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REVIEWS

Innate immunity to Toxoplasma gondii


infection
Felix Yarovinsky

Abstract | Toxoplasma gondii is a protozoan parasite of global importance. In the laboratory


setting, T.gondii is frequently used as a model pathogen to study mechanisms of T helper 1
(TH1) cell-mediated immunity to intracellular infections. However, recent discoveries have
shown that innate type1 immune responses that involve interferon (IFN)-producing
natural killer (NK) cells and neutrophils, rather than IFN-producing Tcells, predetermine
host resistance to T.gondii. This Review summarizes the Toll-like receptor (TLR)-dependent
mechanisms that are responsible for parasite recognition and for the induction of
IFN production by NK cells, as well as the emerging data about the TLR-independent
mechanisms that lead to the IFN-mediated elimination of T.gondii.
Apicomplexan parasite
A member of a large group of
protozoan parasites possessing
a unique apical complex
structure that is involved
in host cell invasion. This
phylogenetic group includes
more than 5,000 species,
all of which are parasitic.
The best known examples of
apicomplexan parasites are
Plasmodium spp. (the cause of
malaria), Toxoplasma gondii
and Cryptosporidium spp.

Department of Immunology,
University of Texas
Southwestern Medical Center,
5323 Harry Hines Boulevard,
Dallas, Texas 75390-9093,
USA.
email: Felix.Yarovinsky@
UTSouthwestern.edu
doi:10.1038/nri3598

Toxoplasma gondii is a protozoan apicomplexan parasite that can infect all mammals and birds, normally
through contaminated food (BOX1). The parasite has
received considerable scientific and medical attention
as it causes severe disease in immunocompromised
individuals, such as patients with AIDS. In otherwise
healthy adults, this intracellular parasite is controlled
by the immune system and remains in a dormant state
in the brain, although in rare instances it can cause
uveitis. However, in patients with AIDS, the development of an immunocompromised state leads to
the reactivation of the T.gondii parasites and to the
development of toxoplasmosis. Uncontrolled parasite
replication ensues, which causes life-threatening brain
damage that is characterized by brain abscesses and
necroticareas.
These clinical observations, coupled with the
knowledge that HIV infection results in CD4+ Tcell
depletion, have led to the development of a model in
which Tcell activation is thought to determine the
outcome of T.gondii infection. As such, the scientific
community has mainly focused on exploring CD4+
and CD8+ Tcell responses to this parasite13. In recent
years, progress has also been made in identifying the
T.gondii virulence factors that regulate host immune
responses 4. However, an appreciation of the role of
the host innate immune system in controlling the
parasite is lagging behind. This Review aims to fill
this gap in knowledge by covering the major innate
immune mechanisms that are essential to promote
interferon (IFN)-mediated host protection against
T.gondii.

Innate sensing of T.gondii


Involvement of TLRs. Recognizing protozoan parasites
is a complicated task for the mammalian immune system. This is partly because the majority of the classical
microbial and viral molecules that are sensed by innate
immune receptors are not found in eukaryotic pathogens5. Instead, the innate immune system has evolved
to recognize T.gondii by sensing a distinct set of molecules that is uniquely present in protozoan parasites69.
In addition, T.gondii is indirectly detected through the
recognition of tissue damage events that are associated
with parasitic infection10. Mice that lack the Toll-like
receptor (TLR) signalling adaptor myeloid differentiation primary-response protein 88 (MYD88) show
defective production of interleukin12 (IL12) and IFN
following T.gondii infection and are highly susceptible
to infection by this parasite11. This observation led to
the idea that TLR-induced MYD88 activation is essential for host resistance to T.gondii11. It was hypothesized
that the TLR-driven activation of MYD88 in dendritic
cells (DCs) leads to the IL12dependent activation
of T helper 1 (TH1) cells, which are required for host
survival during the parasitic infection12,13. However, it
was recently shown that IL12 treatment fails to protect
MYD88deficient mice from T.gondii infection and that
activation of MYD88 in DCs promotes IFN production
from natural killer (NK) cells, but not from Tcells14.
Nevertheless, the initial model for the TLR-driven
activation of DCs was extremely fruitful and initiated a
series of biochemical experiments that aimed to identify
the molecular pathways responsible for the activation
of innate responses to T.gondii. As discussed below,

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Box 1 | Major routes of human infection with T.gondii
There are three major ways of acquiring Toxoplasmagondii infection (see the figure): first, foodborne transmission;
second, animal to human (zoonotic) transmission; and third, mother to child (congenital) transmission. Food-borne
infection is most commonly caused by the ingestion of undercooked meat that contains encysted bradyzoites. However,
both humans and livestock can become infected by accidental ingestion of soil that contains T.gondii oocysts as a result of
poorly washed fruit and vegetables or by drinking water that is contaminated with oocysts. T.gondii oocytes are shed
from the faeces of infected cats into the environment or a litter box. Contaminated litter boxes are of special concern for
pregnant women, as a first-time infection during pregnancy can result in the transmission of the parasite to the fetus
a condition that is known as congenital toxoplasmosis. Although the exposure of adults to the parasite rarely results in
severe disease, congenital toxoplasmosis can lead to severe neurological and ocular diseases in children. Rare forms of
transmission that are not depicted here include parasite transmission during organ transplantation and blood transfusion.
It has been estimated that at least one-third of the worlds human population has been exposed to the parasite, but the
relative importance of the different routes of infection is unknown.
T.gondii is an obligate intracellular parasite. Following exposure to the parasite, infection is divided into acute and chronic
stages. The acute stage of infection is characterized by the replication of tachyzoites, which are the fast-replicating forms of
the parasite. As the infection progresses, tachyzoites differentiate into bradyzoites, which are the slow-replicating forms
of the parasite. Bradyzoites in the form of cysts (encysted bradyzoites) are responsible for establishing the chronic
stages of the infection. In a mouse model of infection, intraperitoneally injected bradyzoites rapidly differentiate into
tachyzoites, disseminate into peripheral tissue and establish a persistent infection within 2 weeks of infection.
Although ingestion of the parasite is a major physiological route of T.gondii infection, experimental injection of cysts
into the peritoneal cavity results in identical infection stages but bypasses the mucosal response to the parasite.
Humans

Domestic animals
Encysted bradyzoites
(food- and water-borne)

Oocysts

Congenital
toxoplasmosis
induced by
tachyzoites

Fetus
Mice

Oocysts
(in faeces)

Encysted
bradyzoites
Cats

Sexual reproduction
in the feline denitive host

Oocysts
Ovoid structures that contain
two sporocysts, each of which
contains four sporozoites. The
sporozoites may then become
tachyzoites or bradyzoites.
Cats shed faecal
Toxoplasmagondii oocysts in
the soil, grass and water. The
oocyst wall is an extremely
resistant multilayer structure
that protects the parasite from
mechanical and chemical
damage, which enables the
parasite to survive for long
periods of time.

