Molecular markers of susceptibility to ocular toxoplasmosis, host and guest behaving badly.

Abstract

Infection with Toxoplasma gondii results in retinochoroiditis in 6% to 20% of immunocompetent individuals. The outcome of infection is the result of a set of interactions involving host genetic background, environmental, and social factors, and the genetic background of the parasite, all of which can be further modified by additional infections or even reinfection. Genes that encode several components of the immune system exhibit polymorphisms in their regulatory and coding regions that affect level and type of expression in response to stimuli, directing the immune response into different pathways. These variant alleles have been associated with susceptibility to immune-mediated diseases and with severity of pathology. We have investigated polymorphisms in several of these genes, identified as candidates for progression to retinochoroiditis caused by toxoplasmosis, namely chemokine (C-C motif) receptor 5 (CCR5), toll-like receptor-2 (TLR2), and TLR4. Furthermore, because interleukin-12 (IL-12) has been shown to be fundamental both in mice and in man to control a protective response against T. gondii, molecules that have a key function in IL-12 production will be emphasized in this review, in addition to discussing the importance of the genetic background of the parasite in the establishment of ocular disease.

Full text

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© 2008 Dove Medical Press Limited. All rights reserved
Clinical Ophthalmology 2008:2(4) 837–848
837
R E V I E W
Molecular markers of susceptibility to ocular
toxoplasmosis, host and guest behaving badly
Adriana Lima Vallochi1
Anna Carla Goldberg2
Angela Falcai3
Rajendranath Ramasawmy4
Jorge Kalil4
Cláudio Silveira5
Rubens Belfort Jr5
Luiz Vicente Rizzo3
1Oswaldo Cruz Institution (IOC),
Oswaldo Cruz Foundation
(FIOCRUZ), Rio de Janeiro, RJ, Brazil;
2NUCEL – Cellular and Molecular
Therapy Center, University of
São Paulo, São Paulo, SP, Brazil;
3Department of Immunology,
Biomedical Science Institute,
University of São Paulo, São Paulo,
SP, Brazil; 4Laboratory of Immunology,
Heart Institute, University
of São Paulo Medical School,
São Paulo, SP, Brazil; 5Department
of Ophthalmology, Federal University
of São Paulo, São Paulo, SP, Brazil
Correspondence: Adriana Lima Vallochi
IOC-FIOCRUZ, Av. Brasil, 4365,
Manguinhos, 21040-900, Rio de Janeiro,
RJ, Brazil
Tel +55 21 3865 8161
Fax +55 21 2590 3495
Email vallochi@ioc.fi ocruz.br
Abstract: Infection with Toxoplasma gondii results in retinochoroiditis in 6% to 20% of
immunocompetent individuals. The outcome of infection is the result of a set of interactions
involving host genetic background, environmental, and social factors, and the genetic background
of the parasite, all of which can be further modifi ed by additional infections or even reinfection.
Genes that encode several components of the immune system exhibit polymorphisms in their
regulatory and coding regions that affect level and type of expression in response to stimuli,
directing the immune response into different pathways. These variant alleles have been associated
with susceptibility to immune-mediated diseases and with severity of pathology. We have
investigated polymorphisms in several of these genes, identifi ed as candidates for progression to
retinochoroiditis caused by toxoplasmosis, namely chemokine (C-C motif) receptor 5 (CCR5),
toll-like receptor-2 (TLR2), and TLR4. Furthermore, because interleukin-12 (IL-12) has been
shown to be fundamental both in mice and in man to control a protective response against
T. gondii, molecules that have a key function in IL-12 production will be emphasized in this
review, in addition to discussing the importance of the genetic background of the parasite in
the establishment of ocular disease.
Keywords: ocular toxoplasmosis, IL-12, TLR, CCR5, immunity
Toxoplasma gondii, an obligate intracellular protozoan parasite, infects close to a
billion people worldwide (Tenter et al 2000) and is an important agent of animal and
human disease worldwide (Dubey and Beattie 1988; Wong and Remington 1993;
Petersen and Dubey 2001).
Toxoplasmosis in humans and other mammals is acquired by oral ingestion of
either tissue cysts in raw or undercooked meat from chronically infected intermediate
hosts or by ingestion of oocysts shed by cats by way of fecal contamination of food or
water (Frenkel 1988, 1990). Once ingested, the cyst wall is digested within the lumen
of the small intestine. The acute phase of infection is characterized by wide-spread
dissemination of rapidly dividing tachyzoites that invade virtually all cell types.
During the infl ammatory process, soluble mediators and cellular components work
together in a systematic manner in the attempt to contain and to eliminate the agents
causing physical distress. The nature and portal of entry of the foreign substance and,
to some degree, the nature and circumstances of a particular individual infl uence the
way in which the infl ammatory process is initiated.
