Acquisition of Adult-Like TLR4 and TLR9 Responses
during the First Year of Life
Muriel Nguyen1., Elke Leuridan2., Tong Zhang1, Dominique De Wit1, Fabienne Willems1, Pierre Van
Damme2, Michel Goldman1, Stanislas Goriely1*
1Institute for Medical Immunology, Universite ´ Libre de Bruxelles, Gosselies, Belgium, 2Centre for the Evaluation of Vaccination, Vaccine and Infectious Diseases Institute,
University of Antwerp, Wilrijk, Belgium
Background: Characteristics of the human neonatal immune system are thought to be responsible for heightened
susceptibility to infectious pathogens and poor responses to vaccine antigens. Using cord blood as a source of immune cells,
many reports indicate that the response of neonatal monocytes and dendritic cells (DC) to Toll-like receptor (TLR) agonists
differs significantly from that of adult cells. Herein, we analyzed the evolution of these responses within the first year of life.
Methodology/Principal Findings: Blood samples from children (0, 3, 6, 9, 12 month old) and healthy adults were stimulated
ex vivo with bacterial lipopolysaccharide (LPS, TLR4 agonist) or CpG oligonucleotides (TLR9 agonist). We determined
phenotypic maturation of monocytes, myeloid (m) and plasmacytoid (p) DC and production of cytokines in the culture
supernatants. We observed that surface expression of CD80 and HLA-DR reaches adult levels within the first 3 months of life
for mDCs and 6–9 months of life for monocytes and pDCs. In response to LPS, production of TNF-a, IP-10 and IL-12p70
reached adult levels between 6–9 months of life. In response to CpG stimulation, production of type I IFN-dependent
chemokines (IP-10 and CXCL9) gradually increased with age but was still limited in 1-year old infants as compared to adult
controls. Finally, cord blood samples stimulated with CpG ODN produced large amounts of IL-6, IL-8, IL-1b and IL-10, a
situation that was not observed for 3 month-old infants.
Conclusions: The first year of life represents a critical period during which adult-like levels of TLR responses are reached for
most but not all cytokine responses.
Citation: Nguyen M, Leuridan E, Zhang T, De Wit D, Willems F, et al. (2010) Acquisition of Adult-Like TLR4 and TLR9 Responses during the First Year of Life. PLoS
ONE 5(4): e10407. doi:10.1371/journal.pone.0010407
Editor: Dominik Hartl, Ludwig-Maximilians-Universita ¨t Mu ¨nchen, Germany
Received December 18, 2009; Accepted March 30, 2010; Published April 28, 2010
Copyright: ? 2010 Nguyen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The Institute for Medical Immunology is sponsored by the government of the Walloon Region and GlaxoSmithKline Biologicals. This study was
supported by the Fonds National de la Recherche Scientifique (FNRS, Belgium) and an Interuniversity Attraction Pole of the Belgian Federal Science Policy. S.G. is a
research associate of the FRS-FNRS. E.L. received a research grant from the University of Antwerp for the conduction of the longitudinal study on maternal
antibodies. 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 characteristics of immune responses in early life are often
held responsible for heightened sensitivity towards infectious
agents and suboptimal responses to vaccination [1,2]. Neonatal
CD4+T cells are indeed unable to mount efficient Th1-type
responses to most stimuli with the exception of BCG vaccine
[3,4]. As recently reviewed , multiple reports have explored
the function of innate immune cells at birth. The capacity of
neonatal monocytes and dendritic cells (DC) to produce cytokines
in response to Toll-like receptor (TLR) agonists differs signifi-
cantly from that of adult cells. Several reports have noted that
production of TNF-a is impaired in early life. This defect is
observed only in certain experimental conditions. It was initially
described in cord blood from preterm infants . More recently,
decreased TNF-a/IL-6 ratio at birth in response to specific TLR
ligands was linked to high adenosine levels in cord blood plasma
. It has also long been noted that production of IL-10 is
elevated in LPS-stimulated cord blood in comparison to adult
samples, which can also down-modulate the production of other
In terms of signaling pathways, neonatal cells were shown to
respond in a qualitatively different manner. TLR4 is the critical
receptor of LPS and is expressed on myeloid cells. TLR4 is coupled
to adaptor proteins that lead to distinct signaling pathways. The
‘‘myeloid differentiation factor 88 (MyD88)-dependent’’ pathway is
generally similar to adults in neonatal cells, although lower MyD88
expression has also been reported in cord blood monocytes and
neutrophils [10,11]. In contrast, the ‘‘MyD88-independent’’
pathway involving TIR-containing adaptor inducing interferon
IFNb (TRIF) and the transcription factor interferon regulatory
factor (IRF)-3  was shown to be less active in early life. Indeed,
impaired interaction of IRF-3 with the coactivator CREB binding
protein (CBP) in neonatal blood cells exposed to LPS was associated
with impaired expression of IFNb, IFN-inducible genes (such as
CXCL10) and bioactive IL-12p70 .
