New Insights into the Modulation of Immune Response by Fasciola hepatica Excretory-Secretory Products

Abstract

Fasciola hepatica is a trematode that affects human and domestic ruminant health, causing significant economic losses in cattle estimated at US$2000 millon per year. Juvenile parasites migrating through the host tissues, as well as adults settle in the biliary ducts, are in contact with different cells from the immune system. Despite those interactions, the persistence of the parasite in the host for many years provides evidence of its ability to prevent or down-modulate the inflammatory response in the infection site. Different strategies have been developed by the parasite to prevent potential damage being induced by the immune response, thus allowing some parasites to reach the adult stage in a safe place such as the biliary ducts. In this review we discuss how excretory-secretory products (ESP) from F. hepatica can affect the functionality of pivotal immune cells, such as eosinophils and macrophages by inducing selective apoptosis pathways and alternative activation of macrophages. Furhermore, the modulatory effects of ESP on dendritic cell activation and lymphocyte proliferation is reviewed as a strategy to facilitate F. hepatica evasion of both innate and adaptive immunity.

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Current Immunology Reviews, 2009, 5, 277-284 277
1573-3955/09 $55.00+.00 © 2009 Bentham Science Publishers Ltd.
New Insights into the Modulation of Immune Response by Fasciola
hepatica Excretory-Secretory Products
Laura Cervi, Marianela C. Serradell, Lorena Guasconi and Diana T. Masih*
Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Centro de
Investigaciones en Bioquímica Clínica e Inmunología, CONICET, Haya de la Torre y Medina Allende, (5000) Córdoba,
Argentina
Abstract: Fasciola hepatica is a trematode that affects human and domestic ruminant health, causing significant
economic losses in cattle estimated at US$2000 millon per year. Juvenile parasites migrating through the host tissues, as
well as adults settle in the biliary ducts, are in contact with different cells from the immune system. Despite those
interactions, the persistence of the parasite in the host for many years provides evidence of its ability to prevent or down-
modulate the inflammatory response in the infection site. Different strategies have been developed by the parasite to
prevent potential damage being induced by the immune response, thus allowing some parasites to reach the adult stage in
a safe place such as the biliary ducts. In this review we discuss how excretory-secretory products (ESP) from F. hepatica
can affect the functionality of pivotal immune cells, such as eosinophils and macrophages by inducing selective apoptosis
pathways and alternative activation of macrophages. Furhermore, the modulatory effects of ESP on dendritic cell
activation and lymphocyte proliferation is reviewed as a strategy to facilitate F. hepatica evasion of both innate and
adaptive immunity.
Keywords: Fasciola hepatica, innate immunity, eosinophils, macrophages, dendritic cells, evasion mechanisms.
INTRODUCTION
The helminth parasite F. hepatica causes liver fluke
disease or fasciolosis, thereby affecting the health of
humans, as well as sheep, cattle and goats, among others.
Fasciolosis causes losses in agriculture estimated at >
US$2000 millon per year, with its now increasing prevalence
in humans [1]. The host acquires the infection by the
ingestion of cysts (metacercariae). Then, once is the parasite
is in the intestine, it is excysted and migrates through the
intestinal wall and liver parenchyma, where it induces
hemorrhagic foci and hyperplasia of bile ducts. The severity
of the infection depends on the number of metacercariae
ingested, as well as on the number of larvae able to survive
attack from the immune response and reach the bile ducts,
where they then develop into adults. Although different hosts
infected with F. hepatica produce an adaptive T helper 2
response, the magnitude of the resistance to infection
depends on the host. For example, sheep and mice are more
susceptible to infection than cattle and rats, which suggests
different roles of innate immune cells in the resistance
mechanisms against infection [2]. After becoming
established in the host tissues, the larvae and adults then
release excretory-secretory products (ESP). This also occurs
in other helminths such as Schistosoma mansoni [3],
Paragonimus westermani [4], and even in some nematodes,
for example Brugia malayi [5] where the products from
different parasite stages modulate the immune response thus
helping to prevent exacerbated inflammation in the host.
*Address correspondence to this author at the Departamento de Bioquímica
Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba,
Centro de Investigaciones en Bioquímica Clínica e Inmunología, CONICET,
Haya de la Torre y Medina Allende, (5000) Córdoba, Argentina; Tel: 54-
351-4344973; Fax: 54-351-4333048;
E-mail: dmasih@fcq.unc.edu.ar
Although some components from ESP from different
heminths are in part involved in the induction of
inmunoevasion mechanisms, the pattern of each individual
ESP is unique thereby making it a hallmark of the particular
immunomodulation. In this review, we have focused on the
involvement of F. hepatica ESP on the prevention of the
effector immune response, by the modulation of innate and
adaptive immune responses.
FIGHTING EOSINOPHILS-HELMINTHS: NO
WINNERS OR LOSERS
One of the features of helminth infections is the
development of eosinophilia. A short time after the helminth
infection occurs, there is an increase in the number of
eosinophils in the blood, which then migrate to the site of the
infection [6]. This recruitment of eosinophils in helminth
infections is partly due to the effect of products derived from
trematodes or nematodes with chemotactic activity. Related
to this, it has been demonstrated that whole worm extract or
excretory/secretory material from ovine nematodes have
chemoattractant activity to eosinophils [7], and a parasite-
derived factor, galectin-like protein with eosinophil
chemokinetic activity in vitro, has been recently identified
[8]. Excretory-secretory products from helminths are also
involved in the maturation of eosinophils, since ESP from F.