Pathogens can initiate infl ammation by a number of distinct and characteristic
mechanisms, including activation of the plasma protease systems by interaction
with degradation products of the pathogens and by secretion of toxins that can
activate the infl ammatory response directly. Injured cells themselves can release
degradation products that initiate one or more of the plasma protease cascades and
augment expression of proinfl ammatory cytokines that promote the infl ammatory

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Vallochi et al
process. The physiological changes in infl ammation are
crucial to maintain the health and integrity of an organism,
however, the infl ammatory process can result in massive
tissue destruction when poorly controlled.
During invasion of T. gondii, the host cell is essentially
passive and no change is detected in membrane ruffl ing,
actin cytoskeleton, or phosphorylation of host cell proteins
(Furtado et al 1992; Manger et al 1998; Ortega-Barria
and Boothroyd 1999; Jacquet et al 2001). The invasion is
an active parasite-mediated process, initiated by contact
between the apex of T. gondii and the host cell surface,
involving many host receptors (proteoglycans like heparin
and heparin-sulphate, β-integrins) and parasite ligands such
as surface antigens (SAG), surface antigen-related sequences
(SRS), microneme proteins (MIC), and laminin (Yap and
Sher 1999).
Intestinal epithelial cells probably are the fi rst cells
infected by T. gondii. Chemokines released by these cells
play a critical role in the initiation and modulation of
immune response to various pathogens (Perez de Lema
et al 2001). They are responsible for the chemoattraction
of polymorphonuclear neutrophils (PMNs), dendritic cells
(DC), macrophages (MØ), and lymphocytes (Mackay 2001).
T. gondii infects and can be disseminated by these cells to
other organs throughout the host, especially to, muscle and
central nervous system.
The disease in animal models
The T. gondii mouse infection model, because of its simplicity
and robust responses it generates, has proven a powerful tool
for studying host-microbe interactions. Because of that,
most of the data published on immune responses and genes
implicated in susceptibility derive from studies employing
inbred mouse strains. Infection of mice leads to lifelong per-
sistence of the parasite. Outcome is variable, depending on
the interaction of many factors like inoculum size (Liesenfeld
1999), virulence of the organism (Su et al 2002), life cycle
stage of the parasite (Johnson 1984; McLeod et al 1989a;
Suzuki et al 1989a, 1993), genetic background (Williams et al
1978; McLeod et al 1989b; Suzuki et al 1996; Lu et al 2005),
gender (Roberts et al 1995), and immunological status. All
these factors seem to affect the course of infection in human
beings and in animal models of toxoplasmosis.
Murine susceptibility to T. gondii or resistance to mortal-
ity following acute oral infection is under multigenic control
by the host (Williams et al 1978; McLeod et al 1989a; John-
son et al 2002). In C57BL/6 mice, genes both within and
outside of the major histocompatibility complex (MHC) are
involved in impaired intracerebral immune response (Brown
and McLeod 1990; Deckert-Schluter et al 1994).
The ability of mice to survive T. gondii infection is
dependent on strong T cell-mediated immunity. Both CD4+ T
helper (Th) 1 cells and CD8+ cytolytic T lymphocytes are
vital in providing protective immunity and long-term survival
during chronic infection, as determined by in vivo depletion
studies and adoptive transfer of CD4+ and CD8+ T cell lines
and clones (Suzuki et al 1988; Gazzinelli et al 1991, 1992;
Parker et al 1991; Kasper et al 1992; Buzoni-Gatel et al
1997). If the host becomes immunosuppressed, chronic infec-
tion can be reactivated, leading to toxoplasmic encephalitis,
which is often fatal if not treated (Suzuki 2002).
The protective competence of these cell types is due to
their ability to produce interferon (IFN)-γ, a pro-infl ammatory
cytokine that has become well known as the major mediator
of resistance to T. gondii (Suzuki et al 1988). Mice that do not
express IFN-γ (IFN-γ knockout mice) are unable to survive
the acute phase of infection (Scharton-Kersten et al 1996).
Antibody-mediated depletion of this cytokine during chronic
infection demonstrates that continued IFN-γ production is
necessary for long-term survival (Gazzinelli et al 1992).
However, pathologic consequences due to overproduction of
cytokines and excessive activation of the immune system are
observed in IL-10 knockout (IL-10 KO) mice infected with
T. gondii (Gazzinelli et al 1996) or wild type mice infected
with highly virulent strains (Mordue et al 2001).
The activation of murine macrophages (MØ) by IFN-γ in
the presence of additional signals, such as lipopolysaccharide
(LPS) or tumor necrosis factor-α (TNF-α), is necessary to
trigger the cytotoxic activity of MØ against T. gondii (Sibley
et al 1993). IFN-γ exerts its antimicrobial activity through
induction of specifi c effector molecules, including reactive
nitrogen intermediates that interfere with key metabolic
enzymes (Khan et al 1997; Scharton-Kersten et al 1997;
Roberts et al 2000), indoleamine 2,3-dioxygenase (IDO)
that induces tryptophan degradation thereby interfering with
viability of the parasite (MacKenzie et al 1999; Fujigaki
et al 2002; Silva et al 2002), and a family of GTP-binding
proteins. IFN-γ-inducible GTP-binding proteins IGTP and
LRP-47 are required for the mice to survive acute T. gondii
infection, while nitric oxide-dependent protection may be
more important during chronic infection (Taylor et al 2000;
Collazo et al 2001).
