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Age-Dependent Maturation of Toll-Like Receptor-
Mediated Cytokine Responses in Gambian Infants
Sarah Burl
1,2
*, John Townend
3
, Jainaba Njie-Jobe
1
, Momodou Cox
1
, Uche J. Adetifa
1
, Ebrima Touray
1
,
Victoria J. Philbin
4
, Christy Mancuso
4
, Beate Kampmann
1,2
, Hilton Whittle
1
, Assan Jaye
1
, Katie L.
Flanagan
1.
, Ofer Levy
4.
1Infant Immunology, Medical Research Council (UK) The Gambia, Fajara, The Gambia, 2Department of Paediatrics, Imperial College London, Paddington, London, United
Kingdom, 3Statistics Department, Medical Research Council (UK) The Gambia, Fajara, The Gambia, 4Division of Infectious Diseases, Department of Medicine, Children’s
Hospital Boston and Harvard Medical School, Boston, Massachusetts, United States of America
Abstract
The global burden of neonatal and infant mortality due to infection is staggering, particularly in resource-poor settings.
Early childhood vaccination is one of the major interventions that can reduce this burden, but there are specific limitations
to inducing effective immunity in early life, including impaired neonatal leukocyte production of Th1-polarizing cytokines to
many stimuli. Characterizing the ontogeny of Toll-like receptor (TLR)-mediated innate immune responses in infants may
shed light on susceptibility to infection in this vulnerable age group, and provide insights into TLR agonists as candidate
adjuvants for improved neonatal vaccines. As little is known about the leukocyte responses of infants in resource-poor
settings, we characterized production of Th1-, Th2-, and anti-inflammatory- cytokines in response to agonists of TLRs 1-9 in
whole blood from 120 Gambian infants ranging from newborns (cord blood) to 12 months of age. Most of the TLR agonists
induced TNFa, IL-1b, IL-6, and IL-10 in cord blood. The greatest TNFaresponses were observed for TLR4, -5, and -8 agonists,
the highest being the thiazoloquinoline CLO75 (TLR7/8) that also uniquely induced cord blood IFNcproduction. For most
agonists, TLR-mediated TNFaand IFNcresponses increased from birth to 1 month of age. TLR8 agonists also induced the
greatest production of the Th1-polarizing cytokines TNFaand IFNcthroughout the first year of life, although the relative
responses to the single TLR8 agonist and the combined TLR7/8 agonist changed with age. In contrast, IL-1b, IL-6, and IL-10
responses to most agonists were robust at birth and remained stable through 12 months of age. These observations
provide fresh insights into the ontogeny of innate immunity in African children, and may inform development of age-
specific adjuvanted vaccine formulations important for global health.
Citation: Burl S, Townend J, Njie-Jobe J, Cox M, Adetifa UJ, et al. (2011) Age-Dependent Maturation of Toll-Like Receptor-Mediated Cytokine Responses in
Gambian Infants. PLoS ONE 6(4): e18185. doi:10.1371/journal.pone.0018185
Editor: Nick Gay, University of Cambridge, United Kingdom
Received November 25, 2010; Accepted February 22, 2011; Published April , 2011
Copyright: ß2011 Burl 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: This work was supported by the Medical Research Council (MRC, UK, www.mrc.ac.uk), The Gambia and by National Institutes of Health (NIH) RO1
AI067353-01A1 and Bill & Melinda Gates Foundation Grand Challenge Explorations and Global Health awards (to OL). The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The laboratory of OL receives sponsored research support from VentiRx Pharmaceuticals, which manufactures certain TLR8 agonists
although distinct from those studied in this manuscript. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.
There are no other known competing interests.
* E-mail: s.burl@imperial.ac.uk
.These authors contributed equally to this work.
Introduction
The greatest burden of morbidity and mortality from infectious
diseases occurs in children under 5 years of age, with the highest
rates occurring in resource-poor countries. This urgent global issue
is the target of United Nations Millennium Goal 4 to reduce
under-five child mortality by two-thirds by 2015 [1]. One of the
most effective measures to prevent infection is vaccination early in
life, in particular at birth as it is the most reliable point of
healthcare contact [2,3]. However, young children often do not
respond to vaccines as efficiently as adults as a result of distinct
features of their immune systems [4–8].
Among the distinct features of neonatal immune system is a
diminished ability of monocytes and antigen-presenting cells
(APCs) to generate Th1-polarizing signals in response to most
stimuli, including reduced production of TNFaand interferon
gamma (IFNc) important for host defence against intracellular
pathogens and for generation of adaptive immune responses [9–
11]. However, the impairment is stimulus-specific and some
stimuli are able to effectively activate neonatal APCs [7,10,12]. In
this context, characterizing the ontogeny of human immune
responses including the contribution of innate immunity, provides
insight into susceptibility of newborns and infants to infection,
assesses immune polarization that may affect risks of allergy and
atopy [13], and may inform development of age-specific
adjuvanted vaccines [7].