hepatica were as capable as IL-5 in stimulating bone marrow
cells from mice to mature as eosinophils as evidenced by
increased eosinophil peroxidase activity [9] (Fig. 1). Once
eosinophils are recruited to the site of infection, they start
releasing many factors, such as IL-4, which is crucial in the
development of the Th2 response, a hallmark of helminth
infection [6]. Mice injected with eggs from S. mansoni
showed a fast IL-4 eosinophil increase, which may
contribute to the Th2 phenotype [10]. Moreover, the Th2
response in a positive loop leads to a maturation of

278 Current Immunology Reviews, 2009, Vol. 5, No. 4 Cervi et al.
eosinophils by the secretion of IL-5 (eosinophil growth
factor) [6] (Fig. 1). Apart from their contribution to Th2
polarization, eosinophils have also been recognized as
effector cells able to kill large and nonphagotizable parasites
such as helminths. Different studies have shown this ability
of eosinophils to kill helminths, either alone or in
conjunction with antibody or complement [11-13]. In in vitro
assays, the killing of parasite larvae from Schistosoma
mansoni [11] and F. hepatica by a antibody dependent
cellular cytotoxicity (ADCC) mechanism has been
demonstrated [2, 14] (Fig. 1). In these assays, specific
antibodies recognize and bind antigens on the surface of F.
hepatica larvae through the Fc receptor, thus provoking the
degranulation of these cells and the release of cytotoxic
proteins able to kill the parasite [15]. Although efficient at
eliminating large parasites, the ADCC mechanism could be
prevented by F. hepatica in different ways, for example by
changing the external glycocalyx during the development of
different stages [16], as well as by releasing proteases able to
cleave the Fc of Immunoglobulins [17, 18].
In both cases the attachment of antibodies to the surface
of the parasite may be avoided, with consequent absence of
eosinophil degranulation. In addition to the ADCC
mechanism, eosinophils can act as effector cells by the
release of oxygen reactive species (ROS) able to damage
large parasites [19]. For example, during H. nana [20] and F.
gigantica [19] infection, ROS produced by eosinophils play
a role in the resistance to re-infection by these parasites.
However, the ROS up-regulation induced by helminth
infection also seems to be detrimental to the eosinophils
themselves, since these molecules can lead to an early
apoptosis, depending on the caspases [21]. The apparent
controversy about the role of ROS in eliminating worms or
inducing eosinophil apoptosis may be explained by the
differences in ROS concentration found in the different
experimental settings. In in vitro assays, the amount of ROS
released by eosinophils is probably higher than that present
under more physiological conditions, such as natural
infection, where lower concentrations of ROS might still be
enough to eliminate parasites, whereas excessive
concentrations can be detrimental to these cells.
Nevertheless, the in vivo importance of ROS, in particular
H2O2, in damaging eosinophils, was evident. A peritoneal
injection of catalase, which abrogated H2O2, in F. hepatica
infected rats, diminished the percentage of apoptotic
eosinophils (unpublished data), suggesting a close
relationship between eosinophil apoptosis and ROS
production. However, the consequence of eosinophil
abrogation during helminth infection is still unclear, since
data concerning eosinophil ablation during the progression
of helminth infection are contradictory. Recently, by using
an eosinophil-deficient mouse model, it has been
demonstrated that mice with a defective eosinophilopoiesis
showed an impaired resistance in early secondary
Nippostrongylus brasiliensis infection [22, 23]. Similarly,
using an anti-CCR3 monoclonal antibody to eliminate
eosinophils, another study demonstrated that eosinophils are
required in the protective innate immune response against
Strongyloides stercoralis [24]. In contrast, eosinophil
ablation in a S. mansoni infected mice model had no impact
on worm burden or on egg deposition [25].
The contribution of eosinophils to host defense could be
related not only to a reduction in the number of pathogens,
but also to contributions to tissue remodeling. Recently,
many reports have focused on the role of eosinophils in
remodeling and reparation of damaged tissue as well as on
cell debris clearance [6, 22]. Related to this, the involvement
of eosinophils has been described in the regulation of
pathogenesis induced by S. mansoni. By using IL-5 knockout
mice, it was demonstrated that eosinophils play an important
role in the induction of liver fibrosis during the pathogenesis
of schistosomiasis by multiple mechanisms. Eosinophils
regulate the inhibition of antifibrotic IFN-, by increasing
IL-5 production (thus helping Th2 polarization), and IL-13
(profibrotic, mediator) as well as the number of
“alternatively activated” macrophages and fibroblasts, with
these latter being important mediators of tissue remodeling
and fibrosis [26]. In fasciolosis, data from our laboratory
showed the presence of eosinophils surrounding the tunnels
generated by fluke migration through the liver [21]. Despite
the lack of information concerning a possible role of
eosinophils in the reparation of the liver damage in this
parasitosis, we cannot rule out a possible participation in this
phenomenon.
HELMINTH INFECTIONS: ALL ROADS LEAD TO
ALTERNATIVELY ACTIVATED AND/OR T
REGULATORY MACROPHAGES
It is well known that macrophages participate as
important effector cells in order to eliminate intracellular
pathogens such as Mycobaterium bovis or Listeria
monocytogenes by an activation dependent on the INF-
produced by Th1 and natural killer cells [29]. Classically
activated macrophages are induced by inflammatory stimuli,
for example INF-, TNF- and microbial products such as
LPS [29]. In contrast, extracellular pathogen helminths such
as Brugia, Nippostrongylus, Litomosoides,
Heligmosomoides, trematodes as Schistosoma and Fasciola
and cestodes such as Taenia, Echinococcus and
Hymenolepis, have been associated with the induction of
alternative activated macrophages (AAM) [30, 31]. IL-4 and
IL-13 are cytokines produced during a Th2 response, and
induce an increase in arginase-I activity, favoring the
metabolization of l-arginine towards proline, polyamines and
urea [31]. However, the induction of AAM can also be
elicited by the innate immune response in the absence of a
specific Th2 response in different ways. Products from
helminths alone [32] or IL-4 production by cells from innate
immunity as mast and NK cells [33] can be sufficient stimuli
to induce AAM. In this way, as early as one day after
infection with F. hepatica, peritoneal macrophages from
infected mice showed an up-regulation of genetic markers of
AAM such as Fizz1 and arginase-I, and by day 7, the
expression of Ym1 may also be induced. In a similar way,
the injection of a purified fraction of ESP from F. hepatica
or a purified protein, peroxiredoxin (Prx) induced AAM in
the peritoneal cavity, which induced the secretion of anti-
inflammatory agents such as IL-10 and PGE2 [34] (Fig. 2).
