Regulatory cells and immunosuppressive cytokines:
parasite-derived factors induce immune polarization.

Ali Ouaissi

► To cite this version:

Ali Ouaissi. Regulatory cells and immunosuppressive cytokines: parasite-derived factors induce immune polarization. Journal of Biomedicine and Biotechnology, 2007, 2007 (4), pp.94971. 10.1155/2007/94971. [hal-00376736]

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## Review Article

# Regulatory Cells and Immunosuppressive Cytokines: Parasite-Derived Factors Induce Immune Polarization

Ali Ouaissi

INSERM, IRD UR008 "Pathogénie des Trypanosomatidés", Centre IRD de Montpellier, 911 Avenue Agropolis, BP 65401, 34394 Montpellier, France

Received 27 November 2006; Accepted 19 March 2007

Recommended by Abdelali Haoudi

Parasitic infections are prevalent in both tropical and subtropical areas. Most of the affected and/or exposed populations are living in developing countries where control measures are lacking or inadequately applied. Although significant progress has been made in our understanding of the immune response to parasites, no definitive step has yet been successfully done in terms of operational vaccines against parasitic diseases. Evidence accumulated during the past few years suggests that the pathology observed during parasitic infections is in part due to deregulation of normal components of the immune system, mainly cytokines, antibodies, and immune effector cell populations. A large number of studies that illustrate how parasites can modify the host immune system for their own benefit have been reported in both metazoan and protozoan parasites. The first line of defense against foreign organisms is barrier tissue such as skin, humoral factors, for instance the complement system and pentraxin, which upon activation of the complement cascade facilitate pathogen recognition by cells of innate immunity such as macrophages and DC. However, all the major groups of parasites studied have been shown to contain and/or to release factors, which interfere with both arms of the host immune system. Even some astonishing observations relate to the production by some parasites of orthologues of mammalian cytokines. Furthermore, chronic parasitic infections have led to the immunosuppressive environment that correlates with increased levels of myeloid and T suppressor cells that may limit the success of immunotherapeutic strategies based on vaccination. This minireview briefly analyzes some of the current data related to the regulatory cells and molecules derived from parasites that affect cellular function and contribute to the polarization of the immune response of the host. Special attention is given to some of the data from our laboratory illustrating the role of immunomodulatory factors released by protozoan parasites, in the induction and perpetuation of chronic disease.

Copyright © 2007 Ali Ouaissi. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## 1. INTRODUCTION

There is increasing evidence that immune mechanisms are involved in the pathogenesis of many parasitic infections. The initial stages of the disease are generally characterized by the induction of a nonspecific lymphoproliferation, which is believed to disrupt antigen recognition and interfere with protective immune responses. Paradoxically, in most cases a state of immunosuppression can be evidenced. This hyporesponsiveness to antigen-specific and polyclonal stimuli in chronic parasitic infections could be related to immunosuppressive cytokines (i.e., IL-10 and TGF-β) secreted by antigen presenting cells and regulatory T cells (Treg cells). A growing list of parasite-derived molecules able to exert immunomodulatory activities on the cells of the innate immunity leading to such polarized cytokine secretion has been reported [1, 2]. Interestingly, these immunosuppressive regulatory responses

resulting from repeated exposure to pathogens and/or their released products have been postulated to be responsible for protection against inflammatory diseases such as allergy or autoimmunity leading to the germless theory of allergic diseases and the hygiene hypothesis [3, 4]. This has led investigators to search for parasite molecules which could be used as a new therapy for immunological disorders [5].

In fact, complex feedback loops could explain the properties of a suppressor activity seen in parasitic infections. For instance, the parasites cannot only induce the production of host immunomodulatory lipids, the best characterized being the endogenous eicosanoids, but are also able to synthesize/secrete their own glycoproteins and lipids, which in turn activate cells of innate immunity towards the anti-inflammatory cytokine response. This physiological microenvironment may favor the development of Treg cells. A number of excellent reviews were devoted to the Treg cells
and their physiological role at the level of peripheral self-
tolerance, avoidance of autoimmune diseases, tumor, and in-
fectious disease immunology [6–13].

The purpose of this minireview is to analyze some of
the current data related to the regulatory components or
processes originating from the parasite (protozoa and some
helminth pathogens) that affect host cellular functions lead-
ing to an immunosuppressive state that counteract proin-
flammatory cytokine production which may lead to excessive
host tissue damage. The outcome of a parasitic infection will
depend on the final balance of the protective and pathologi-
cal properties of the cytokine network.

