Ascaris lumbricoides pseudocoelomic body uid induces a partially activated

dendritic cell phenotype with Th2 promoting ability in vivo

David J. Dowling
a
, Cariosa M. Noone
a
, Paul N. Adams
a
, Krisztina V. Vukman
a
, Sile F. Molloy
b
,
Jessica Forde
a
, Samuel Asaolu
c
, Sandra M. ONeill
a,
a
Parasite Immune Modulation Group, School of Nursing, Faculty of Science and Health, Dublin City University, Glasnevin, Dublin 9, Ireland
b
School of Zoology, Trinity College Dublin, Dublin, Ireland
c
Obafemi Awolowo University, Nigeria
a r t i c l e i n f o
Article history:
Received 26 May 2010
Received in revised form 14 September
2010
Accepted 15 September 2010
Available online 23 October 2010
Keywords:
Ascaris lumbricoides
Dendritic cell
Helminth
Pseudocoelomic body uid
Th2 immune responses
a b s t r a c t
Dendritic cells (DCs) matured with helminth-derived molecules that promote Th2 immune responses do
not follow conventional denitions of DC maturation processes. While a number of models of DC matu-
ration by Th2 stimuli are postulated, further studies are required if we are to clearly dene DC maturation
processes that lead to Th2 immune responses. In this study, we examine the interaction of Th2-inducing
molecules from the parasitic helminth Ascaris lumbricoides with the maturation processes and function of
DCs. Here we show that murine bone marrow-derived DCs are partially matured by A. lumbricoides
pseudocoelomic body uid (ABF) as characterised by the production of IL-6, IL-12p40 and macrophage
inammatory protein 2 (MIP-2) but no enhanced expression of cluster of differentiation (CD)-14, T-cell
co-stimulatory markers CD80, CD86, CD40, OX40L and major histocompatibility complex class II was
observed. Despite these phenotypic characteristics, ABF-stimulated DCs displayed the functional hall-
marks of fully matured cells, enhancing DC phagocytosis and promoting Th2-type responses in skin-
draining lymph node cells in vivo. ABF activated Th2-associated extracellular signal-regulated kinase-1
and nuclear factor-kB intracellular signalling pathways independently of toll-like receptor 4. Taken
together, we believe this is the rst paper to demonstrate A. lumbricoides murine DC-Th cell-driven
responses shedding further light on DC maturation processes by helminth antigens.
2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction
Dendritic cells (DCs) are crucial in the development of immune
responses, in particular the initiation and polarisation of T helper
cell responses (Banchereau and Steinman, 1998; Banchereau
et al., 2000). While much is known about DC biology, a substantial
gap exists between the well dened mechanisms of how DCs drive
Th1 immune responses and the less dened characteristics of DCs
that drive Th2 responses associated with helminth infection
(Maizels and Yazdanbakhsh, 2003). This discrepancy has lead to
a growing interest in understanding and dening the effects of
helminth-derived molecules upon DC function and their subse-
quent T helper responses.
It is well established that DCs matured with Th2 stimuli do not
follow conventional denitions of DC maturation processes
(Maizels et al., 2004). The induction of Th1 immune responses re-
quires three signals delivered by mature DCs. These signals include
the expression of major histocompatibility complex (MHC) class
IIpeptide complexes to nave T-cells (signal 1), the up-regulation
of co-stimulator molecules (CD40, CD80 and CD86; Signal 2) and
the secretion of IL-12 (Signal 3) (Iwasaki and Medzhitov, 2004).
However, the signals required for maturation of DCs by Th2-stim-
uli are less clearly dened and in a review by MacDonald and
Maizels (2008), three alternative models of selective Th2 induction
by DCs are described.