More recently, it has been demonstrated that Prx stimulates
the expression of Ym1 in macrophages, independently of IL-
4 and IL-13 signaling. In addition, Prx has been involved in
Th2 polarization through a mechanism involving AAM,
since the antibody mediated neutralization of Prx during

Fasciola hepatica Excretory-Secretory Products Current Immunology Reviews, 2009, Vol. 5, No. 4 279
infection with F. hepatica, blocked the Ym1 expression and
Th2 polarization [32] (Fig. 2). The alternative activation
phenotype in macrophages in response to an injury might be
independent of the adaptive immune response; but to be
sustained over time, for example in a chronic infection, they
require IL-4 and/or IL-13 Th2 producing cells in order to
mediate wound healing [35]. AAM contribute to wound
healing by the clearing matrix and cell debris as well as the
releasing of different factors that promote fibroplastia and
angiogenesis [6]. Overall, these data suggest that in helminth
infections, the recognition of molecules released by the
parasite could be sufficient to promote an alternative profile
in macrophages, leading to the development of a Th2
response with an expansion and maintenance of this
alternative profile favoring the wound healing in the chronic
stage of infection.
In agreement with this concept, it has been demonstrated
that AAM play a crucial role in the reduction of
inflammation and host survival during murine
schistosomiasis [36]. The presence of AAM contribute to
protect the mice against organ injury through down
regulation of egg-induced inflammation, by the clearance of
Fig. (1). Interaction F. hepatica-eosinophils. The effector function of eosinophils against F. hepatica, include the ADCC mechanisms as well
as ROS releasing, both able to kill the parasite. At the same time the parasite can prevent the damage produced by eosinophils, inducing the
apoptosis of these cells [21, 27], preventing ROS effect by the secretion of detoxifying enzymes [28], or through Igs cleavage to block
ADCC. Alternatively, ESP induce the maturation of eosinophils, which up-regulate the secretion of IL-4, IL-5 and ROS, favoring Th2
polarization, which increases the number of fibroblasts and macrophages, mediators of tissue remodeling and fibrosis.

280 Current Immunology Reviews, 2009, Vol. 5, No. 4 Cervi et al.
cellular debris from dead cells, and by promoting tissue
reparation through IL-13 secretion, with the latter favoring
proline synthesis, a precursor of collagen [36].
Although some evidence supports the fact that helminth
products are able to induce AAM, little information is
available about the receptors involved in the recognition of
inflammatory stimuli and their relation with the alternative
pattern induction. In some cases, the recognition of some
specific molecular pattern by macrophages stimulates TLR
signaling. For example, ES-62 from filaria signals through
TLR4 in an unconventional way, thus interfering with the
LPS signaling [37]. Similarly, we have found that peritoneal
cells from rats cultured in the presence of ESP or a purified
protein-glutathione transferase, inhibit nitric oxide
production induced by the TLR4 ligand, LPS [38].
Coincidently, other authors have shown that ESP from F.
hepatica reduce the ability of bovine macrophages to
produce nitric oxide (NO) or IFN– induced by LPS [39].
Also, the purified protein derivatives from Mycobacterium
bovis (PPD–B), which are TLR 4 and 2 ligands, induce a rise
in IL–10 production [39] (Fig. 2). Additionally, ESP
treatment of bovine macrophages increases the arginase and
chitinase activities, two markers of alternative activation
[39]. From these evidences it seems clear that helminth
products have the ability to both modulate the phenotype of
macrophages to an alternative phenotype and to exert control
on the inflammatory signals initiated by different pathogen
associated molecular patterns (PAMPs). However, it is still
not known whether both events are related or if the
acquisition alternative activated phenotype could condition
the activation initiated by different PAMPs. Parasite
products may interfere with the signaling involved in NO or
arginase production through the transcription factor STAT6,
which is required to induce arginase [40]. Its role in the
inhibition of NO production was demonstrated in murine
macrophage expression stimulated with IL-4 and IL-13 [40].
