand) superfamily, member 11
tumor necrosis factor receptor superfamily, member 8
tumor necrosis factor receptor superfamily, member 18
Of the 35 interleukin and respective receptor genes analyzed, interleukin genes, such as IL1b, IL6, IL15, and IL16, showed significantly attenuated expression levels 6 dpi, whereas most were expressed at relatively attenuated levels 3 dpi in PR8-infected p53KO mice compared with PR8-infected p53WT mice (Table 6). Analysis of the expression levels of chemokines and their respective receptor genes indicated that many were significantly changed between PR8-infected p53WT and p53KO mice. Notably, Ccl2 (encoding MCP-1), Cxcl9, and Tnf showed remarkably higher expression levels 3 dpi and significantly attenuated expression levels 6 dpi in PR8-infected p53KO mice compared to PR8-infected p53WT mice (Table 6). For instance, Ccl2, which is significantly expressed in H5N1-infected primary human cells and in IAV-infected highly susceptible mice [29, 32], showed an upregulated expression 3 dpi in PR8-infected p53KO mice with a FC of 130.8, which was 11-fold higher than that (FC = 11.8) in PR8-infected p53WT mice, whereas it displayed attenuated expression 6 dpi in PR8-infected p53KO mice with a FC of 9.89, which was 2.3-fold lower than that (FC = 22.32) in PR8-infected p53WT mice. Similar expression patterns were also observed for Ccl3, Ccl7, Ccl11, Ccl19, Cxcl10, Cxcl13 and Cxcl14 (Table 6). The differences in expression levels of IL1b, IL6, Ccl2, Ccl19, Cxcl10, and Tnf between PR8-infected p53WT and p53KO mice were confirmed by qRT-PCR (Fig. 2). These observations suggested dysregulated expression of cytokines and chemokines in the absence of p53 during IAV infection.
Tumor suppressor p53 is ubiquitously expressed in cells and plays an important role in host defense against tumor development. A growing body of evidence has indicated that p53 is involved in regulation of immune responses against viral infections [7–10]. In this study, we observed that p53-deficient mice infected with PR8 virus showed increased mortality, severe weight loss, and higher viral loads in the infected lungs compared to PR8-infected p53WT mice (Fig. 1), suggesting that p53 was involved in host defense mechanisms against IAV infection. These observations were in good agreement with a previous description that p53 serves as a host antiviral factor against IAV infection .
The major mechanism by which p53 functions is as a transcription factor that regulates, both positively and negatively, the expression of a large and disparate group of responsive genes . We comparatively analyzed the global expression profiles of immune-related genes between IAV-infected p53WT and p53KO mice, which could gain an insight into the basis of susceptibility differences to IAV infection between p53WT and p53KO mice. We observed that a number of immune-related genes showed a significant change in expression levels between PR8-infected p53WT and p53KO mice (Table 2). Notably, a considerable number of genes that showed significantly attenuated expression in PR8-infected p53KO mice compared with PR8-infected p53WT mice belonged to the GO category “immune system process” (Tables 2 and 3). These data indicated that the expression of a range of immune-related genes was impaired in the absence of p53 during IAV infection.
The IFN signaling pathway and especially, IFN-induced antiviral genes, plays a key role in regulating the immune response against IAV infection . In this study, we found that several anti-IAV genes, including Mx2, Oas2, Oas3, Eif2ak2 (encoding PKR), Gbp1, Ifitm1, and Bst2 (encoding tetherin) [19–23] and other antiviral genes, including Ifi44, Nampt, Rtp4, Trex1, and Daxx [24, 25] were expressed at significantly attenuated levels in PR8-infected p53KO mice compared to PR8-infected p53WT mice (Table 5). We thought that this impaired expression of antiviral genes in the absence of p53 during IAV infection was responsible for the high level of viral replication in the lungs of PR8-infected p53KO mice. In addition, the expression of several genes, such as Irf7, Ifng, Stat4, and Stat6, which play important roles in IFN-mediated immune response, was detected at significantly attenuated levels in PR8-infected p53KO mice (Table 5), suggesting that the IFN-mediated immune response against IAV infection was impaired in the absence of p53.
During IAV infection, unbalanced cytokine and chemokine responses lead to uncontrolled inflammation and unfavorable disease outcomes . A comparison in cytokine and chemokine expression levels between PR8-infected p53WT and 53KO mice showed that several were significantly different (Table 6), suggesting a dysregulated cytokine and chemokine response in the absence of p53 during IAV infection. It is known that the upregulated expression of proinflammatory cytokines and chemokines, including Ccl2 (encoding MCP-1), Ccl3 (encoding MIP-1α), Ccl4 (encoding MIP-1β), Cxcl10 (encoding IP-10), and Tnf, was observed during IAV infection and thought to be associated with unfavorable disease outcomes , such as the significant expression of Ccl2 in H5N1-infected cells and in IAV-infected highly susceptible mice [29, 32]. We observed that Ccl2 was upregulated 3 dpi in PR8-infected p53KO mice with a FC of 130.8, which was 11-fold higher than that in PR8-infected p53WT mice (FC = 11.8) (Table 6). The p53 protein is a suppressor of inflammation . The upregulated expression of proinflammatory cytokine and chemokine genes, such as Ccl2, Ccl3, Cxcl9, Cxcl10, and Tnf, suggested aberrant inflammation conditions in PR8-infected p53KO mice during early IAV infection and that the absence of p53 was responsible for the upregulated expression.
In addition to inducing inflammatory responses, cytokines and chemokines are immunological messengers that play important roles in the development of innate and adaptive immunity against IAV infection [31, 34]. For example, IFN-γ, the most important cytokine in cell-mediated immunity, mediates expression of major histocompatibility complex classes I and II and stimulates antigen presentation and cytokine production . IFN-γ treatment at early stages of IAV infection protects mice from death . IL1b and IL6 play crucial roles in the regulation of immune responses against IAV infection. IL-1b-deficient mice infected with IAV exhibited greater mortalities than wild-type mice . IL-6 is involved in the development of influenza-specific memory CD4 T cells . Cxcl10 is a potent chemoattractant for activated Th1 lymphocytes and natural killer cells and is thought to play a role in the temporal development of innate and adaptive immunity in concert with type I and II IFNs . We observed that a range of cytokines and chemokines, such as Ifng, IL-1b, Il6, Ccl2, and Cxcl10, showed significantly attenuated expression 6 dpi in PR8-infected p53KO mice compared to PR8-infected p53WT mice (Tables 5 and 6). The rapid decline in the expression of cytokine and chemokine genes in the absence of p53 might be associated with an impairment of innate and adaptive immunity against IAV infection.
Lack of p53 resulted in an increased susceptibility of mice to IAV infection, which was associated with significantly altered expression of a range of immune-related genes in IAV-infected p53-deficient mice. The significantly attenuated expression of Ifng, Irf7, and antiviral genes, such as Mx2 and Eif2ak2, suggested an impaired IFN-mediated immune response against IAV infection in the absence of p53. On the other hand, dysregulated expression of cytokines and chemokines, such as Ccl2, Cxcl9, Cxcl10, and Tnf, has been observed, reflecting aberrant inflammation conditions in p53-deficient mice during early IAV infection. The impaired IFN-mediated antiviral response and the aberrant inflammatory response in the absence of p53 suggested an essential role of p53 in the regulation of antiviral and inflammatory responses during IAV infection.
This work was sponsored by the National Natural Science Foundation of China (no. 81171547 and 81201266). We would like to thank Prof. Xianzhu Xia (Institute of Veterinary Science, Academy of Military Medical Science, China) for providing experimental materials.
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