The rst, a maturation model, suggests DCs are activated into
semi-mature cells, characterised by the lack of a third signal. The
induction of Th2 cells by DCs is therefore a default mechanism that
occurs in the absence of conventional maturation processes. The
second, an alternative pathway model, suggests that Th2 stimuli
activate DC maturation following recognition by distinct pattern
recognition receptors such as C-type Lectin receptors that activate
an alternative pathway favouring IL-10 induction. This differs from
conventional activation of DCs via toll-like receptors (TLRs) by Th1
stimuli that activate the release of pro-inammatory cytokines
such as IL-12. Finally, the inhibition model suggests that Th2 stim-
uli induce pathways that inhibit processes such as IL-12 secretion
0020-7519/$36.00 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijpara.2010.09.007

Corresponding author. Tel.: +353 1 700 5455; fax: +353 1 700 7919.
E-mail address: Sandra.ONeill@dcu.ie (S.M. ONeill).
International Journal for Parasitology 41 (2011) 255261
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which is important for conventional maturation processes. While
there is evidence to support all three models they are not consid-
ered to be mutually exclusive and a unique combination of all
pathways may be at play for each individual helminth. Therefore,
more studies are required if we are to clearly dene how DC mat-
uration processes by Th2 stimuli drive Th2 immune responses
(Terrazas et al., 2010).
The human parasitic nematode Ascaris lumbricoides is of major
medical importance, infecting 1.5 billion people worldwide
(OLorcain and Holland, 2000; Elliott et al., 2007). Infection by
A. lumbricoides causes acute effects such as intestinal obstruction
and chronic infection impacts upon growth, appetite, physical t-
ness, work capacity and cognitive development in populations
who are already compromised by poor nutrition, and other educa-
tional and health disadvantages (Bradley and Jackson, 2004;
Holland, 2009). Ascaris infection is characterised by a predominant
Th2 immune response and resistance to infection is associated
with high levels of the Th2 cytokines, IL-4 and IL-5, and low levels
of Th1 cytokines (Cooper et al., 2000; Geiger et al., 2002). However,
there are few studies to date that have examined the interaction of
A. lumbricoides-derived molecules with DCs.
Ascaris lumbricoides pseudocoelomic uid (ABF) is a metabol-
ically active uid which provides precursor molecules for mem-
brane and cuticular synthesis (Kennedy and Qureshi, 1986). It
serves as a cavity for circulation, aids in digestion and acts as
an internal hydrostatic skeleton that functions in locomotion.
ABF is easily obtained from the body cavity of adult worms
and studies have shown it to contain a number of immunogenic
antigens since ABF-specic antibody responses can be observed
in several different mammalian species, including mice (Kennedy
and Qureshi, 1986; Urban et al., 1988). The focus of this paper is
to determine the effect of ABF, a Th2 inducing helminth antigen
(van Riet et al., 2009), upon DC maturation and function.
2. Materials and methods
2.1. Animals and preparation of A. lumbricoides pseudocoelomic uid
C57BL/6j and BALB/c mice, 68 weeks old, were purchased from
Harlan UK Ltd. (Bicester, UK). The DO11.10 mice, that carry the MHC
class II restricted rearranged T-cell receptor transgene (DO11.10)
which react to ovalbumin (OVA) peptide (TCR) (on a BALB/c back-
ground) were purchased from Jackson Laboratory (USA). TLR4KO
bone marrowcells (ona C57BL/6j background), were a gift fromPro-
fessor Padraic Fallon (Trinity College, Dublin, Ireland). All mice were
maintained according to the requirements of the Department of
Health (Ireland) and approval for animal experiments was obtained
from Dublin City University Ethics Committee.
ABF was extracted from 10 female adult worms obtained from
infected children in Nigeria who were participating in a random-
ised control trial. Ethics approval was obtained from the Ethics
and Research Committee, Obafemi Awolowo University Teaching
Hospitals Complex, IleIfe, Nigeria (Kirwan et al., 2010). To remove
the uid, a sterile needle was injected into the body cavity of each
worm (Christie et al., 1993). ABF from all 10 worms were pooled
and aliquots of 500 ll were stored at 70 C. Protein concentra-
tions were measured using a BCA protein assay kit (Pierce) and
endotoxin levels were assessed using a Pyrogene

endotoxin
detection system (Cambrex, Walkersville, USA) which utilises re-
combinant Factor C, an endotoxin-sensitive protein. ABF gave
endotoxin levels similar to background and to complete RPMI
1640 medium (supplemented with 5% heat-inactivated FCS,
100 U/ml penicillin, 100 lg/ml streptomycin, 2 mM L-glutamine
and 50 lM 2-mercaptoethanol) and were less than the lower limit
of detection in this assay (<0.01 EU/ml).