Oocysts
Nature Reviews | Immunology

multiple TLRs are involved in sensing T.gondii and it


is now apparent that TLR11 is a principal innate sensor for the T.gondii parasite in rodents (FIG.1). TLR11
recognizes the unconventional actin-binding protein
profilin, which is released by the parasites through an
unknown mechanism6,7. Blocking protein secretion in
T.gondii did not prevent the DCmediated recognition
of profilin, which suggests that TLR11 senses profilin

that is passively released from parasites. T.gondii profilin is an essential protein for the parasite7: conditional
deletion of profilin in T.gondii revealed that this protein
regulates parasite motility and host cell invasion7. As
T.gondii is an obligatory intracellular pathogen, failure
to invade host cells because of profilin deficiency results
in the loss of parasite viability. Thus, TLR11mediated
sensing of T.gondii profilin fits the general idea that

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Prolin

TLR12

TLR11
UNC93B1

UNC93B1
Endolysosome

TLR12
UNC93B1

UNC93B1
DNA

RNA

TLR7

TLR1

UNC93B1

UNC93B1

TLR9

MYD88
UNC93B1

UNC93B1
TLR2
GPI

IRF8
TLR4

IL-12

NF-B
p50 p65

IL-12
and IFN

Dendritic cell

Figure 1 | TLRs involved in the recognition of T. gondii. Toll-like receptor 11 (TLR11) and TLR12 are the major
Reviews | Immunology
receptors for the Toxoplasmagondii-derived protein profilin. Although many cells expressNature
TLR11 and
TLR12,
IFN-regulatory factor 8 (IRF8)+ dendritic cells (DCs), in particular CD8+ DCs, have a crucial role in detecting T.gondii
profilin and in the subsequent induction of IL12 production downstream of myeloid differention primary-response
protein 88 (MYD88). A novel MYD88dependent signalling pathway that depends on activation of IRF8 is an
explanation for the potent induction of IL12 expression by CD8+ DCs that have been exposed to T.gondii, but the
direct connection between MYD88 and IRF8 has not been established. In addition, both TLR7 and TLR9 have been
implicated in detecting T.gondii RNA and genomic DNA, respectively. UNC93 homologue B1 (UNC93B1), an
endoplasmic reticulumresident protein, has a central role in the function of all of the depicted endosomal TLRs
and is essential for resistance to T.gondii. In addition to endosomal TLRs, TLR2 and TLR4 are involved in detecting
T.gondii glycosylphosphatidylinositol (GPI) during infection. IFN, interferon-; NF-B, nuclear factor-B.

microbes are detected through the direct recognition


of essential molecules that are uniquely present in the
microorganisms5,15. Once inside the host cells, T.gondii
profilin is mainly dispensable for parasite replication or
egress from the infected cells7. Thus, the innate immune
system detects an early event that is associated with parasite invasion and motility. Furthermore, it is of interest to
note that the DCmediated recognition of T.gondii profilin occurs before direct interaction with the parasite,
using the detection at a distance strategy 16,17. Humans
do not have the gene to encode functional TLR11 (BOX2),
and the principal sensors that are responsible for T.gon
dii detection by human innate immune cells are yet

to be revealed. It is also not known whether T.gondii


profilin can be recognized in the absence of TLR11 by
other classes of receptors or whether T.gondii profilin
can induce inflammasome activation.
Cooperation of endosomal TLR11 and TLR12. So far,
at least three models have been suggested for TLR11regulated immunity to T.gondii (FIG.1). Experiments
from our own laboratory revealed that direct interaction of TLR11 and T.gondii profilin within endolysosomes leads to the recruitment of MYD88 and to the
initiation of downstream immune signalling cascades
and that this depends on the association of TLR12 with

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Box 2 | Species-specific differences in TLR11, TLR12 and TLR13
The Toll-like receptor 11 (TLR11) family includes three closely related TLRs, TLR11,
TLR12 and TLR13, all of which localize within endosomal compartments. Although all
of the TLR11 family members are expressed in mice, human TLR11 is a nonfunctional
pseudogene and humans completely lack TLR12 and TLR13. An important point relating
to the sequences of TLR11 and TLR12 is that the National Center for Biotechnology
Information (NCBI) database contains a DNA sequence for TLR11 that is actually the
sequence for TLR12 and vice versa.

Parasitophorous vacuole
A vacuole surrounded by a
parasitophorous vacuolar
membrane (PVM), which forms
as a result of the invasion of
Toxoplasma gondii into the
host cell. The PVM is formed
during active invasion of the
host cell and depends on actin
polymerization in T.gondii, but
not in the host. The physical
force created by the parasite
initiates the formation of a
membrane, which surrounds
the intracellular parasite within
the parasitophorous vacuole
and which differs from
endosomal or phagolysosomal
membranes.

