The Toll-Like Receptor Gene Family Is Integrated into
Human DNA Damage and p53 Networks
Daniel Menendez1., Maria Shatz1., Kathleen Azzam2, Stavros Garantziotis3, Michael B. Fessler2,
Michael A. Resnick1*
1Chromosome Stability Group, Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle
Park, North Carolina, United States of America, 2Host Defense Group, Laboratory of Respiratory Biology, National Institute of Environmental Health Sciences, National
Institutes of Health, Research Triangle Park, North Carolina, United States of America, 3Clinical Research Unit, National Institute of Environmental Health Sciences, National
Institutes of Health, Research Triangle Park, North Carolina, United States of America
In recent years the functions that the p53 tumor suppressor plays in human biology have been greatly extended beyond
‘‘guardian of the genome.’’ Our studies of promoter response element sequences targeted by the p53 master regulatory
transcription factor suggest a general role for this DNA damage and stress-responsive regulator in the control of human
Toll-like receptor (TLR) gene expression. The TLR gene family mediates innate immunity to a wide variety of pathogenic
threats through recognition of conserved pathogen-associated molecular motifs. Using primary human immune cells, we
have examined expression of the entire TLR gene family following exposure to anti-cancer agents that induce the p53
network. Expression of all TLR genes, TLR1 to TLR10, in blood lymphocytes and alveolar macrophages from healthy
volunteers can be induced by DNA metabolic stressors. However, there is considerable inter-individual variability. Most of
the TLR genes respond to p53 via canonical as well as noncanonical promoter binding sites. Importantly, the integration of
the TLR gene family into the p53 network is unique to primates, a recurrent theme raised for other gene families in our
previous studies. Furthermore, a polymorphism in a TLR8 response element provides the first human example of a p53
target sequence specifically responsible for endogenous gene induction. These findings—demonstrating that the human
innate immune system, including downstream induction of cytokines, can be modulated by DNA metabolic stress—have
many implications for health and disease, as well as for understanding the evolution of damage and p53 responsive
Citation: Menendez D, Shatz M, Azzam K, Garantziotis S, Fessler MB, et al. (2011) The Toll-Like Receptor Gene Family Is Integrated into Human DNA Damage and
p53 Networks. PLoS Genet 7(3): e1001360. doi:10.1371/journal.pgen.1001360
Editor: Derry C. Roopenian, The Jackson Laboratory, United States of America
Received August 31, 2010; Accepted March 1, 2011; Published March 31, 2011
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This work was supported by intramural research funds from NIEHS as follows: to MAR, project Z01-ES065079; to MBF, project Z01 ES102005. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
. These authors contributed equally to this work.
The p53 master regulator is responsive to a variety of DNA
metabolic stresses resulting in induction or repression of over 200
genes as well as several LINC- and micro-RNAs [1,2]. In its role as
tumor suppressor and ‘‘guardian of the genome’’ many of the
target genes in humans influence cell cycle progression or
apoptosis. Over the past decade the p53 network has been
extended to transcriptional regulation of genes associated with a
wide variety of biological functions including DNA repair,
angiogenesis, cellular metabolism, autophagy, stem cell renewal,
fertility, differentiation and cellular reprogramming. To better
understand the broad role that p53 can play in human biology, we
have pursued ‘‘functionality rules’’ for identifying target response
element (REs) sequences where p53 can directly influence
transactivation. Using in vivo transactivation systems based in yeast
and human cells as well as binding in human cell extracts [3,4], we
recently found that functionality of the binding consensus
RRRCWWGYYYnRRRCWWGYYY (R, pyrimidine; Y, pyrim-
idine; W, A or T; n, spacer of 0 to 13 bases) is greatest when
there is at most a single base spacer and if ‘‘WW’’ is ‘‘AT.’’
Furthermore, we defined functionality for half-sites (RRRCWW-
GYYY) and found in cis synergy when another transcription factor,
estrogen receptor, was bound nearby (for a description of
noncanonical REs including half-sites see [3,5] and summary in
). Based on these functionality rules, we found that the evolution
of p53 control of at least one gene family, DNA metabolism and
repair, is limited to primates . Furthermore, we identified single
nucleotide polymorphisms in REs that are predicted to modify
responsiveness of genes to p53 mediated stress .
Our functionality studies of canonical and noncanonical
promoter p53 REs suggested that p53 may have a role in human
Toll-like receptor (TLR) expression. We had reported that a
single nucleotide polymorphism (ChrX:12923681, rs3761624 A/
G) in the promoter of the TLR8 gene creates an RE that can be
targeted by p53 in yeast and cell line reporter systems 
although we could not detect endogenous expression of the
TLR8 gene. In addition, the TLR3 gene in epithelial cancer cell
lines was found to be induced by p53 following exposure to 5-
fluorouracil (5FU) .
PLoS Genetics | www.plosgenetics.org1 March 2011 | Volume 7 | Issue 3 | e1001360
Innate immunity is paramount to infection and tissue injury
responses . The TLR gene family mediates innate immunity to
a wide variety of pathogenic threats. The TLRs recognize
conserved exogenous pathogen-associated molecular patterns
(PAMPs) and endogenous danger-associated molecular patterns
(DAMPs) , while adaptive immune receptors ensure specific
antigenic responses through clonal expansion. TLR function is
associated with circulating immune cells such as monocytes and
dendritic cells, tissue phagocytes that include alveolar macrophag-
es and with nonimmune cells (such as epithelial cells of gut, skin,
lung, etc.) that are exposed to environmental injury or pathogens.
The roles of TLRs in mammalian biology are continuously
expanding and they are now understood to function in such
diverse processes as inflammation, cell differentiation and cell
survival . Altered TLR function is implicated in human
diseases such as systemic lupus erythematosus, inflammatory
bowel disease (IBD) and cancer [11,12], and agonist/antagonist
manipulations of the TLR system are being pursued to alleviate
various diseases (reviewed in ).
Given the varied functions of TLRs, factors that regulate their
expression are expected to shape immune responses .
However, there are few examples of TLR gene induction and
these are limited to specific TLR-stimulus interactions [15,16].
The TLR-dependent innate immune response is thus generally
considered to be ‘‘hard-wired.’’ Although environmental stress can
influence gene expression indirectly, e.g., through epigenetic
mechanisms, to our knowledge there are no reports of TLR
induction by environmental factors in primary human cells
These collected observations led us to investigate the respon-
siveness of TLR genes as a class to common DNA stressors. We
have examined expression of the entire TLR gene family following
exposure to anti-cancer agents that induce the p53 network in
primary immune cells obtained directly from human subjects as
well as the impact on downstream cytokines. Agents were applied
to T-lymphocytes and alveolar macrophages ex vivo. Prior to this
there were no reports that we are aware of addressing DNA
damage-induced responses of any TLR genes in cells directly
associated with innate immunity. As part of this study, we establish
that in the evolution of the TLR responses, the p53-mediated
expression of TLRs is unique to primates.
DNA stress induces TLR gene family expression
We selected ionizing radiation (IR), 5FU and Doxorubicin
(Doxo) because these agents represent a cross-section of DNA
metabolic stressors, such as damage or replication inhibition, that
may occur endogenously or environmentally and are well-known
to activate p53 and its network of target genes. Furthermore, these
agents are often employed in cancer treatments. To address TLR
responses ex vivo in primary human immune cells, T-lymphocytes
were expanded by phytohemagglutinin (PHA) stimulation of
peripheral blood mononuclear cells (PBMC) freshly isolated from
the blood of healthy human volunteers (see Figure S1A;
for demographics of volunteers see Table 1); alternatively,
T-lymphocytes were obtained from the PBMC fraction using
anti-CD3 antibody-based purification as described in Figure S1B.