2.2. Isolation, maturation and characterisation of bone
marrow-derived DCs
Bone marrow-derived immature DCs (BMDCs) were prepared by
culturing bone marrow cells, isolated from the femurs and tibias of
mice, in complete RPMI 1640 with recombinant mouse GM-CSF
(20 ng/ml; R&D Systems), at 37 C. On days 3 and 6 of culture, fresh
medium with GM-CSF (20 ng/ml) was added to the cells. On day 8,
cells were harvested, counted and stained with CD11c (Caltag Lab-
oratories, Burlingame, CA, USA) which was analysed by owcytom-
etry to determine purity (>90%). DCs were seeded into 24-well
plates (Nunc) at 1 10
6
/ml in complete RPMI 1640 plus 5 ng/ml
GM-CSF and were treated with medium, ABF (10 lg/ml), lipopoly-
saccharide (LPS) (Alexa; 100 ng/ml), zymosan A (SigmaAldrich;
5 lg/ml) or with ABF (10 lg/ml) for 2.5 h prior to stimulation with
LPS for 18 h.
Supernatants from cultured DCs were tested for the production
of IL-6, IL-10, IL-12p40, IL-12p70, TNF-a, IL-23, macrophage
inammatory protein (MIP)-1a, MIP-2 (BD OptEIA ELISA sets;
BD Biosciences) and Prostaglandin E
2
(PGE
2
) (EIA Kits; Cayman
Chemical, Michigan, USA) by sandwich ELISA. Nitrite was mea-
sured using a Griess reagent system (Promega). Expression of cell
surface markers on DCs was quantied by three-colour ow
cytometry using phycoerythrin (PE)- or uorescein isothiocyanate
(FITC)-conjugated antibodies specic for cluster of differentiation
(CD)-14, T-cell co-stimulatory markers CD80, CD86, CD40, OX40L
(BD Biosciences) or MHC II (eBiosciences). Appropriately labelled
isotype-matched antibodies were used as controls. Acquisition
was performed using a FACSCalibur ow cytometer (BD Biosci-
ences), and analyses of results performed using FlowJo software
Version 7.24 (TreeStar Inc., Ashland, USA).
2.3. Phagocytosis assay
The phagocytic ability of DCs was measured using the CytoSe-
lect 96-well Phagocytosis Assay (Cell Biolabs Inc., Cambridge
Bioscience Ltd., Cambridge, UK). Briey, DCs from C57BL/6 mice
were plated at 0.5 10
6
/ml in complete RPMI and incubated
overnight at 37 C to allow adherence to plate. On day 2, DCs
were treated with ABF (10 lg/ml), LPS (100 ng/ml) or zymosan
A (5 lg/ml) for 2.5 h before the addition of sheep erythrocytes
opsonised by IgG at a ratio of 50:1. Supernatants were aspirated
after 1 h and adherent cells washed with sterile PBS to remove
non-phagocytosed erythrocytes. Adherent DCs were then lysed,
substrate solution added and the amount of engulfed erythro-
cytes determined by colorimetric assay at an absorbance of
610 nm. Negative control cells where treated with 2 lM Cyto-
chalasin D to block phagocytosis.
2.4. In vivo T-cell assays and cytokine measurements
For assays of in vivo T-cell priming, DCs isolated from BALB/c
mice were stimulated with or without ABF (10 lg/ml) in the pres-
ence of ovalbumin (OVA) peptide (323-ISQAVHAAHAEINEAGR-
329; 100 nm; GenScript, New Jersey, USA) for 24 h. After being
washed with sterile endotoxin-free PBS, DCs (3 10
5
) were deliv-
ered over the sternumof naive DO11.10 mice by s.c. injection. After
7 days, skin-draining lymph nodes (sdLN) were removed and a sin-
gle cell suspension of cells plated with medium, OVA peptide
(500 nm) or phorbol 12-myristate 13-acetate (PMA) (25 ng/ml;
SigmaAldrich)/anti-CD3e monoclonal antibody (mAb) (1 lg/ml;
clone 145-2C11; BD Biosciences). After 72 h, supernatants were re-
moved for measurement of IL-4, IL-5, IL-10 and IFN-c by commer-
cial assays (R&D Systems).