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Plasmacytoid DCs (pDCs) represent a major source of type I
IFNs especially in the course of viral infections or exposure to
TLR7 or TLR9 ligands. Although pDCs are present in significant
numbers in human cord blood, there is evidence that they produce
less IFN-a upon exposure to unmethylated CpG-rich oligonucle-
otides (CpGs) . At the molecular level, this observation was
linked to impaired nuclear translocation of IRF-7 .
Due to ethical and technical limitations, essentially all these
observations were done using cord blood as a source of neonatal
immune cells. Very few studies analyzed the evolution of innate
immune cells function in the first months of life. This has major
implication in terms of vaccination strategy as TLR4 agonists are
now used in newly-developed vaccines targeting this age group.
Reports indicate that the capacity of mononuclear cells to produce
adult-like levels of cytokines in response to LPS/IFN-c is reached
only later in life [8,16].
Herein, we assessed different parameters of the response of
monocytes and dendritic cell subsets to LPS and CpG during the
first year of life. We used a ‘‘whole-blood’’ assay in order to take
into account the possible implication of plasma factors on TLR
responses. We observed a stepwise development of the response to
TLR4 and TLR9 stimulation during this period.
Materials and Methods
A prospective multi-centre study was conducted in the Province
of Antwerp, Belgium, in accordance with the Helsinki Declaration
and procedures established by Belgian law. The study was
approved by the local ethic committees (University Hospital
Antwerp, Wilrijk, St Vincentius ziekenhuis vzw, Antwerp, St
Augustinus ziekenhuis, Wilrijk and ZNA middelheim ziekenhuis,
Antwerp). The main goal was the description of the duration of
maternal antibodies against measles in two groups: children born
from vaccinated women or from women with naturally acquired
immunity (Leuridan E, Hens N, Hutse V, Ieven M., Aerts M, Van
Damme P. How long are neonates protected by maternal
antibodies? ESPID 2009, Brussels, abstract nu 728). Written
informed consent was obtained from all participants and from
both parents for the participating infants. Healthy pregnant
women and their healthy offspring were included starting April
2006 and follow-up lasted until November 2008. Exclusion criteria
were impaired immunology in mother or child, administration of
immunoglobulins or blood products during the study period,
preterm delivery (,36 weeks) and low birth weight (,2400 g). A
questionnaire was completed on demographics, validated vacci-
nation history and medical history. Growth parameters, breast-
feeding, day-care attendance, immunization data, episodes of
illness and medication used were registered for the participants at
each visit, as well as medical histories for all household members.
Additionally to the phlebotomy planned for the maternal antibody
study, extra venous whole blood was collected from cord blood
(10 ml, n=13) and in some infants (0.5–2 ml) at 3 months (84–99
days, n=20), 6 months (175–189 days, n=8), 9 months (267–282
days, n=9) and 12 months (358–372 days, n=14). Healthy adults
and mothers (3 months post-partum) were also included (n=10
and n=20, respectively). All samples, except for cord blood and
adult controls, were collected during home visits. All women and
children were Caucasian. Parameters at birth were within the
normal ranges for gestational age, weight and length at birth. as
expected due to the exclusion criteria (see table 1). The weight and
length were followed though the complete study as well as type of
feeding. Almost all children received breastfeeding with a mean
duration of 15 weeks. Due to limited amount of blood, all
parameters were not measured in all samples. Hence, for some
parameters, values obtained from different age groups were pooled
as indicated in the legends of the figures.