Alternatively, up-regulation in arginase may be a result of
the inhibition in NO production due to competition for the
same substrate: l-arginine [40]. Furthermore, helminth
products may interact with TLR or non-TLR such as C type
lectins [41]. Regarding th is, it has been described that the
cytokines interleukin IL-4 and IL-13, which are involved in
the development of AAM, induce an increase in the
expression of the macrophage mannose receptor in murine
peritoneal macrophages [42]. Additionally, a macrophage
galactose-type-C-type lectin was induced during mouse
infection with Taenia crassiceps [43]. The increased of C-
type lectins receptors by helminth products in AAM
macrophages, could be a consequence of an interaction
between receptors, as it happen after the stimulation with
products from fungus as -glucans [41]. Illustrating this
concept, it has been demonstrated that the cross talk between
the -glucan receptor Dectin-1 with TLR-2 induced by
zymosan, promotes an increase in TNF and IL-12
production, as well as in IL-2 and IL-10 [44]. However, a
recent work demontrated that depending on dose of
zymosan, dendritic cells produce high level of IL-10 and
tolerogenic dendritic cells, or IL-12 and inflammatory
dendritic cells. These authors observed that concentrations of
zymosan above 200 μg/ml stimulated higher levels of IL-6
and IL-12(p70) while the induction of IL-10, appeared to be
largely unaffected by these higher concentrations of
zymosan [45]. Considering these observations in the light of
a recent review proposing a new grouping of macrophage
populations, it seems that helminth infection or its derived
products could act to induce AAM, (referred as wound
healing macrophages), with an increased expression of YM1,
(a chitinase-like molecule with a carbohydrate and matrix-
binding activity [46], with an ability to accommodate longer
chitin polymers and involved in matrix reorganization and
wound healing). Although in that review, the main inducer
of wound healing macrophages was described as IL-4, the
ability of helminth products to drive the polarization of
AAM or wound healing macrophages is becoming
increasingly evident. Despite the apparent role of IL-4
provided by innate immunity and Th2 cells inducing AAM
polarization, in an experimental setting with macrophages in
the complete absence of IL-4, helminth products were
sufficient to produce the same effect [32]. In the particular
case of fasciolosis, tissue migration of the flukes through the
liver, the main pathogenic event of this disease, coincides
with an increased expression of Fizz, Arginase1 and Ym1 [1,
32], which could be mediators to the additional hepatic
fibrosis described in this infection. Furthermore, we and
others have shown the ability of ESP to interfere with TLR
activated macrophages to produce NO, and also increase IL-
10 production [39], unpublished data). Both these features
are similar to those described for T regulatory macrophages
[46], which are producers of high levels of IL-10 and capable
of interfering with inflammatory signals such as TLR
ligation. Whether the features developed in macrophages by
ESP treatment correspond to a different profile of
macrophages, or alternatively to a change in macrophage
population over time, is not yet clear, but both alternatives
could be possible and form a target for future studies.
Finally, some evidence shows an unexpected role for
AAM as a protective mechanism against some nematodes
[47]. The participation of AAM in the clearance of
helminths, was demonstrated in infected mice with
Heligmosomoides polygyrus, showing the recruiting of AAM
in the intestinal lumen depending on the generation of IL-4
by memory CD4+ T cells. In this work, was also shown that
AAM diminished the larval viability by an arginase
dependent mechanism, which probably contributed to the
elimination of the worm [6]. In summary, the presence of
macrophages with an alternative activation or regulatory
phenotype, could be beneficial, to the host to prevent a
massive infection playing a role in the diminution of the
parasite burden, and to reduce the damage to tissues, as well
as inhibiting danger signals from exogenous or endogenous
stimuli which help to create an unhostile environment.
HELMINTH ANTIGENS MODULATE DENDRITIC
CELLS MATURATION INDUCED BY TLR
LIGATION AND PROMOTE TH2 AND T
REGULATORY CELL DEVELOPMENT
In the initiation of an adaptive immune response,
dendritic cells are crucial for providing signals to naïve T
cells and are responsible for the polarization of these cells
[48]. Depending on the nature and the intensity of the
stimuli, dendritic cells can achieve different activation
statuses [49, 50]. Associated with the differential maturation,
dendritic cells have the capacity to prime polarized T helper
(Th) cell responses. Thus, depending on the initial

Fasciola hepatica Excretory-Secretory Products Current Immunology Reviews, 2009, Vol. 5, No. 4 281
maturation signal, dendritic cells can prime Th cells to
differentiate toward the Th1, Th2, Th17 or T-regulatory type
(T reg) [49, 51-53]. In a quite simplifed view, a mature
phenotype of dendritic cells is related to the induction of Th1
response, and in contrast, immature dendritic cells have been
associated to Th2 polarization [54]. However, new evidence
shows that the activation of dendritic cells with the thymic
stromal lymphopoietin (TSLP) produced by intestinal
epithelial cells, lead to a Th2 polarization promoting
protection against an intestinal parasite such as Trichuris
muris. TSLP mature dendritic cells can also prevent Th1
polarization by the inhibition of IL-12 production [6, 55, 56].
Increasingly research shows the lack of ability of helminth
antigens as soluble antigens from S. mansoni eggs (ESA) to
induce “classical dendritic cell maturation” [57]; or to
selectively up-regulate some activation markers [58]. In
addition, ESA, as well as NES, inhibit the activation initiated
by a TLR ligand such as LPS, by down-regulating the
production of pro-inflammatory cytokines and costimulatory
molecules [57, 58]. In a similar way, ESP from F. hepatica
failed to induce maturation of these cells or suppress the up
regulation of TNF production and CD40 expression in
response to TLR ligands (TLR4, TLR3 and TLR7)
(unpublished data). However, although helminth products
seem to prevent TLR initiated maturation, an interesting
question arising is whether they can their selves act as
PAMPs to TLR. In this sense, some purified molecules from
helminths such as phosphatidyl serine, (a schistosomal lipid)
or LNFPIII have been proposed as ligands for TLR2, and
TLR4 respectively [59-61]. However, none of these ligands
work in an identical way, compared to the case of classical
TLR ligands, such as peptidoglycan or bacterial LPS, with
schistosomal lipid antigen acting on dendritic cells in a way
so that these cells generate regulatory cells or LNFPIII,
which then induces a transient translocation and activity of
NF-kB [61]. This differs from the persistent activation
induced LPS necessary to activate proinflammatory genes
[57, 58].