## 2. INDUCTION OF SUPPRESSOR CELLS

Immunosuppression involves an act that reduces the activa-
tion or efficacy of the immune system. Some components
of the immune system itself have immunosuppressive ef-
fects on other parts of the immune system, and immunosup-
pression may occur as an adverse reaction to treatment or
other conditions like infection processes. The ability of par-
asites to survive in the immune hosts depends on a variety
of escape mechanisms. One of these is the inhibition or the
suppression of the immune responses of their hosts. Several
possible explanations have been put forward, such as anti-
genic competition, acquired tolerance, as well as the possible
blocking role of soluble antigens or circulating immune com-
plexes. The release by the parasites themselves of excretory-
secretory products, which have potent immunosuppressive
activity, represents another possible explanation. Moreover, a
large number of reports have described the presence of sup-
pressor cells (T lymphocytes and macrophages) in humans
and animals infected with various parasites.

For instance, during onchocerciasis due to a pathogenic
human filarial worm, *Onchocerca volvulus*, a state of cellu-
lar immune unresponsiveness develops in patients with the
generalized form of the disease [14]. Moreover, it has been
reported that parasite antigens are present in breast milk of
*O. volvulus*-infected women which activated cells that sup-
pressed the proliferative response of autologous lymphocytes
to mitogens and antigens, suggesting therefore that this may
induce tolerance and/or suppression in infants born of in-
fected mothers [15].

A number of years ago, we have shown that total antigens
from *O. volvulus* (OVA) markedly inhibited the proliferation
of normal human lymphocytes stimulated with polyclonal
activators such as phytohaemagglutinin (PHA). The inhibi-
tion was not due to a cytotoxic effect of OVA and was not ab-
rogated by removal of the adherent cell population. Interest-
ingly, we showed that the in vitro response of normal human
lymphocytes was suppressed by coculture with allogeneic or
syngeneic lymphocytes, which had previously been exposed
to OVA. A significant reduction of the suppression was how-
ever observed when OVA pretreated cells were depleted of T
cells by centrifugation of E rosettes. Moreover, the passage
of OVA through an immunoadsorbant column containing a
monoclonal antibody to OVA epitope abrogated its immuno-
suppressive effect. These observations allowed us to postulate

that a parasite antigen(s) was responsible for the induction of
T suppressor cells [16]. Since the molecular mechanisms of
suppressor cells were difficult to characterize, interest in such
cells was lost.

Recent progress in the identification of CD4⁺ T cell pop-
ulations, together with the use of genetically modified an-
imal models have led to significant advances in the under-
standing of the immunosuppression phenomenon at the cel-
lular and molecular levels. The concept of T regulatory cells
(Treg) suppressing immune responses via cell-cell interac-
tions and/or the production of suppressor cytokines are cur-
rently well documented [6–9]. At least two main Treg cell
populations were defined: “naturally” occurring regulatory
T cells (Foxp3⁺ CD4⁺ CD25⁺) and the “adaptive” regula-
tory T cells (e.g., T<sub>R1</sub> or T<sub>H3</sub>) [10–12]. Although some con-
troversy has been reported in the literature, evidence which
accumulated over the years has undoubtedly shed light on
the importance of Treg cells in health and disease. Thus, in
the case of onchocerciasis, a series of reports has shown that
the hyporesponsiveness in individuals with the generalized
form of the disease is not due to a shift towards a T<sub>H2</sub> re-
sponse. Rather, it results from *O. volvulus* antigen-specific T
cells having a cytokine profile with no IL-2 and high IL-10
and TGF-β production similar to the adaptive Treg cells also
known as T<sub>R1</sub> and T<sub>H3</sub> which suppress ongoing inflamma-
tion [17, 18]. Cloning procedures allowed obtaining T cell
clones bearing T<sub>R1</sub> suppressor cytokine profile producing
significant amounts of IL-10 but no IL-2 or IL-4 and express-
ing high levels of cytotoxic T lymphocyte antigen (CTLA-4)
after stimulation.

Although the examination of T cell lines or clones de-
rived from the peripheral blood mononuclear cells of in-
fected individuals has the great advantage that the cells pro-
ducing the cytokines can be accurately defined, in this case
the cytokines derived from “neighboring” cells in vivo are no
longer represented in the system. Nevertheless, the genera-
tion of T cell clones has provided valuable information on
human responses in general and to infections in particular.