Soluble tachyzoite antigen


(STAg). A soluble extract
from Toxoplasma gondii that
is obtained by sonication of
the parasites.

both TLR11 and T.gondii profilin6,16,18. In an alternative model it was suggested that both TLR11TLR12
heterodimers and TLR12 homodimers are sufficient for
DC responsiveness to T.gondii profilin19. Finally, in a
third model it was suggested that, although the TLR11
TLR12 heterodimer promotes the activation of MYD88
in response to T.gondii, TLR7 and TLR9 can induce sufficient MYD88 activation to compensate for a lack of
TLR11mediated parasite recognition20. It is of interest to
note that, although TLR11 is a pseudogene in the human
genome, TLR12 is not present in the human genome.
Thus, although the endosomal TLR11dependent and
TLR12dependent recognition system is central for host
defence in mice, it is not applicable to human innate
immune defence against T.gondii.
The models from the Gazzinelli20 and Yarovinsky6,18
laboratories are in agreement that the TLR11TLR12
heterodimer complex is a central sensory unit for the recognition of T.gondii profilin by mouse DCs. Biochemical
experiments using the highly purified extracellular
domains of TLR11 and TLR12 have shown that there is a
direct receptorligand interaction between T.gondii profilin and TLR11 or TLR12, and that the formation of profilinTLR11 and profilinTLR12 complexes occurs18. An
alternative model jointly suggested by the Ghosh and Sher
laboratories suggests that TLR12 alone is sufficient for the
initiation of immune signalling cascades in the absence
of TLR11 (REF.19). However, this model is in contrast to
experiments showing that DCs from TLR11deficient
mice fail to produce IL12 and other cytokines, including IFN, in response to T.gondii extracts or to highly
purified T.gondii profilin6,7,16,21. Furthermore, TLR11 but
not TLR12 recruits MYD88 to initiate MYD88dependent
signalling events downstream of profilin recognition18.
How the TLR12 signalling receptor complex alone
contributes to cytokine production in the absence
of MYD88 recruitment has not been explained.
Roles for other endosomal TLRs: TLR7 and TLR9.
In addition to TLR11 and TLR12, several other TLRs
have been implicated in the recognition of T.gondii. It
has been suggested that the combined action of two
additional endosomal receptors, TLR7 and TLR9, can
induce protective MYD88 activation in the absence of
TLR11 (REF.20) (FIG.1). The involvement of these endosomal TLRs in mediating host defence to T.gondii is consistent with the observed role for UNC93 homologue
B1 (UNC93B1), a chaperone-like protein that regulates
the trafficking of endosomal TLRs, in the regulation of
IL12dependent host resistance to the parasite16. An
alternative TLR-independent function of UNC93B1

in cell-autonomous parasite killing has also been suggested on the basis of the colocalization of UNC93B1
and the parasitophorous vacuole22, but this mechanism
was recently dismissed by the original authors20.
TLR7 is a receptor for single-stranded RNAs that
are produced by RNA viruses23,24. In addition, TLR7
can detect bacterial RNA in the endolysosomes of conventional DCs25. Uridine and ribose, which are the two
defining features of RNA, are both necessary and sufficient for TLR7 stimulation, and short single-stranded
RNAs function as TLR7 agonists in a sequence-independent manner as long as they contain several uridines in close proximity 26. Thus, it is not surprising that
T.gondii RNA is capable of inducing TLR7 activation, as
even host RNA can trigger TLR7 activation27. However,
it is not clear how TLR7 regulates immunity to T.gondii
and why its function is only apparent in the absence of
TLR11. TLR7 is not expressed in CD8+ DCs28, which
are the main DC subset responsible for T.gondii recognition. Furthermore, CD8 conventional DCs that
express high levels of TLR7 (REF.28) fail to produce IL12
invitro or invivo when stimulated with T.gondii or with
T.gondii soluble tachyzoite antigen (STAg)6,29.
These unanswered questions are also pertinent to
TLR9, which was discovered as a receptor for unmethylated DNA that has CpG motifs derived from bacteria
and viruses30. Although the CpG motif was thought
to be essential for TLR9 stimulation, the DNA sugar
backbone of 2 deoxyribose also mediates TLR9 recognition31. TLR9 can also recognize host DNA32,33. Thus, it
is expected that synthetic oligonucleotides that contain
sequences present in the T.gondii genome are capable
of inducing TLR9 activation20. Furthermore, T.gondii
lacks detectable DNA cytosine methylation34, which
highlights it as a putative target for TLR9mediated
recognition invivo. Nevertheless, for reasons we do not
understand, activation of TLR9 in response to T.gon
dii is not readily detected in DCs that express TLR9,
and the IFN-mediated priming of DCs is essential
for the induction of TLR9mediated responsiveness to
T.gondii DNA20. Furthermore, non-infected DCs are
the major source of IL12 during T.gondii infection16.
Future studies are needed to understand how T.gon
dii RNA and DNA can access the endolysosomes of
non-infectedDCs.
It is worth mentioning that TLR9 has a broad influence on immune responses to protozoan parasites other
than T.gondii. In addition to DNA, TLR9 also recognizes haemozoin a crystalline metabolite of haemoglobin that is produced by an apicomplexan parasite
Plasmodium falciparum, which is phylogenetically
related to T.gondii35. However, whether TLR9 directly
interacts with haemozoin is currently under debate, as
it has been suggested that haemozoin only transports
malarial DNA to the endosome, where TLR9 is present36.
Furthermore, intestinal damage that is caused by parasitic infection can result in TLR9 activation by intestinal
bacteria10,37,38. This mechanism of bystander TLR9 activation during T.gondii infection has a role in coordinating DCmediated IL12 production and subsequent
IFN secretion by CD4+ Tcells10,39.

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Congenital toxoplasmosis
A disease that occurs when a
developing child is infected
with the parasite Toxoplasma
gondii. The developing
child can be infected during
pregnancy, labour or delivery.