Table 1. Demographics of subjects in this study and
genotype information for TLR8 p53RE SNP of donor
TLR8 p53RE SNP
BS#1 31Male CaucasianG
BS#2 57 FemaleCaucasian A/A
BS#4 52 FemaleAfrican AmericanG/G
BS#5 25 FemaleCaucasianG/A
BS#7 25Female CaucasianG/G
BS#8 35 FemaleCaucasian G/A
BS#9 42 FemaleCaucasianA/A
BS#11 25Female CaucasianA/A
BS#1242 Male CaucasianA
BS#1340 Male CaucasianA
BS#16 38Male CaucasianG
BS#19 22 Male CaucasianG
BS#20 23Male CaucasianA
BS#25 30Female African AmericanA/A
BS#2647 Male CaucasianA
CRU116121 Female CaucasianA/A
CRU116322 Male CaucasianG
CRU116934Male African AmericanA
CRU1173 19 MaleCaucasianA
*The TLR8 SNP is located in the promoter region of the gene and the A allele
impairs functionality of a p53 response element (rs3761624),
GGCAAGATGAAACAT(G/A)TCA (SNP is in italics and underlined bases) .
Reported allele frequency for the G allele is 0.44 and for the A allele is 0.56 (the
common polymorphism). The TLR8 gene is located on the X chromosome.
Among the most prominently studied regulators of gene
function is the p53 tumor suppressor, which has many
roles in human biology. The transcriptional master
regulator p53 directly targets expression of .200 genes.
Previously, we sought to define the p53 network in terms
of functionality, specifically the ability of target response
element sequences (REs) to support p53 transactivation.
Here we identify p53 target canonical and noncanonical
REs in the family of Toll-like Receptor (TLR) innate immune
response genes and establish p53 regulation of most TLR
genes. We address p53 responsiveness in primary human
lymphocytes and alveolar macrophages collected from
healthy volunteers. Notably, all TLR genes show responses
to DNA damage, and most are p53-mediated. However,
there is considerable variability between individuals,
suggesting that DNA and p53 metabolic stresses can
markedly differ in impact on the innate immune system as
well as downstream appearance of cytokines. Indeed, we
report a SNP in a p53 RE within the TLR8 promoter that
alters p53 responsiveness in primary human cells. Further-
more, the p53-mediated expression of TLRs is unique to
primates. Overall, these findings identify a new, pivotal role
for the well-known human tumor suppressor p53, namely,
integration of DNA damage and innate immune responses.
p53 Regulates Expression of TLR Genes
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Presented in Figure 1 are the expression responses of the entire
family of TLR genes in stimulated lymphocytes for 18 subjects
(except where noted) following exposure to IR (4 Gray), 5FU
(300 mM), and Doxo (0.3 mg/ml) (Figure 1A, 1B, and 1C,
respectively). These responses are relative to no treatment controls
and normalized to 18S ribosomal DNA. As additional controls, we
also examined the expression of the beta-glucuronidase, GUSB,
and actin genes (Figure S2) that are considered to be nonrespon-
sive to chromosomal and/or p53 stress; they showed little variation
after doxorubicin and nutlin exposure. The doses chosen were
similar to therapeutic doses or doses commonly used in the
literature. By way of comparison, we also examined the response
of the cyclin-dependent kinase inhibitor gene p21WAF1(CDKN1A),
a prototypical p53 target gene induced by these agents in a variety
of human cells. Notably, these results establish that expression of
all TLRs can be responsive to DNA metabolic insults, even
exceeding p21 induction. However, there is considerable variabil-
ity in the individual responses between subjects, TLRs, and
treatments as summarized in the ‘‘box and whiskers’’ presentation
of Figure S2. For example, the IR induction of the ten TLR genes
varies from 1- to over 4-fold for each TLR except TLR3 (Figure 1
and Figure S2), which is not detected in the PHA-stimulated
lymphocytes of most subjects. This agrees with the large
differences in expression of IR-inducible genes between human
lymphoblastoid cell lines . The variability in gene expression
among the 18 subjects can represent a continuum of responses (e.g.,
TLR4 expression after IR), or exhibit more of a binary induction
pattern (e.g., TLR8 response after IR; see Figure 1). Specifically, for
the TLR8 gene, approximately half the subjects respond strongly
and half exhibit a much lower response, a finding that appears to
be genetically determined , as discussed below. Different agents
also elicit differential TLR gene expression responses. For example,
TLR1 is responsive to IR by more than 2-fold in most subjects, but
generally there is only a small level of induction in response to 5FU
To better assess each TLR gene response across subjects, we
portrayed the induction of TLRs for each subject in a format akin
to a heat map, as described in Figure 1E for IR, where 2.5-fold
induction is indicated in red and ,2.5 fold in black (this value
corresponds to the minimal p21WAF1response for nearly all agents
and subjects; see Figure S3 for other agents). Among three subjects
(BS4, 5, 21), all TLRs were induced at least 2.5-fold by IR
exposure, while only 1 (BS2), 2 (BS9) or 3 (BS8, 13) TLRs were
induced in four others. There is no obvious pattern to the
differences between TLRs or subjects for IR as well as for 5FU and
Doxo, although it is clear that TLR1 is generally much less
responsive to the last two agents (Figure 1, Figures S2 and S3).
Consistent with TLR3 induction by DNA damage in cancer cell
lines , its expression was induced in 5 out of 7 subjects; however,
TLR3 gene expression was not detected in 11 subjects (Figure 1
and Figure S3). Even though all TLRs are responsive in at least one
subject, only subjects BS4, 5 and 21 exhibited high responses for
most TLRs for all agents tested (Figure S3).
We also addressed statistically the responsiveness of the
population as a whole (18 subjects, Figure 1A, 1B, 1C, except
TLR3 which was expressed only in 7 subjects) to IR, 5FU, and
Doxo employing a t-test (see Material and Methods) to assess the
ability of each agent to induce expression of each TLR gene in the
population. As expected, induction of the p21 gene in the
population was highly significant for all the treatments (p,0.0001).
While there was variation between individuals, all the TLR genes
in the population were responsive to IR (p,0.0001). A similar
likelihood of responsiveness (p,0.0001) was observed for 5FU and
Doxo treatment for all but the TLR1 and 7 genes (p,0.003) and
TLR9 (p=0.011 for 5FU and 0.053 for Doxo); however, there
were substantial responses for the TLR 9 genes of several
individuals. Collectively, these results suggest that multiple path-
ways may influence TLR expression after DNA damage, and these
are specific to the mode of chromosomal stress.
p53 directly enhances TLR expression
Since p53 mediates many DNA damage responses and given
our finding that all TLRs respond to at least one DNA metabolic
disruptor, we sought to examine in more detail the ability of p53 to
induce TLRs. PHA-stimulated lymphocytes were exposed to the
p53 activator nutlin (10 mM) to increase p53 levels. Stabilization of
p53 normally occurs through stress-induced post-translational
modifications affecting both p53 and MDM2 . Nutlin can
directly prevent p53 destruction by interfering with the MDM2-
p53 interaction . As expected, all treatments activated the p53
pathway in the lymphocytes of all individuals as assessed by
immunodetection of p53 and p21 proteins (Figure S4; Figure 1F is
a representative example). As shown in Figure 1D, Figures S2 and
S3, nearly all TLR genes in most subjects are induced over 2.5 fold
after nutlin treatment, except for TLRs 1 and 7. The TLR1 gene
much less responsive while the TLR7 gene is generally repressed
by p53 induction. Interestingly, TLR7 is induced by the DNA
damaging treatments, suggesting p53-independent induction
mechanisms. While TLR3 is not detected in 11 subjects, it is
induced by nutlin in 5 of the remaining 7 subjects. Although there
was variation between individuals, we found that most of the TLR
genes in the population were responsive to nutlin. There was no
statistically significant induction of the TLR9 gene (p=0.11);
however, there was statistically significant repression of the TLR7
gene (p=0.006). The expression of all the remaining TLR genes
was significantly induced in the population (p,0.003).