Modifying the innate response may increase the strength of
adaptive responses required for inducing efficient immunological
memory in infants. Until recently, the only approved adjuvant
component in vaccines was alum. However, the discovery that
microbial products activate host cells via pattern recognition
receptors such as Toll-like receptors (TLRs) and enhance adaptive
immune responses by triggering cytokine production and dendritic
cell maturation, has led to TLR agonists being developed as
PLoS ONE | www.plosone.org 1 April 2011 | Volume 6 | Issue 4 | e18185
13
vaccine adjuvants [14–18]. The TLR4 agonist monophosphoryl
lipid A (MPL) has been combined with alum (AS04) in hepatitis B
and human papilloma vaccines[19–21]. Agonists of several other
TLRs, including TLR3, 7, 8 and 9, are in clinical development as
vaccine adjuvants targeting mycobacterial, parasitic and diseases
[22] [7,23–27]. Of note, several existing vaccines trigger TLRs:
Bacillus Calmette Guerin (BCG), the most widely used vaccine against
TB with an established safety and efficacy profile activates TLR2, -
4 and -8 [28–30] and the meningococcal outer membrane protein
complex used to adjuvant the Haemophilus conjugate vaccine,
was subsequently shown to be a TLR2 agonist [31]. These
examples suggest that in certain contexts TLR agonists can be safe
and effective as vaccine adjuvants and highlight the importance of
characterizing ontogeny of TLR-mediated responses in target
populations, including newborns and infants.
Age-dependent changes in TLR function have been demon-
strated in humans by natural deficiencies in TLR signaling
molecules including a deficiency in IRAK-4 (a kinase involved in
the TLR signaling cascade) [32,33] and MyD88 (a TLR adaptor
molecule) [11] that can result in life-threatening infections in
infancy and childhood. Of note, infections decrease in IRAK-4-
deficient children .8 years of age, suggesting a greater
dependency early in life on TLR signaling for protection against
infection. Although newborns demonstrate similar basal expres-
sion of monocyte TLRs at birth [34], neonatal blood mononuclear
cell responses to TLR agonists are distinct from those of adults,
with impairment of Th1 cytokine responses (e.g., TNFa, IFNc) but
similar or greater production of cytokines with Th2 (IL-6, IL-1b),
Th17 (IL-23) and anti-inflammatory (IL-10) activity [4,34–42]. By
contrast, cord blood responses to TLR8 agonists, including
imidazoquinoline compounds and single stranded RNAs
(ssRNAs), induced robust adult-like TNFaresponses raising the
possibility that TLR8 agonists may serve as effective vaccine
adjuvants for newborns and young infants [34,43].
Because infant peripheral blood is more difficult to obtain than
cord blood, relatively less is known about TLR function during
infancy. The few infant studies available have been conducted in
resource-rich settings and indicate that there is a marked increase
in TLR-mediated Th1 cytokine production within the first 6
months of life [13,37,44,45]. To our knowledge, there are no
published studies of TLR function in newborns and infants from
resource-poor countries and none from the African continent.
Such data may be of considerable importance as these populations
are at high risk of infection, and may demonstrate distinct innate
response profiles compared to other populations with lesser
burdens of disease. For example, infants from resource-poor
countries often respond quite differently to immune stimuli
including to BCG vaccination [46,47]. In addition to providing
insight into age-dependent development of TLR-mediated innate
immunity, characterizing the ontogeny of TLR responses in an
African country may also inform development of adjuvanted
vaccines for high-risk newborns and infants. We therefore
characterised the production of Th1, Th2, and anti-inflammatory
cytokines in response to agonists of TLRs 1–9inwhole blood
cultures derived from Gambian infants during the first year of life.
We found that cord blood Th1-polarising cytokine responses were
generally impaired, that TLR8 agonists gave the strongest Th1-
cytokine responses at birth and that by 1 month of age the pro-
inflammatory responses to most TLR agonists were increased,
with predominance of responses to TLR4, -5 and -8 agonists that
changed across the first 12 months of life.
Materials and Methods
Study Design
This cross-sectional study was approved by the Joint Gambia
Government/Medical Research Council Ethics Committee and the
London School of Hygiene and Tropical Medicine Ethics
Committee. Between June and November 2009, 120 Gambian
infants, up to 12 months of age, were recruited at the Sukuta Health
Centre (a peri-urban (area surrounding an urban town with
characteristics of a rural setting) population 30 minutes from the
coast of The Gambia). Infants were recruited into 8 age groups:
birth and 1-, 2-, 3-, 4-, 6-, 9- or 12- months of age (or +2 weeks of
age stated; n = 15 per group). For every study subject, written
informed consent of a parent/guardian was obtained. Children
were excluded if they had any signs of intercurrent infection.
Neonates were also excluded if they had a low birth weight (,
2.5 kg) or were a twin. Each child within an age group had received
comparable vaccines according to the Gambian Extended Pro-
gramme of Immunisation (EPI) schedule, and had not received any
vaccine within the previous 7 days. Infants were also excluded if
their weight was outside the target range for age stated on the Infant
Welfare Card. Ten millilitres of umbilical cord blood (collected
before delivery of the placenta) and 3 mL of infant venous blood
was collected into tubes containing heparin (sodium salt from
porcine intestinal mucosa, Sigma-Aldrich, Poole, UK) at 7.5 U/mL
blood and transferred to the laboratories within 6 hrs of collection.
Blood was acquired prior to administration of any EPI vaccine that
may have been indicated on the day of recruitment.