Sensing of T.gondii by cell surface TLRs. In addition to


endosomal TLRs, cell surface TLR2 and TLR4 have also
been implicated in the recognition of T.gondii. TLR2
functions as a general innate immune receptor for the
glycosylphosphatidylinositol (GPI) anchors that cover
the cell surface of multiple protozoan parasites, including T.gondii9,40,41. Although TLR2 deficiency does not
have a major effect on IL12 production during T.gon
dii infection11, TLR2 regulates tumour necrosis factor
(TNF) and CCchemokine ligand 2 (CCL2) production
by macrophages and neutrophils, respectively 9,42, and
both of these soluble mediators are essential for host
resistance to T. gondii4347. TLR2 expression is not limited
to macrophages, and activation of this innate immune
sensor in epithelial cells also triggers the production of
chemokines during T.gondii infection48. Nevertheless,
mice that are deficient in TLR2 are only susceptible to
infection with very high doses of T.gondii49 and mount
an unimpaired immune response when infected with
conventional parasite loads11. These observations call
into question the biological relevance of TLR2 in host
defence against T.gondii infection invivo. TLR2 is
involved in the recognition of a broad range of pathogens, mainly Gram-positive bacteria5. Furthermore,
multiple host-derived molecules that are released during tissue damage can trigger TLR2 activation 50. The
limited susceptibility of TLR2deficient mice to T.gon
dii infection suggests that the observed phenotype is
caused by impaired responsiveness to host-derived molecules that are released during T.gondii infection. This
possibility is supported by similar observations that
have been made following infections with other pathogens, including Mycobacterium tuberculosis. TLR2 deficiency has little effect on host resistance at low infection
doses, but infection with high doses of M.tuberculosis
results in rapid mortality in TLR2deficient mice51.
Thus, further studies are required to understand the
precise biological function of TLR2 in host resistance
to T.gondii. It is also unknown whether TLR1TLR2
or TLR2TLR6 heterodimers mediate the recognition
of T.gondii GPI, despite the fact that the structural
similarities between T.gondii and P.falciparum GPI52
suggest that the TLR1TLR2 heterodimer could mediate the recognition of T.gondii53. Finally, in addition
to TLR2, TLR4 can also be activated by T.gondii GPI
invitro9. Both TLR2 and TLR4 can also be activated
by T.gondii-derived heat shock protein 70 (HSP70)54.
The relevance of these observations to the regulation
of host defence to T.gondii is unclear, as deficiency in
TLR4 has no obvious effects on host resistance or on the
production of pro-inflammatory cytokines in response
to T.gondii infection11.
Overall, it seems that a range of TLRs, including
two cell surface TLRs (that is, TLR2 and TLR4) and
four endosomal TLRs (that is, TLR7, TLR9, TLR11 and
TLR12), are involved in the innate sensing of T.gondii
infection in mice. The function of innate receptors is
not only to detect the presence of a pathogen but also
to decipher its nature. Furthermore, it seems that only
recognition of T.gondii profilin by TLR11 and TLR12
provides innate specificity towards T.gondii and related

apicomplexan parasites, whereas the remaining TLRs


contribute to the immune response by sensing more
generic inflammatory signals during infection. These
results raise an important question about the sensing of
T.gondii by human innate immune cells. Although it is
possible that cell surface TLR2 and TLR4 or endosomal
TLR7 and TLR9 have a more important role in T.gondii
recognition in humans than in mice, it is likely that, in the
absence of TLR11, a distinct TLR-independent mechanism for parasite recognition functions in human DCs,
monocytes and neutrophils.
Sensing of T.gondii through TLR-independent mechanisms. Inflammasomes are intracellular sensor systems
that cooperate with TLRs in pathogen recognition, but
that primarily function to detect microbial ligands in the
cytosolic compartment. Activation of caspase 1 and caspase 11 downstream of inflammasome activation results
in the induction of a pyroptotic cell death that eliminates the niche for T.gondii invasion and replication. In
addition, caspase 1 has a central role in processing IL1
and IL18, which are pro-inflammatory cytokines that
are involved in the host defence against the parasite. In
addition, genetic studies revealed that the intracellular
sensor NLRP1 (NOD-, LRR- and pyrin domaincontaining 1) influences susceptibility to the development of human congenital toxoplasmosis and that
this NOD-like receptor (NLR) family member can be
activated in T.gondii-infected human monocytes55,56.
Although the upstream events that are associated with
NLRP1 activation by T.gondii are poorly understood,
recent invitro and invivo experiments using mouse
and rat cells have formally established NLRP1 as an
inflammasome sensor for T.gondii that is capable of
inducing cell death and the production of IL1 during an infection57. Although the parasite itself failed to
induce complete NLRP1 activation and required additional priming that was mediated by lipopolysaccharide
(LPS), deficiency in NLRP1 resulted in a small increase in
susceptibility to T.gondii57. Further studies are needed to
understand how NLRP1 cooperates with TLRs in T.gon
dii sensing, as well as to understand other potential intracellular recognition systems that could be involved in the
initial detection of T.gondii.

Effector immune responses to T.gondii


CD8+ DCs drive IL12mediated immunity to T.gondii. A major downstream effect of TLR-mediated parasite recognition that is essential for host resistance is
the production of IL12. Blocking IL12 (REFS5860)
or genetically ablating the IL12 subunits IL12p40
(REF.61) or IL12p35 (REF.62) results in the development
of acute susceptibility to T.gondii, which is similar to
that seen in MYD88deficient mice11. CD8+ DCs are
the most important cell type for the induction of TLRdependent IL12 production in response to T.gondii
infection6,11,29. It was shown that splenic CD8+ DCs, but
not macrophages or other DC subsets, produce IL12
within several hours of STAg injection29. Experiments
in vitro suggested similar conclusions and showed that
CD8+ DCs initiate the IL12 response downstream of

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Bradyzoites
A slow replicating form of
Toxoplasmagondii. Bradyzoites
in the form of cysts are
responsible for the chronic
stage of T.gondii infection.

Cysts
Structures that contain
bradyzoites. Cysts grow and
remain intracellular despite
variations in their size. Young
cysts may be as small as
5m and may contain only
two bradyzoites, whereas older
cysts may contain hundreds
of bradyzoites.