The intraocular infl ammatory response to the parasite is
also mediated primarily by the CD4+ T- and CD8+ T-cell
responses. CD8+ T cells recognize and destroy parasite-
infected cells in an MHC class I restricted manner. However,

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Host and parasite genetics in ocular toxoplasmosis
as part of the local immune privilege, cells in the eye
express low levels of MHC class I while infected mice
usually show increased expression (Lyons et al 2001).
This would, in turn, permit the precise targeting of
T. gondii-infected cells by CD8+ cytolytic T lymphocytes
and unlike other killing mechanisms that rely on soluble
mediators, would limit collateral damage to uninfected
cells. Indeed, depletion of the CD8+ cell subset resulted in
increased lesion formation and cyst burden in an acquired
disease model of infection (Gazzinelli et al 1994a), and
a severe necrosis accompanied by higher ocular parasite
burdens, in CD8-defi cient mice immunized and challenged
by a intraocular inoculum (Lu et al 2004). B cells may also
contribute by limiting tachyzoite proliferation in the eyes
(Lu et al 2004).
Programmed cell death is an important protective strategy
used in immune-privileged organs. Fas is expressed on a
variety of immune and nonimmune cells, whilst FasL is
evident on T cells as well as in immune-privileged tissues.
Ligation of these two surface molecules results in apop-
tosis of the Fas-expressing cell. In this way, the eye can
prevent the damage caused by infi ltrating lymphocytes and
activated T cells can induce apoptosis in infected cells.
T. gondii induces increased Fas and FasL expression on the
cells of mouse retina. When intracamerally inoculated with
tachyzoites of one virulent strain, Fas- and FasL-defective
mice exhibit greater infl ammatory scores and a more severe
histopathology than similarly infected wild-type mice, added
to a reduced ability to limit parasite proliferation early in
infection (Hu et al 1999, 2001). However, when intraperi-
toneally inoculated with a less virulent strain, the same mice
showed no difference in the degree of ocular infl ammation
and apoptosis. The higher level of IFN-γ and NO apparent
early in these mice must have provided an alternative
means to control parasitemia in the absence of Fas or FasL
expression (Shen et al 2001).
In the immunosuppressed environment of the eye with
high levels of TGF-β antigen-presenting cells can produce
IL-10 when activated, which would stimulate the expansion
of a regulatory T cell subset able to counteract the Th1 type
pro-infl ammatory responses and thus maintain the immune
privilege. Nevertheless, the ocular pathogenesis in C57BL/6
mice is more severe than that of BALB/c and CBA/J mice and
the serum levels of IFN-γ and TNF-α in C57BL/6 mice are
signifi cantly higher than those in BALB/c and CBA/J mice
following ocular infection with a virulent strain of T. gondii
(Lu et al 2005). There are higher levels of IFN-γ mRNA
expression in the retinas of C57BL/6 mice than in those of
BALB/c mice infected with T. gondii (Norose et al 2003).
Furthermore, a decline in IFN-γ production in CD4-KO mice
protects mice from mortality due to an exacerbated immune
response (Casciotti et al 2002). IFN-γ has been shown to
regulate the T. gondii load and inter-conversion between the
bradyzoite and tachyzoite stages of T. gondii in the murine
eye (Norose et al 2003). Therefore, IFN-γ mediates immuno-
pathology and contributes to early death following T. gondii
infection (Liesenfeld et al 1996).
Toxoplasmosis
Some recent studies support the idea that T. gondii is a
master manipulator of host immune responses. The parasite
simultaneously triggers the secretion of protective cytokines
(IFN-γ and IL-12) and paradoxically suppresses the same
type of response (Denkers et al 2004; Gaddi and Yap 2007).
This dual capacity of the parasite could actually be benefi cial
and allow the establishment of a stable host–parasite interac-
tion. Failure to successfully synchronize these responses has
negative consequences for both. Antimicrobial protection is
ensured by the coordinated action of the innate and adaptative
immune systems. Molecular communication between the
host innate immune system and the intracellular protozoan
T. gondii is fast emerging as a dramatic example of these key
principles in action.
In the immunocompetent host the acute infection is usu-
ally asymptomatic or causes a infl uenza-like syndrome, and
rarely leads to severe symptoms such as interstitial pneu-
monia, pericardial effusions, myositis, myocarditis, and
neurological disorders (retinochoroiditis, Guillain-Barré
syndrome, mental confusion) (Bossi and Bricaire 2004).
Encephalitis is most commonly seen in immunocompro-
mised individuals whereas uveitis is the most common
lesion appearing in immunocompetent individuals (Hill
and Dubey 2002). The infection incites a quasieffi cient
immune response, which controls parasitemia but allows
tissue cysts to form in the brain, eyes, and muscles, lead-
ing to lifelong persistence of the parasite and variable
medical outcome.