All samples were stimulated within 5 h after blood collection.
Whole blood (200 ml) was incubated at 37uC with PBS (8 ml), LPS
(20 ng/ml, Sigma-Aldrich, Bornem, Belgium. We assessed the
specificity for TLR4 by gene reporter assay in 293 cells stably
transfected with TLR2 and TLR4) or a combination of CpG
ODN 2006 and 2216 (25 mg/ml each, synthesized by Tib Molbiol,
Berlin, Germany). After overnight incubation (16–18 h), culture
supernatant was collected by centrifugation and stored at 220uC
for cytokine determination. Blood cells were resuspended in PBS
for further FACS analysis.
To analyze the phenotype of circulating DC, whole blood cells
were first pre-incubated with FcR-blocking reagent (6 ml/200 ml,
Miltenyi Biotec, Bergisch Gladbach, Germany) for 10 min and then
for CD3, CD19, CD20 and CD56 (2 ml each), 3 ml of allophycocya-
nin (APC)-conjugated anti-CD11c mAb, 2.5 ml PeCy5-conjugated
CD123, 2.5 ml of Pacific blue-conjugated CD14 and 3 ml PE-
conjugated anti-CD80 (all from Becton Dickinson, Mountain View,
CA) and 3 ml of PE/Texas Red-conjugated anti-HLA-DR mAb
(Immunotech, Marseilles, France). After this first incubation, blood
cells were incubated for 10 min at room temperature in the dark with
3 ml of FACS Lysis 1X (Becton Dickinson). Cells were then
centrifuged at 1500 rpm for 10 min and resuspended in 200 ml
Cytofix buffer (Becton Dickinson). Samples were then analyzed using
an LX-9 cytometer (Dako Cytomation). Compensation beads
(CompBeads, BD biosciences) were used for each experiment to
standardize voltage settings and to generate a compensation matrix.
IL-1b, IL-6, IL-8 TNF-a, IFN-c, IL-12p70, IP-10, IL-10 and
CXCL9 (MIG) levels in samples were diluted 1/4 (and 1/10 or 1/
20 to fall within the range of the standard curve) and assessed in
Table 1. General characteristics of the participants.
Mean age in years (SD) 29 (4)
Vaccinated against measles 13
Naturally immune to measles17
Mean gestational age (95% CI) 39 weeks (60,012)
Mean birth weight in grams (SD)3182 (466) g
Mean length at birth in cm (SD)50(61)
Breastfeeding (mean duration)15 weeks
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duplicate by multiplex bead array from Bio-Rad (Bio-Plex Pro
human cytokine, Bio-Rad Laboratories, Hercules, CA) according
to manufacturer’s instructions. Assays were read on the Bio-Plex
200 system (Bio-Rad).
All data were analyzed using the Graphpad Prism software.
Cytokine and flow cytometry data were compared using one-way
ANOVA (using the non parametric Kruskal-Wallis test) with
Dunns post test for multiple comparison using adult values as a
reference. All p values are two-sided and were considered
significant when p,0.05.
Surface expression of CD80 and HLA-DR molecules
reaches adult levels within the first 3 months of life for
monocytes and mDCs and 6–9 months of life for pDCs.
We first evaluated the phenotype of circulating APCs in samples
obtained at birth, within the first year of life and from adult
controls. Using 6-color flow cytometry, we analyzed the expression
of CD80 and HLA DR molecules at the surface of monocytes
(Lin-, CD14+, CD11c+), mDCs (Lin-, CD14-, CD11c+, CD123-)
and pDCs (Lin-, CD14-, CD11c-, CD123+). We first noted that in
cord blood, basal expression of HLA-DR molecules on monocytes,
mDCs and pDCs was lower than in adult samples (Fig. 1). Within
the first 6 months of life, basal HLA-DR expression gradually
increased at the surface of these three populations. In LPS and
CpG-stimulated adult samples, we observed a further increase in
HLA DR expression at the surface of monocytes, mDCs and
pDCs. For mDCs, adult-level HLA-DR expression was already
reached in 3-month old infants. In contrast, for both monocytes
and pDCs, HLA DR levels were significantly decreased in
stimulated samples from 3-month old infants as compared to the
adult group. HLA DR upregulation upon TLR4 or TLR9
stimulation in older infants (.6months) was comparable to adults.