Cell culture conditions
From each blood sample, 100 ml of undiluted whole blood (per
condition) was cultured overnight (18–24 hours) without stimula-
tion (negative control), with phorbol 12-myristate 13-acetate
(PMA; 0.1 mg/mL, Sigma)/ionomycin (calcium salt, from Strepto-
myces conglobatus,1mg/mL, Sigma) (positive control) and with each
of the 9 TLR agonists (Table S1). TLR agonists were purchased
from InvivoGen (San Diego, CA, US) and included: Pam3CSK4
(synthetic tripalmitoylated lipopeptide that mimics bacterial
lipoproteins; TLR1/2, 1 mg/mL), Poly (I:C) (a synthetic analog
of double-stranded RNA (dsRNA) associated with viral infection;
TLR3, 100 mg/mL), LPS (Escherichia. coli K12 Lipopolysaccharide;
TLR4, 1 mg/mL), flagellin (from Salmonella typhimurium; TLR5,
10 mg/mL), FSL-1 (synthetic lipoprotein of Mycoplasma salivarium;
TLR2/6, 10 mg/mL), ssRNA40 (20-mer phosphothioate protect-
ed single-stranded RNA oligonucleotide; TLR8, 10 mg/mL),
CL075 (thiazoloquinolone derivative; TLR8, 10 mg/mL), Gardi-
quimod
TM
(an imidazoquinoline amine analogue to guanosine;
TLR7, 10 mg/mL), ODN M362 (synthetic type C unmethylated-
CpG dinucleotide-containing oligonucleotide; TLR9, 1 mM)
(Table S1). TLR concentrations were selected based on those
that induced maximal cytokine responses from previous studies
[34,43] and from preliminary studies in 9 months old Gambian
infants (unpublished data). After overnight culture, 100 mL of ice-
cold serum-free RPMI media was added to the wells and
centrifuged at 1,500 rpm for 10 mins. 120 mL of supernatant
was collected and stored at 220uC for subsequent analysis. For
RNA studies, 300 mL of whole blood were cultured in each of four
conditions (unstimulated, ssRNA, Gardiquimod
TM
or CLO75) for
4 hours then added to 860 mL PaxGene lysing reagent (Pre-
AnalytiX, Becton Dickinson, France) prior to storage at 270uC
ahead of RNA extraction.
TLR Responses in Gambian Infants
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Cytokine measurement
Supernatants were thawed and centrifuged at 1500 rpm for
5 mins to pellet any precipitation in the sample. Concentrations of
TNFa, IL-1b, IL-6, IFNcand IL-10 (Th1 cytokine kit, Bio-Rad,
Hercules, California, US) were measured for all samples. The bead
array assays were conducted according to the manufacturer’s
instructions (Bioplex Reagent kit, Bio-Rad) using 50 mL of serial
(1:4) dilutions of the standard in RPMI medium containing 10%
human serum and 50 ml of samples. Standard curve outliers were
eliminated by identifying samples where the coefficient of variance
(CV) was greater than 10% and observed/expected x 100 (obs/
exp*100) was outside the range of 100620. Cytokine concentra-
tions below the level of detection (out of range (OOR) ,) were
assigned a value of half the lowest value recorded in that assay.
Similarly, all samples with values above the level of detection
(OOR.) were assigned a value of twice the largest value recorded
in that assay.
RNA extraction
Samples were thawed and left at room temperature (15 –
25uC) .4 hours for complete lysis prior to centrifugation for
10 mins at 5000 rpm. RNA was extracted according to manufac-
turer’s instructions with modification for the small blood volumes
(PaxGene Blood RNA kit, QIAGEN, Germany). Briefly, the pellet
was resuspended in 500 mL of RNase-free water and centrifuged
again at 5,000 rpm for 10 mins. The pellet was then resuspended in
360 mL of BR1 buffer (PaxGene Blood RNA kit). The samples were
further processed according to the manufacturer’s instructions and
eluted in a final volume of 80 mL of Tris/EDTA (TE)-based elution
buffer (BR5). RNA was then purified further using the RNeasy Mini
Elute Clean Up kit (QIAGEN, Germany) and resuspended in 12 mL
of BR5. The RNA yield was measured on the Nanodrop (Thermo
Scientific, Wilmington, Delaware, US) and purity was based on
absorption values at 280 nm (protein) and 230 nm (detection of
contaminating organics/proteins) using the following criteria:
260 nm/280 nm .1.8 and 260 nm/230 nm .1.8.
SA Bioscience Microarrays
0.1 mg total RNA was reverse transcribed to cDNA using RT
2
First Strand Kit (C-03) (SA Biosciences, Frederick, MD, US)
according to the manufacturer’s instructions and diluted with
RNase-free water into a final volume of 111 mL. A customized 6-
gene real time PCR array (SABiosciences, Frederick, MD) was
employed, containing primers specific for TNF, IL-6, IL-10, IL12A,
IL12B and IFNG and using the RT-SYBR Green/ROX qPCR
Master Mix according to the manufacturer’s instructions (SABios-
ciences, Frederick, MD) using approximately 1 ng of original RNA
per gene. mRNA expression was calculated as a copy number (Ct)
value and normalized using multiple housekeeping genes (B2M,
RPL13A, GAPDH, DC
t
). Fold change (DDC
t
) in gene expression
was used to display the data and calculated between the
unstimulated control cells in comparison to TLR agonist-stimulated
cells applying the following equation: if X .1, X, -1/X, if X = DC
t
therefore all values ,0 represented down-regulated genes and all
values .0 represented up-regulated genes. DC
t
values were used for
all statistical analysis.
Statistical analysis
Net cytokine responses were calculated by subtracting the
cytokine concentrations in the medium control wells from those in
supernatants derived form samples stimulated with TLR agonists.
For each TLR agonist/cytokine combination the resulting datasets
of net responses (all ages combined) were dichotomised into $
median value, or ,median value. Due to a number of responses
above the level of detection, comparisons of responses at difference
ages were made by comparing the proportions of values above and
below the median using Fisher’s exact test. Tests for a trend with
age were carried out using logistic regression with above or below
the median as the response variable and age as the independent
variable. Comparisons between the response to medium and the
gross responses to each of the TLR agonists in cord blood were
made using Sign tests which are robust to the influence of out of
range values, in accordance with the paired nature of the data.