Dissemination of T. gondii in immunocompromised hosts,
such as AIDS patients with very low CD4 counts, patients
under immunosuppression to prevent or treat transplant
rejection, and fetuses, is common, and often represents a reac-
tivation of an earlier infection, rather than a newly acquired
one. In these individuals the parasite can, besides encephalitis
and retinochoroiditis, cause carditis, pneumonia, and men-
ingitis, amongst other manifestations (Hill and Dubey 2002;
Mele et al 2002; Montoya and Liesenfeld 2004).

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Vallochi et al
Ocular lesions may result from congenital or after
birth-acquired infections. The lesion is often necrotic,
destroying the architecture of the neural retina and some-
times involving the choroid (retinochoroiditis). Typical
fi ndings of toxoplasmic retinochoroiditis include white
focal lesions with an intense overlaying and vitreal
infl ammatory reaction. Toxoplasmic retinochoroiditis is
the most common lesion caused by infection with this
protozoan and it may occur either immediately or long
after the initial infection (Silveira et al 1988; Couvreur
and Thulliez 1996; Montoya and Remington 1996) or
reactivation (Bosch-Driessen et al 2002).
Recurrent lesions are usually identifi ed at the borders
of the retinochoroidal scars, are typically found in clusters,
and have been attributed to the rupture of tissue cysts within
these old lesions or eventually coming from other tissues due
to activation of memory immune cells. It is thought that the
satellite lesions occurring both in the brain and the eye are
caused by rupture of local cysts (Holland 2003, 2004), but the
origin of active infections in remote tissues has been credited
to either clinically unapparent tissue cysts or to re-infection
(Hill and Dubey 2002). In this last case, it is conceivable that
the immune response by memory cells might be triggered by
circulating parasites or even by other infections, through the
activation of IFN-γ and other infl ammatory mediators.
Many questions about the disease still remain that
confound ophthalmologists (Vallochi et al 2002; Holland
2003, 2004). The response to antibiotic therapy alone or
in combination with corticosteroids varies widely among
patients (Stanford et al 2003). Clinical presentation also
varies, with some patients presenting only one episode of
mild infl ammation whereas others have multiple recurrences
of severe uveitis leading to loss of eyesight. The immune
response against retinal antigens does not explain the exten-
sive damage to the eye in patients with ocular toxoplasmo-
sis. Furthermore, we have presented data suggesting that a
controlled autoimmune response may develop after infection
with T. gondii in some patients and, in addition, may help to
prevent the parasite from spreading through the retina thus
associated with a milder disease (Vallochi et al 2005a).
Familial ocular toxoplasmosis is found in southern Brazil,
where the incidence of ocular toxoplasmosis is higher than
in most regions of the globe, and accounts for 70% to 90%
of all uveitis cases seen in rural areas. A household survey in
the endemic area showed 17,7% (186/1,042) of the popula-
tion had toxoplasmic scars in the retina (Silveira et al 1988;
Glasner et al 1992), a fi nding that cannot be explained by
congenital infection alone (Holland 2003, 2004). The same
population was reassessed 7 and 11 years later and showed
that seroconversion was more frequent in individuals under
17 years of age or above 50 years of age, with a risk of 10%
of ocular involvement (Silveira et al 2001; Silveira 2002).
Finally, acquired infection causing late necrotizing retino-
choroiditis is seen not only in Brazil (Nussenblatt and Belfort
1994), but also in Europe (Brezin and Cisneros 1999) and in
the USA (Montoya and Remington 1996).
Analysis of systemic cellular response to T. gondii antigen
in 136 subjects with a diagnosis of ocular toxoplasmosis,
no ocular disease or no infection by T. gondii suggested
that resistance to the development of ocular toxoplasmo-
sis has been associated with the ability to mount a lasting
T-helper (Th) 1-type response (IL-2 and IFN-γ production)
to the parasite antigens. On the other hand, susceptibility
has also been associated with an infl ammatory response,
mostly mediated by cytokines (IL-1 and TNF-α) possibly
produced by activated macrophages (MØ), which may them-
selves take part in the pathological features of the disease
(Yamamoto et al 2000).
A recent study performed in France is in opposition to our
study. Analyzing the induction of CD25 expression, no sig-
nifi cant difference was observed in immune response among
16 individuals with a diagnosis of congenital, acquired, or
undetermined ocular toxoplasmosis or between patients
with active or inactive ocular lesions. Furthermore, higher
levels of IFN-γ were detected in stimulated blood cultures
from infected patients than in those from controls, with no
difference between patients with asymptomatic or ocular
toxoplasmosis (Fatoohi et al 2006).
TNF-α also plays an important role in murine resistance
to acute and chronic toxoplasmosis (Chang et al 1990;
Johnson 1992; Langermans et al 1992). However, high
levels of this cytokine produced during any lethal infection
can potentially contribute to harmful cerebral and hepatic
effects (Black et al 1989; Beaman et al 1992; Marshall
et al 1999). Furthermore, TNF-α may aid the intracerebral
dissemination of T. gondii in mice (Grau et al 1992) and
may be increased in toxoplasmic retinochoroidal in humans
(Yamamoto et al 2000).