However, when considering a wide range of autoimmune
and inflammatory manifestations, the mechanisms by which
regulatory Treg cells exert their activity remain unclear. For
instance, studies in vivo have demonstrated that regulation
is dependent on cytokines such as IL-10 and TGF-β as well
as the expression of CTLA-4 molecule [19, 20]. However, in
vitro studies have shown that neither soluble cytokines nor
CTLA-4 is required for the suppressive effects of Treg [21–
23]. Moreover, it has been shown that regulation of the ileal
inflammatory process resulting from *Toxoplasma gondii* in
murine model is dependent on TGF-β producing intraep-
ithelial lymphocytes suggesting therefore that these cells rep-
resent an essential component in gut homeostasis after oral
infection with this parasite [24]. Furthermore, in the sus-
ceptible BALB/c mouse experimental model of filarial in-
fection with *Litomosoides sigmodontis*, a parasite closely re-
lated to *Brugia* and *Wuchereria* species causing human lym-
phatic filariasis, the Treg cells have been shown to be re-
sponsible for susceptibility to parasite. However, although
treatment of infected mice with antibodies to CD25 and
glucocorticoid-induced TNF receptor family-related gene re-
duced Treg activity and led to increased antigen-specific im-
mune responses, this results in the significant reduction of
parasite numbers, in vivo neutralization of IL-10 receptor,
but did not restore the ability of the immune system to kill
parasites, supporting the notion that Treg cells act in an IL-
10-independent manner [25].

Nevertheless, a recent study has shown that in a murine model of coinfection with a gastrointestinal nematode parasite *Heligmosomoides polygyrus* and the blood-stage malaria parasite *Plasmodium chabaudi* increased levels of IL-10. This occurred in concurrent infections, whereas high levels of TGF-β were seen during *P. chabaudi* single infections [26]. Interestingly, anthelminthic drug treatment of mice before *P. chabaudi* infection reduced TGF-β levels and restored antimalarial immunity. Thus, induction/expansion of the suppressor function is a complex process depending on multiple factors, among which concurrent infections are highly prevalent in many endemic tropical and subtropical regions of the world.

Hyporesponsiveness also occurs during human schisto-
somiasis. In fact, the infection downregulates both T<sub>H1</sub> and
T<sub>H2</sub> cytokines [27]. In more recent studies, it has been shown
that in a mouse schistosomiasis, Treg cells and IL-10 inhib-
ited T<sub>H1</sub> development [28] and egg-induced pathology [29].
However, a number of studies have pointed to the role of
IL-10 and TGF-β (two cytokines being released by adaptive
Treg cells) as immunomodulatory cytokines in helminth in-
fections. Parasite-specific activation of natural Treg cells has
been reported in mice *Leishmania major* infection [30]. The
cells were positive for Foxp3, produce IL-10 in response to
*Leishmania*-infected dendritic cells, and exerted strong sup-
pressive activity in vitro. In fact, previous observations have
shown that in the case of *L. major* infection of genetically
resistant C57BL/6 mice which spontaneously heal their der-
mal lesions with persistence of latent parasites, CD4<sup>+</sup> T cells
are the main producers of IFN-γ and IL-10 in the dermis,
although CD8<sup>+</sup> T cells were also able to produce either cy-
tokine with appropriate stimuli [31]. Similar to T<sub>R1</sub> cells, the
majority of CD4<sup>+</sup> T cells in the dermis and a proportion of
CD4<sup>+</sup> T cells in the draining lymph nodes were able to pro-
duce both IL-10 and IFN-γ. Thus, in the chronic sites of in-
fection, the release of IL-10 and IFN-γ by T cells led to the
establishment of a latency with persistence of low number of
viable parasites within lymphoid tissue and skin lesion after
self-cure.

Although the T cell network seems to play a key role
in the immunosuppression process, the existence of other
T-cell-independent mechanisms has been clearly demon-
strated. Indeed, recent investigations have shown that
helminth and protozan infections can elicit a myeloid popu-
lation characterized as Gr-1<sup>+</sup>/CD11b<sup>+</sup> cells that substantially
impaired antigen-specific T cell responses [32–34].