Analyses that compared two time points or two ages were done
using a non-parametric Mann U Whitney test and correlations
were calculated on log-transformed data using a Pearson
correlation. Data were analysed using Stata version 11.0
(StataCorp, Texas, US) and GraphPad Prism version 5.01
(GraphPad Software Inc., US). Real time PCR analysis used
software from SA Biosciences as stated above. Box and whisker
plots demonstrate inter-quartile ranges indicated by boxes, median
values by horizontal bars, and 10 and 90 percentiles indicated by
whiskers. P-values ,0.05 were taken to indicate statistical
significance.
Results
Reactivity to TLR4, 5 and 8 agonists predominate at birth
The positive control PMA/ionomycin induced cytokine re-
sponses that were largely independent of age (TNFap = 0.603, IL-
6 p = 0.758, IL-1bp = 0.257, IFNcp = 0.167). The lone exception
was IL-10, whose production diminished with increasing age
(p = 0.005; data not shown).
Compared to the unstimulated control, there was a significant
induction of TNFa, IL-6, IL-1band IL-10 production in cord
blood to all the TLR agonists studied with the exception of FSL-1
(TLR2/6) that did not induce a significant TNFaresponse
(Table 1).
Of note, the efficacy with which TLR agonists induced these
cytokines, differed both between agonists and with age. Agonists of
TLR4, -5, and, in particular TLR8 (including the combined
TLR7/8 agonist) induced the highest levels of most cytokines, with
agonists of TLR1/2, -3, -2/6, -7 and -9 eliciting a reduced
cytokine response (Table 1 and Figure 1A). The combined TLR7/
8 agonist (the thiazoloquinolone CL075) induced the highest levels
of TNFacompared to agonists of TLR4 (p = 0.0202) and TLR5
(,0.0001). Moreover CL075 (TLR 7/8) was the only TLR agonist
that induced IFNcin cord blood compared to the unstimulated
control (p = 0.002, Table 1 and Figure 1B). In agreement with
previous studies [43], the TLR8 agonists (ssRNA and CLO75)
induced significantly more cytokine production in cord blood
cultures compared to the TLR7 agonist (Gardiquimod
TM
)
(Figure 2).
TLR-mediated IL-6 and IL-1bproduction is high from
birth, while TNFaand IFNcproduction peak at 1 month
Each cytokine studied demonstrated a distinct age-dependent
profile in response to each TLR agonist during the first year of life.
An assessment of effect with age for each cytokine response to each
agonist revealed that TNFaresponses to FSL-1 (TLR2/6;
p,0.001), LPS (TLR4; p = 0.009), Flagellin (TLR5; p = 0.001),
Gardiquimod
TM
(TLR7; p ,0.001), ssRNA (TLR8; p,0.001),
and CL075 (TLR7/8; p ,0.001) all demonstrated age-dependent
effects (Table S2A). Graphic representation of these data suggested
this effect was due to a predominant increase in TNFaproduction
between cord blood and 1 month of age in response to most
agonists (Figure 3). This impression was confirmed by repeating
TLR Responses in Gambian Infants
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the analysis between ages 1 to 12 months (excluding the cord
blood) (Table S2B) and comparing the individual responses
between birth and 1 month of age (Figure 4). After the initial
increase in TNFaresponses by 1 month, TNFaresponses to most
agonists were stable from 1 month to 12 months of age (Table S2B
and Figure 3).
Responses to the TLR8 agonist (ssRNA) and the combined
TLR7/8 agonist (CLO75) were notable exceptions to this pattern.
The initial increase in reactivity to CLO75 from birth to 1 month
of age (p =0.005), was then followed by a decline from 1 to 12
months of age (trend analysis p,0.001, from a median at 1 month
of 13,852 pg/mL, to a median at 12 months of 2,771 pg/mL),
though nevertheless remained higher than those for TLR2, -3, -2/
6 and -9 (Table S2 and Figure 3). In contrast, TNFaresponses to
ssRNA (TLR8) did not reveal a significant trend from 1 to 12
months of age (Figure 3). The relatively low TNFaresponse to
Pam3CSK4 (TLR1/2), Poly (I:C) (TLR3) and ODN M362
(TLR9) at birth failed to increase in any age group to 12 months,
in fact the TLR9-mediated TNFaresponse significantly decreased
from the 1 month to 12 month age groups (trend analysis
p = 0.008, Table S2 and Figure 3).
While TLR-mediated IFNcproduction at birth was very limited
(Figure 1 and Table 1), most TLR agonists elicited greater IFNc
production by 1 month of age: Pam3CSK4 (TLR1/2; p = 0.003
(Figure 5A), Poly I:C (TLR3; p,0.001), LPS (TLR4; p,0.001),
Flagellin (TLR5; p,0.001), FSL-1 (TLR6/2; p = 0.002), Gardi-
quimod
TM
(TLR7; p,0.001; Figure 5B), ssRNA (TLR8;
p = 0.017; Figure 5C), CL075 (TLR7/8; p,0.001; Figure 5D),
ODN M362 (TLR9; p = 0.066; Figure 4 and Table S2). TLR-
mediated IFNcproduction then remained stable from 1 to 12
months of age, with the exception of responses to the TLR1/2
agonist that decreased with age (trend analysis; p = 0.003,
Figure 5A).