A role for p53 in TLR induction is further indicated by the
finding that co-treatment with the p53 inhibitor pifithrin-alpha
 represses the induction of TLRs by IR and other agents
(Figure 1G for IR; Figure 2 for Doxo, 5FU and nutlin). Notably,
samples taken on two occasions from the same individuals in a 4
month-interval, blind study revealed a striking consistency in TLR
gene expression patterns (Figure 3), thereby excluding technical or
temporal variables as significant sources of variability in findings.
The p53 inducibility of most TLRs led us to investigate if p53
could act directly on transcription. As discussed in the Introduc-
tion, the commonly accepted consensus target RE consists of two
decamers composed of (RRRC A/T A/T GYYY) separated by up
to 13 bases (reviewed in [25,26]). We had established rules for
predicting in vivo functionality of p53 REs including separation of
,2 bases and dependence on a core CATG (summarized in [3,6]).
Among several potential p53REs identified by in silico search and
bioinformatic tools, we found that each TLR has at least one p53
target sequence within 65 kb of the transcription start site that is
predicted to provide at least a weak-to-modest p53 responsiveness
(Table 2 contains the sites predicted to have the greatest functional
responses). As shown in Figure 4A, each of these target sequences
(TLR7 was not examined because we did not find any p53-like
binding sequences) could support p53-driven transcription of a
luciferase reporter co-transfected, along with a p53 expression
plasmid, into p53 null H1299 cells. For half of the TLRs, the
induction levels were comparable to those obtained with the
moderately responsive p53 target response element of AIP.
Based on these results, we examined whether the functional REs
of Figure 4A are indeed targets of induced p53 using chromatin
immunoprecipitation (ChIP) analysis. We chose a representative
subject BS4 whose lymphocytes exhibited strong Doxo-induced
expression (Figure 1) for all TLRs except TLR1 and TLR7. As
p53 Regulates Expression of TLR Genes
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Figure 1. Induced expression of the TLR gene family in primary human T-lymphocytes by DNA stressors and activation of the p53
pathway in cells from healthy subjects. Human peripheral blood mononuclear cells (PBMC) freshly isolated from healthy subjects (n=17–18)
were incubated with PHA to stimulate T-lymphocyte expansion. After 48 h incubation, cells were exposed to (A) ionizing radiation (IR);
(B) 5-fluorouracil (5FU); (C) doxorubicin or (D) nutlin. Cells were harvested following 24 h treatment in the presence of PHA. In the case of IR, they
were harvested 24 h after exposure to 4 Gy. (Robust responses were observed under these conditions; however, further studies might reveal more
optimum times and dose for primary cells.) Presented in panels A to D are mRNA expression levels of TLRs and p21WAF1as compared to expression in
untreated cells for each subject (the dashed red line corresponds to no change with treatment, i.e., a value of 1; horizontal bars correspond to the
median). Gene expression was analyzed by qPCR and normalized to 18S ribosomal RNA. Statistical analysis in A–D. Unless noted, the TLR genes of the
population as a whole exhibited statistically significant change in expression induced by an agent (vs. untreated) at the p,0.006 level (see text). Black
arrows indicate changes that are not significantly different from the control for specific TLR genes in the population (p$0.05). (E) Changes in TLR
expression after IR of cells from all subjects (BS#) presented as a heat map. The subjects are grouped according to IR responsiveness (i.e., purple, for
nearly all TLRs being inducible and blue for people with only a few TLRs induced by IR). (F) p53 and p21 activation analyzed by Western blot in a
representative experiment for subject BS#7 with actin as a loading control. (G) Inhibition of IR-induced TLR gene expression by the p53 inhibitor
pifithrin-a (PFTa, 40 mM) added to cells 2 h prior to IR and kept there after exposure. Values are relative to cells treated with DMSO represented as the
dash red line, i.e., a value of 1. Presented is a representative experiment with T-lymphocytes from subject BS#19.
p53 Regulates Expression of TLR Genes
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Figure 2. Inhibition of p53 activity by pifithrin-alpha dramatically reduces p53-dependent TLR induction by DNA damage and p53
activation. The p53 inhibitor pifithrin-alpha (PFTa, 40 mM) or DMSO (control) were added to PHA-stimulated T-lymphocytes 2 h prior to Doxo
(0.3 mg/mL), 5FU (300 mM) or nutlin (10 mM) exposure. Following 24 h of exposure, gene expression was assessed by qPCR. Presented is the mRNA
fold-change compared to untreated cells for subjects BS#7 and BS#19. Each bar represents an average of 3 PCR replicates with its standard
p53 Regulates Expression of TLR Genes
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shown in Figure 4B, there is clear binding following exposure of
cells to Doxo at the established p53RE on p21WAF1as well as at the
TLR2, 4, 5, 6, 8, 9 and 10 sequences described in Figure 4A. Thus,
all TLR genes are subject to DNA-damage associated transcrip-
tional regulation and most are directly targeted by p53. (We also
identified other p53-like sequences in the analyzed regions;
however, these were predicted to be less functional than the
sequences examined). Furthermore, a direct role for p53 in TLR
gene expression was demonstrated using p53 null SaOS2
osteosarcoma cells that have p53 under the control of a
tetracycline inducible promoter . Expression of wild-type
p53, but not the transcriptionally inactive G279E mutant protein,
results in induction of all TLRs except TLR7 (TLR8 was not tested
because it is not expressed by SaOS2 cells; Figure S5).
We also determined whether p53 induction of TLR genes by
nutlin can lead to a corresponding increase in protein. (Nutlin was
chosen because it typically led to the largest increase in p53, as
shown in Figure S3.) Using western blot analysis with TLR specific
antibodies (see Materials and Methods), the levels of TLR2 and
TLR5 proteins were examined in the membrane fraction from
stimulated lymphocytes of volunteers BS25 and B26 (sufficient
cells were obtained from these subjects to enable the protein
Figure 3. Induced TLR expression in primary human T-lymphocytes obtained from the same subjects on different days. TLR gene
expression assessed by real time-PCR was measured in PHA-stimulated lymphocytes from 2 volunteers who were sampled twice, separated by 4
months, in a blind study as indicated in the figures (i.e., 12-2009 and 04-2010). The cells were exposed to DNA stressors or nutlin. Presented is the
mRNA fold-change compared to untreated cells for patients BS#19 (A) and BS#20 (B).
p53 Regulates Expression of TLR Genes
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measurements; the TLR2 and TLR5 proteins were only detected
in the membrane fraction as noted in the Material and Methods).
Nutlin treatment resulted in a substantial increase in both proteins
in the membrane fraction which corresponded well with induced
expression of the TLR 2 and TLR5 genes, as described in Figure 5.
To address the generality of DNA damage-induced TLR
expression and the role of p53, we also examined alveolar
macrophages since they are well-known to play a pivotal role in
pulmonary innate immunity and are susceptible to DNA damage
from environmental exposures including cigarette smoke and
particulate matter [28–30]. The cells were collected by broncho-
alveolar lavage of healthy human subjects and treated ex vivo.