The myeloid suppressor cell-induced immunosuppres-
sion is mediated by nitric oxide production (NO), a mes-
senger known to be involved in diverse signaling pathways
including smooth muscle relaxation, platelet inhibition, neu-
rotransmission, immune regulation, and destruction of mi-

crobes and tumor cells [35]. The participation of NO in the
suppression of T cell activation has been reported in a num-
ber of biological systems (reviewed in [36]). In fact, NO pro-
duction during toxoplasmosis in C57BL/6 mice has two op-
posite effects being protective against *Toxoplasma gondii* and
downregulating the immune response, suggesting its possi-
ble contribution in the establishment of chronic infections
[37]. In the case of *Trypanosoma cruzi*, previous studies have
shown that IFN-γ and nonoxidative molecules (TNF-α and
NO) could play a role in the control of *T. cruzi* infection
in mice [36]. Furthermore, a series of experiments supports
the notion that IFN-γ and TNF-α mediated activation of
macrophages which leads to increased production of NO,
and in turn suppresses T cell activation [38]. The involve-
ment of NO in apoptosis of thymocytes and macrophages
has also been documented [39, 40] and NO markedly in-
hibits the induction of IL-2 promoter, which can account
for most of the reduction in IL-2 production, and weakly
increases the activation of IL-4 promoter [41]. This mech-
anism could be involved in the downregulation of IL-2 gene
expression observed during *T. cruzi* infection [42]. Therefore,
it is likely that NO production during the initial phase of
acute infections might participate in the clearance of para-
sites by macrophages, whereas its overproduction during the
late phase of acute infection would account for the immuno-
suppression observed.

Although significant advances have been made regard-
ing the Treg subsets, a recent exiting review pointed to the
importance of B cells possessing regulatory functions and
suggested these be called Breg cells (reviewed in [43]). In-
deed, in addition to the pathogenic role of B cells, which
produce autoantibodies that contribute to the development
of autoimmune diseases, the existence of regulatory B cells
capable of inhibiting inflammatory responses through the
production of regulatory cytokines IL-10 and TGF-β has
been demonstrated in a number of experimental models of
chronic inflammation [43].

In the case of parasitic infections, a number of studies
have reported several alterations in B cell functions. For in-
stance, in the mouse model of *Schistosoma mansoni* infection,
splenic B cells have been shown to proliferate in response to
an oligosaccharidic antigen (see also the next section) and
were triggered to secrete high levels of IL-10 [44]. Further-
more, spleen B cells from mice infected with the L3 larval
stage of filarial nematode *Brugia pahangi* contributed sub-
stantially to IL-10 production that in turn downregulated the
expression of B7 molecules on the B cell surface [45]. This
led to the decrease of their efficiency as antigen presenting
cells to CD4+ T cells and restricting their expansion, suggest-
ing therefore that B-cell-derived IL-10 could participate in
the regulation of proinflammatory CD4 responses as in other
various models of autoimmune diseases [46].

2.1. Induction of apoptosis in the host immune cells

Another mechanism leading to the homeostasis disorder
in the host is the fact that the invading parasites can re-
lease factors which kill the cells of the immune system by
activating the cellular death machinery, thus inducing apoptosis. Therefore, apoptosis seems to represent a fundamental feature with particular relevance in the maintenance of protozoan infections [47]. Indeed, studies in experimental mouse *T. cruzi* infections have shown that apoptosis of T cells play an important role in the immunossupression that occurs during the acute phase of Chagas' disease [48]. In a canine model of acute Chagas myocarditis, Zhang *et al.* [49] have reported that programmed cell death (PCD) occurs in cardiac myocytes, endothelial cells, macrophages, interstitial dendritic cells, and lymphocytes, suggesting that parasite-derived factors could be responsible of the apoptosis observed in the immune cells. In the same way, a correlation between the extent of PCD and the level of suppression of CD4⁺ T-cell proliferative responses was observed [50]. Moreover, it has been suggested that the upregulation of Fas and Fas ligand play an important role in the induction of CD4⁺ T cell death. This pathway is able to control and modulate the immune response against *T. cruzi* [51].