All the TLR agonists studied induced high levels of IL-6 at birth
(Table 1) and generally TLR-mediated IL-6 production remained
high for the first year of life (Table S2). Similarly, IL-1band IL-10
production was high at birth in response to TLR4, -5, and -8
agonists and remained high for the first year of life with the
exception of the combined TLR7/8 agonist (CL075) that induced
less IL-1bwith age (Table S2). In accordance with prior reports of
an early life bias towards a high ratio of IL-6/TNFaproduction
[40,41], in our cohort, ratios of TLR-mediated IL-6/TLR-
mediated TNFawere initially high at birth (cord) and then
decreased with age, reflecting a relatively constant and robust IL-6
production and an increase in TLR-mediated TNFaby 1 month
of age (data not shown).
TLR8 agonists elicit distinct early age-dependent Th1
responses
In accordance with a prior cord blood study [43], TLR8
agonists induced greater Th1-polarising cytokine responses in
early life compared to agonists of other TLRs. CLO75 (TLR7/8)
and ssRNA (TLR8) differed with respect to the dynamics across
age of cytokine responses. By one month of age, CL075 induced
higher concentrations of TNFathan ssRNA (p = 0.006), but by 2
months of age the two agonists induced similar concentrations
(p = 0.735) which remained constant up to 12 months (Figure 6A).
IFNcresponses to these two agonists were similar from birth up to
6 months of age, from which point ssRNA induced greater levels
of IFNcthan CL075 through 12 months of age (p = 0.001)
(Figure 6B). In contrast, CL075-induced IL-10 production
exceeded that induced by ssRNA at all ages (p,0.05, data not
shown).
TLR8-mediated IFNcprotein production correlates with
gene transcription
As the ability to induce IFNcis of particular importance to host
defence and generation of adaptive immunity, we further
characterised TLR-mediated IFNcinduction in blood samples
from 5 individuals for which longitudinal samples were available
from birth, 1 and 12 months of age. In addition, we also measured
IL-12A and IL-12B that encode components of IL-12p70, a
cytokine that is important to IFNcproduction [48,49] and to Th1
adaptive immune responses but can be challenging to detect at the
protein level. Total RNA was purified from blood samples that
were stimulated for 4 hours with Gardiquimod
TM
(TLR7), ssRNA
(TLR8) and CL075 (TLR7/8) and mRNA levels measured by real
time PCR. Cytokine mRNAs were significantly up-regulated at
each time point for each TLR agonist-stimulated condition
compared to the unstimulated control (data not shown).
In accordance with the protein data, cytokine mRNA
expression patterns appeared similar with lower responses to
Gardiquimod
TM
(TLR7) compared to ssRNA (TLR8) and CL075
(TLR7/8) for most cytokines at each age (Figure 7) except IL-12A
expression that appeared to be expressed at similar levels in
response to each of the agonists at a given age (Figure 7). In
addition, the pattern of protein concentrations of IL-10 demon-
strated similar expression patterns to IL-10 mRNA with greater
responses to CL075 than to ssRNA for all ages (Figure 7).
Correlations between mRNA expression levels and protein
levels for the same cytokines were analysed as pooled ages. In
accord with greater IFNcresponse to TLR8 versus TLR7
agonists, significant correlations were noted between TLR
Table 1. TLR agonists induce cytokine production in cord blood of Gambian newborns.
Unstim.
Control Pam. (TLR1/2)
PolyI:C
(TLR3) LPS (TLR4) Flag. (TLR5)
FSL-1
(TLR2/6)
Guard.
(TLR7)
ssRNA
(TLR8)
CL075
(TLR7/8) ODN (TLR9)
TNFa1.309 3.98* 6.34* 1,117.17*** 133.52*** 4.29 12.13* 5,003.68*** 5,180.04*** 10.74**
IL-6 245.9 7,161.75*** 1,831.22** 22,529.47*** 25,209.28*** 8,811.02*** 5,390.73*** 14,412.48** 21,262.00*** 3,802.29**
IL-1b3.11 36.42** 15.31** 1,293.61*** 1,522.90*** 30.17** 59.92** 3,069.16*** 2,557.96*** 36.92**
IL-10 10.36 151.20*** 35.05** 966.90*** 1,145.50*** 145.06*** 156.36*** 745.06*** 1,647.61*** 33.06**
IFNc3.335 3.34 2.83 6.37 6.38 3.24 3.34 23.36 59.17** 3.34
100 ml cord whole blood was cultured overnight with each of the TLR agonists and cytokine production (pg/mL) in supernatants was measured as described in the
Materials and Methods. Concentrations are absolute values in pg/mL. Comparisons between unstimulated and stimulated values, n = 12–15; *p = 0.05 – 0.019, **p = 0.01
– 0.001, ***p,0.001.
doi:10.1371/journal.pone.0018185.t001
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PLoS ONE | www.plosone.org 4 April 2011 | Volume 6 | Issue 4 | e18185
agonist-induced mRNA expression of IFNG and IFNcprotein
levels induced by ssRNA (p = 0.005; Figure 8B) and CL075
(p = 0.027; Figure 8C), but not Gardiquimod
TM
(p = 0.075;
Figure 8A). Of note, TLR-mediated increases in IL12A and
IL12B mRNA also correlated with TLR-mediated IFNcprotein
production, including ssRNA-induced IL-12A and IL-12B expres-
sion (p = 0.017, p = 0.001 respectively) as well as CL075- and
Gardiquimod
TM
-induced IL-12B expression (p = 0.027 and
p = 0.035, respectively). For the other cytokines studied, the
ssRNA- and CL075-induced cytokine mRNA levels at 4 hours of
stimulation did not correlate with the corresponding cytokine
protein levels at 24 hours.