256 D.J. Dowling et al. / International Journal for Parasitology 41 (2011) 255261
2.5. Protein extraction and Western blot analysis
Total protein was extracted from cell lysates using RIPA buffer
containing 50 mM TrisHCl, pH 8.0, 150 mM NaCl, 1.0% NP-40,
0.5% sodium deoxycholate, 0.1% SDS, and protease and phospha-
tase inhibitor cocktails (SigmaAldrich). Cells were incubated in
the extraction buffer on ice for 5 min before being centrifuged at
8000 g for 10 min at 4 C. Supernatants were transferred to clean
tubes and protein concentrations were determined using a BCA
protein assay kit (Pierce). Protein samples (1040 lg) and pre-
stained protein markers (Precision Plus Protein Standards; Bio-
Rad) were separated by SDSPAGE and blotted onto 0.45 lM
Immobilon-P polyvinylidene diuoride membrane (Sigma
Aldrich). Membranes were blocked for 1 h at room temperature
in 5% non-fat dried milk in PBS and incubated overnight at 4 C
with anti-extracellular signal regulated kinase (anti-ERK), anti-
phosphorylated (p)-ERK, anti-cFos, anti-p-cFos or anti-p-nuclear
factor-kB (NFkB)p65 (Santa Cruz Biotechnology, Inc., California,
USA; 1:1000). Membranes were washed in PBS with 0.05%
Tween-20 (PBS-T) and incubated for 1 h at room temperature with
peroxide-conjugated anti-rabbit IgG (SigmaAldrich; 1:2000).
After further washing, proteins were visualised with supersignal
(Pierce), exposed to lm for 1030 min, and processed using an
FPM 100A Processor (FujiFilm). Protein bands were quantied
using the GeneSnap acquisition and GeneTools analysis software
(GeneGenius Gel Documentation and Analysis System; Syngene,
Cambridge, UK). Levels of p-ERK and p-cFos were normalised to to-
tal ERK and total cFos, respectively, and expressed in arbitrary
units as a percentage increase over the medium control. Levels of
p-NF-jBp65 were expressed in arbitrary units as a percentage in-
crease over medium controls.
2.6. Statistics
All data were analysed for normality prior to statistical testing.
Where multiple group comparisons were made, data were
analysed using one-way ANOVA. For comparisons between two
Fig. 1. Ascaris lumbricoides pseudocoelomic uid (ABF) induces partial activation of dendritic cells (DCs). (A) DCs from C57BL/6 mice were cultured with medium (Med), ABF
(10 lg/ml) or lipopolysaccharide (LPS) (100 ng/ml) for 24 h, and the production of IL-12p40, IL-6, IL-12p70, prostaglandin E
2
(PGE2), tumour necrosis factor (TNF)-a, IL-10,
nitrate, macrophage inammatory protein (MIP)-1a and (MIP)-2 in supernatants were determined by ELISA. Data are presented as the mean SEM and are representative of
three experiments. *P 6 0.05; **P 6 0.01; ***P 6 0.001 compared with Med. (B) Cells were harvested and analysed by three-colour ow cytometry for of cluster of
differentiation (CD)-14, T-cell co-stimulatory markers CD80, CD86, CD40, OX40L and major histocompatibility complex class II. Med (grey line), ABF (dotted black line, 10 lg/
ml), LPS (black line, 100 ng/ml) and isotype (shaded). Mean uorescent intensity (MFI).
D.J. Dowling et al. / International Journal for Parasitology 41 (2011) 255261 257
groups, the Students t test was used. In all tests, P < 0.05 was
deemed signicant.