Fig. (2). F. hepatica infection or ESP lead to an alternative polarization of macrophages. F. hepatica infection induce the expression of
arginase, Fizz 1 and Ym1. In a similar way peroxiredoxin, stimulates the expression of Ym1, which is involved in Th2 polarization and up-
regulates the secretion of IL-10 and PGE2. At the same time, Th2 polarization through the secretion of IL-4 and IL-13 increases the arginase
expression. ESP from F. hepatica or glutathione transferase inhibit the ability of macrophages to produce NO induced by TLR 4 and 2
ligands. The transcription factor STAT6 is required to induce arginase and the up-regulation in arginase may be result in NO production.

282 Current Immunology Reviews, 2009, Vol. 5, No. 4 Cervi et al.
In the infection scenario, these data are difficult to
interpret, since the antigens recognized by antigen presenting
cells (APC) are much more complex than purified
molecules, and their composition could be closer to
excretory-secretory or total soluble antigens. The failure of
ES or soluble antigens from helminths, as well as ESP from
F. hepatica to activate dendritic cells, suggests that in spite
of the presence of PAMPs among the helminth products,
their final effect on APC is the inhibition of TLR or other
signaling. Thus, some “stimulatory” molecules from
helminths lead to a “tolerogenic” phenotype to promote Th2
and/or regulatory response. However, more work is
necessary to dissect whether the inhibitory components
isolated from unpurified ES antigens, are able to negatively
modulate the effect of activator PAMPs present in those
mixtures. Overall, although in different settings a mixture of
helminth antigens or their purified derived molecules can
partly activate dendritic cells, these cells still act as a Th2
response driver.
It has been well documented that a polarization of
adaptive immune response to a Th2 profile occurs in mice,
rats, bovine and ovine infected with F. hepatica [32, 39, 62-
64]. This ability has been explored in a study showing the
modulatory effect of the Th2 response induced by F.
hepatica infection on Th1 induced by unrelated antigens
[64]. Thus, co-infected mice with F. hepatica and Bordetella
pertusis showed an impaired bacterial specific Th1 response
and bacterial clearance in the lungs. In contrast, the Th2
profile induced by the parasite was not modified by the Th1
response induced by B. pertussis. In agreement with this
study we observed, that the inoculation of ESP in rats
induces a population of non-adherent cells to nylon wool
splenocytes which when transferred to normal recipient rats,
inhibits the ability of these animals to respond to an
unrelated or related antigen as measured by the DTH
response [65].
More recently, we found that the treatment of bone
marrow derived murine dendritic cells with ESP, target the
dendritic cells differentiation and function to convert these
cells into tolerogenic dendritic cells, capable of leading naive
T cells to Th2 and T regulatory phenotypes. Also, ESP-
treated dendritic cells expanded a population of T cells
which match with a regulatory phenotype, since these cells
showed an increased expression of CD25, Foxp3 and IL-10
(unpublished data). These results prompt us to think that the
injection of ESP in rats might induce in vivo a population of
T regulatory cells (Treg), which could explain the
suppressive effect on the DTH response. However, whether
the Foxp3 positive cells are also capable of secreting Th2
cytokines remains unanswered.
There is evidence showing that during a helminth
infection, a population of natural Treg cells (CD4 CD25
Foxp3) is expanded together with Th2 cells [66].
Furthermore, as documented by Pearce et al. [67], natural
Treg and Th2 cells both contribute to make IL-10 which
suppresses the development of the Th1 profile in response to
the schistosome egg Ag. However, an additional role for
Treg emerged during helminth infection, which prevented an
exacerbate Th2 response. Supporting this idea, it has been
demonstrated that S. japonicum egg antigens induced
CD4+CD25+ regulatory T cells, contributing to the
inhibition of mediated asthma development [68].
The singular property of helminth parasites to
simultaneously induce Th2 and T regulatory responses has
been used beneficially to prevent or ameliorate auto-immune
diseases [61]. Human infection with T. suis ameliorated the
severity of Crohn’s [69, 70], as well as reducing ulcerative
colitis [71, 72]. The severity of EAE was diminished by S.
mansoni infection and was also mediated by STAT6,
suggesting an involvement of this molecule in the induction
of the Th2 response [73]. Dendritic cells have been largely
involved in the generation of tolerance or Treg cells, and it is
well known that immature dendritic cells induce a
tolerogenic immune response and stimulate naïve T cells into
regulatory cells [74-76]. The ability of ESP-treated dendritic
cells to induce Treg cells together with the impaired
allogeneic responses induced by LPS-treated dendritic cells
(unpublished data), suggest that the identification of new
molecular targets in ESP could be useful in designing new
therapeutic strategies to reduce excessive and harmful
inflammatory responses.
CONCLUDING REMARKS
In conclusion, F. hepatica as in the case of other
helminths, has a strong effect on the functionality of the cells
from innate immunity such as eosinophils, macrophages as
well as dendritic cells. Once the parasite enters the host
tissues, a delicate balance between the host effector
mechanism and the defense by the parasite is established,
allowing the survival of a number of flukes that escape from
the immune attack, and as long as some parasites persist, are
able to act as effectors to regulate immune responses. The
understanding of the molecular basis by which ESP or other
helminth products modulate the functionality of eosinophils,
macrophages and dendritic cells, has an enormous potential
since the modulation of these cell activities has a strong
impact on the type of the adaptive immune response as well
as in tissue repair. This information could be also exploited
in the prevention of diseases with excessive inflammatory
responses, such as the autoimmune diseases or
immunopathologies. The characterization of helminth
molecules which interfere with inflammatory signals will
help in the designing of new drugs.
ACKNOWLEDGEMENTS
This work was supported by grants from Consejo
Nacional de Investigaciones Científicas y Técnicas de
Argentina (CONICET) and the Agencia de Promoción
Científica y Tecnológica (PICT 2005-32027 and 33326),
MCS and LG are Ph.D fellows of CONICET. L.C. and
D.T.M. are members of the Scientific Career of CONICET.