We have shown that individuals with ocular toxoplasmosis
from congenital infection have a diminished immune
response towards T. gondii antigens when compared with
individuals with ocular disease due to acquired infection
(Yamamoto et al 2000). Interestingly, unlike mice, the
diminished response is not caused by a superantigen
(Vallochi et al 2001). This means that, despite the similarity
at macroscopic and histopathologic level, the lesions seen on

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Host and parasite genetics in ocular toxoplasmosis
ocular toxoplasmosis may be generated by different mecha-
nisms, therefore explaining the wide range of responses to
available therapy.
Toxoplasma genotype and pathology
Variation of clinical presentation and severity of disease in
susceptible individuals has been attributed to several factors,
including host genetic heterogeneity and parasite genotype
(Boothroyd and Grigg 2002). We have typed T. gondii strains
in Brazil (Vallochi et al 2005b) and our fi ndings reveal that
the highly clonal population structure of T. gondii seen in
North America and Europe does not predominate in South
America (Khan et al 2006). Identifi cation of the direct players
in host–pathogen interactions during the initial infection and
chronic phases of the disease is perhaps the best hope for
better clinical treatment in this region.
Population studies analyzing more than 50 different
genetic markers have identifi ed a limited number of T. gondii
genotypes in nature, despite the presence of a sexual phase
in the life cycle, a wide geographical distribution and a
broad range of intermediate hosts. Phylogenetic analysis
detects 2 main groups in T. gondii population. From a
practical point of view, 3 main lineages (type I, type II,
and type III) that despite sharing about 98% genetic iden-
tity differ in virulence and epidemiological pattern of
occurrence (Darde 2004; Switaj et al 2005), are usually
considered. For most groups, allelic polymorphism is low
(2 to 4 alleles) (Darde et al 1992; Howe and Sibley 1995;
Ajzenberg et al 2002), indicating that they descend recently
from two closely-related parents that underwent a small
number of genetic recombination events. Less than 1% of
the previously studied strains contain unique genotypes and
high divergence of DNA sequence, and have therefore been
considered ‘exotic’ or ‘atypical’ strains (Su et al 2003).
However, it was demonstrated that sexual recombination
combining polymorphisms in two of these distinct and
competing clonal lines, can be a powerful force driving
the natural evolution of virulence in this highly successful
pathogen (Grigg et al 2001).
The apparently low genetic diversity in T. gondii may
have been underestimated because most parasite strains
in previous studies were collected from human patients
and domestic animals in North America and Europe. The
choice largely employed by researchers is using the SAG2
locus for rapid typing since it is capable of distinguishing
all three alleles from a single locus (Howe et al 1997). This
method is also widely used in other genes where the popula-
tion structure has not been studied so extensively, therefore
genetic diversity is under-represented. Several studies have
examined the distribution of genotypes in chickens from
countries such as Egypt, Argentina, India, and Brazil (Dubey
et al 2002, 2003a, 2003b, 2003c; Sreekumar et al 2003;
Lehmann et al 2004), but the genetic studies were limited
by the use of only one marker (SAG2) (except [Sreekumar
et al 2003; Lehmann et al 2004]), and because chickens in
farms are indicators of strain prevalence in a domestic or
peridomestic environment. Strains isolated from humans and
animals (rabbit, mouse, goat, chickens, and dogs) in Minas
Gerais, Brazil were analyzed by polymerase chain reaction-
restriction fragment length polymorphism (PCR–RFLP) at
eight independent loci and all strains carried recombinant
genotypes, with typical types I, II, or III in almost all loci
assessed (de Melo Ferreira et al 2006).
We also have identified type I T. gondii strains
by PCR-based typing at the SAG2 locus in retinochoroidal
specimens from Brazil (Vallochi et al 2005b). When ana-
lyzed by multilocus PCR-RFLP, our recent fi ndings reveal
that the highly clonal population structure of T. gondii
seen in North America and Europe does not predominate
in South America (Khan et al 2006). Consistent with this
observation, T. gondii strains isolated from French Guiana
patients are also highly diversifi ed and not characteristic of
a clonal organism as seen in North America strains (Ajzen-
berg et al 2004).
In the Brazilian strains, genotyping showed no cases of
mixed infection (two alleles at a given locus). Unfortunately,
the true extent of sequence divergence is not captured
by multilocus RFLP and microsatellite analysis tends
to overestimate genetic divergence. The UPRT-1 intron
sequence was compared, and all except one strain from Brazil
proved to have multiple additional polymorphisms not seen
in the clonal lineages. This analysis suggested the presence
of more than one predominant haplotype in Brazil along with
less common unique genotypes (Khan et al 2006).