Basal CD80 expression was low in all age groups (Fig. 2). We
observed marked upregulation of CD80 surface expression on
monocytes and mDCs from LPS and CpG-treated adult samples.
For pDCs, CpG but not LPS treatment led to an increase of CD80
expression in adult samples. This phenotypic maturation was
strongly reduced in cord blood samples. For both monocytes and
mDCs, LPS- and CpG-mediated upregulation of CD80 expression
was comparable to adult cells for 3-, 6-, 9- and 12-months old
infants. In contrast, CpG-mediated phenotypic maturation of
pDCs did not reach adult-levels before the age of 6 months. These
results indicate that circulating APCs acquire adult-like phenotype
during the first 6 months of life. These data also suggest that this
maturation process is more rapid for mDCs and monocytes than
Age-dependent upregulation of LPS-induced TNF-a
IP-10, IL-12p70 and IFN-c production.
We next analyzed LPS-stimulated cytokine production in whole
blood. As previously described [6,8,9,13,17–19], we noted that
cord blood cells produced reduced amounts of TNF-a, IP-10, IL-
12p70 and IFN-cas compared to adult controls (Fig. 3). Adult-
levels were reached at 6 months of age for TNF-a and IL-12p70.
For IP-10, production significantly increased in samples collected
from 3- and 6-month infants but only reaches adult levels at 9
months of age. Interestingly, IFN-c production in response to LPS
was still reduced at 1 year of age as compared to adult samples.
These results indicate that the reduced capacity of cord blood cells
to produce these inflammatory cytokines is not restricted to the
neonatal period. However, under these experimental conditions,
adult levels were reached rapidly during infancy.
The capacity to produce type I IFN-dependent
chemokines in response to CpGs is acquired
progressively during the first year of life
We next evaluated the production of cytokines induced by
TLR9 agonists. We noted that production of two type I IFN-
inducible chemokines, IP-10 and MIG, was strongly reduced in
cord blood as compared to adult samples (Fig. 4). This is consistent
with the fact that cord blood pDCs display a major defect in their
capacity to produce type I IFNs . We observed an age-
dependent increase in the production of IP-10 and MIG but levels
at 1 year of age were still significantly lower than that of adult
controls. Taken together with phenotypic markers, this result
suggests that maturation of pDCs function is delayed compared to
that of LPS-responsive cells such as monocytes and mDCs.
Hyperproduction of IL-6, IL-8 and IL-10 in the first
months of life.
In line with previous reports [7,8,19], we observed that
production of several cytokines, including IL-6, IL-8 and IL-10
was significantly increased in LPS-stimulated cord blood as
compared to adult samples (Fig. 5). For IL-6, production
decreased to adult levels in the group of 3-month old infants
and remained low in the other groups. For IL-10, production was
still significantly higher in 6-, 9- and 12-month old infants as
compared to adults. A similar persisting trend was also observed
for IL-8 but variations between individuals were very important in
these groups. Finally, no difference between the age groups was
noted for IL-1b production.
Activation of adult blood through TLR9 induces low levels of
inflammatory cytokines such as IL-6 and IL-8. Unexpectedly, very
high levels of these cytokines were detected in CpGs-stimulated
cord blood samples. This was accompanied by increased
production of IL-1b and IL-10. Hyper-responsiveness to CpGs
was found to be transient as it was not observed in samples
collected from 3-month or older infants.
Over the last few years evidence has accumulated indicating
that innate immune responses of newborns and adults differ
significantly. Several mechanisms may contribute to these
observations. It is therefore difficult to identify the developmental
processes that will lead to the acquisition of adult-like responses.