We would like to thank native speaker, Dr. Paul Hobson for
revision of the manuscript.
REFERENCES
[1] McManus DP, Dalton JP. Vaccines against the zoonotic trematodes
Schistosoma japonicum, Fasciola hepatica and Fasciola gigantica.
Parasitology 2006; 133(Suppl): S43-61.
[2] Piedrafita D, Parsons JC, Sandeman RM, et al. Antibody-
dependent cell-mediated cytotoxicity to newly excysted juvenile
Fasciola hepatica in vitro is mediated by reactive nitrogen
intermediates. Parasite Immunol 2001; 23: 473-82.

Fasciola hepatica Excretory-Secretory Products Current Immunology Reviews, 2009, Vol. 5, No. 4 283
[3] Trottein FS, Nutten V, Angeli P, et al. Schistosoma mansoni
schistosomula reduce E-selectin and VCAM-1 expression in TNF-
alpha-stimulated lung microvascular endothelial cells by interfering
with the NF-kappaB pathway. Eur J Immunol 1999; 29: 3691-701.
[4] Shin MH, Kita H, Park HY, Seoh JY. Cysteine protease secreted by
Paragonimus wetermani attenuates effector functions of human
eosinophils stimulated with immunoglobulin G. Infect Immun
2001; 69: 1599-604.
[5] Semnani RT, Liu AY, Sabzevari HJ, et al. Brugia malayi
microfilariae induce cell death in human dendritic cells, inhibit
their ability to make IL-12 and IL-10, and reduce their capacity to
activate CD4+ T cells. J Immunol 2003; 171:1950-60.
[6] Anthony RM, Rutitzky LI, Urban JF, Jr. Stadecker MJ, Gause WC.
Protective immune mechanisms in helminth infection. Nat Rev
Immunol 2007; 7: 975-87.
[7] Wildblood LA, Kerr K, Clark DA, Cameron A, Turner DG, Jones
DG. Production of eosinophil chemoattractant activity by ovine
gastrointestinal nematodes. Vet Immunol Immunopathol 2005;
107: 57-65.
[8] Turner DG, Wildblood LA, Inglis NF, Jones DG. Characterization
of a galectin-like activity from the parasitic nematode,
Haemonchus contortus, which modulates ovine eosinophil
migration in vitro. Vet Immunol Immunopathol 2008; 122: 138-45.
[9] Milbourne EA, Howell MJ. Eosinophil differentiation in response
to Fasciola hepatica and its excretory/secretory antigens. Int J
Parasitol 1993; 23: 1005-9.
[10] Sabin EA, Kopf MA, Pearce EJ. Schistosoma mansoni egg-induced
early IL-4 production is dependent upon IL-5 and eosinophils. J
Exp Med 1996; 184: 1871-8.
[11] David JR, Butterworth AE, Vadas MA. Mechanism of the
interaction mediating killing of Schistosoma mansoni by human
eosinophils. Am J Trop Med Hyg 1980; 29: 842-8.
[12] Haque AA, Ouaissi M, Joseph M, Capron A. IgE antibody in
eosinophil- and macrophage-mediated in vitro killing of
Dipetalonema viteae microfilariae. J Immunol 1981; 127: 716-25.
[13] Kazura JW, Grove DI. Stage-specific antibody-dependent
eosinophil-mediated destruction of Trichinella spiralis. Nature
1978; 274: 588-9.
[14] Van Milligen FJ, Cornelissen JB, Hendriks IM, Gaasenbeek CP,
Bokhout BA. Protection of Fasciola hepatica in the gut mucosa of
immune rats is associated with infiltrates of eosinophils, IgG1 and
IgG2a antibodies around the parasites. Parasite Immunol 1998; 20:
285-92.
[15] Duffus WP, Thorne K, Oliver R. Killing of juvenile Fasciola
hepatica by purified bovine eosinophil proteins. Clin Exp Immunol
1980; 40: 336-44.
[16] Duffus WP, Franks D. In vitro effect of immune serum and bovine
granulocytes on juvenile Fasciola hepatica. Clin Exp Immunol
1980; 41: 430-40.
[17] Chapman CB, Mitchell GF. Proteolytic cleavage of
immunoglobulin by enzymes released by Fasciola hepatica. Vet
Parasitol 1982; 11: 165-78.
[18] Carmona C, Dowd AJ, Smith AM, Dalton JP. Cathepsin L
proteinase secreted by Fasciola hepatica in vitro prevents antibody-
mediated eosinophil attachment to newly excysted juveniles. Mol
Biochem Parasitol 1993; 62: 9-17.
[19] Piedrafita D, Estuningsih E, Pleasance J, et al. Peritoneal lavage
cells of Indonesian thin-tail sheep mediate antibody-dependent
superoxide radical cytotoxicity in vitro against newly excysted
juvenile Fasciola gigantica but not juvenile Fasciola hepatica.
Infect Immun 2007; 75: 1954-63.
[20] Niwa A, Miyazato T. Reactive oxygen intermediates from
eosinophils in mice infected with Hymenolepis nana. Parasite
Immunol 1996; 18: 285-95.
[21] Serradell MC, Guasconi L, Cervi L, Chiapello LS, Masih DT.
Excretory-secretory products from Fasciola hepatica induce
eosinophil apoptosis by a caspase-dependent mechanism. Vet
Immunol Immunopathol 2007; 117: 197-208.
[22] Jacobsen EA, Taranova AG, Lee NA, Lee JJ. Eosinophils:
singularly destructive effector cells or purveyors of
immunoregulation? J Allergy Clin Immunol 2007; 119: 1313-20.