3. Results
3.1. ABF induces partial activation of murine BMDCs
Antigens derived from helminths do not induce conventional
activation of DCs and therefore to determine whether ABF de-
rived from A. lumbricoides shares similar properties with other
helminth-derived antigens, BMDCs were cultured with ABF
(10 lg/ml) for 18 h and the supernatants analysed for cytokine
and chemokine responses. As a positive control DCs were stimu-
lated with LPS.
DCs stimulated with ABF displayed signicant levels of IL-6 and
IL-12p40 but not IL-12p70, PGE
2
, TNF-a and IL-10 (Fig. 1A). ABF
also induced DCs to secrete the chemokine MIP-2 (CXCL2) but
not MIP-1a (Fig. 1A). As expected, treatment of DCs with LPS in-
duced classical maturation of DCs, evident by highly elevated
levels of all cytokines and chemokines measured.
The ability of ABF to induce enhanced expression of CD14,
CD40, CD86, CD80, OX40L and MHC II DCs was also investigated.
Results demonstrated that ABF did not signicantly alter the
expression of any of the cell surface markers tested (Fig. 1B). As ex-
pected, treatment of DCs with LPS enhanced the expression of all
cell surface markers measured (Fig. 1B).
3.2. ABF modulates DC cytokine production following LPS stimulation
Given that exposure of DCs to helminth antigens can render
them hypo-responsive to LPS stimulation, we investigated whether
ABF could inuence cytokine secretion of DCs following exposure
to LPS. Here, we demonstrate that treatment of DCs with ABF prior
to LPS stimulation resulted in a signicant suppression of IL-12p70
(P 6 0.01) but had no signicant effect upon IL-12p40, IL-6 or MIP-
2 production (Fig. 2).
3.3. ABF activates DC phagocytosis
The ability of DCs to phagocytose is a functional hallmark of
their maturation, allowing these key cells of the innate immune
system to ingest foreign pathogens and present them to naive T-
cells, thus driving the hosts response to infection. While matura-
tion of DCs ultimately leads to the down-regulation of their
phagocytotic ability, studies have shown that phagocytosis is
initially up-regulated during the rst few hours following TLR
stimulation (West et al., 2004). To determine whether ABF could
enhance phagocytosis by DCs during this initial stage, DCs were
cultured with ABF prior to exposure to opsonised erythrocytes.
DCs were also stimulated with medium as a negative control and
Fig. 3. Ascaris lumbricoides pseudocoelomic uid (ABF) activates DC phagocytosis.
DCs from C57BL/6 mice were cultured with medium (Med), ABF (10 lg/ml),
lipopolysaccharide (LPS) (100 ng/ml) or zymosan (Zym) for 2.5 h before the
addition of opsonised sheep erythrocytes. After 1 h, phagocytosis was stopped;
cells were washed to remove non-opsonised erythrocytes, lysed and the amount of
engulfed erythrocytes determined by a colorimetric assay. Negative control cells
where treated with 2 lM Cytochalasin D to block phagocytosis (data not shown).
Data are presented as the mean (SEM) and are representative of three experi-
ments. **P 6 0.01 compared with untreated Med control group.
Fig. 2. Ascaris lumbricoides pseudocoelomic uid (ABF) modulates lipopolysaccha-
ride (LPS)-stimulated dendritic cell (DC) cytokine production. DCs were treated
with ABF (10 lg/ml) or medium (Med) for 2.5 h before stimulation with LPS
(100 ng/ml) for 18 h. IL-12p70, IL-12p40, IL-6 and monocyte-chemoattractant
protein (MIP)-2 were measured in the supernatants by ELISA. Data are the mean
(SEM) of three individual experiments. ***P 6 0.001 compared with LPS group.
Fig. 4. Dendritic cells (DCs) activated with Ascaris lumbricoides pseudocoelomic
uid (ABF) can promote a Th2 polarised response in vivo. DCs from DO11.10 mice
were cultured with ovalbumin peptide (OVA) (100 nm) in the presence or absence
of ABF (10 lg/ml) overnight at 37 C. Stimulated DCs were thoroughly washed and
s.c. injected over the sternum of nave DO11.10 mice. After 7 days, skin-draining
lymph node (sdLN) cells were removed for re-stimulation in vitro with OVA (0
1000 nm) or phorbol 12-myristate 13-acetate (PMA) (25 ng/ml)/anti-CD3 (1 lg/ml).