the TLR11mediated and TLR12mediated activation of


MYD88 (REFS6,11). Other DC subsets and macrophages
are capable of producing IL12 in response to T.gondii,
but their responsiveness is only apparent after they are
stimulated with IFN a process known as priming 63.
As IL12 production invivo is mainly unimpaired in the
absence of IFN29,64, it can be concluded that CD8+ DCs
are the major primary sensors of T.gondii infection that
initiate IL12dependent host resistance to the parasite.
The importance of CD8+ DCs as the early source
of protective IL12 was shown by the analysis of mice
that lack the transcription factors IFN-regulatory factor 8 (IRF8)65 or those that lack basic leucine zipper
transcriptional factor ATF-like 3 (BATF3)66. Deficiency
in IRF8 blocks CD8+ DC development, and it was
shown that when IRF8deficient mice were challenged
with T.gondii they failed to produce IL12 or IFN and
they rapidly succumbed to the infection, despite producing normal levels of TNF, IL6, IL1 and IL165.
It seems that the ability of naive CD8+ DCs to initiate IL12 production in response to T.gondii occurs
because IRF8 is an indispensable transcription factor
that regulates both IL12p40 and IL12p35 expression
downstream of TLR11 and MYD88 activation18. How
exactly IRF8 is activated by TLR11 is not yet known
as, in contrast to IRF5 and IRF7, which directly interact with MYD88 (REFS67,68), IRF8 does not seem to be
engaged in the proximal TLR receptor complex. The
IRF8 signalling pathway, in addition to nuclear factor-B
(NFB)mediated signal transduction, can be used to
explain the selective loss of IL12 but not of the other
pro-inflammatory cytokines that occurs in the absence
of IRF8 (REF.65) (FIG.1). Although IFN does not influence the levels of TLR11 or TLR12 expression, it induces
IRF8 expression in macrophages and inCD8 DCs
and enables these cells to respond to T.gondii profilin18.
This model is consistent with the ability of recombinant IL12 to compensate for a deficiency of CD8+
DCs; IL12 triggers NK cell secretion of IFN69,70, which
leads to the induction of IRF8 expression in macro
phages and in CD8 DCs, and drives these cells to
become potent IL12secreting cells that compensate
for the deficiency in the CD8+ DC subset. Overall, IRF8
functions as a master regulator of IL12 expression in
response to T.gondii infection downstream of TLR11
and TLR12 activation18 (FIG.1).
A necessary role forCD8+ DCs in secreting hostprotective IL12 has been independently shown in
T.gondii-infected BATF3deficient mice. BAFT3 is
expressed by both CD8 and CD8+ conventional
DCs, but a lack of BATF3 expression selectively alters
the development of the CD8+ conventional DC subset without affecting the plasmacytoid DC (pDC)
subset, which is missing in IRF8deficient mice 7174.
Reconstitution of BATF3deficient mice with recombinant IL12 was shown to protect mice from the infection66. However, IL12 treatment also partially rescues
the appearance of CD8 + DCs in BATF3deficient
mice74,75, which complicates the idea that a role for
CD8+ DCs in host resistance to T.gondii is limited to
their production of IL12.

Regulation of IFN by MYD88 signalling in DCs and


Tcells. A major role for DCs in the priming of Tcell
responses suggests that there is a central role for DC
TLR signalling in the induction of both CD4+ and CD8+
Tcell responses. This is partly because the TLR-mediated
activation of DCs regulates the DC expression of co
stimulatory molecules and cytokines76, and is also indispensable for efficient antigen presentation77. Experiments
using purified TLR agonists revealed that TLR-mediated
MYD88 activation results not only in rapid DC maturation and production of pro-inflammatory cytokines
but also enhances the presentation of microbial antigens that are physically associated with TLR ligands77,78.
This is particularly evident for T.gondii profilin13 and
bacterial flagellin79,80. The use of mice in which MYD88
is selectively inactivated in DCs revealed an unforeseen determinate of TLR-dependent host resistance to
T.gondii. Although these mice are highly susceptible
to T.gondii infection14, they show only minor deficiencies in IFN secretion by both CD4+ and CD8+ Tcells14.
Thus, although TLR activation in DCs is essential for host
resistance to T.gondii, this type of innate immunity does
not regulate Tcell responses to T.gondii (FIG.2). MYD88
signalling in other antigen-presenting cells (APCs),
including in macrophages and in Bcells, was also dispensable for TH1 cell responses during the parasitic infection81. Mice that have deficiencies of MYD88 in both DCs
and macrophages or in both DCs and Bcells were used to
dismiss the possibility that there is a redundancy in the
requirement for TLR signalling in these cell types to drive
Tcell responses81. This is in contrast to studies using
mice that have complete MYD88 deficiency, which
showed that these animals are severely compromised
in TH1 cell polarization and in the activation of CD8+
Tcells11,12,82. We found that Tcell-intrinsic MYD88 signalling is indispensable for TH1 cell responses to T.gon
dii81, which is an explanation for why MYD88deficient
mice are compromised in their Tcell production of IFN.
A surprising conclusion from these experiments is that
robust Tcell activation is not sufficient for host resistance
to T.gondii if parasite recognition by DCs is compromised14. Furthermore, TLR signalling in DCs, but not in
other cell types, is essential for early IFN production by
NK cells, but Tcell-intrinsic MYD88 signalling regulates
IFN production by CD4+ Tcells.
Effect of failed innate detection on chronic infection.
Unexpectedly, the impaired innate recognition of T.gon
dii also has a major effect on the outcome of the chronic
stage of the infection. Despite showing potent IFN production by both CD4+ and CD8+ Tcells, mice that have a
DCspecific ablation of MYD88 have significantly higher
parasite burdens in the form of encysted bradyzoites than
wild-type mice14. This may be a result of the higher parasitaemia levels during acute infection, which results in
the production of more cysts, or it may be a direct effect
of innate recognition affecting chronic infection. As a
potential mechanism, this may indicate that there is a
requirement for DCs to initiate an appropriate Tcell
response to the parasite; however, the mechanisms by
which parasite-derived antigens are presented to Tcells

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TLR11
IL-12
Intracellular
T. gondii infection

TLR12

CD4+ T cell

Parasitophorous
vesicle

NK cell

Parasite
destruction

DC
Prolin
IFN
Free
tachyzoite

Primed for
intracellular
killing

Neutrophil

Macrophage
Priming of
CD8+ T cell

CD8+ T cell
Infected DC

Figure 2 | Cellular sources of IFN during T.gondii infection. Interferon (IFN) is crucial for survival during
Nature Reviews
| Immunology
Toxoplasma gondii infection. Production of this cytokine by natural killer (NK) cells is dependent
on the Toll-like
+
receptor 11 (TLR11)-mediated recognition of T.gondii profilin by dendritic cells (DCs). Both CD4 T cell-derived
and CD8+ Tcell-derived IFN is essential for resistance to T.gondii during the chronic stage of infection, but TLR
signalling in DCs is dispensable for this Tcell-derived IFN. Instead, the CD4+ Tcell-intrinsic myeloid differentiation
primary-response protein 88 (MYD88) pathway regulates T helper 1 (TH1) cell responses to the parasite, and the
activation of CD8+ Tcells is mediated by T.gondii antigens that are expressed on infected DCs. Neutrophils provide
an important innate source for IFN; the mechanisms that regulate neutrophil-derived IFN are not well understood
because this IFN is not regulated by TLRs or by interleukin12 (IL12).