Herein, we focused on specific aspects of neonatal TLR responses
that have previously been analyzed using cord blood. As
summarized in table 2, we analyzed the ontogeny of these
parameters over the first year of life. A consistent finding is the
decreased capacity of cord blood-derived cells to produce bioactive
IL-12p70 by monocytes and mDCs. In response to LPS, we
detected low but reproducible IL-12p70 production in adult
samples. By the age of 6 months, adult levels of IL-12p70 were
observed in some LPS-stimulated samples. Our data is in line with
a recent report that indicates that LPS+IFNc-induced IL-12p70
production in whole blood was significantly increased between
birth and 1 month of age but still low compared to adult samples
. A previous report indicated that decreased capacity to
produce IL-12p70 was observed throughout childhood (samples
from 5- and 12-yr-old children were analyzed) . There are
major differences in the design of the experiments that could
account for this apparent discrepancy. In their work, Upham et al
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Figure 1. HLA DR expression on the surface of circulating monocytes, myeloid and plasmacytoid DCs. Blood samples were incubated
with PBS or the indicated stimulant. The different subpopulations were gated as described in the Material and Methods section. Expression was
compared in samples from the different age groups: Cord blood (CB, n=10), 3-month (3 m, n=10), 6&9-month (6–9 m pooled data from n=4 and 8,
respectively), 12-month (12 m, n=9) old infants and healthy adults (n=16). Data are represented as median+interquartile range. *p,0.05, **p,0.01,
***p,0.001 as compared to stimulated adult samples.
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Figure 2. CD80 expression on the surface of circulating monocytes, myeloid and plasmacytoid DCs. The different subpopulations were
gated as described in the Material and Methods section. Expression was compared in samples from the different age groups: Cord blood (CB, n=10),
3-month (3 m, n=10), 6 and 9-month (6–9 m, pooled data from n=4 and 8, respectively), 12-month (12 m, n=9) old infants and healthy adults
(n=16). Data are represented as median+interquartile range. *p,0.05, **p,0.01, ***p,0.001.
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Figure 3. Age-dependent upregulation of LPS-induced production of TNF-a, IP-10, IL-12p70 and IFN-c. Whole blood samples were
stimulated with LPS and culture supernatants were collected after 16–18 h. Production in the different infant groups was compared to adult values.
Cord blood (CB, n=13), 3-month (3 m, n=15), 6-month (6 m, n=8), 9-month (9 m, n=9), 12-month (12 m, n=9) old infants and healthy adults
(n=10). Data are represented as median+interquartile range. *p,0.05, **p,0.01, ***p,0.001 as compared to stimulated adult samples.
Figure 4. Age-dependent upregulation of CpG-induced production of IP-10 and MIG. Whole blood samples were stimulated with CpG
A+B combination and culture supernatants were collected after 16–18 h. Production in the different infant groups was compared to adult values.
Cord blood (CB, n=13), 3-month (3 m, n=10), 6&9-month (6 m, n=3, 9 m, n=5), 12-month (12 m, n=11) old infants and healthy adults (n=10).
Data are represented as median+interquartile range. *p,0.05, **p,0.01, ***p,0.001 as compared to stimulated adult samples.
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analyzed IL-12p70 production in isolated PBMC, cultured in
FCS-containing medium and stimulated by LPS and IFNc
combination. Here, we analyzed IL-12p70 production in LPS-
stimulated whole blood. Multiple parameters that differ between
these experiments could affect the capacity of APCs to produce IL-
12p70. For example, circulating plasma factors and the presence
of red blood cells affect cytokine production [5,22]. The cellular
source of IL-12 could also differ. Indeed, when monocytes are
primed with IFN-c, they gain the ability to produce IL-12p70
through upregulation of IRF1 and IRF8 expression [23,24].
Taken together, these data indicate that the capacity of circulating
APCs to produce IL-12p70 in response to LPS gradually increases
within the first months of life. However, this might not reflect the
capacity of cells to produce IL-12 under more intense stimulation
conditions. Reduced production of IFN-c in response to LPS was
observed throughout the first year of life. This is consistent with
the fact that it reflects both the capacity of TLR4-expressing cells
to produce IL-12 and of lymphocytes and NK cells to produce
IFN-c. Indeed, upon direct stimulation of lymphocytes by
phytohemagglutinin, production of IFN-c was found to be low
in children until the age of 18 months .
