p53 has been extensively studied as a tumor suppressor and more recently shown to be an inflammation suppressor. In the present study, we extend the domain of this master regulatory transcription factor to that of suppression of the host defense response, showing that p53-deficient mice have enhanced clearance of bacterial pneumonia associated with coordinate disinhibition of macrophage and PMN function. We propose that enhanced activation of NF-κB and induction of cytokines in the infected p53-null lung, originating at least in part from an expanded, TLR-hyperresponsive alveolar macrophage population, further augments recruitment of p53-null PMNs to the alveolus beyond a migratory advantage they already display in response to alveolar chemokines. p53−/−
PMNs, in turn, display coordinate enhancement of a suite of antimicrobial functions, including phagocytosis, bacterial killing, oxidant generation, and granule release. That said, our data do not exclude possible important host defense roles for p53 in nonhematopoietic cells in the lung, such as alveolar epithelial cells. However, in the end, reminiscent of the case for some other gene deletions (e.g., Abcg1
[Li et al., 2009
; Draper et al., 2010
]), enhanced pathogen clearance in the p53-null mouse is associated with worsened survival. p53’s integrated role may thus be to serve as a beneficial brake on the lung’s response to infection. We provide correlative data suggesting that p53−/−
mice may suffer lower survival as the result of lung injury from an overexuberant immune response; however, it is very possible that p53 deletion may engender additional maladaptive responses that compromise survival.
We speculate that the p53-null lung is poised for a more robust host defense response at least in part through genome-wide effects of NF-κB disinhibition. Our finding of an NF-κB response element–enriched transcriptional signature of inflammation in the naive p53-null lung is reminiscent of a recent study showing that p53 inhibition and TNF treatment elicit strikingly similar NF-κB target gene–enriched transcriptional profiles in LNCaP prostate cancer cells (Komarova et al., 2005
). We are unaware, however, of any previous report that p53 deletion induces inflammatory gene programs in vivo in the steady-state. Of note, NF-κB can be activated by both ROS and DNA damage, and it has been reported that p53−/−
mice have increased DNA damage in multiple tissues caused by increased ROS (Sablina et al., 2005
). It is thus interesting to speculate that the genomic signature of the p53−/−
lung in our study may in part stem from oxidative stress and that p53 may thus exert coordinate control over cancer and inflammation through regulation of ROS.
Alveolar macrophages were also increased in number in the naive p53−/−
lung and displayed a reduced rate of constitutive apoptosis, consistent with a report that p53 promotes apoptosis of macrophages in other settings (Mercer et al., 2005
). Given the equivalent expression (<1.5-fold difference) of monocyte/macrophage-attracting chemokines (CCL1, -2, -3, -4, -5, -7, -8, and -12) in naive p53−/−
lungs (unpublished data) and the normal number of circulating monocytes in p53−/−
mice, we speculate that the increase in alveolar macrophages stems, at least in part, from reduced local apoptosis, rather than increased trafficking to the lung. p53 has also been reported to suppress macrophage proliferation (Merched et al., 2003
). Although we were unable to detect the proliferation marker PCNA in naive alveolar macrophages by either immunoblotting or flow cytometry, bone marrow–derived p53−/−
macrophages expressed significantly increased PCNA compared with WT counterparts (unpublished data), suggesting that increased local proliferation may also possibly contribute to increased numbers of alveolar macrophages in the p53−/−
lung. However, of interest, our finding of a normal number of splenic macrophages in p53−/−
mice suggests that this regulatory effect of p53 on steady-state macrophage populations is tissue selective.