It has been suggested previously that such divergent
strains are more ancient in origin than the recently derived
clonal lineages and that T. gondii expansion is linked to
enhanced oral transmission between intermediate hosts, a
trait not shared by other closely related parasites (Su et al
2003). Despite of high serologic prevalence and level of
recurrent ocular disease in Erechim, Brazil (Glasner et al
1992), it is premature to say that sexual reproduction plays a
central role in the population structure of T. gondii, since there
were no cases of mixed infection. Given that the samples
are from two small outbreaks and one of them due to food
borne. It is likewise early to describe the epidemiology of

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Vallochi et al
toxoplasmosis in Brazil, since it is a country of continential
size, boasting tropical and subtropical areas.
Recent studies in mice have identified some genes
responsible for the strain-specifi c differences in virulence
(Taylor et al 2006; Saeij et al 2007), where type I strains
are more virulent than types II and III. As virulence is a
multigenic trait, crossing the less virulent type II and type
III strains generates some F1 progeny in which the virulence
is greatly enhanced. The use of QTL mapping in such prog-
eny identifi ed a total of fi ve virulence loci, including one
on chromosome VIIa, which was found to correspond to
ROP18, and one on chromosome VIIb, which corresponded
to a gene encoding another rhoptry kinase, ROP16 (Saeij
et al 2007). ROP18 encodes the ROP18 protein, which is
a functional serine kinase and is secreted into host cells
(Taylor et al 2006). ROP16 is a key modulator of the host
response and the three clonal lineages differ in their level
of activation of the STAT signaling pathway. As this is the
pathway to the crucial cytokine interleukin 12, ROP16 is
involved in this effect (Saeij et al 2007). It seems that these
eukaryotic pathogens can secrete protein kinases into host
cells to subvert host-cell signaling pathways and that this
explains many of the differences in virulence among the
three dominant clonal lineages.
Our fi ndings reveal that the genetic makeup of T. gondii
is more complex than previously recognized and are in
agreement that unique or divergent genotypes may contribute
to different clinical outcomes of toxoplasmosis in different
localities. Further strain comparisons based on a wider set
of sequence based markers will be necessary to defi ne the
global population structure of T. gondii and to resolve the
relationships between major strain types seen in different
geographic regions.
Host genotype and the immune
response to T. gondii
A wide array of studies has further demonstrated
differences in genotype and allele frequencies of cytokine
gene polymorphisms depending on ethnicity and race
(Golovleva et al 1997; Cox et al 2001; Uboldi de Capei
et al 2003). Cytokines and chemokines and Toll-like
receptors play a key role in the regulation of the type and
magnitude of immune response, and the polymorphic
nature of these genes may confer further fl exibility to the
immune response (Schroder and Schumann 2005). We
have analyzed some of the genes involved in the response
against T. gondii infection, whose functions are detailed
in the following pages.
Toll-like receptors
Toll-like receptors (TLRs) have been recently defi ned as
important transmembrane proteins that can confer a cer-
tain degree of specifi city to the cells of the innate immune
compartment. TLRs, also referred to as pattern recognition
receptors (PRRs), have been implicated in recognition of
every known category of pathogen that causes human disease
and whose signaling through the common adaptor mol-
ecule myeloid differentiation primary-response protein 88
(MyD88) is essential in proinfl ammatory cytokine responses
to many microbial pathogens.
TLRs can recognize traces of microbial components and
orchestrate an early defense, largely dependent on the activa-
tion of nuclear factor-kappaB (NF-κB), which will lead to
the production of proinfl ammatory cytokines and triggering
of microbiostatic/microbicidal effector mechanisms (Iwasaki
and Medzhitov 2004). While major advances have been
made in the assignment of individual TLRs to defi ned roles
in bacterial infections (Takeda et al 2003), such identifi cation
has only recently begun to emerge in protozoan parasites
(Gazzinelli et al 2004).
Mice that are deficient in the TLR adaptor protein
MyD88 were found to have a pronounced decrease in IL-
12 production by peritoneal macrophages, neutrophils, and
splenic DC in response to soluble tachyzoite antigen (STAg)
stimulation in vivo and in vitro being highly susceptible to
the acute infection with low doses of the infective stage of
T. gondii (Scanga et al 2002). At this lower parasite dose,
neither TLR2 nor TLR4 were important for mouse resistance
to infection with T. gondii. In addition, DC from TLR2- or
TLR4-KO mice developed normal IL-12 responses to STAg
(Scanga et al 2002) just as PMNs from TLR2-KO animals
did (Del Rio et al 2004). TLR2-KO mice produce suffi cient
IL-12 to mediate partial protection that is nonetheless not
enough to provide complete resistance to high dose infec-
tion, as compared to the wild type or TLR4-KO mice (Mun
et al 2003). Hence, TLR2 might be involved in the early
activation of the innate immune system during infection with
T. gondii to be partially involved in the parasite recognition
and MyD88 activation during early stages of infection with
T. gondii. It seems that the defect seen in TLR2-KO mice is
due to ineffi cient activation of microbicidal functions, with
diminished nitric-oxide production by macrophages (Mun
et al 2003).
