Our present study demonstrates that in the innate immune response to pulmonary IAV infection, the absence of p53 severely impairs early expression of antiviral genes and pro-inflammatory chemokines, including p53-direct target genes such as IRF9 (9
) and MCP-1 (15
). These findings are consistent with a growing body of evidence indicating that the p53-dependent transcriptional program, in addition to its known influence on apoptosis and cell cycle arrest, enhances the expression of key regulators of innate immunity pathways (9
). Indeed, a recent study revealed that innate immune responses in the metazoan C. elegans
are dependent on p53 function (31
). All of these findings suggest that the highly conserved nature of p53 among eukaryotes, may rely more on its role in host immunity than in its functions as a tumor suppressor gene.
Previous studies have indicated that p53-dependent apoptosis plays a role in inhibiting viral replication of several viruses in vitro
). However, the role of apoptosis in the pathogenesis of and the host response to IAV infection in vivo
, is not fully understood. Our study demonstrates that p53 promotes the expression of antiviral genes and pro-inflammatory chemokines in the bone marrow, which has been previously shown to contribute to the establishment of a systemic antiviral state (21
). Replication of low pathogenic IAV in mice is restricted to the respiratory tract (21
) and thus, bone marrow cells are not exposed to the virus. This evidence, indicates that the observed p53-dependent effects on antiviral gene induction in the bone marrow, cannot be dependent on its ability to promote virus-induced apoptosis. Thus, our studies suggest that the role of p53 in the antiviral response to IAV in vivo
, is dependent on its ability to transcriptionally up-regulate pulmonary cytokine production rather than on its pro-apoptotic functions.
The defective cytokine responses in lungs and bone marrow observed in p53−/− mice, were associated with reduced pulmonary monocyte infiltration in comparison to their wt counterparts. These findings are consistent with evidence that production of pro-inflammatory mediators is necessary for leukocyte infiltration at inflammation sites (8
). The recent discovery that MCP-1, a known monocyte chemoattractant, is a bona fide p53 target gene (15
) also raises the possibility that direct effects of p53 on MCP-1 expression may account to some extent for the differences observed in pulmonary monocyte infiltration. In fact, recent studies indicate that mice knockout for the MCP-1 receptor, CCR2, show defective monocyte migration to inflammation sites (35
A previous report indicated that p53 deficiency leads to accelerated immunosenescence and accumulation of memory T cells in CBA/N aged mice, an effect that was particularly obvious in mice older than 15 weeks (36
). Of note, we used male mice between 8-10 weeks of age in the C57BL/6 background, which showed an entirely normal immune system as assessed by analysis of leukocyte cellularity in bone marrow and spleens of 9 week-old naïve mice (Supplementary Fig. 4
). A slight decrease in splenic CD8+
T cells as well as accumulation of bone marrow monocytes was observed in p53−/− mice (Supplementary Fig. 4
). These differences are presumably attributable to a deficiency in type I IFN signaling and MCP-1 production in p53−/− mice, since both type I IFN and MCP-1 have been previously shown to regulate peripheral CD8+
T cell maintenance and bone marrow monocyte emigration (35
). Further studies are required to evaluate the influence of aging on the ability of p53 to enhance host antiviral immunity.
Pulmonary IAV infection has been shown to promote infiltration of blood monocytes that differentiate into inflammatory CD11b+
moDCs both in lungs (23
) and mLNs (39
), and that these CD11b+
DCs have full potential to present microbial antigens and stimulate T cell responses (39
). Moreover, pulmonary accumulation of monocytes and CD11b+
DCs has been shown to be essential to ensure viral clearance after infection with respiratory viruses by both T cell dependent and independent mechanisms (21
). We observed that p53 absence significantly impaired moDC accumulation in the lungs of infected mice, causing increased viral replication and delayed viral clearance. These findings strongly suggest that a rapid production of IFN-stimulated genes and cytokines during the early phase of IAV infection, is needed for an effective control of viral replication, and that p53-dependent transcription is required for this effect.
p53 absence was associated with defective IAV-specific T cell immunity. These findings further suggest that defects associated with the innate antiviral response also ultimately impact the effectiveness of specific T cell antiviral immunity. A previous study by Grayson et al. reported that p53 was not involved in antiviral T cell responses to Lymphocytic choriomeningitis virus (LCMV)(41
). However it should be noted that IAV and LCMV induce very different inflammatory responses. Whereas IAV induces inflammation through pathways influenced by p53 status such as RIG-I and TLR3 (6
), LCMV has been shown to primarily engage the TLR2-MyD88/Mal pathway (43
). In any case, further studies will be required to evaluate the extent to which the impact of p53 on the immune response to IAV may be translatable to other viral infections.
Since p53 status affected CTL but not IAV-specific antibody responses, it is conceivable that helper CD4+
T cell function is not affected by p53 status. This hypothesis is in agreement with recent reports indicating that innate immune responses to several viruses modulate CTL activity without the involvement of CD4+
T cells or cognate antigen stimulation (7
Our findings demonstrate that the antiviral effects exerted by p53 rely primarily on its functions in non-hematopoietic cells and are likely dependent on its ability to promote more rapid pro-inflammatory and antiviral gene expression. Of note, mixed-bone marrow chimeric mice showed significantly higher engraftment of p53−/− hematopoietic cells compared to wt cells. Although further experiments will be needed to determine the basis for such differences, we hypothesize that apoptotic defects associated with p53 loss of function may provide an advantage in adaptation to the host environment (46
). Even though bone marrow-derived p53−/− cells outnumbered wt cells in both wt and p53−/− recipients, we still observed that the absence of p53 in non-hematopoietic cells inhibited migration of DCs carrying IAV antigens to the draining lymph nodes and prevented efficient T cell immunity. These findings strongly suggest that antiviral gene expression and cytokine production in non-hematopoietic cells infected with IAV play a major role in the recruitment of inflammatory cells, the activation of DC functions, and the onset of specific T cell immunity. This may help to explain the multifunctional role of cytokines such as type I IFNs and chemokines such as RANTES or MCP-1, which have been recently shown to fine-tune different aspects of the antiviral adaptive immune response, such as T cell cross-priming (38
), peripheral CD8+
T cell maintenance (37
), and Th2 polarization (48
Our present studies strongly support the concept, that enhancement of p53 functions as a host resistance factor against IAV infection, may be used as a host-targeted therapeutic strategy to develop anti-IAV antiviral therapies and vaccine adjuvants. Indeed, recent studies indicate that the enforcement of innate immune responses through the use of TLR ligands, serves to enhance IAV-specific antibody responses (4
). These studies strongly suggest that modulation of innate immunity can increase the magnitude and persistence of adaptive immunity. A potential advantage of p53-based therapies in contrast to TLR-directed strategies, is that p53, is expressed by both the epithelial cells primarily infected with IAV, and the immune cell subsets involved in the antiviral response.