Parasite-derived substances are believed to be key factors in the immunossupression phenomena observed by exerting a proapoptotic activity against some immune cells. Thus, it has been reported that the *T. cruzi*-secreted trans-sialidase (TS) was able to induce apoptosis features in cells of the immune system in vivo [52]. Furthermore, evidences reported support that TS is a virulence factor responsible for thymic alterations via apoptosis of “nurse cell complex” [53]. Moreover, the glycoinositolphospholipid (GIPL) from *T. cruzi*, in the presence of IFN-γ, induced murine macrophage apoptosis leading to increased parasite release from these cells [54]. Surprisingly, internalization of apoptotic T lymphocytes by macrophages increased the replication of *T. cruzi* amastigotes inside macrophages [55]. Another additional mechanism that may amplify the immune suppression process is the fact that apoptotic cells release their TGF-β during their suicidal act. They flip intracellular phosphatidylserine onto the outer leaflet of their membranes rendering them target of phagocytic cells signaling the macrophages to release significant amounts of TGF-β which in conjunction with IL-10 and PGE-2 may act as a feedback amplification loop mediating immune suppression.

## 2.2. PD-1 as active suppressor of T regulatory cells

Programmed death-1 (PD-1) [56], a member of the CD28 family, is an immunoreceptor tyrosine-based inhibitory-motif- (ITIM-) containing receptor induced on T, B, and myeloid cells upon activation in vitro [57]. Interaction of PD-1 ligands (PD-L1 and PD-L2, members of the B7 family) with PD-1 may lead to the inhibition of proliferation and cell division of activated T cells expressing PD-1. Thus, the absence of PD-1 induced proliferation of effector T cells in the adenovirus-infected liver and resulted in rapid clearance of the virus. The blockage of the PD-1 pathway can augment antiviral immunity [58]. Recent investigations have reported that PD-1/PD-L systems may play a role during parasitic infections. Indeed, it has been shown that PD-L1 and PD-L2 have distinct roles in regulating host immunity to cutaneous

leishmaniasis [59]. In fact when compared to wilt-type mice (WT), the PD-L1⁻/⁻ and PD-L2⁻/⁻ exhibited distinct disease outcomes following infection with *L. mexicana*.

PD-L⁻/⁻ mice developed resistance, whereas PD-L2⁻/⁻ showed exacerbated disease. Although both PD-L1⁻/⁻ and PD-L2⁻/⁻ produced similar levels of IFN-γ as the WT mice, the development of IL-4 producing cells was reduced in PD-L1⁻/⁻ mice suggesting that impairment of T_H2 response due to PD-L1 deficiency could be related to increased resistance to *L. mexicana* infection. Furthermore, in the case of CBA/J mouse *Schisitosoma mansoni* chronic infection, the parasites can induce increased expression of PD-L2 on splenic CD11c⁺/B220⁻ dendritic cells leading to moderate morbidity [60]. Taken together, these observations suggest that PD-1/PD-L systems may play a role in negative regulation of immune responses during parasitic infections.

## 2.3. Parasite released molecules as immunoregulatory factors

### 2.3.1. Lipids

A number of reviews pointed to the importance of endogenous as well as parasite-derived lipids as immunoregulatory factors [2, 62, 63]. It is well known that eicosanoids such as prostaglandins PGE2, PGD2, and LipoxinA4 can act not only on antigen-presenting cells through defined or putative surface receptors and strongly modify their pattern of cytokine synthesis (IL-1, TNF-α, IL-12, and IL-10), but also on T cells at the level of IL-2 synthesis, IL-2 receptor expression, and cellular proliferation [2]. Glycoinositolphospholipids (GIPLs) are some of the major glycoconjugates present on the cellular surface of *Leishmania* [63] and different strains of *T. cruzi* [64]. *T. cruzi* GIPL blocks T cell responses induced by different polyclonal activators, the suppressive domain being assigned to the ceramide portion of the molecule. Indeed, purified GIPLs from *T. cruzi* inhibit in vitro CD4⁺ and CD8⁺ T cell proliferation induced by bacterial superantigen and anti-TCR;CD3 antibodies. The inhibition leads to loss of IL-2 responsiveness, with inhibition of CD25 expression on both CD4⁺ and CD8⁺ subsets [65].

Dendritic cells (DCs) are crucial in the initiation of the immune response and are distinguishable from the other antigen presenting cells by their highly efficient antigen presentation. DCs are specialized to acquire and process antigen in peripheral nonlymphoid sites, and to transport the antigen to the secondary lymphoid organs where the stimulation of naïve lymphocytes occurs. During their migration, DCs enter a process of maturation that determines whether adaptive immune response occurs and the nature of that immune response. Studies with the glycoinositolphospholipid (GIPL) from *T. cruzi* have demonstrated that this molecule led to a downregulation of human DC surface antigens, such as CD80, CD86, HLA-DR, CD40, and CD57 that are important for T cell activation [66]. These observations allowed investigators to propose a novel efficient mechanism leading to the alteration of DC function and maturation that may be used by *T. cruzi* to escape the host immune response. However, although these investigations are interesting, it is important
to remember that the GPI anchors express biological activi-
ties similar to those of lipopolysaccharides (LPS). Given the
fact that LPS induces the maturation of dendritic cells, one
would expect that *T. cruzi*-derived LPS-like substances could
activate rather than inhibit DC maturation [67]. Therefore,
the observations showing DC inhibition await further expla-
nation. In this regard, it is noteworthy that GPI anchors and
GIPLs from *T. cruzi* are potent activators of the human and
mouse macrophage toll-like receptor 2 (TLR2) [68].