Among 6 subjects, there were considerable differences between
TLRs with little or no induction of TLR1 and TLR6 by Doxo or
nutlin, as described in Figure 6, and a dramatic induction of TLR9
by Doxo, suggesting cell-specific factors that determine damage-
Table 2. The p53RE-related sequence that is predicted to have the greatest function in the promoter regions (65 kb from
transcription start site) of human TLR genes.
sequences * Predicted most functional p53 RE
TLR111 (2)RE#4 AGGCATGagC-tAcCATGCCC
4: 38807969-38807950NO YES
TLR2 10 (3) RE#4 AAACAAGaTCtacccctatatGGGCATGTCC
4: 154621975-154622005 NOYES
TLR3 13 (2)RE#4 AGGCATGCaC-cAACATGCCC
4: 186988380-186988399NO YES
TLR415 (4)RE#8 AGGCATGCTCcaGAGCAAaTCT
TLR511 (3)RE#4 GGGCATGgTg-GYACATGCCT‘
1: 223311442-223311461NO YES
4: 38832459-38832492 NOYES
TLR75 (2)RE#2 GGGCATGTCacaatttcagattaatcaAcGCTTGCTC
X: 12885856-12885892 NO NO
TLR8 10 (7) RE#6 AGGCAAGaTg-AAACATRTCa‘
X: 12923665-12923684 NO YES
TRL9 17 (3) RE#14 AGGCATGgTgGtGCATGCCT
3: 52273914-52273933NO YES
TLR106 (1)RE#4 AGACATGTTT-GtAtATGTTT
In lower case characters are the mismatches relative to the consensus p53 response element (RE).
The bold characters are half-site p53REs without mismatches relative to the consensus p53 RE.
The italic lower case characters correspond to the spacer sequence between p53 decamers. A lack of spacer between decamer sequences is denoted by ‘‘-’’.
TLR5 SNP rs2192617 C/T
TLR6 SNP rs167251 G/A
TLR8 SNP rs3761624 A/G
*Number of noncanonical p53 REs is indicated inside the parenthesis.
**Chromosome coordinates based on USC Genome Browser Human Feb. 2009 (GRCh37/hg19) http://genome.ucsc.edu/.
Indicates that the predicted RE contains a single nucleotide polymorphism (SNP). SNPs are indicated in bold and underline characters.
Figure 4. p53 drives expression of TLR family through direct binding to regulatory regions. (A) Functionality of presumptive p53 RE
sequences associated with TLRs (sequences are described further in Table 2). H1299 cells were transfected with RE::luciferase reporter constructs in
the presence (solid bars) or absence (open bars) of a vector expressing wild type p53. At 48 h post-transfection, induction of the luciferase reporter
was compared with cells containing the pGL4.26 plasmid lacking p53. Presented are the average and standard deviations of 3 independent
experiments. p53REs corresponding to TLRs 5, 6 and 8 contain SNPs as described in Table 2. (B) Occupancy of p53 at promoters of p21 and TLRs 2, 4, 5,
6, 8, 9 and 10 assessed by ChIP analysis of PHA-stimulated T-lymphocytes from subject BS#4 following 24 h of doxorubicin (0.3 mg/ml) treatment.
p53 Regulates Expression of TLR Genes
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TLR response profiles. In general, fewer of the TLR genes in the
macrophages responded to Doxo and nutlin as compared to
lymphocytes and the response levels were not as large.
Activation of p53 influences PAMP-induced TLR cytokine
Induction of cytokines is a prototypical functional response that
is downstream of TLR activation [11,31,32], and increases in TLR
expression can enhance PAMP or DAMP-induced signaling and
innate-immune mediated effects. As shown in Figure 7, pretreat-
ment of freshly isolated CD3+ T-lymphocytes (see Figure S1) with
nutlin to induce TLR2 by p53 resulted in 2- to 5-fold increased
expression of interleukin IL-1 and IL-8 by the TLR2 ligand
PAM3CSK4, suggesting that TLR induction by p53 can directly
affect innate immune function. (The TLR2 induced cytokine
response was examined because of our observation that T-
lymphocytes from all subjects experience nutlin-induced TLR2
expression, as shown in Figure 1 and Figure S3). The magnitude of
cytokine induction varied between subjects (see Figure S6),
suggesting that other factors, besides p53-induced gene expression,
affect innate immune responsiveness.
TLR8 promoter SNP determines nutlin and IR
Our previous report  of a SNP in a potential p53 response
element of the TLR8 promoter (AAACAT(G/A)TCa; see Table 1)
provided a unique opportunity to directly assess the relationship
between p53 and TLR expression. While we had established large
differences in the potential p53-responsiveness of the two SNP
sequences ; see Figure 4A for the ‘‘positive’’, p53-responsive
G-allele), their cellular impact could not be assessed because the
TLR8 gene was not expressed in the human cell systems
previously examined. Unlike the results with cell lines, we
observed a dichotomous response in TLR8 expression in
lymphocytes following IR and nutlin treatment (Figure 1), and
in alveolar macrophages following nutlin exposure (Figure 6),
with some subjects showing low TLR8 induction and others
robust induction. We, therefore, determined which p53 response
element alleles were present and their relationship to TLR8
induction. Since TLR8 is located on the X-chromosome, males
carry only a single copy and females 2 copies (the specific alleles
for each volunteer are described in Table 1). As shown in Figure 8
and Figure S3, the ability of nutlin and IR to induce TLR8
correlates well with the presence of the G-allele. The frequency of
this allele in our study is 0.43 (13/28, which corresponds to the
frequency in the general population (Table 1). Importantly, TLR8
induction was always high when only G-alleles were present (both
alleles in females or the single allele in males) and absent if there
were only A-alleles. However, among the 3 female subjects that
were heterozygous for these alleles, only one responded poorly to
both nutlin and IR. Possibly, the variability between female
subjects heterozygous for the SNP is related to X chromosome
Figure 5. Nutlin induces TLR2 and TLR5 mRNA expression as well as proteins. (A) Induction of TLR2 and 5 protein expression in stimulated
lymphocytes cells from subjects BS25 and BS26 by nutlin (10 mM) after 24 hr post-treatment. Proteins were determined by western blot analysis using
antibodies specific to TLR2 and TLR5 in the membrane fractions. Actin protein provided a loading control. (While there is little change in the amount
of actin protein detected, there may be differences in the nonspecific bands.) (B) Induction of TLR2 and 5 mRNA expression in stimulated
lymphocytes from subjects BS25 and BS26 (data obtained from Figure 1) by nutlin.
p53 Regulates Expression of TLR Genes
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While these results demonstrate differences in the impact of the
polymorphic alleles, the differences do not extend to 5FU or Doxo,
suggesting that other p53-related target sequences are responsible
for the induction or involvement of additional p53-independent
mechanisms (Figure 8 and Figure S3). Notably, these findings with
TLR8 provide the first direct demonstration, to our knowledge, in
humans of the ability of a specific RE in a p53 target gene to drive
transcription. The SNP-associated differences in expression may
provide opportunities to identify other factors in human primary
tissue that determine the ability of specific REs to support p53-
driven transcription [33,34]. Although there are SNPs in the REs
of TLR5 and TLR6, as described in Table 2, they are predicted to
have no functional impact (see [6–8]).