After 72 h, supernatants were analysed by ELISA for IFN-c, IL-4, IL-5 and IL-10. Data
are the mean (SEM) of three individual wells for four individual mice, and are
representative of two experiments. *P 6 0.05; **P 6 0.01; ***P 6 0.001 compared
with DCs-medium (Med).
258 D.J. Dowling et al. / International Journal for Parasitology 41 (2011) 255261
LPS or zymosan as positive controls. All treatments resulted in a
signicant increase (P 6 0.01) in the number of erythrocytes en-
gulfed by DCs and no signicant differences between ABF and
LPS/zymosan was observed (Fig. 3).
3.4. DCs activated with ABF drive a Th2 phenotype in vivo
Since ABF induced partial maturation of DCs and induced DC
phagocytosis, we investigated the ability of ABF to differentially
activate DCs to prime T-cell responses in DO11.10 mice. DCs were
cultured with OVA (100 nm) in the presence or absence of ABF
(10 lg/ml) and then s.c. injected over the sternum of nave
DO11.10 mice. After 7 days sdLN cells were re-stimulated in vitro
with OVA peptide (0, 100, or 1000 nm) or PMA/anti-CD3 for 72 h.
DCs primed with medium induced IFN-c, IL-4, IL-5 and IL-10 pro-
duction from sdLN cells in response to OVA stimulation (Fig. 4).
DCs primed with ABF induced signicantly more OVA-specic
IFN-c (Fig. 4; P 6 0.01), IL-4, IL-5 and IL-10 (Fig. 4; P 6 0.01
0.001) fromsdLN cells than control DCs primed with medium, indi-
cating that DCs exposed to ABF can promote local Th2 responses
in vivo.
3.5. Enhanced expression of NF-jB and p-ERK is observed in DCs
stimulated with ABF and expression of cytokines by ABF-stimulated
DCs is ERK-dependent
The intracellular mitogen activated protein kinase (MAPK), ERK
and phosphorylation of NF-jBp65 are known to be activated dur-
ing Th2 immune responses (Harnett and Harnett, 2010). More spe-
cically, ERK is thought to be important in promoting Th2 immune
responses in vivo. In this experiment ABF induced ERK expression
at all time points with signicant enhancement at 60 min. As ex-
pected, LPS caused the activation of ERK comparison with medium,
with distinct kinetics similar to previously published data (Fig. 5A)
(Dowling et al., 2010).
To further determine the role of ERK in ABF-stimulated DCs, we
pre-treated DCs with medium or specic ERK inhibitors (U0126)
prior to ABF stimulation and measured IL-6 and IL-12p40 cytokine
production. As previously shown, ABF induced IL-6 and IL-12p40
cytokine secretion from DCs at signicantly higher levels com-
pared with untreated DCs. Pre-treatment with ERK inhibitor re-
sulted in a marked reduction in ABF-induced IL-6 and IL-12p40
(Fig. 5B), thus supporting a role for ERK in Ascaris-induced DC
signalling.
Mechanistically, it has been suggested that the up-regulation of
p-ERK during Th2 responses is associated with stabilisation of the
transcription factor c-fos which results in an intracellular block on
IL-12 production (Agrawal et al., 2003). We have shown that ABF
suppresses IL-12p70 from LPS stimulated dendritic cells but in this
study we show that it does not induce p-cFos in DCs at the time
points tested, while our control LPS as previously shown can en-
hance p-cFos expression after 1 h.
The transcription factor NF-jB is primarily associated with typ-
ical DC maturation, and is involved in regulating the expression of
a large number of inammatory mediators. The phosphorylation of
the DNA binding subunit p65 is an indicator of NF-jB activation
(Hayden and Ghosh, 2008; Sun and Ley, 2008). In this experiment
the level of NF-jBp65 in DCs following treatment with ABF was
determined by Western blot using LPS as a positive control. DCs
stimulated with LPS exhibited enhanced phosphorylated NF-
jBp65 (p-NF-jBp65) expression at 15, 30 and 60 min (Fig. 5A
and C). In contrast, ABF induced only a transient increase in the lev-
els of p-NF-jBp65 at 15 but not at 30 or 60 min (Fig. 5A).