Levels of TLR-mediated IL-12A and IL-12B mRNA expression
were age-dependent for all three TLR agonists whereas IFNG
showed similar levels between birth, 1 and 12 months of age
(Figure 9 and Figure 7). In response to CL075 (TLR7/8 agonist),
expression of IL-12A increased from birth to 1 month of age
(p = 0.008) but subsequently decreased by 12 months of age
(p = 0.008). These mRNA patterns corresponded to a similar
pattern of IFNcprotein production by the same agonist
(comparing for the same subjects): birth vs. 1 month of age
(p = 0.002), and 1 month vs. 12 months of age (p = 0.018).
Gardiquimod
TM
also demonstrated age-dependent expression of
mRNAs encoding IL-10, IL-6 and TNF mainly due to low
expression levels at birth that increased with age: birth to 1 month
of age: IL-10 (p = 0.008), and birth to 12 months of age: IL-10
p = 0.3095, IL-6 p = 0.032, TNF p = 0.008 (Figure 9 and Figure 7).
Discussion
Our study evaluated the ontogeny of TLR-mediated in vitro
whole blood cytokine responses during the first year of life in
infants from The Gambia employing agonists of TLRs 1-9 and
blood samples from 8 different age groups. We found that cytokine
responses of Gambian infants varied between TLR agonists, with
the greatest responses to TLR8 agonists (ssRNA and CL075),
substantial responses to TLR4 and TLR5 agonists, and relatively
weak responses to TLR1, 2, 3, 7 and 9 agonists at all ages. Thus
the ontogeny of responses is TLR-specific with distinct differences
across age groups in the first 12 months of life. Most of the
previous studies exploring TLR-mediated responses of newborns
have focused solely on comparisons between cord blood and adults
[4,34,40]. The few studies that have also examined infant
responses are limited to resource-rich setting and did not include
the wide range of agonists tested in our study [37,39,44,45].
We found that CL075 (TLR7/8) was the only TLR agonist to
induce IFNcat birth but that in general, cord blood pro-
Figure 1. TLR-mediated cytokine production in cord blood. 100 ml of whole cord blood was cultured overnight with each of the TLR agonists
and supernatants recovered for measurement of (A) TNFaand (B) IFNcas described in Materials and Methods (pg/mL) and presented as box and
whisker plots illustrating 10 and 90 percentile error bars, n = 12–15. Comparisons between cytokine levels in the stimulated samples and the
unstimulated samples were analysed using a paired Sign test at 5% significance (* represent significant differences).
doi:10.1371/journal.pone.0018185.g001
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Figure 2. TLR7 and 8 agonist-induced cytokine induction in cord blood. 100 ml of whole cord blood was cultured overnight with each of the
TLR agonists and supernatants recovered for measurement of IFNc, TNFa, IL-10, IL-1band IL-6 as described in Materials and Methods (pg/mL), n = 12–
15. Unstimulated values are subtracted from stimulated values and presented as box and whisker plots, n = 12–15.
doi:10.1371/journal.pone.0018185.g002
Figure 3. Net TLR-mediated TNFaresponses differ between agonists and across the first year of life. 100 ml of whole blood was cultured
overnight with each of the TLR agonists at birth, 1, 2, 3, 4, 6, 9 and 12 months of age (x axis) and TNFacytokine production (y axis) was measured as
described in the Materials and Methods (pg/mL). Unstimulated values are subtracted from stimulated values and presented as box and whisker plots,
n = 12-15.
doi:10.1371/journal.pone.0018185.g003
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PLoS ONE | www.plosone.org 6 April 2011 | Volume 6 | Issue 4 | e18185
inflammatory/Th1-polarizing cytokine responses (i.e., TNFaand
IFNc) were lower than at later age groups, in accordance with prior
studies [4,34,39]. In contrast, TLR-mediated neonatal IL-6, IL-1b
and IL-10 production was higher or similar at birth compared to later
age groups, suggesting that neonatal TLR-mediated responses are
biased towards acute phase (IL-1band IL-6) and anti-inflammatory
(IL-10) cytokines. We have previously shown in 5-day cultures that
IL-10 and IL-6 responses to mycobacterial antigens were present at
birth [50]. It has been speculated that the bias towards high IL-10
production may reflect the need to dampen potentially over-
exuberant responses to the numerous new antigens to which an
infant is exposed in early life [51]. Greater IL-6 to TNFaratios have
been found in cord blood compared to adults, both in response to in
vitro stimulation [41] and with respect to basal serum levels during the
first week of life [40]. Our findings indicate a similar polarisation in
our cohort with greater IL-6 to TNFaratios at birth compared to 12
months of age. Relatively high IL-6 production in newborns likely
contributes to initiation of an acute phase response at birth that may
serve to clear perinatally-acquired microbes [52]. In addition, IL-6
enhances both differentiation of Th17 cells and production of IL-17,
Figure 4. TLR agonists induce greater cytokine production in Gambian newborns at 1 month compared to at birth. 100 ml whole blood
was cultured overnight with each of the TLR agonists and cytokine production (pg/mL) in supernatants was measured as described in the Materials and
Methods. Comparisons between cytokine levels at birth and 1 month of age were calculated using a non-parametric Mann Whitney test. *(light grey square)
p = 0.05 – 0.019, **(medium grey square) p= 0.01 – 0.001, ***(dark grey square) p,0.001. Pam3. = Pam
3
CysSerLys
4,
Gard. = Ga rdiquimod.