Evolution of TLR gene family responsiveness to p53
Our results with primary human cells have been confirmed for
DNA damage induction of TLRs (to be presented elsewhere) in
several human cell lines including the previously reported TLR3
. However, as shown in Figure S7, they do not extend to murine
cells. The TLR responses to Doxo, 5FU and IR were found to be
Figure 6. Induced expression of TLR gene family in human alveolar macrophages by DNA stressors and activation of the p53
pathway. Alveolar macrophages obtained by bronchoalveolar lavage of normal, healthy human subjects were incubated for 24 h in the presence of
(A) nutlin or (B) Doxo. TLR gene expression was analyzed by qPCR and is presented as fold-change compared to untreated cells. Horizontal bars
correspond to the median. (C) Activation of p53 pathway (p53 and p21) in alveolar macrophages following 24 h of incubation with nutlin or Doxo as
analyzed by Western blots.
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small in mouse peritoneal elicited macrophages, bone marrow-
derived macrophages, and embryonic fibroblasts (MEFs; except
for TLR9 in MEFs). The low induction level appears to be p53-
independent based on results with nutlin and on the similar
responses in p53-positive and -null MEFs. These results are
consistent with the lack of sequence conservation between humans
and rodents of functional p53 response elements in TLR genes
(Figure S8) even though the coding sequences are well-conserved.
Although we were able to identify p53 RE-related sequences in the
vicinity of the transcription start site of several mouse TLRs, none
were predicted to be functional p53 targets.
Cellular stress and the inflammatory response are intricately
linked pathways subject to endogenous and exogenous challenges.
Here, we provide the first evidence that DNA stressors may also
modulate inflammatory responses at a fundamental level in
primary human cells, namely, by altering TLR expression. All
members of the human TLR gene family tested (TLR1-10) are
responsive to at least one disruptor of chromosome metabolism.
While there are considerable variations between individuals, for
each TLR gene there is a group of several subjects that is
responsive to at least one of the stressors (5FU, doxorubicin, IR
and/or nutlin). Note, for example, that there is at most a low level
of TLR1 induction by 5FU and nutlin in the primary cells from 18
subjects and there is even a general repression of TLR7 by nutlin.
We thus propose that, contrary to the paradigm that innate
immunity is hard-wired, the TLR system in humans actually has a
complex, robust responsiveness to environmental conditions that
challenge the integrity of the genome. We establish that induction
of TLRs by DNA damage is a class effect that in many cases can be
mediated by p53 (including repression of TLR7) and prevented by
the p53 inhibitor pifithrin. In support of these findings, several
potential p53 binding sites were identified in the proximity of the
transcription start sites for all TLRs, except TLR7. The new
p53REs were identified using functionality rules to predict p53RE
responsiveness [6,7]. These were confirmed with reporter assays
and by the binding of p53 in human lymphocytes following stress
activation. In addition the functional TLR8 SNP in the p53 target
response element directly confirms a role for p53. Interestingly, for
TLR2 and TLR10, the most functional sites were predicted to be
noncanonical, containing only a half–site p53RE (i.e., one
decamer). Previously, the genes FLT1  and RAP80  were
shown to be directly controlled by p53 through half-site REs and
several other target genes have been identified that are predicted
to be regulated by noncanonical p53 RE (summarized in ).
The inclusion of TLR genes expands the universe of genes and
biological functions that fall within control of the p53 master
regulator. The addition of the set of TLR genes identifies a
biological gene niche regulated by the p53 master regulator that is
distinct from rodents whose TLR genes appear to lack functional
p53 REs. It is interesting that a similar niche was identified for all
the DNA metabolic genes that are p53-responsive in humans in
that none of them respond to p53 in rodents [7,36]. p53 provides a
rapid, integrated signaling mechanism for increasing or maintain-
ing gene responses to acute and chronic DNA damage stress. In a
more general sense, we suggest that the evolutionary inclusion of
sets of genes with related functions may provide an efficient means
of dealing with sudden, temporary challenges that can result from
DNA damage and/or DNA damage itself may be a modulator of
broader biological threats, as for the case of infection.
We speculate that there may be feedback loops that integrate
this newly identified role for p53 in DNA damage and innate
immune responses, as described in Figure 9. In this scheme, DNA
damage from environmental agents or potentially from TLR-
elicited reactive oxygen species (ROS) may amplify the respon-
siveness of the innate immune system by promoting TLR
upregulation. p53 itself displays a variety of roles in mediating
ROS signals [37,38]. On the other hand, TLR upregulation may
also sensitize tissues to maladaptive aseptic inflammation in the
setting of environmental injury. For example, tissue injury is
reported to induce inflammation through release of DAMPs that
act upon TLR2, TLR4, and TLR9 [39,40]. We speculate that,
during tissue injury, upregulation/activation of TLRs may serve as
a cell-fate counterbalance to p53-mediated pro-apoptotic respons-
es by leading to activation of the pro-survival factor NF-kB .
This may be particularly relevant to cancer therapy, as stimulation
of TLR5, 7, 8, and 9 have been shown to modify cellular radio-
and chemoresistance [41,42]. These findings demonstrating that
p53 can increase an inflammatory response differ from the
generally held view relating to the antagonistic affect of p53 on
inflammation directed by NF-kB . However, the mechanism
here is quite different in that it involves the p53-mediated increase
in a receptor that translates ligand interactions into cytokine
responses. Our results may be particularly relevant to diseases in
which variations in T-cell function can impact pathogenesis, such
as autoimmune disease, asthma, and IBD. For example, recent
reports suggest that intestinal inflammation can induce genotoxi-
city in circulating leukocytes , while increased DNA damage is
also detected in lymphocytes obtained from rheumatoid arthritis
patients . The heightened pro-inflammatory status in such
Figure 7. Induction of p53 sensitizes freshly isolated CD3+ + cells
to PAMP stimulation. (A) TLR2 and cytokine (IL-1 and IL-8) expression
in CD3+ lymphocytes incubated for 20 h with nutlin (10 mM) or DMSO
and then exposed for 4 h to the TLR2 ligand PAM3CSK4 (1 mg/ml).
Presented are representative results for subject BS#37. Gene expres-
sion was analyzed by qPCR and presented as fold-change compared to
untreated cells. (B) Activation of p53 pathway by 24 h of nutlin
treatment in CD3+ cells isolated from peripheral blood as determined
by Western blot analysis.
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patients, as well as the common finding of systemic or multi-organ
inflammation during exacerbations of autoimmune disease, might
be mediated by circulating immune cells which have suffered
DNA damage during passage through inflamed tissues.
Beyond the TLR gene and cell subset variability in response to
DNA damage and p53 activation, we also demonstrate consider-
able inter-individual variation. This variability may be relevant to
inter-individual differences in susceptibility to a wide spectrum of
diseases and therapies. Our results suggest that various anti-cancer
agents may yield different patterns of responses across TLRs and
between subjects. Since TLR ligands are increasingly used as
adjuvants for vaccines (TLR4 and TLR9) and cancer treatment
(TLR3/7/9) (reviewed in ), the ability to detect and predict
genetically determined inter-individual variability in TLR induc-
tion may prove a useful therapeutic tool. Future studies are
warranted to determine whether single agents such as nutlin, or
even factors that induce chromosome stress, may serve as useful
immune adjuvants through manipulation of TLR expression in
Materials and Methods
Healthy adult volunteers were recruited to the NIEHS Clinical
Research Unit and underwent phlebotomy. Subjects were
excluded if they had a history of recent infection, were on anti-
inflammatory medications, or tested positive for hepatitis B, C or
HIV. Up to 300 ml of whole blood were withdrawn from an
antecubital vein into citrated tubes. Lymphocytes were isolated
using percoll (Sigma) and anti-CD3-coupled Magnetic Beads
(Miltenyi Biotec) as per manufacturer’s protocol. Cell purity was
.98% after percoll/magnetic bead isolation based on flow
cytometry. We maintained lymphocytes in RPMI supplemented
with 10% FBS. For T cell stimulation, cells were activated with
phytohemagglutinin-M (PHA, Invitrogen, 3% vol/vol) for 72 h.