2.3.2. Polysaccharides

A number of saccharides (from oligosaccharides to complex polysaccharides) derived from parasites have been identified and were shown to be implicated in host cell signaling systems. Complex polysaccharides for example which are not digested by the macrophage lysosomal enzymes can be retained intracellularly for a long period of time, interfering therefore with the presentation of peptide antigens to T cells [69]. Moreover, these molecules can act directly on the cell of the immune system. For instance, lacto-*N*-fucopentaose III (LNFP-III) and lacto-*N*-neotetraose (LNnT), sugars of egg antigens of *S. mansoni*, also found in human milk were shown to participate in the *T*<sub>H2</sub> polarization and immune suppression. Indeed, intraperitoneal injection of LNnT-Dex into mice expanded a cell population, phenotypically defined as Gr1<sup>+</sup>/CD11b<sup>+</sup>/F4/80<sup>+</sup> producing high levels of IL-10 and TGF-β<sup>ex vivo</sup> [32].

Gr1⁺ cells suppressed naïve CD4⁺ T cell proliferation
in vitro. Suppression involved cell contact and was dependent on
IFN-γ and NO, with a discrete role played by IL-10 [32]. Fur-
thermore, LNFP-III stimulated splenic B cells from parasite-
infected mice to proliferate and produce IL-10 and PGE2,
two molecules known to downregulate T<sub>H</sub>1 cells [44]. The
major source of IL-10 was the B-1 subset (CD5⁺ B220<sup>+</sup>) [70].

Oligosaccharide structures from other parasites have
been shown to modulate B cell activity. Indeed, the *T. cruzi*
GIPL was found to be a stimulatory factor for B cells, induc-
ing the production of IgG3 in the absence of any costimuli,
the active portion being present in the oligosaccharide frac-
tion [71].

In the case of *Leishmania* parasites, the major cell sur-
face molecule, phosphoglycan (PG), has been shown to selec-
tively inhibit the synthesis of IL-12 (p. 40, p. 70) by activated
murine macrophages. The inhibition was dependent on the
galactose (beta1-4) mannose (alpha1)-PO4 repeating units
and not the GPI lipid anchor of lipophosphoglycan [72].

2.3.3. Polypeptides

In addition to lipids and polysaccharides, parasite-derived
proteins and even small RNA molecules could interfere with
the cell of the immune system. Thus, the glutathione-S-
transferases (GSTs), which are ubiquitous housekeeping en-
zymes found in nearly all animals and some parasites, ap-
peared to have immunomodulatory functions [73]. Indeed,
the dimeric form of GST present in the excretory-secretory

products of *Fasciola hepatica* exerted a significant inhibi-
tion of rat T cell proliferation in vitro and a downregula-
tion of NO production by normal peritoneal macrophages.
Furthermore, in *O. volvulus*, a novel type of GST possess-
ing the characteristic of secreted protein has been identified.
In fact, the parasite has two GSTs (ovGST1 and ovGST2),
the ovGST2 functions as an intracellular cytosolic house-
keeping enzyme, whereas the ovGST1 is found in the media
surrounding adult worms maintained in culture, suggesting
therefore that the enzyme is released from the parasite. Re-
cent investigations have shown that *O. volvulus* extracellular
GST produces PGD2, a known anti-inflammatory molecule
[74].