Alternatively, MyD88-dependent neutrophil production of
CCL2 (monocyte chemoattractant protein 1), but not IL-12, is
dependent upon TLR2 signaling and defective CCL2 release
could confer partial susceptibility to infection (Del Rio et al

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Host and parasite genetics in ocular toxoplasmosis
2004). Of note, neutrophil IL-12 production is not dependent
upon parasite cyclophilin-18 (C-18), a T. gondii protein shown
to trigger DC IL-12 through CCR5 ligation (Aliberti et al
2003). Thus, T. gondii possesses multiple molecules triggering
distinct MyD88-dependent signaling cascades, and as these
pathways are independently regulated, they may lead to dis-
tinct profi les of cytokine production (Del Rio et al 2004).
MyD88 signaling occurs also through IL-1R or IL-18R,
but ICE-KO mice (lacking functional IL-1 and IL-18)
exhibit no increased susceptibility to infection suggesting
that signaling through IL-1R and IL-18R is not essential for
responses against T. gondii infection. Importantly, TLR1-,
TLR2-, TLR4-, TLR6-, and TLR9-KO mice exhibited similar
resistance to infection compared with wild-type mice sug-
gesting that these receptors are not independently essential
for control of T. gondii infection (Hitziger et al 2005).
A recent study from our laboratory indicates that there is
no association between polymorphisms in TLR2 (Arg677Trp
and Arg763Gln) and TLR4 (Asp299Gly) and ocular toxoplas-
mosis in Brazilian patients (unpublished observations), a fi nd-
ing in keeping with the observations in the murine model.
On the other hand, of the whole family of TLRs, two
distinct TLR have been implicated in the recognition of
protein ligands. TLR5 has been shown to be triggered by
bacterial flagellin while TLR11 signaling is stimulated
by protease sensitive molecules in uropathogenic bacteria
(Hayashi et al 2001; Zhang et al 2004). TLR11 was impli-
cated in resistance to T. gondii when an additional ligand in
the parasite, T. gondii profi lin (PTG), that apparently triggers
high DC IL-12 production by a MyD88 dependent but CCR5
independent pathway, was discovered (Yarovinsky et al
2005). Interestingly, while DC IL-12 production appeared to
be almost totally impaired in TLR11-KO mice infected with
T. gondii, these animals, unlike either MyD88 or IL-12-KO
mice (Reis e Sousa et al 1997) retained partial resistance to
challenge and survived the acute phase of the infection, most
likely due to a residual IFN-γ response (Yarovinsky et al
2005). It is important to note however, that human TLR11
is nonfunctional because of the presence of a stop codon in
the gene (Zhang et al 2004). At present it is not clear whether
TLR11 recognition of T. gondii is of importance in limiting
infection in other mammalian species or whether humans
utilize alternative pattern recognition receptors in the innate
response to T. gondii.
Interleukin-12
Immunity to T. gondii involves acute infl ammatory responses
and antigen-specifi c adaptive immunity (Denkers and Gazzi-
nelli 1998; Cai et al 2000). Central to host resistance is the
generation of IFN-γ by innate-type natural killer (NK) cells
and adaptive CD4 and CD8 T lymphocytes (Suzuki et al
1988, 1989b). IL-12 has been identifi ed as critical for driv-
ing prompt IFN-γ production and proper differentiation of
Th1 lymphocytes during the immune response to T. gondii
(Gazzinelli et al 1994b; Yap et al 2000).
IL-12 is produced by PMNs, DC, B cells and other
antigen-presenting cells (APCs). PMNs store cytokines
in pre-formed pools namely IL-12, IL-6, MIP-2 in mouse
neutrophils in the absence of infection (Terebuh et al 1992;
Bliss et al 2000; Matzer et al 2001), and IL-4 in PMN from
healthy human donors (Brandt et al 2000). T. gondii induces
secretion of IL-12 directly and rapidly by both human and
mouse neutrophils, DCs as well as MØ (Gazzinelli et al
1993; Reis e Sousa et al 1997; Bliss et al 1999a, 1999b;
Denkers et al 2003; Robben et al 2004). Release of IL-12
does not require IFN-γ priming since responses are intact
in gene-targeted mice lacking this cytokine. On the other
hand, recombinant TNF-α alone stimulates CCL2 and CCL4
production by PMN, and therefore, the ability of T. gondii to
trigger TNF-α may provide an important positive feedback
loop for release of neutrophil CCL2 and CCL4, important
chemokines which recruit macrophages and other cells
to infected tissue. T. gondii also induces up regulation of
RANTES (CCL5), MIP-3α (CCL20) and macrophage
chemoattractant protein-1 (MCP-1; CCL2) gene transcription
by PMN (Bennouna et al 2003), which recruited to the site
of infection by chemokines such as IL-8 and MCP-1, are
themselves equipped to immediately release cytokines and
chemokines that contribute further to cell recruitment and
activation.