doi:10.1371/journal.pone.0018185.g004
Figure 5. Net TLR-mediated IFNcresponses differ between agonists and across the first year of life. 100 ml of whole blood was cultured
overnight with (A) Pam3CSK4 (TLR1/2 agonist), (B) Gardiquimod
TM
(TLR7 agonist), (C) ssRNA (TLR8 agonist) and (D) CL075 (TLR7/8 agonist) at birth
(cord), 1, 2, 3, 4, 6, 9 and 12 months of age (x axis) and IFNccytokine production (y axis) was measured in supernatants (pg/mL) as described in
Materials and Methods. Unstimulated values are subtracted from stimulated values and data were presented as box and whisker, n = 12–15.
doi:10.1371/journal.pone.0018185.g005
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and thereby may enhance neutrophil- and antimicrobial peptide-
based host defence at neonatal mucosal and epithelial barriers [52–
56]. That this neonatal bias towards TLR-mediated IL-6 and IL-10
responses is evident in vivo was recently demonstrated in a field trial of
alum-adjuvanted pneumococcal conjugate vaccine [3].
For most TLR agonists the major effect of age occurred within
the first month of life during which time TLR-mediated TNFa
and IFNccytokine production increased, suggesting early
maturation in the ability to mount these responses. Whether this
maturation in cytokine production reflects changes in the
adenosine system that serves to limit production of TNFain
human newborn cord blood [41], will be the subject of future
studies. The only other study that reported responses in 1 month
old infants was conducted by Belderbos et al in Holland, which also
showed increased pro-inflammatory responses (e.g., IL-12p70 and
IFNa) from birth to 1 month of age in response to TLR3, -4, -7
and -9 agonists but similar levels of IL-10 production in response
to TLR3, -4 and -9 [39]. That study found that loxoribine (TLR7)-
induced IL-10 responses increased by 1 month of age, in accord
with our observation of a borderline increase at 1 month of age
(p = 0.05) using the TLR7 agonist Guardiquimod
TM
. A Belgian
study by Ngyuen et al also found that LPS-induced cord blood
production of IL-10 and IL-6 was greater than in later age groups
(6-9 months and 12 months of age). Taken together, our current
study and those by Belderbos et al [39], and Vosters et al [44]
indicate that for most TLR agonists, IL-10 production is largely
similar from birth through to 18 months of age.
A comparison of the few studies of infant TLR function
[37,39,44,45] and our current study indicates that Gambian
infants have broadly similar TLR-mediated responses to those
found in a Western European environment. However, Ngyuen et
al studying a Belgian cohort found a slower postnatal increase in
TNFaproduction in response to LPS from birth compared to our
study, such that adult levels were not reached until 6 months of age
[37], whereas our Gambian cohort demonstrated LPS-induced
TNFaresponses that peaked at 1 month of and then remained
stable to 12 months of age. Earlier maturation of LPS-induced
TNFaresponses in Gambian infants than in the European infants
[37,39,45] may reflect more rapid polarisation to Th1 responses in
a resource-poor setting, in accord with the hygiene hypothesis
[13,57,58], and may also suggest that TLR4 agonists, currently
used in several vaccine formulations (e.g., MPL[59]), may be
useful vaccine adjuvants in early infancy.
Although TLR8 agonist-induced cord blood TNFaresponses
have been shown to be greater than TNFaresponses to agonists of
TLRs 1–7 [43], little is known regarding cytokine responses to
TLR8 agonists such as ssRNA (TLR8 only) and CL075 (TLR7/8
agonist) during the first year of life. Our study confirmed the
Figure 6. TLR8 agonists induce distinct patterns of TNFaand IFNcproduction across the first year of life. 100 ml of whole blood was
cultured overnight with ssRNA (TLR8 agonist) and CL075 (TLR7/8) at birth, 1, 2 and 12 months of age. (A) TNFaand (B) IFNccytokine (pg/mL) was
measured in supernatants as described in the Materials and Methods. Unstimulated values are subtracted from stimulated values and data are
presented as box and whisker plots, n = 12–15.
doi:10.1371/journal.pone.0018185.g006
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greater responsiveness to TLR8 agonists in cord blood and
extended the finding by demonstrating that TLR8 agonists were
the predominant inducers of pro-inflammatory cytokines up to 12
months of age in Gambian infants. We also found that the
combined TLR7/8 agonist (CL075) induced more TNFathan the
TLR7 selective agonist Gardiquimod
TM
or the TLR8 selective
agonist ssRNA up to 1 month of age. By 12 months of age
however, there was a trend towards reduced TNFaproduction in
response to the combined agonist compared to the single TLR8
agonist. Likewise, IFNcproduction was similar between the two
agonists up to 6 months of age after which CL075-induced IFNc
diminished relative to ssRNA-induced IFNcand remained lower
up to 12 months of age. It should be noted however, that in
addition to differences between TLR7 and TLR8 selectivity,
differences in biochemical structures between ssRNA and the low-
molecular weight thiazoloquinolone, CL075 may affect TLR-
independent variables (e.g., solubility, protein binding and cell
penetration) that may also contribute to distinct bioactivities. The
correlations between IFNG mRNA and IFNcprotein in response
to ssRNA and CL075 indicate that the ontogeny of TLR-mediated
IFNcproduction is manifest at the transcriptional level. Given the
importance of IL-12 to IFNcproduction [49,60,61] and the
observed correlations of TLR-mediated IFNcprotein with IL12B
message, TLR8-mediated IFNcresponses may also involve
Figure 7. TLR7- and TLR8-mediated cytokine mRNA expression varies between agonists and across the first year of life. 300 mlof
whole blood was cultured for 4 hours with Gardiquimod
TM
(TLR7), ssRNA (TLR8 agonist) and CL075 (TLR7/8) at (A) birth, (B) 1 month and (C) 12
months of age. RNA was purified and cytokine mRNA was quantified by real time PCR as described in Materials and Methods. Ct values were
normalised against the housekeeping genes and compared to the unstimulated control (DCt) and fold differences of DCt values between
unstimulated and stimulated cultures were calculated (DDCt). Fold changes are presented as box and whisker plots, n = 5.