In accordance with these findings, we have also demon-
strated the ability of a *T. cruzi* released protein, Tc52, con-
taining a tandemly repeated structure characteristic of glu-
tathione S-transferases (GSTs) to induce nonspecific sup-
pression of T lymphocyte activation [75]. Furthermore, our
studies have provided evidence demonstrating that puri-
fied Tc52 acted directly on macrophages to increase IL-10
gene expression [76]. In addition, experiments carried out
with murine macrophages harboring a eukaryotic plasmid
carrying Tc52 gene showed increased IL-10 mRNA levels
[77]. Moreover, using synthetic peptides spanning the amino
terminal or carboxy-terminal domain of Tc52 protein, we
found that the sequence encapsing the carboxy-terminal
residues 432-445 when coupled to a carrier protein, ovalbu-
min, exhibited increased inhibitory activity on T lymphocyte
activation and significantly downregulated IFN-γ and IL-2
secretions [78].

The in vivo immunomodulatory effect of Tc52 has been
investigated in mice. Given that we have already estab-
lished by genetic manipulation *T. cruzi* clones lacking a Tc52
protein-encoding allele (Tc52⁺/⁻) [79], we decided to exam-
ine the disease phenotype in Tc52⁺/⁻-infected BALB/c mice,
during the acute and chronic phases of the disease. The re-
sults obtained are in agreement with the observations made
when using in vitro experimental models. Indeed, these stud-
ies showed a reversion of the suppressive phenotype in vivo
during the infection with mutant parasites lacking one Tc52
gene allele. Moreover, a lack of increased secretion of IL-
10 correlates with decreased in vivo Tc52 production [80].
Therefore, it is reasonable to suggest that this reduction by
gene targeting which in turn downregulates the IL-10 synthe-
sis could be among the immunregulatory mechanisms oper-
ating during *T. cruzi* infection. It is tempting to speculate that
Tc52-inducing increased IL-10 secretion might participate in
the downregulation of IL-2 production. This is in agreement
with previous studies showing that murine IL-10 can down-
regulate the host immune response by decreasing the pro-
duction of IL-2 and inhibiting mitogen-driven T cell prolifer-
ation [81, 82]. Furthermore, the effect of Tc52 on the allergic
airway inflammation induced by OVA in the BALB/c strain
of mouse was evaluated. While the OVA challenge induced
increased cellular infiltrates in the bronchoalveolar lavage
fluid, simultaneous injection of Tc52 with OVA significantly
reduced inflammation (Lamkhioued, personal communica-
tion).
<figure><img src="image_2.png" /><figcaption>FIGURE 1: A model for parasite-derived factors-host cell interaction and signaling pathways.</figcaption></figure>

In view of the fact that increased NO production by splenic macrophages has been involved in the suppression of lymphocytes proliferation in mice infected with *T.cruzi* [38], we have investigated whether Tc52 modified macrophage NO production. We first showed that Tc52 could be bound to the macrophage surface as evidenced by FACS analysis. Moreover, while Tc52 alone had no effect, addition of IFN-γ induced the production of high amounts of NO by macrophages correlating with increased levels of *iNOS* transcripts [76]. It is known that IFN-γ by itself is a relatively poor stimulator of NO production by macrophages and the addition of a second signal can significantly enhance NO production [83]. Since high levels of IFN-γ have been recorded during acute phase mouse *T.cruzi* infection, and that Tc52 could be detected in the blood of infected mice, it is reasonable to suggest that Tc52 may act as a “secondary signal” for NO secretion by macrophages in vivo, which in turn could modulate T cell function. This kind of mechanism also occurred in *T. brucei* infection. Indeed, it has been reported that NO-mediated suppression of T cells during *T. brucei* infection could result from a synergistic effect of soluble trypanosome products and IFN-γ on *iNOS* expression [84].

Modulation of *iNOS* gene and NO production by soluble proteins has also been reported in the case of *Entamoeba histolytica* [85].

Although Tc52 could induce the secretion of the suppressive cytokine IL-10 by macrophages, recent investigations have shown that the protein could trigger human and

mouse DC maturation as evidenced by an upregulation of costimulatory surface antigens such as CD54, CD86, and HLA-DR molecules. Moreover, incubation of DC with Tc52 led to increased inflammatory chemokine synthesis (IL-8, monocyte chemoattractant protein-1, and macrophage-inflammatory protein-1α). Interestingly, binding experiments showed complex molecular Tc52-DC interactions that involved toll-like receptor 2 and Tc52 glutathione-binding site which mediated intracellular signaling, whereas another unidentified portion of the Tc52 molecule is involved in its binding to DC [86]. In fact, the Tc52 is made of two homologous domains comprising a glutathione binding site (G-site) and a hydrophobic C-terminal region (H-site). The molecule may act as a dimeric-like complex where the two “pseudo-subunits” areas are arranged in an antiparallel fashion separated by a strong β-turn motif (Ala225-Pro-Gly-Tyr228). The Tc52 G-site binds to TLR2, the other portion of the molecule, likely the H-site, interacts with a putative DC surface structure. Binding to the TLR2 activates the signaling cascade leading to NF-κB nuclear translocation and regulation of nuclear gene expression. It might be that the H-site interacts first with the still unknown DC surface structure, the membrane-bound receptor-Tc52 complex, then moves to reach the TLR2 and binds to it through the Tc52 G-site resulting in the activation of intracellular signaling cascades leading to NF-κB nuclear translocation and regulation of DC gene expression.