[23] Knott ML, Matthaei KI, Giacomin PR, Wang H, Foster PS, Dent
LA. Impaired resistance in early secondary Nippostrongylus
brasiliensis infections in mice with defective eosinophilopoeisis. Int
J Parasitol 2007; 37:1367-78.
[24] Galioto AM, Hess JA, Nolan TJ, Schad GA, Lee JJ, Abraham D.
Role of eosinophils and neutrophils in innate and adaptive
protective immunity to larval strongyloides stercoralis in mice.
Infect Immun 2006; 74: 5730-8.
[25] Swartz JM, Dyer KD, Cheever AW, et al. Schistosoma mansoni
infection in eosinophil lineage-ablated mice. Blood 2006; 108:
2420-7.
[26] Reiman RM, Thompson RW, Feng CG, et al. Interleukin-5 (IL-5)
augments the progression of liver fibrosis by regulating IL-13
activity. Infect Immun 2006; 74: 1471-9.
[27] Serradell MC, Guasconi L, Masih DT. Involvement of a
mitochondrial pathway and key role of hydrogen peroxide during
eosinophil apoptosis induced by excretory-secretory products from
Fasciola hepatica. Mol Biochem Parasitol 2009; 163: 95-106.
[28] McGonigle S, Curley GP, Dalton JP. Cloning of peroxiredoxin, a
novel antioxidant enzyme, from the helminth parasite Fasciola
hepatica. Parasitology 1997; 115 ( Pt 1): 101-4.
[29] Gordon S. Alternative activation of macrophages. Nat Rev
Immunol 2003; 3: 23-35.
[30] Reyes JL, Terrazas LI. The divergent roles of alternatively
activated macrophages in helminthic infections. Parasite Immunol
2007; 29: 609-19.
[31] Choi BS, Martinez-Falero IC, Corset C, et al. Differential impact of
L-arginine deprivation on the activation and effector functions of T
cells and macrophages. J Leukoc Biol 2009; 85: 268-77.
[32] Donnelly S, Stack CM, O'Neill SM, Sayed AA, Williams DL,
Dalton JP. Helminth 2-Cys peroxiredoxin drives Th2 responses
through a mechanism involving alternatively activated
macrophages. FASEB J 2008; 22: 4022-32.
[33] Martinez FO, Helming L, Gordon S. Alternative activation of
macrophages: an immunologic functional perspective. Ann Rev
Immunol 2008.
[34] Donnelly S, O'Neill SM, Sekiya M, Mulcahy G, Dalton JP.
Thioredoxin peroxidase secreted by Fasciola hepatica induces the
alternative activation of macrophages. Infect Immun 2005; 73: 166-
73.
[35] Loke P, Gallagher I, Nair MG, et al. Alternative activation is an
innate response to injury that requires CD4+ T cells to be sustained
during chronic infection. J Immunol 2007; 179: 3926-36.
[36] Herbert DR, Holscher C, Mohrs M, et al. Alternative macrophage
activation is essential for survival during schistosomiasis and
downmodulates T helper 1 responses and immunopathology.
Immunity 2004; 20: 623-35.
[37] Goodridge HS, Marshall FA, Else KJ, et al. Immunomodulation via
novel use of TLR4 by the filarial nematode phosphorylcholine-
containing secreted product, ES-62. J Immunol 2005; 174: 284-93.
[38] Cervi L, Rossi G, Masih DT. Potential role for excretory-secretory
forms of glutathione-S-transferase (GST) in Fasciola hepatica.
Parasitology 1999; 119 ( Pt 6): 627-33.
[39] Flynn RJ, Mulcahy G. Possible role for Toll-like receptors in
interaction of Fasciola hepatica excretory/secretory products with
bovine macrophages. Infect Immun 2008; 76: 678-84.
[40] Rutschman R, Lang R, Hesse M, Ihle JN, Wynn TA, Murray PJ.
Cutting edge: Stat6-dependent substrate depletion regulates nitric
oxide production. J Immunol 2001; 166: 2173-7.
[41] Smits HH, Yazdanbakhsh M. Chronic helminth infections
modulate allergen-specific immune responses: protection against
development of allergic disorders? Ann Med 2007; 39: 428-39.
[42] Prieto-Lafuente L, Gregory WF, Allen JE, Maizels RM. MIF
homologues from a filarial nematode parasite synergize with IL-4
to induce alternative activation of host macrophages. J Leukoc Biol
2009.
[43] Raes G, Brys L, Dahal BK, et al. Macrophage galactose-type C-
type lectins as novel markers for alternatively activated
macrophages elicited by parasitic infections and allergic airway
inflammation. J Leukoc Biol 2005; 77: 321-7.
[44] Rogers NC, Slack EC, Edwards AD. Reis e Sousa. Syk-dependent
cytokine induction by Dectin-1 reveals a novel pattern recognition
pathway for C type lectins. Immunity 2005; 22: 507-17.
[45] Dillon S, Agrawal S, Banerjee K, et al. Yeast zymosan, a stimulus
for TLR2 and dectin-1, induces regulatory antigen-presenting cells
and immunological tolerance. J Clin Invest 2006; 116: 916-28.
[46] Mosser DM, Edwards JP. Exploring the full spectrum of
macrophage activation. Nat Rev Immunol 2008; 8: 958-69.
[47] Patel N, Kreider T, Urban JF Jr, Gause WC. Characterisation of
effector mechanisms at the host:parasite interface during the

284 Current Immunology Reviews, 2009, Vol. 5, No. 4 Cervi et al.
immune response to tissue-dwelling intestinal nematode parasites.
Int J Parasitol 2009; 39: 13-21.
[48] Steinman RM, Hemmi H. Dendritic cells: translating innate to
adaptive immunity. Curr Top Microbiol Immunol 2006; 311:17-58.