The total number of lymphocytes available per treatment
conditions after PHA was typically around 10 million cells or less.
Cells were treated starting at 48 h post PHA addition and cell
cultures were harvested 24 h later. Freshly isolated CD3+ cells
were treated with nutlin for 20 h or DMSO as a vehicle control,
then washed and exposed to TLR1/2 ligand PAM3CSK4 (1 mg/
ml) at 16106cells/ml. Total yield of CD3+ cells from a single
subject was typically around 10–15 million cells or less. Protocol
and procedures were approved by the NIEHS Institutional
Alveolar macrophage isolation
Healthy, nonsmoking male volunteers, 18 to 40 yr of age,
underwent fiberoptic bronchoscopy with lavage to procure
alveolar macrophages. The screening procedures for each subject
included a medical history, physical examination, and routine
hematologic and biochemical tests. None of the subjects had a
history of asthma, allergic rhinitis, chronic respiratory disease, or
cardiac disease. Subjects were excluded from the study if they had
Figure 8. Ability of p53 to drive TLR8 expression in primary human cells depends on SNP in p53 response element of TLR8
promoter. The regulatory region of TLR8 was amplified by PCR and the product was analyzed for the presence of the A and/or the G version of the
p53 RE. Presented are SNP genotypes and the expression levels of TLR8 following 24 h of treatment with (A) nutlin, (B) IR, (C) Doxo and (D) 5FU. Since
the gene is X-linked, males have one copy and females have two copies of the allele. Presented are results with stimulated lymphocytes (black
symbols) or alveolar macrophages (red symbols).
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suffered a recent acute respiratory illness and were asked to avoid
exposure to air pollutants such as tobacco smoke and paint fumes.
A fiberoptic bronchoscope was wedged into a segmental bronchus
of the lingula. Six 50-ml aliquots of sterile saline were instilled and
immediately aspirated. The procedure was repeated on the right
middle lobe, again using 300 ml of saline. Samples were put on ice
immediately after aspiration and centrifuged at 300 x g for 10 min
at 4–8uC. Cells from all aliquots were pooled, washed twice with
RPMI 1640, and re-suspended in RPMI 1640 at 1.06106/mL.
Total yield was typically around 10–15 million cells or less.
Around 2.06106cells per well were seeded in a 12 well plate. After
2 hr, cells were washed twice with warm PBS and 2 ml of growing
media was added to each well. Cells were then treated with nutlin
or doxorubicin. Cells were harvested 24 hr post-treatment. The
protocol and consent form were approved by the University of
North Carolina School of Medicine Committee on the Protection
of the Rights of Human Subjects. Prior to participation in the
study, subjects were informed of the procedures and potential risks
and each signed a statement of informed consent.
Cancer cell cultures
H1299 lung cancer cells (American Type Culture Collection)
were routinely maintained following standard conditions and
procedures for culturing mammalian cells. All cultures were
incubated at 37uC with 5% CO2. p53 tetracycline inducible
SaOS2 TET-off cell lines expressing the wild-type or the G279E
mutant protein were cultured as described previously . p53
expression was kept ‘‘off’’ by 2 mg/ml doxycycline (Clontech). To
induce the p53 expression, cells were washed 3X with phosphate-
buffered saline (PBS) and placed in medium lacking doxycyline
during 24 h.
Reagents and treatment conditions
For drug treatment and p53 activation, cells were incubated
with doxorubicin (Sigma 0.3 mg/mL), nutlin3 (Sigma, 10 mM) and
5-fluorouracil (Sigma, 300 mM). For ionizing radiation treatment,
cells were irradiated at 1.56 Gy/min from a Shepherd cesium
irradiator in PBS at room temperature at final dose of 4 Gy.
Where indicated cells were also pretreated 2 h with p53 inhibitor
pifithrin-alpha (Sigma, 40 mM).
RNA isolation and gene expression analysis
Total RNA was isolated by RNEasy kit (Qiagen). Real-time
PCR was performed in triplicate with Taqman PCR Mix (Applied
Biosystems) in the 7000 ABI sequence Detection System (Applied
Biosystems). All human and mouse primers were purchased from
Applied Biosystems (information available upon request). For PHA
stimulated lymphocytes and alveolar macrophages expression of
TLR genes was normalized to 18S ribosomal RNA gene while for
freshly isolated CD3+ T cells, the glucuronidase-beta gene was
used for normalization.
Luciferase reporter assays
Pairs of complimentary oligonucleotides for the desired p53RE
from selected TLRs and containing restriction sites were cloned
into the open reading frame of firefly luciferase pGL4.26 plasmid
(Promega) previously double digested by Xho I/Kpn I restriction
enzymes. The identity of the inserts was confirmed by DNA
sequencing. Luciferase activity was measured 48 h after Fugene6-
mediated co-transfection of the TLR p53RE constructs in the
presence of p53 (pC53-SN3) or empty vector pCMV NEO-BAM3
along with pRL-TK Renilla as a transfection efficiency control
into p53 null H1299 cells, as previously described . Forty-eight
hours post-transfection extracts were prepared using the Dual
Luciferase Assay System (Promega) following the manufacturer’s
protocol and luciferase activity was measured on a Victor Wallac
multilabel plate reader (PerkinElmer). Relative luciferases activities
for each construct was defined as the mean value of the firefly
luciferase/Renilla luciferase rations obtained from 4 independent
experiments performed in triplicate.
Whole cell extracts were quantified using the Bradford protein
assay kit and gamma globulin as a reference standard (BioRad).
For TLR protein detection, cellular pellets were subjected to
subcellular protein fractionation (Thermo Scientific) following the
manufacturer’s instructions and protein was quantified using BCA
protein assay kit (Thermo Scientific). For TLR western blot
analysis ,30 mg of total membrane fraction was used, while for
the analysis of other proteins ,25 mg of total cell extract were
used. As expected, we did not detect TLR2 and TLR5 in the
cytosolic fractions; therefore, those data are not included. Proteins
were resolved on 4–12% BisTris NuPAGE and transferred to
polyvinylidene difluoride membranes (Invitrogen) and were
visualized with primary antibodies followed by horseradish
peroxidase–conjugated goat anti–mouse or donkey anti-goat
immunoglobulin (Santa Cruz Biotechnology) through the use of
enhanced chemiluminescence reagents (Amersham Biotechnolo-
gy). The primary antibodies used in these studies were against p53
(DO1, Santa Cruz Biotechnology,), p21 (SXM30, BD Biosciences
Pharmigen) and Actin (C-11 Santa Cruz Biotechnology). The
following is the list of TLR antibodies tested in this study in order
Figure 9. Model describing expression loop between p53 and
the TLR gene family in response to chromosome and inflam-
mation stresses. DNA metabolic stress is induced by both environ-
mental factors and endogenous sources, such as host-derived reactive
oxygen species (ROS) generated downstream of Toll-like Receptors and
pro-inflammatory cytokines. DNA damage/stress activates p53-depen-
dent and independent (‘‘X’’) pathways that in turn induce expression of
TLRs. Increased TLR expression sensitizes the cell to both exogenous,
pathogen-associated molecular patterns (PAMPs) and endogenous,
damage-associated molecular patterns (DAMPs) released during tissue
injury which, in this loop, lead to further ROS.
p53 Regulates Expression of TLR Genes
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to detect TLR protein expression in whole cell extracts as well as
membrane and cytosol protein fractions: TLR8 ab24185 and
TLR10 ab45088 from Abcam, Inc.; TLR1#2209, TLR2#2229,
TLR7#2633 and TLR9#2254 from Cell Signaling. We also used
a Toll-like receptor detection kit that includes antibodies for all
human TLRs (TLR1 to TLR10 antibodies from ProSci, Inc., as
well as TLR3-4H270 and TLR5-H1-27 antibodies from Santa
Cruz Bitoechnology. The TLR3-IMG-315A, TLR4-IMG6370A
antibodies were from IMGENEX. Only the TLR2 (Cell
Signalling) and TLR5 (Santa Cruz) gave clear results. Attempts
to detect other TLRs with these antibodies were unsuccessful and
appear to be a general problem with TLR antibodies from our
collective experience. One of the antibodies enabled us to detect
induction of full length TLR4 by nutlin; however, those results are
not presented due to the appearance in both untreated and treated
samples of nonspecific bands.