On first examination, the fact that Tc52 activity resulted in the induction of gene encoding both anti-inflammatory
(IL-10) and proinflammatory (IL-6, IL-8) mediators may appear paradoxical. Yet, in common with other physiological systems, it is apparent that a counter-regulatory mechanism is essential to provide the balance and regulation that are necessary to control the inflammation cascade. Within the site of an inflammation reaction, IL-10 produced locally would act to counter the stimulatory effects of IL-8 and IL-6, and thereby enable the balance to be established.

Other parasite-derived molecules which subvert immune regulation have been described: the *T. cruzi* antigen molecule SAPA (shed acute phase antigen) which exhibited a neuraminidase-transsialidase activity downregulated T lymphocyte proliferation as a consequence of T suppressor/cytotoxic cell activation and secretion of PGE2 [87]. A *T. cruzi* membrane glycoprotein inhibited the expression of IL-2 receptor chains and secretion of cytokines by subpopulations of activated human T lymphocytes [88] among others.

Moreover, parasite-derived polypeptides could act directly on B cells either as specific or nonspecific activators. Indeed, we have shown that in vivo treatment of mice with a flagellar $Ca^+$-binding protein, Tc24 from *T. cruzi*, induced a quick increase in the number of B cell secreted immunoglobulins of IgM isotype, suggestive of a mitogenic activity of Tc24 on B cells that is T cell independent [89]. Moreover, we have identified an *L. major* gene encoding a protein sharing significant homology to mammalian ribosomal protein S3a named LmS3a exhibiting dual activity being stimulatory and inhibitory towards T and B cells, respectively [90]. Analysis of cytokine production revealed a significant downregulation of IFN-$\gamma$, IL-2, and IL-12 secretion by LmS3a. These results are compatible with mitogenic induction of the immune system accompanied by a state of immunosuppression.

Another intriguing aspect in the parasite relationship is the fact that parasites could release factors that mimic host cytokines. For instance, (1) hydatid fluid fractions mimicked IL-1, IL-2, and IL-6 [91]; (2) an IFN-$\gamma$ homolog that binds to the IFN-$\gamma$ receptor and induced change in lymphoid cells has been identified in an intestinal nematode [92]; (3) two homologs of the human macrophage migration inhibitory factor (MIF) have been characterized in the human parasitic nematode *Brugia malayi* and termed Bm-MIF-1 and Bm-MIF-2, both having functional properties similar to the MIF human counterpart [93]; (4) *Toxoplasma gondii* releases cyclophilin-18 (C-18) that signals through the chemokine receptor CCR5 leading to the IL-12 synthesis by dendritic cells and a strong protective response [94]; (5) the tapeworm *Hymenolepis diminuta* has been shown to express an IL-12-like peptide, one of the suggested hypothesis being that the peptide could act as a competitive antagonist for the IL-12 receptor, thus contributing to the general immunosuppression [95].

Taken together, these examples raise the distinct possibility that the production of parasite factors that interact with cell surface receptors may be one mechanism whereby the parasite is able to interfere with the regulation of the induction/initiation phase of the host immune response that may protect the host from excessive inflammation and may potentiate the parasite's own survival.

## 2.4. Concluding remarks

The soluble parasite factors can elicit a complex series of cellular interactions leading to an immunosuppression state (Figure 1). The fact that immunoregulatory parasite-derived substances may have additional roles in driving early immunological events towards $T_{H2}$-type or anti-inflammatory responses has opened new areas of investigation looking for molecules that may represent novel potential therapeutic agents for the treatment of $T_{H1}$-mediated inflammatory and autoimmune diseases.

## ACKNOWLEDGMENTS

Part of the studies described in this review was supported by grants from INSERM, IRD, and ACI Microbiology no. MIC 03 29. The author thanks M. John Bolger for editorial assistance.

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