doi:10.1371/journal.pone.0018185.g007
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activation of IL-12B (that encodes for IL-12p40)[49]. However,
expression of IL-12A (that encodes for IL-12p35) was very low at
all three ages tested, in agreement with Western European studies
that demonstrate deficiency in TLR-mediated IL-12p35 produc-
tion by neonatal DCs, contributing to impaired neonatal IFNc
responses [62]. Analysis of TLR-mediated production of addi-
tional cytokines that may influence adaptive immune responses,
including IL-4 (Th2) and IL-17 (Th17), is of importance and
should be included in future studies.
Overall, characterizing the ontogeny of innate immune responses
may eventually inform selection of adjuvant vaccine formulations that
are tailored for certain age groups. Indeed, recent studies highlight
that appropriate immunologic signatures can predict vaccine efficacy
[17,63].TotheextentthatTNFaand IFNcmay be markers of
strong Th1-polarising responses required for induction of cell-
mediated immunity, and that the whole blood in vitro responses
measured in our study may reflect those that would pertain in vivo,our
data suggest that vaccine adjuvants based on TLR4, -5 or -8 agonists,
may be particularly effective in protecting newborns and infants
against pathogens requiring cell-mediated immunity.
Supporting Information
Table S1 Summary of the TLR agonists used in the study.
(DOC)
Table S2 Analysis for age-dependent evolution of cyto-
kine responses to TLR agonists. 100 ml of whole blood was
cultured overnight with Pam3CSK4 (TLR1/2), Poly (I:C) (TLR3),
LPS (TLR4), Flagellin (TLR), FSL-1 (TLR6/2), Gardiquimod
TM
(TLR7), ssRNA (TLR8) and CL075 (TLR7/8) or ODN M362
(TLR9) at birth (cord), 1, 2, 3, 4, 6, 9 and 12 months of age. IFNc,
TNFa,IL-1b,IL-10and IL-6 cytokine concentrations (pg/mL) were
measured in supernatants as described in Materials and Methods.
Comparisons of responses were made (A) from birth to 12 months
of age and (B) from 1 to 12 months of age by comparing the
proportions of values above and below the median using Fisher’s
exact test. All grey squares represent significant effects with age
(p,0.05) while dark grey squares indicate the effects with age
corresponding to a significant decline in cytokine production from
1 to 12 months of age using trend analysis.
(DOC)
Acknowledgments
We would like to thank the field workers and nurses at the MRC clinic in
Sukuta, and Sally Savage and her staff at the Sukuta Government Health
Centre. We also acknowledge the support of the mothers and newborns
without which this study could not take place. OL acknowledges the
mentorship of Drs. Michael Wessels, Richard Malley, Christopher Wilson
Figure 8. Correlations between ssRNA-induced cytokine mRNA and protein expression. 300 ml of whole blood was cultured for 4 hours
with (A) Gardiquimod
TM
(TLR7), (B) ssRNA (TLR8) and (C) CL075 (TLR7/8). RNA was purified and cytokine mRNA was quantified by real time PCR as
described in Materials and Methods. Comparisons between IFNcprotein and IFNG mRNA levels were calculated where stimulated values (Ct and
cytokine concentrations) were divided by unstimulated values. The values were log transformed and compared using Pearson correlation test, linear
regression line presented in graph, n = 5. At birth (black circles), 1 month (open circles), or 12 months (crosses) of age.
doi:10.1371/journal.pone.0018185.g008
Figure 9. Age-dependent effects of cytokine transcription in
response to TLR agonists. 300 ml whole blood was cultured for
4 hours with Gardiquimod
TM
(TLR7 agonist), ssRNA (TLR8 agonist) and
CL075 (TLR7/8 agonist) at birth (cord), 1 and 12 months of age and IL-
12A, IL-12B, IFNG,IL-6, IL-10 and TNF cytokine mRNA gene transcription
was measured. Ct values were normalised against the housekeeping
genes and compared to the unstimulated control (DCt). Comparisons of
responses from birth, 1 and 12 months of age were made using the
Kruskall Wallis test. Grey squares represent significant effects with age,
p.0.05. Gard. = Gardiquimod.
doi:10.1371/journal.pone.0018185.g009
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PLoS ONE | www.plosone.org 10 April 2011 | Volume 6 | Issue 4 | e18185
and Gary Fleisher. We thank the Gambian government for their support,
in particular the National EPI programme.
Author Contributions
Conceived and designed the experiments: SB KLF OL AJ HW VJP.
Performed the experiments: SB JNJ MC CM. Analyzed the data: SB JT
OL. Contributed reagents/materials/analysis tools: SB JT KLF OL.
Wrote the paper: SB OL KLF BK. Acquired field data and ran field team:
SB UJA ET. Contributed to review and editing of manuscript: SB JT JNJ
MC UJA ET VJP CM BK HW AJ KLF OL. Interpretation of the data: SB
OL KLF BK.
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TLR Responses in Gambian Infants
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