Chromatin Immunoprecipitation (ChIP) assays
ChIP assays were done as previously described  using ChIP
kits (Millipore). Approximately 406106PHA stimulated lympho-
cytes were used for each experimental sample. Cell lysates were
sonicated using conditions that yield chromatin fragments 200–
500 bp long. One microgram of DO-7 p53-specific monoclonal
antibody (BD Biosciences Pharmigen) was used per ChIP assay. As
a negative control, mouse Ig (Santa Cruz Biotechnology) was used.
PCR amplifications were performed on immunoprecipitated
chromatin using primers to amplify specific regions on the TLRs
promoters (sequence information available upon request). The
PCR cycles were as follows: initial 10 min Taq polymerase
(Invitrogen) at 95uC followed by 40 cycles of 95uC for 15 s and
60uC for 1 min. The PCR products were then run on a 1.8%
Flow cytometry analysis
Cells were resuspended in 100 ml of PBS and incubated with
5 ml of fluorescent antibody per sample for 30 min, then washed
and fixed with 0.5% paraformaldehyde. The fluorescence intensity
was evaluated using a Becton Dickinson LSR II Flow Cytometer.
All antibodies used for FACS were from BD Pharmigen.
SNPs were assessed by three different approaches. In RFLP
assays, genomic DNA was extracted from Percoll-isolated
lymphocytes by DNeasy kit (Qiagen). For the SNP in the TLR8
p53RE#6 (AGGCAAGATGAAACAT(G/C)TCA), the G-SNP
creates a unique restriction cutting site for NspI (R CATG Y).
PCR was performed with 100 ng of DNA, 50 pmol of each
primer, 1.5 mM MgCl2, 1 mL 106PCR buffer, and 0.0125 U of
Taq (Invitrogen). After 10 min at 94uC, 35 cycles were repeated as
follows: 94uC 30 s, 60uC 30 s, and 72uC 35 s; this cycling was
followed by a final extension at 72uC for 10 min. The PCR
product was digested with 5 U Nsp I (New England Biolabs,
Ipswich, MA), at 37uC for 4 h. Since Nsp I recognizes the
polymorphic sequence, a G allele is demonstrated by the presence
of two fragments 109 and 69 bp in a gel. The A allele is revealed
by the presence of a single 177 bp band. The following primers
were used for amplifying the region containing the p53RE on
TLR8 promoter region:
The status of this SNP was determined also by using a Taqman
SNP genotyping assay. All primers were purchased from Applied
Biosystems (Assay ID:C_27497635_10). For direct sequencing the
region containing the p53RE was first PCR amplified using the
following pairs of primers:
This was followed by running the samples on a TEA-agarose
gel. The expected product (397 bp) was cut out and cleaned using
QIAquick gel extraction kit (QIAGEN). The sequencing reactions
used Big dye (Applied Biosystems) per manufacturer recommen-
dations and the following primers:
Mouse primary cell culture
p53+/+ and p532/2 mouse embryonic fibroblasts (MEFs) were
cultured in DMEM media and 10% of FBS. Female C57BL/6
mice were purchased from Jackson Laboratories. All experiments
were performed in accordance with the Animal Welfare Act and
the U.S. Public Health Service Policy on Humane Care and Use
of Laboratory Animals after review of the protocol by the Animal
Care and Use Committee of the National Institute of Environ-
mental Health Sciences. For murine peritoneal macrophage
harvests, mice were injected i.p. with 2 ml of 4% Brewer’s
thioglycollate and euthanized 96 h later. The peritoneum was
washed with 10 ml ice cold PBS three times. Cells were
centrifuged (1,000x RPM, 6 minutes, 4uC) and washed twice with
sterile PBS. Peritoneal exudate macrophages were resuspended in
DMEM/0.1% FBS, counted, and plated at 26106cells/well in a
12-well plate. Cells were allowed to settle for 2 h (37uC/5% CO2)
before replacing media with DMEM complimented with 10%
For bone marrow-derived macrophages (BMM), marrow was
flushed from femoral and tibial bones using bone marrow media
(DMEM/2 mM L-glutamine/10% L929-conditioned medium/
10% FBS). Cells were spun down (2200x RPM, 5 min, 4uC),
brought up in 1 ml sterile ACK buffer, incubated 4uC for 1 min
after which 10 ml PBS was added. Cells were spun as above,
resuspended in bone marrow medium, counted, and plated at
16106cells/well in a 12-well plate. Cells were cultured at 37uC
and 10% CO2 in 2 ml bone marrow medium/well and fed on
Day 5 with addition of 1 ml medium/well. Experiments were
performed on Day 6. At 24 h post-treatment, cells were harvested
for RNA extraction.
each locus in the population sampled differed from 1 for the various
exposures, we applied one-sample Student’s t tests to log-transformed
values of mRNA fold change. The logarithmic transformation helps
the data meet the distributional assumptions for the t test. This
procedure, in effect, tests the null hypothesis that the geometric mean
mRNA fold change at the locus is equal to 1 against the two-sided
alternative that the geometric mean differs from 1.
peripheral blood of healthy subjects. Presented are FACS profiles
of peripheral blood mononuclear cells (PBMC) obtained by percoll
gradient separation from subject BS#8. Cells were either
stimulated by PHA, yielding predominantly T-lymphocytes (A)
or (B) the CD3+ cells were isolated from PBMC by magnetic beads
conjugated to anti-CD3 antibody. The amount of monocytes in
these preparations based on CD14 staining did not exceed 1.4%.
Cells were stained with fluorescent anti-CD3 antibody to visualize
Characterization of cell populations obtained from
p53 Regulates Expression of TLR Genes
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surface marker expression. The X-axis identifies the CD3
Found at: doi:10.1371/journal.pgen.1001360.s001 (0.54 MB TIF)
human T-lymphocytes via DNA stressors and activation of the p53
pathway. Freshly isolated human peripheral blood mononuclear
cells (PBMC) from healthy subjects (n=17–18) were incubated with
PHA to stimulate T-lymphocyte expansion. After 48 h, cells were
exposed to (A) ionizing radiation (IR); (B) 5-fluorouracil (5FU); (C)
doxorubicin or (D) nutlin. Cells were harvested following 24 h
treatment (24 h after IR exposure). Presented in panels A–D are
mRNA expression levels as compared to expression in untreated
cells (the dashed line is a value of 1; i.e., no change) measured by
qPCR and normalized to 18S ribosomal RNA. In this ‘‘boxes and
whiskers’’ diagram, the limits of the 2nd and 3rd quartiles of
observed values (i.e., the middle 50% of observations)are at the ends
of the box; the median is the horizontal line within the box; ‘‘+’’ is
the average. Maximum and minimum values are at the ends of the
‘‘whiskers’’ and outliers (see Figure 1) are presented as dots. For
TLR3 only subjects that expressed TLR3 (see Figure 1) are
presented. The outliers were determined using the method of
Grubbs (also called the Extreme Studentized Deviate, or ESD,
method which determinesa value that is unlikely to havecome from
the same Gaussian population as the other values in the group
[Barnett et al.]. There is little difference in the expression of
housekeeping genes GUSB and actin following treatment with
doxorubicin (S2E) or nutlin (S2F). (Only 14 of the 18 subject
samples in Figure 1 had sufficient material to be able to include
GUSB and actin analysis.) Furthermore, for TLR genes that are not
responsive to DNA damage or p53 induction (i.e., TLR1 with nutlin
treatment), there is a nearly constant ratio across the individuals
tested, as seen in these graphs. The basal levels of expression of the
TLR genes, while low, are clearly measurable. [Barnett V, Lewis T,
Rothamsted V. ‘‘Outliers in statistical data’’ in the Wiley Series in
Probability and mathematical statistics. Applied Probability and
Statistics, John Wiley & Sons, 1994.]