Macrophage apoptosis has been reported to be essential not only for resolution of inflammation but also for successful clearance of S. pneumoniae
(Dockrell et al., 2003
; Marriott et al., 2006
). Although the reduced apoptosis of alveolar macrophages in the infected p53−/−
lung was not associated with impaired microbial clearance, it is possible that sustained macrophage survival in the p53−/−
lung nonetheless contributed to amplifying inflammation and organ injury. A prior report that p53−/−
macrophages have defective efferocytosis (Komarova et al., 2005
) may offer a unifying mechanism for the p53−/−
pneumonia phenotype in our study, as efferocytosis impairs bacterial clearance (Medeiros et al., 2009
) and represses inflammation (Huynh et al., 2002
). Suggesting cell type specificity, and consistent with a prior report that Bcl-2 family members play a dominant role in regulation of PMN apoptosis (Dzhagalov et al., 2007
), PMN apoptosis was unaltered in vivo in infected p53−/−
We report a critical role for the molecule NO in the enhanced bacterial clearance phenotype of the p53−/− lung. Treatment of p53−/− mice with the NO synthase inhibitor L-NAME markedly impaired clearance of K. pneumoniae from the lung, whereas no such effect was seen in p53+/+ mice. In addition to PMNs, several lung-resident cell types have the capacity to generate NO. In support of the possibility that alveolar macrophages may be the responsible cell type, we found that p53−/− bone marrow–derived macrophages produce elevated NO after in vitro stimulation. Thus, in addition to contributing to increased PMN influx during pneumonia via augmented cytokine induction, p53−/− alveolar macrophages may also contribute more directly to microbial killing through enhanced NO generation. Further studies are warranted to identify whether additional cell types in the p53−/− lung, including the alveolar epithelium, may also contribute to increased bacterial killing through augmented NO generation.
Our findings indicate a novel role for p53 as a master regulator of multiple hallmark host defense functions of the PMN. Remarkably, a single systemic injection of PFTα enhanced bacterial killing by PMNs and bacterial clearance in vivo, suggesting potential for p53 inhibitors as immunostimulatory adjuvants. Our use of bone marrow–purified PMNs suggests that at least some of these p53-regulated functions (e.g., O2−
and elastase release) are, moreover, cell autonomous. Although others have demonstrated that p53 buffers ROS through induction of antioxidants (Sablina et al., 2005
), we are unaware of prior reports that p53 regulates Nox-dependent generation of O2−
in any cell type. p53 may modulate Nox function at least in part through directly repressing Nox2 complex genes. However, the finding that PFTα enhances PMN bacterial killing comparably in Nox2-sufficient and -deficient PMNs indicates that enhanced O2−
does not, at least in isolation, explain the enhanced killing conferred by p53 deficiency. p53−/−
PMNs also released increased elastase, a critical executor protease of bacterial killing (Belaaouaj et al., 1998
). We provide evidence that the killing advantage of p53-deficient PMNs derives from enhanced serine protease and, likely, elastase activity. As PMN elastase has been implicated in a wide array of lung disorders and shown to induce p53-dependent lung epithelial apoptosis (Suzuki et al., 2009
), we speculate that p53 may represent an under-recognized central regulator of lung disease.
p53 in the lung is indeed broadly responsive to environmental stressors. Diesel exhaust, silica, and cigarette smoke all activate p53 in macrophages and/or other lung cell types (Wang et al., 2005
; Yun et al., 2009
; Damico et al., 2011
), suggesting that these exposures may alter host defense functions at least in part through p53. p53 is also up-regulated in the alveolar epithelium in chronic obstructive lung disease (Siganaki et al., 2010
). As small molecule p53 agonists are under development for human cancer therapy, future studies are urgently needed to define the effects of pharmacologic activation of p53 on the human innate immune response in vivo. Moreover, as chemotherapeutics already in wide clinical use as well as radiation therapy also activate p53, it is incumbent to determine whether these agents modify innate immunity and pneumonia risk in cancer patients via effects on p53. Finally, studies are warranted to determine whether genetic polymorphisms leading to hypofunction of the p53 pathway are associated with increased risk for lung injury and/or mortality during human pneumonia.
The recent recognition that bacteria and their products induce DNA damage and compromise DNA repair (Koturbash et al., 2009
; Güngör et al., 2010
) and, conversely, that DNA repair enzymes regulate the innate immune response (Haskó et al., 2002
) has suggested that defense of the host and of the genome may be intrinsically interconnected to a degree not previously appreciated. We speculate that p53 may be centrally positioned to integrate these two fundamental responses to the environment and that this carries wide-ranging implications for human disease.