High circulating adenosine levels contribute to the low capacity
of cord blood cells to produce TNF-a in response to LPS . We
observed that by the age of 6 months, TNFa production reached
adult levels, suggesting that alteration in plasma factors last for
more than 3 months after birth. Differences in plasma factors also
contribute to low IP-10 production in LPS-stimulated cord blood.
However, we showed that cell intrinsic factors are also important.
Indeed, we previously showed that incomplete IRF3 activation in
cord blood cells led to decreased IFNb production and induction
of IFN-dependent genes such as IP-10 . IP-10 production
reached adult levels by the age of 9 months. This result suggests
that the differences in signalling pathways are gradually overcome
before that age.
We previously reported that neonatal pDCs produce less type I
IFNs . As a consequence, IFN-inducible genes, such as IP-10
and MIG levels are strongly reduced upon TLR9 stimulation of
cord blood. With age, we observe a gradual increase in production
of these 2 chemokines. However, in 1-year old infants, levels were
still significantly lower than in adults. This result strongly suggests
that pDC reach adult-like function later in life than monocytes or
mDCs. We observed a similar trend for phenotypic markers
As previously reported [8,19,26], we observed high production
of IL-6, a pleiotropic cytokine, in cord blood samples as compared
to adult samples. LPS-induced IL-6 production was comparable to
adult levels by the age of 3 months. In contrast, we detected high
IL-8 and IL-10 production in some samples from 3- to 12-month
old infants. We observed a dramatic increase in IL-6, IL-8, IL-10
and IL-1b production in CpG-stimulated cord blood but not
samples from older infants or adults. Interestingly, production of
these cytokines by purified cord blood pDCs was not found to be
increased . This result suggests that an alternative cellular
source of these cytokines could be more responsive to TLR9
stimulation (in a direct or indirect fashion) in cord blood. Cytokine
production was found to be affected by the mode of delivery
[27,28]. However, no differences in CpG-induced cytokine levels
were observed between children that were delivered naturally or
through C-section. This transient overexpression of these inflam-
matory cytokines should be kept in mind in the context of
vaccination of young infants with TLR ligand-containing
adjuvants, such as monophosphoryl lipid A (MPL).
In conclusion, our study indicates that most of the parameters of
TLR responses we analyzed progressively reach adult-like levels
within the first year of life. Clearly, intra-uterine environment
conditions the function of APCs. It remains unknown whether
‘‘maturation’’ of these responses reflects the normal turn-over of
APCs and the disappearance of these immunomodulators or if it is
an active process that requires education of immune cells by
environmental exposure to microbial compounds. It would
therefore be helpful to assess the impact of specific factors
(vaccination, intercurrent infections, atopic background etc.) and
Figure 5. Hyperproduction of specific cytokines in early life. Whole blood samples were stimulated with the indicated TLR ligand and culture
supernatants were collected after 16–18 h. Production in the different infant groups was compared to adult values. Cord blood (CB, n=13), 3-month
(3 m, n=10), 6&9-month (6 m, n=3, 9 m, n=5), 12-month (12 m, n=11) old infants and healthy adults (n=10). Data are represented as
median+interquartile range. *p,0.05, **p,0.01, ***p,0.001 as compared to stimulated adult samples.
Table 2. Summary of the main results.
ParameterStimulation Response in CB Reaches Adult level at:
CD80/DR expression (Monocytes)LPS
CD80/DR expression (mDC)LPS
CD80/DR expression (pDC)CpG
TLR Responses in Early Life
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settings (resource-rich vs. developing countries) on the ontogeny of Download full-text
TLR responses in early life.
The authors would like to thank all participating women and their lovely
children. We thank Aline Bontenakel for the very valuable technical
support in taking the blood samples.
Conceived and designed the experiments: DDW FW PVD MG SG.
Performed the experiments: MN TZ DDW. Analyzed the data: MN SG.
Wrote the paper: EL SG. Recruited the patient cohort: EL.
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TLR Responses in Early Life
PLoS ONE | www.plosone.org9 April 2010 | Volume 5 | Issue 4 | e10407