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Induced expression of TLR gene family in primary
damage stress and p53 activation in PHA-stimulated human T
lymphocytes. A modified microarray heat map graphic style was
used to represent the fold-change in mRNA expression for TLR
and P21 genes in PHA-stimulated human lymphocytes from 18
subjects (BS#) after 24 h of treatment with nutlin, ionizing
radiation, 5-fluorouracil (5FU) or doxorubicin at the doses
indicated. The subjects were grouped by colors according to their
responses to nutlin as indicated in the top part of each heatmap.
An arbitrary cut-off value for induction, no change and repression
was based on P21 gene expression responses to nutlin treatment.
BS#1 sample was not treated with nutlin (yellow squares). The
genotype for the SNP associated with the putative p53RE in the
promoter of TLR8 is also presented for each subject.
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Heat map presentation of TLR expression after DNA
lymphocytes from 18 healthy subjects. T-lymphocytes generated
from PBMC, as described above, were treated with nutlin
(10 mM); doxorubicin (Doxo, 0.3 mg/mL); 5-fluorouracil (5FU,
300 mM); ionizing radiation (IR, 4Gy); DMSO (vehicle control) or
left untreated (NT). Following 24 h treatment, cells were harvested
and subjected to Western blot analysis. Activation of the p53
pathway was assessed by induction of p53 and p21 protein levels.
Actin levels provided loading control. The ‘‘BS#’’ corresponds to
individual subjects as described in Figures S2 and S3.
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Activation of p53 pathway in PHA-stimulated T-
TLR gene expression was assessed by real-time PCR as previously
described  using SaOS2 TET-off inducible cell lines expre-
ssing WT or the transcriptionally inactive mutant G279E p53
protein after removing doxycycline from the media to induce p53.
The gray bar corresponds to cells grown in the presence of
doxycycline (i.e., no p53 expression). Presented is the mRNA fold-
change compared to the parental SaOS2 TET off cells lacking p53
expression vectors, represented by the dashed line, i.e., a value of 1.
Each bar represents an average of 3 replicates with its standard
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TLR expression in p53 inducible SaOS2 cell line.
CD3+ cells to stimulation by TLR2 ligand PAM3CSK4. Presented is
cytokine gene expression in isolated CD3+ lymphocytes incubated for
20 h with nutlin or DMSO and then exposed for 4 h to the TLR2
ligand PAM3CSK4 (1 mg/ml). Gene expression changes for
lymphocytes from subjects BS#19, #22, #26, and #29 were
analyzed by qPCR and are presented as fold-change compared to
untreated cells. Each bar represents an average of 2 replicates.
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Induction of p53 pathway sensitizes freshly isolated
response to DNA stressors and activation of the p53 pathway. A
panel of mouse cells was exposed to doxorubicin (0.3 mg/ml); 5-
fluorouracil (5FU, 300 mM); ionizing radiation (IR, 4Gy); nutlin
(10 mM); DMSO (vehicle control); transfected with p53 expression
plasmid or left untreated (NT). TLRs and P21 gene expression
were analyzed by qPCR and presented as fold-change compared
to untreated cells in (A) peritoneal macrophages, (B) bone marrow-
derived macrophages, (C) MEF p53 +/+, and (D) MEF p53 2/2.
Presented is the average of 2 or 3 experiments. Each experiment
was done in triplicate; presented is the standard deviation.
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Expression of the TLR gene family in mouse cells in
for putative p53 response elements that are predicted to be
functional in the human toll-like receptor genes. Presented are
multispecies alignments for 9 primates (blue box) and 8 rodents (red
box). Sequence alignments were derived from the BLAT alignment
tool of the USC genome browser (Human Feb. 2009 (GRCh37/
hg19); http://genome.ucsc.edu/). The human p53 response
element flanked by 10 bp was used as reference sequence. For the
human reference p53 RE sequence (second line), mismatches with
respect to the commonly accepted p53 consensus binding sequence
(p53CBS) site are in red characters. For each species, the characters
highlighted in red are mismatches relative to the reference human
p53RE sequence. Mismatches relative to the reference human
p53RE sequence but matches relative to the p53CBS are shown in
highlighted characters. Highlighted in green are base transition
changes (i.e., purine to purine or pyrimidine to pyrimidine) relative
to the human p53RE, while highlighted in blue are changes relative
to a mismatch in the original human p53RE that restore the base
relative to the p53CBS. Characters in light blue color are the bases
corresponding to the spacer between p53 decamers. Mismatches
versusthehuman referencespacersequence arehighlighted inpink.
Based on display chains between alignment configurations from the
USC genome browser, which enables the display of gaps between
alignment blocks in the pairwise alignments, the following
conventions are used: 1) Single dashed line: no bases in the aligned
species. This may be due to a lineage-specific insertion between
the aligned blocks in the human genome or a lineage-specific
deletion between the aligned blocks in the aligning species. 2)
Double dashed line: aligning species has one or more unalienable
bases in the gap region. This may be due to excessive evolutionary
Sequence conservation between primates and rodents
p53 Regulates Expression of TLR Genes
PLoS Genetics | www.plosgenetics.org14 March 2011 | Volume 7 | Issue 3 | e1001360
distance between species or independent indels in the region Download full-text
between the aligned blocks in both species. 3) Blank space: sequence
is not available in the database for the corresponding species. ‘‘N’’
denotes a gap in the genomic sequence. Additional details: 4) SNPs
in the p53RE arehighlighted inlight green.Forp53REscontaining
SNPs, additional information about the SNP frequencies are
presented. 5) The corresponding chromosome coordinates for each
p53 RE are provided. For the TLR5, TLR6 and TLR9 genes,
different transcription start sites (TSS) of the associated transcripts
are shown (Ensembl, current release 57 - Mar 2010, http://www.
Found at: doi:10.1371/journal.pgen.1001360.s008 (0.21 MB PDF)
We thank Andrew J. Ghio, MD, for providing alveolar macrophages and
Joyce Snipe, Brenda Yingling, Annette Rice, and Jamie Marshburn for
excellent technical assistance. We greatly appreciate the statistical analysis
by Dr. David Umbach and further advice provided by Dr. Pierre Bushel.
Conceived and designed the experiments: DM MS SG MBF MAR.
Performed the experiments: DM MS KA. Analyzed the data: DM MS SG
MBF MAR. Contributed reagents/materials/analysis tools: DM SG MBF
MAR. Wrote the paper: DM MS SG MBF MAR.
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