3.6. The partial activation of DCs induced by ABF is not
TLR4-dependent
Since ERK and p-NF-jBp65 are common components of the TLR
pathway (Harnett and Harnett, 2010) we proceeded to determine
whether the partial activation of DCs by ABF is dependent upon
TLR4. Wild-type TLR4 DCs (on a C57BL/6 background) produced
Fig. 5. Activation of dendritic cells (DCs) by Ascaris lumbricoides pseudocoelomic uid (ABF) is extracellular signal regulated kinase (ERK)-dependent. (A) DCs from C57BL/6
mice were cultured with culture medium (Med, 1), ABF (10 lg/ml, 2) or lipopolysaccharide (LPS) (100 ng/ml, 3). At 15, 30 and 60 min expression of phosphorylated ERK
(p-ERK), total ERK (t-ERK), p-cFos, t-cFos and p-NF-jBp65 was measured by Western blot. Densitometric analysis is representative of three experiments. Values of p-ERK and
p-cFos were normalised to t-ERK and t-cFos, respectively, and are expressed in arbitrary units as percentage increases over Med control while p-NF-jBp65 are expressed in
arbitrary units as percentage increases over Med control. (B) Cells were cultured as in A, but in the presence or absence of a specic ERK inhibitor (U0126; SigmaAldrich,
5 lM) which was added 1 h prior to antigen stimulation. Cells were incubated for a further 18 h, and the production of IL-12p40 and IL-6 in supernatants determined by
ELISA. Data are presented as the mean SEM and are representative of three experiments. ***P 6 0.001 compared with control mice.
D.J. Dowling et al. / International Journal for Parasitology 41 (2011) 255261 259
signicantly increased levels of IL-6, IL-12p40 and MIP-2 in re-
sponse to ABF, LPS and zymosan compared with medium alone
(Fig. 6). TLR4 knock-out (KO) DCs failed to respond to LPS, produc-
ing little or no IL-6, IL-12p40 or MIP-2, however stimulation with
the TLR4-independent ligand zymosan, which signals through
TLR2 (Gantner et al., 2003), remained unaffected, conrming the
integrity of TLR signalling in the TLR4 KO DCs. There was no de-
crease observed in IL-6, IL-12p40 or MIP-2 levels in TLR4 KO DCs
stimulated with ABF, indicating that cytokine production is not
dependent upon TLR4 (Fig. 6).
4. Discussion
ABF and its derived antigens can polarise a Th2 response in hu-
man DCs when co-cultured with LPS (van Riet et al., 2009). Here we
demonstrate that ABF induces partial maturation of DCs as charac-
terised by the increased production of signicant amounts of IL-6,
IL-12p40 and MIP-2. While ABF does not enhance expression of co-
stimulatory molecules on DCs, ABF-activated DCs displayed an
ability to promote a Th2 response in vivo. These observations sup-
port the DC maturation model in which Th2 differentiation can oc-
cur through a default pathway where Th2 immune responses are
initiated by DCs in a semi-mature state. However in contrast to
the model described by MacDonald and Maizels (2008) we
observed an absence of enhanced co-stimulatory molecule expres-
sion but instead observed strong to moderate cytokine production
(MacDonald and Maizels, 2008). This study also supports the inhi-
bition model of DC maturation since ABF suppresses LPS-induced
IL-12p70.