Non-professional APCs
Cells that do not constitutively
express MHC class II
molecules, but that upregulate
MHC class II following
stimulation with certain
cytokines, particularly
interferon-. Important
examples of non-professional
APCs include fibroblasts and
thymic epithelial cells.

by DCs are not completely understood. The parasitophorous vacuole that contains T.gondii in infected APCs
does not fuse with lysosomes83,84, thus T.gondii-infected
DCs and macrophages do not degrade the parasite and
are not likely to function as efficientAPCs.
Restricted antigen-presentation is further exaggerated by the active suppression of maturation of DCs and
macrophages by the parasite8587. The inability of the
infected APC to activate endolysosomal machinery can
be overcome by previous stimulation with IFN (that
is, priming), which provides evidence that early IFN is
required for the potent induction of Tcell responses to
the parasite. CD8+ DCspecific recognition of parasites
leads to IL12dependent NK cell activation and production of IFN, which enables DCs to degrade T.gondii
and to present parasite antigens to Tcells. In addition,
antigen presentation as a result of uptake of the killed
parasite is indistinguishable from a conventional pathway of antigen presentation88. Overall, although not
formally examined invivo, it is likely that the induction
of CD4+ Tcell responses during T.gondii infection is
initiated by IFN-primedDCs.
Detailed analysis has been carried out to understand
the induction of CD8+ Tcell responses to the parasite1. It
was shown that infected DCs and non-professional APCs
can prime CD8+ Tcell responses against secreted T.gon
dii antigens89,90. T.gondii-containing parasitophorous
vacuoles in infected cells, including DCs, establish a
close contact with the host endoplasmic reticulum (ER),

which leads to the direct exchange of antigens between


the host and the parasite compartments9193. Although
the term cross-presentation is widely used to describe
this process, it is likely that T.gondii antigens are actively
secreted by the parasite, transferred from the membrane
of parasitophorous vacuoles into the host ER and then
effectively presented to CD8+ Tcells94. In addition, degradation of phagocytosed parasites is not sufficient to
prime of CD8+ Tcell responses94. Intravital imaging of
DCCD8+ Tcell interactions has shown that DCs are the
major APCs that are required to prime a CD8+ Tcell to
respond to T.gondii invivo, but these studies have also
unexpectedly suggested that bystander DCs rather than
infected DCs are the major APCs involved in promoting immunity to the parasite during toxoplasmosis95.
The discovery of a large number of uninfected cells that
were in close contact with the parasite and that had been
injected with T.gondii-secreted proteins may provide
an explanation for the apparent discrepancy between
in vivo and in vitro experiments and may further
emphasize that T.gondii-secreted proteins are selective
targets for host CD8+ Tcell responses96.
Overall, there is an appreciation that NK cells, CD4+
T cells and CD8+ Tcells mediate IFN-dependent host
protection to T.gondii and are themselves mainly regulated by the MYD88 signalling that occurs in both DCs
and Tcells (FIG.2). Although NK cells and CD4+ Tcells
are primarily involved in host resistance during the acute
stages of the infection, CD8+ Tcells and to a lesser extent

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CD4+ Tcells are the major IFN-producing cells that
control parasites in the brains of chronically infected
mice. IL12 is indispensable for IFN production during both acute and chronic stages of the infection97,98 and,
although TLR11dependent activation of MYD88 regulates early IL12 production by CD8+ DCs6, the mechanisms that are responsible for the maintenance of IL12
production during the chronic stage of the infection are
mostly unknown. Following infection with attenuated
parasites, IL12independent and MYD88independent
IFN production by CD8+ Tcells is sufficient to control
and eliminate the parasites17,99, but this mechanism is not
sufficient to control the T.gondii strains that are typically responsible for human infection and that are widely
studied in experimental animals17.
IFN

IDO

Tryptophan

Inhibition of
T. gondii growth

iNOS

Arginine

IRGs and GBPs

NO

NO-mediated
toxicity

T. gondii

Destruction of
parasitophorous
vesicle

Parasitophorous
vesicle

Macrophage

Figure 3 | Effector mechanisms of IFN-mediated parasite elimination in infected


cells. Interferon (IFN) has pleiotropic effects on infectedNature
cells and
results| in
reduced
Reviews
Immunology
parasite replication by inducing the expression of the inhibitory proteins indoleamine 2,3
dioxygenase (IDO) and inducible nitric oxide synthase (iNOS) as well as the expression of
the effector proteins immunity-related GTPases (IRGs) and guanylate-binding proteins
(GBPs). IDO depletes tryptophan, which is an essential amino acid for Toxoplasmagondii
growth. iNOS, in addition to generating the highly toxic metabolite nitric oxide (NO),
restricts parasite replication by depleting arginine, which is another essential amino
acid for T.gondii growth. IRGs and GBPs are responsible for the destruction of the
parasitophorous vacuole, which is a special organelle containing T.gondii that protects it
from the endolysosomal mechanism of microbe elimination. Once the parasitophorous
vacuole is damaged, T.gondii parasites are vulnerable to rapid elimination from the
cytoplasm of infected cells. The IFN-inducible effector mechanisms are differently
expressed in various cell types, but expression of the IFN receptor on both bone
marrow-derived cells and stromal cells is essential for host protection, as T.gondii can
infect any nucleated cell.

Role for neutrophils in IFN-mediated immune responses.