[49] Sher A, Pearce E, Kaye P. Shaping the immune response to
parasites: role of dendritic cells. Curr Opin Immunol 2003; 15:
421-9.
[50] Pulendran B. Variegation of the immune response with dendritic
cells and pathogen recognition receptors. J Immunol 2005; 174:
2457-65.
[51] Reis e Sousa C, Diebold SD, Edwards AD, Rogers N, Schulz O,
Sporri R. Regulation of dendritic cell function by microbial stimuli.
Pathol Biol 2003; 51: 67-8.
[52] Cools N, Ponsaerts P, Van Tendeloo VF, Berneman ZN. Balancing
between immunity and tolerance: an interplay between dendritic
cells, regulatory T cells, and effector T cells. J Leukoc Biol 2007;
82:1365-74.
[53] Rutella S, Danese S, Leone G. Tolerogenic dendritic cells: cytokine
modulation comes of age. Blood 2006; 108:1435-40.
[54] MacDonald AS, Straw AD, Bauman B, Pearce EJ. CD8- dendritic
cell activation status plays an integral role in influencing Th2
response development. J Immunol 2001; 167:1982-8.
[55] Taylor BC, Zaph C, Troy AE, et al. TSLP regulates intestinal
immunity and inflammation in mouse models of helminth infection
and colitis. J Exp Med 2009; 206: 655-67.
[56] Rimoldi M, Chieppa M, Salucci V, et al. Intestinal immune
homeostasis is regulated by the crosstalk between epithelial cells
and dendritic cells. Nat Immunol 2005; 6: 507-14.
[57] Kane CM, Cervi L, Sun J, et al. Helminth antigens modulate TLR-
initiated dendritic cell activation. J Immunol 2004; 173: 7454-61.
[58] Balic A, Harcus Y, Holland MJ, Maizels RM. Selective maturation
of dendritic cells by Nippostrongylus brasiliensis-secreted proteins
drives Th2 immune responses. Eur J Immunol 2004; 34: 3047-59.
[59] Thomas PG, Carter MR, Atochina O, et al. Maturation of dendritic
cell 2 phenotype by a helminth glycan uses a Toll-like receptor 4-
dependent mechanism. J Immunol 2003; 171: 5837-41.
[60] van der Kleij D, Latz E, Brouwers JF, et al. A novel host-parasite
lipid cross-talk. Schistosomal lyso-phosphatidylserine activates
toll-like receptor 2 and affects immune polarization. J Biol Chem
2002; 277: 48122-9.
[61] van Riet E, Hartgers FC, Yazdanbakhsh M. Chronic helminth
infections induce immunomodulation: consequences and
mechanisms. Immunobiology 2007; 212: 475-90.
[62] Wang CR, Qiu JH, Zhu XQ, et al. Survey of helminths in adult
sheep in Heilongjiang Province, People's Republic of China. Vet
Parasitol 2006; 140: 378-82.
[63] Tliba O, Moire N, Le Vern Y, Boulard C, Chauvin A, Sibille P.
Early hepatic immune response in rats infected with Fasciola
hepatica. Vet Res 2002; 33: 261-70.
[64] Brady MT, O'Neill SM, Dalton JP, Mills KH. Fasciola hepatica
suppresses a protective Th1 response against Bordetella pertussis.
Infect Immun 1999; 67: 5372-8.
[65] Cervi L, Rubinstein H, Masih DT. Involvement of excretion-
secretion products from Fasciola hepatica inducing suppression of
the cellular immune responses. Vet Parasitol 1996; 61: 97-111.
[66] Diaz A, Allen JE. Mapping immune response profiles: the
emerging scenario from helminth immunology. Eur J Immunol
2007; 37: 3319-26.
[67] McKee AS, Pearce EJ. CD25+CD4+ cells contribute to Th2
polarization during helminth infection by suppressing Th1 response
development. J Immunol 2004; 173: 1224-31.
[68] Yang J, Zhao J, Yang Y, et al. Schistosoma japonicum egg
antigens stimulate CD4 CD25 T cells and modulate airway
inflammation in a murine model of asthma. Immunology 2007;
120: 8-18.
[69] Reddy A, Fried B. An update on the use of helminths to treat
Crohn's and other autoimmunune diseases. Parasitol Res 2009; 104:
217-21.
[70] Reddy A, Fried B. The use of Trichuris suis and other helminth
therapies to treat Crohn's disease. Parasitol Res 2007; 100: 921-7.
[71] Summers RW, Elliott DE, Urban JF Jr, Thompson RA, Weinstock
JV. Trichuris suis therapy for active ulcerative colitis: a
randomized controlled trial. Gastroenterology 2005; 128: 825-32.
[72] Hsu SJ, Tseng PH, Chen PJ. Trichuris suis therapy for ulcerative
colitis: nonresponsive patients may need anti-helminth therapy.
Gastroenterology 2005; 129: 768-9; author reply 769.
[73] Sewell D, Qing Z, Reinke E, et al. Immunomodulation of
experimental autoimmune encephalomyelitis by helminth ova
immunization. Int Immunol 2003; 15: 59-69.
[74] Coquerelle C, Moser M. Are dendritic cells central to regulatory T
cell function? Immunol Lett 2008; 119:12-6.
[75] Lange C, Durr M, Doster H, Melms A, Bischof F. Dendritic cell-
regulatory T-cell interactions control self-directed immunity.
Immunol Cell Biol 2007; 85: 575-81.
[76] Steinman RM, Hawiger D, Liu K, et al. Dendritic cell function in
vivo during the steady state: a role in peripheral tolerance. Ann NY
Acad Sci 2003; 987: 15-25.
Received: March 3, 2009 Revised: May 5, 2009 Accepted: May 14, 2009