The secretion of IL-6, IL-12p40 and MIP-2 by ABF-stimulated
DCs has not to our knowledge been previously reported, however
it is a characteristic observed in studies investigating the interac-
tions of other helminth-derived antigens with DCs (Harnett and
Harnett, 2010). Previous studies examining antigens derived from
the closely linked pig nematode, Ascaris suum, demonstrated that
glycophingolipid isolated from its pseudocoelomic uid induced
DC and macrophage secretion of IL-12p40 and TNF-a (Kean et al.,
2006). Similarly, Schistosoma larvae-derived products (Jenkins
and Mountford, 2005), Nippostrongylus brasiliensis excretory/secre-
tory antigens (Balic et al., 2004) and more recently Fasciola hepatica
Cathepsin L and sigma class GST (Dowling et al., 2010) were shown
to induce the secretion of IL-12p40, IL-6 or MIP-2 from DCs. These
cytokines have a role in promoting Th2 immune responses where
IL-6 and IL-12p40 promote Th2 immune responses by negative
regulation of Th1/Th17 inammatory responses (La Flamme
et al., 2000; Brahmachari and Pahan, 2008) and MIP-2 is thought
to inuence a Th2 micro-environment by directly recruiting Th2
cells to the site of infection (Perrigoue et al., 2008). However, in
contrast to this study these molecules also induced co-stimulatory
molecule expression on DCs.
Under our experimental conditions the induction of cytokine
production by DCs in response to ABF is dependent upon ERK sig-
nalling. This is similar to other studies as Schistosoma egg antigen
(SEA) and the sugar lacto-N-fucopentaose III (LNFPIII) isolated from
SEA which conditions DCs to induce strong Th2 responses also in-
duced the phosphorylation of ERK by DCs (Agrawal et al., 2003;
Thomas et al., 2003). The propensity of ERK-decient mice to exhi-
bit Th1 polarisation further supports a role for ERK in modulating
the behaviour of DCs and the consequences this has on the promo-
tion of Th2 responses (Agrawal et al., 2006).
While ABF stimulation of DCs activated phosphorylation of TLR-
associated signalling components ERK and NF-jB, the secretion of
IL-6, IL-12p40 and MIP-2 was not dependent upon TLR4, suggest-
ing that other TLRs may be involved (Jenkins et al., 2005). Both
Schistosoma mansoni and A. lumbricoides-derived phosphatidylser-
ine have been shown to have TLR2 activating capacity (van der
Kleij et al., 2002; van Riet et al., 2009). However, other studies have
shown that S. mansoni SEA can modulate both TLR-induced DC
activation and induce Th2 responses in nave mice independently
of TLR2, TLR4 or MyD88 (Kane et al., 2008).
In summary, we believe this study was the rst to investigate
ABF interactions with murine DCs. While our observations do not
t one particular model of DC maturation by Th2 stimuli, it does
support current observations in studies examining the maturation
of DCs by helminth antigens and supports the hypothesis by
MacDonald and Maizels (2008) who suggest that there is no single
mechanism that primes Th2 immune responses but rather a com-
bination of pathways that may vary for individual helminths. By
adding to the growing body of knowledge in this eld we can test
current hypotheses so that we can clearly dene the mechanism
involved in the activation of Th2 cells by helminths.
Fig. 6. Ascaris induction of IL-6 and IL-12p40 and is not toll-like receptor 4 (TLR4)-
dependent. Dendritic cells (DCs) from C57BL/6j (control, wild-type (WT)) and TLR4
knockout (KO) mice were cultured with medium only (Med), Ascaris lumbricoides
pseudocoelomic uid (ABF) (10 lg/ml), zymosan (Zym, 5 lg/ml) or lipopolysac-
charide (LPS, 100 ng/ml) for 24 h, and the production of IL-12p40, IL-6 and
macrophage inammatory protein (MIP)-2 in supernatants determined by ELISA.
Data are presented as the mean SEM and are representative of three experiments.
***P 6 0.001; **P 6 0.01 compared with control mice.
260 D.J. Dowling et al. / International Journal for Parasitology 41 (2011) 255261
Acknowledgments
This work was supported by the Dublin City University (DCU)
Faculty of Science (Ireland) and Health Targeted Research Develop-
ment Fund, European Union (DELIVER: Grant FOOD-CT-2005-
023025); and Biotechnology and Biological Sciences Research
Council (Grant BB/C503638/2). We thank Carolyn Wilson (DCU)
for technical support.
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