In addition to NK cells and Tcells, it is becoming recognized that there is a role for early IFN that is produced by neutrophils100. This previously unappreciated
cellular source of IFN provides an explanation for why
mice that lack both NK cells and Tcells are not as susceptible to T.gondii infection as IFN-deficient mice70.
Although combined deficiency in NK cells and Tcells
does result in high susceptibility to the infection, mice
that are deficient in IFN are far more susceptible to
T.gondii infection11,64,70. It seems that neutrophil-derived
IFN is independent of IL12 or TLR activation and,
instead, is regulated by TNF and IL1100. The central
role for IL1 in the regulation of IFN-producing neutrophils is an explanation for why MYD88deficient mice
cannot be rescued by the addition of large quantities of
exogenous IL12 (REF.14). Furthermore, until recently it
was unclear how TLR11deficient mice, which do not
produce IL12 in response to T.gondii, can produce
large quantities of IFN6,101, which provides partial protection during the acute stage of infection6. Depletion of
neutrophils or blocking IFN in TLR11deficient mice
results in acute susceptibility to the parasitic infection
and provides evidence for the biological importance
of neutrophil-derived IFN, especially in the absence of
TLR11 (REF.100) (FIG.2).

IFN-mediated innate effector mechanisms


The ability of IFN to eliminate T.gondii in infected cells
was shown very early on in experiments using human
macrophages that were stimulated with recombinant
IFN102. Partial purification of soluble fractions that were
produced by human leukocytes in response to toxoplasma antigens or to concanavalin A, complemented
with IFN-blocking experiments, established that IFN
is the principal factor that enhances the capacity of human
macrophages to kill toxoplasma102. Additional experiments using macrophages that had been isolated from
patients with AIDS showed that these cells were capable
of T.gondii elimination if stimulated by IFN103. Thus,
IFN is a major effector molecule that is required for host
resistance to the parasite and at least three IFN-mediated
protective mechanisms have been identified (FIG.3).
Induction of IDO. It has previously been established that
IFN suppresses the growth of T.gondii via the induction of tryptophan degradation104. The antiproliferative
effect of IFN on T.gondii is mediated by the induction of indoleamine 2,3dioxygenase (IDO)105, which
converts tryptophan into Nformylkynurenine (FIG.3),
resulting in cell starvation because of the deficiency of
this essential amino acid; the tryptophan auxotrophy
of T.gondii makes it vulnerable to this host defence
pathway 104,105.
However, the lack of susceptibility of IDO-deficient
mice to T.gondii infection markedly diminished interest in this mechanism of IFN-mediated protection.
Nevertheless, identification of IDO2 (REFS106,107),
which is encoded by a gene that is closely related to
IDO (now known as IDO1) may be an explanation for
the resistance of IDO1deficient mice to the parasite108.

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This is particularly evident from studies that involve the
chemical inhibition of both IDO1 and IDO2 in T.gondiiinfected mice: these mice did not show acute susceptibility to T.gondii, but were unable to control T.gondii
parasites during the chronic stage of the infection108.

Tachyzoites
Fast-replicating forms of
Toxoplasmagondii that are
responsible for the acute
stage of infection.

NO production. A better understood mechanism of


IFN-mediated parasite killing depends on the biochemical pathway that results in nitrogen oxide (NO)
production from l-arginine109. NO has potent microbicidal effects on a broad range of phylogenetically unrelated intracellular pathogenic microorganisms, including
T.gondii. Although most NO targets have been identified in bacterial pathogens, they are likely to be shared
by T.gondii. NO intermediates are nonpolar uncharged
molecules that can readily cross membranes and directly
interact with the parasite in the parasitophorous vacuole.
NO modifies proteins at reactive thiols, haem groups,
ironsulphur clusters, phenolic or aromatic amino acid
residues, tyrosyl radicals and amines. The inhibition
of metabolic enzymes by NO constitutes an important
mechanism of NOmediated cytostasis110.
A less well-characterized host defence mechanism
that is mediated by inducible nitric oxide synthase
(iNOS) depends on the depletion of arginine during
NO synthesis. T.gondii is a strict arginine auxotroph
and obtains this amino acid from the cytoplasm of the
infected cells via an uncharacterized amino acid transporter 111. Synthesis of NO results in arginine deprivation
and efficiently blocks T.gondii replication111. Arginine
starvation was primarily studied as a metabolic trigger
for the differentiation of tachyzoites into dormant bradyzoites111, but has also been implicated as a host defence
mechanism invivo112,113.
Genetic analysis of mice that are deficient in iNOS
revealed a more complex role for NOmediated host
resistance to T.gondii invivo. In contrast to IFNdeficient mice that succumb to the infection during the
acute stage of parasite replication, iNOS-deficient mice
are able to control the replication of the parasite during this stage, although these mice eventually succumb
during the chronic stage of the infection61. These results
led to several important conclusions. First, although
NO has a role in parasite elimination, it is not the major
downstream effector molecule that is triggered by IFN
during the acute stage of the infection in mice. Second,
the requirement for NO to control the chronic but not the
acute stages of the parasitic infection suggests that the
key effector mechanism responsible for tachyzoite elimination compared with bradyzoite elimination may be
different and may warrant further investigation. Finally,
NO may mediate a protective effect in the central nervous system, which is the main reservoir for bradyzoites,
but may have a limited role in the peripheral tissues.
These are unexpected results because, although responsiveness to IFN by both bone marrow-derived and
radiation-resistant cells is required for host resistance
to the parasite, iNOS expression by macrophages that
originated in the bone marrow, but not by radiationresistant stromal cells, contributes to NOmediated
parasite killing 114.