IL-12 is composed of two subunits, IL-12 p40 and
IL-12 p35. In the absence of the IL-12 p40 gene or the
IL-12-receptor-associated signal transducers Tyk2 and
STAT-4, IFN-γ production is severely impaired resulting
in enhanced susceptibility to acute T. gondii infection (Yap
and Sher 1999; Cai et al 2000; Shaw et al 2003). In contrast,
the IL-12-related cytokine, IL-23, which is composed of
the shared IL-12 p40 subunit and a unique p19 subunit is
not required or essential for mouse immunity to T. gondii
infection (Lieberman et al 2004). Thus, IL-12 p40 production
by DC and via a MyD88-dependent recognition mechanism
is critical and perhaps suffi cient for initiating IFN-γ-medi-
ated immunity to T. gondii (Reis e Sousa et al 1997; Scanga
et al 2002). Related cytokines such as IL-18 and IL-23 may
instead play a pathogenic role (Mordue et al 2001; Gaddi
and Yap 2007).

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Clinical Ophthalmology 2008:2(4)
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Vallochi et al
Studies of differential production of IL-12 in BALB/c
and DBA/2 mice suggest that strain-specifi c variation not
encoded by MHC genes affects IL-12 production with a
skewing of antigen-specifi c recall responses to either a Th1 or
a Th2 direction (Gieni et al 1996). In humans, deletions
of IL-12 p40 or IL-12 receptors lead to serious impairment
of immunity against intracellular bacteria (Altare et al 1998;
de Jong et al 1998). A complete genomic sequence analysis
of the IL-12 gene encoding its p40 subunit (IL-12B) identi-
fi ed several intronic polymorphisms, a single-nucleotide
polymorphism (SNP) at position +16974 (+16974 A/C) in
the 3’-untranslated region (UTR) of IL-12B (Hall et al 2000;
Huang et al 2000) and a promoter polymorphism (IL-12Bpro)
(Morahan et al 2002).
Association between genotype (+16974 A/C) and
IL-12p40 production by stimulated PBMC was observed
and depend on the stimuli used. There is a signifi cantly
decreased IL-12 p40 secretion for the following order of
genotypes: AA ? CA ? CC, after stimulation of PBMC
with C3-binding glycoprotein (C3bgp) in contrast to
lipopolysaccharide, phytohaemagglutinin and pokeweed
mitogen (Stanilova and Miteva 2005). Furthermore, poly-
morphisms in the IL12B 3’UTR gene have been show to
infl uence the secretion of IL-12 and susceptibility to Type
1 diabetes (Windsor et al 2004), and the promoter polymor-
phism may infl uence the outcome of malaria infection in at
least one African population (Morahan et al 2002).
CCR5
Although low-level IFN-γ production occurs in the absence
of IL-12 signaling, optimal induction is strictly IL-12-
dependent (Cai et al 2000). Therefore, much effort has been
dedicated to disclose the mechanism(s) by which T. gondii
tachyzoites elicit the production of IL-12 during the initial
stages of infection.
The best defi ned mechanism by which T. gondii induces
the production of IL-12 is through the parasite-derived
cyclophilin-18, which signals murine DCs to produce IL-
12 via the chemokine receptor CCR5 (Aliberti et al 2000;
Aliberti et al 2003). The biological relevance of the unusual
requirement for a chemokine receptor to participate in micro-
bial recognition by DCs is supported by the observation that
IL-12 production is decreased during acute infections of
CCR5-KO animals (Aliberti et al 2000), although defects
in cell migration could also contribute to this susceptible
phenotype. In theory, cell-surface CCR5 on DCs could
deliver signals derived from stimulation with autologous
chemokines that have been induced following microbial
stimulation or could be directly triggered by pathogen
derived ligands.
In humans, a mutant allele of the CCR5 gene, bearing
a 32-bp deletion, results in reduced CCR5 cell surface
expression (Blanpain et al 2000). However, we couldn’t
observe association between the allele CCR5Δ32 and
ocular toxoplasmosis in Brazilian patients (unpublished
observations). Incidentally, we and others have found this
variant to be quite rare in Brazilian population samples
(unpublished results).
The IL-12 levels observed after stimulation of murine
DCs with C-18 are much lower than those seen after stimula-
tion with whole-parasite lysate or with a pool of tachyzoite-
secreted proteins, indicating that pathways other than those
initiated by CCR5 and C-18 might also be important for IL-12
production by DCs (Aliberti et al 2003).
Conclusion
While the data summarized in this review demonstrate
that the binomial IL-12 and IFN-γ production is critical to
resistance to T. gondii infection in mice, and that an intricate
mechanism involving simultaneous activation of different
signaling pathways could be required for induction and
control of IL-12 during acute infection with T. gondii, much
remains to be learned about the mechanisms involved and
their relevance to parasite and host interactions during natu-
ral infection in humans. We had discussed the importance
of the genetic background of both the parasite and the host
in the establishment of ocular disease and the major ques-
tions concerning the specifi c receptors and parasite ligands
required for innate immunity response triggering and how
these interactions result in resistance to T. gondii.
Acknowledgments
Supported by Fundação de Amparo a Pesquisa do Estado de
São Paulo (FAPESP), Conselho Nacional de Desenvolvimento
Científi co e Tecnológico (CNPq), and the Pan-American
Association of Ophthalmology (PAAO).
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