In addition, the requirement for NOdependent host


resistance to T.gondii in mice is strain specific. C57BL/6
mice require iNOS expression to prevent fatal cerebral
toxoplasmosis, but production of NO is dispensable for
T.gondii control in BALB/c mice115. Even in C57BL/6
mice, the requirement for iNOS-mediated production of
NO can be overcome following vaccination with highly
attenuated T.gondii parasites. These studies show that
IFN-producing CD8+ memory Tcells can provide
long-term protection against T.gondii in the absence of
iNOS116. It is interesting to note that expression of mRNA
for iNOS can also be detected in the brain of T.gondiiinfected Ifng/ mice117, which indicates that there are
IFN-independent mechanisms for NO production.
However, the expression of iNOS in IFN-deficient mice
failed to provide protection against T.gondii, and the
cell types that express iNOS in the absence of IFN are
unknown117.
Finally, experiments that were carried out using
human cells have shown that NO production by human
phagocytes is not required for T.gondii control118. This
is probably explained by a dominant effect of alternative IFN-dependent pathways, particularly IFNmediated induction of IDO, in eliminating T.gondii from
humancells.
ROS. In addition to NO, reactive oxygen intermediates
have been implicated in the IFN-mediated elimination of
T.gondii in a cell type-specific and species-specific manner. In particular, reactive oxygen species (ROS) have a
role in parasite elimination in human monocytes and in
activated mouse macrophages119. However, the production of ROS was not induced in human macrophages that
were infected with the parasite120. Whether different levels
of ROS induction in different cell types determines cell
susceptibility to the infection remains to be tested. It has
been shown that human monocytes that produce high
levels of ROS are able to kill the parasite and to inhibit
the intracellular replication of those few organisms that
remained alive. By contrast, the failure of macrophages
to produce ROS correlates with the inability of these cells
to kill or to inhibit the replication of intracellular T.gon
dii120. It is interesting to note that mononuclear cells isolated from patients with Xlinked chronic granulomatous
disease have an impaired ability to kill T.gondii120. Despite
this, the role for ROS in T.gondii elimination remains controversial. Other work suggests that the killing of intra
cellular T.gondii in activated mouse macrophages does
not seem to be oxygen dependent because toxic oxygen
fails to reach the vacuoles containing the parasite121.
IFN-inducible IRGs and GBPs. The immunity-related
GTPases (IRGs) are a family of IFN-inducible proteins
that are essential for host resistance against a variety of
intracellular pathogens, including T.gondii122. There
are 21 members of this family in mice and only one in
humans, which is another example of species-specific
differences in IFN-mediated immunity to intracellular
pathogens123. It has been suggested that the IRG resistance system has been lost in humans as a result of the
divergent evolution of primates123.

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Sexual cycle
Sexual replication of
Toxoplasma gondii occurs in
the gut of the cat during the
enteroepithelial stage of the
parasite life cycle, which takes
about 310days. Sexual
replication leads to the
production of oocysts. A host
in which parasites sexually
reproduce is known as the
definitive, final or primary host.

Mice that lack IFN-induced IRG family M protein 3 (IRGM3; also known as IGTP) or IRGM1 (also
known as LRG47) are highly susceptible to T.gondii
infection and succumb to lethal disease with equal or
even more striking kinetics than those seen in IFNdeficient mice124,125, despite showing normal induction
of IL12, IFN and iNOS production; this suggests a
cell-autonomous role for these IRGs in mediating parasite destruction. Deficiency of IRGD (also known as
IRG47)125, IFN-inducible IRGA6 (also known as IIGP1
and GTPase 1)126 or IRGB6 (also known as TGTP)127,128
also increases susceptibility to T. gondii infection,
although not to the same extent as deficiency in either
IRGM1 or IRGM3. Under steady-state conditions, IRG
proteins are distributed in different cellular compartments, including in the Golgi (in the case of IRGM1
and IRGM2)129131, in the endolysosomal compartment
(in the case of IRGM1)132, in the endoplasmic reticulum (in the case of IRGA6, IRGB6 and IRGM3)130,133
135
or within the cytosol (in the case of IRGB6 and
IRGD)129,135. Infection with T.gondii results in the highly
coordinated loading of IRGs onto the T.gondii parasitophorous vacuole136. The process is initiated by IRGB6
and IRGB10, and is then followed by the recruitment
of IRGA6, IRGD and IRGM2 (REF.136). This assembly of
IRG proteins on the parasitophorous vacuole leads to
vesiculation of the vacuole membrane, disruption of the
vacuole and elimination of the parasite by lysosomemediated degradation137,138. Such coordinated assembly
of the IRG proteins may be an explanation for the strong
susceptibility to infection of cells that are deficient in a
single member of thefamily.
Despite its importance in mediating host resistance
to T.gondii125, IRGM1 does not localize to the T.gondiicontaining parasitophorous vacuole136. Instead, IRGM1
seems to function as a master regulator of the IRGmediated host defence programme139,140. Lack of IRGM1
leads to uncontrolled and mislocalized activation of
other IRG family members, which results in cytopathic
effects because of overactivation of the endolysosomal and the autophagic pathways in macrophages141,
Tcells142 and haematopoietic stem cells143.
The importance of IRGs in determining the outcome ofhostparasite interactions is evident from the
analysis of virulence factors in T.gondii4. T.gondii can
be subdivided into three main strain types (BOX3) and,
although many typeII and typeIII strains of T.gon
dii are controlled by IRGs, typeI strains of T.gondii
counteract this host defence system. Genetic mapping
strategies identified ROP5 and ROP18 as the major virulence factors produced by typeI strains of T.gondii that
inhibit the loading of IRGs onto the parasitophorous
vacuole membrane144,145 and thus prevent IRG-mediated
T.gondii elimination127,146,147.
In contrast to mice, a role for IRGs in human cells
is still under debate148. First, only one IRG homologue,
IRGM, is present in the human genome123. Furthermore,
IRGM is severely truncated compared with other IRG
gene products and is not regulated by IFN123. However,
human IRGM participates in phagosome maturation
and in autophagy of intracellular bacteria. In addition,

Box 3 | T.gondii strain types


The sexual cycle of Toxoplasmagondii is limited to feline
hosts and does not occur in other mammals. This sexual
cycle in cats is responsible for the formation of distinct
strains of the parasites, the majority of which in North
America belong to one of three major genotypes: I, II and
III. The acute virulence of the strain strongly correlates
with the genotype of the parasite; a single typeI parasite
is sufficient to induce a 100% death rate in mice (lethal
dose 100% (LD100)=1 organism), but much higher
numbers of type II or type III tachyzoites are required for
lethality (LD100 >103 organisms). It seems that only a few
virulence factors are responsible for the different
susceptibility of mice to the parasitic infection. These
virulence factors have recently been extensively
reviewed elsewhere4,156.

the functionality of human IRGM is evident from the


association of polymorphisms immediately upstream of
IRGM with Crohns disease149. Whether these mechanisms are applicable to T.gondii elimination in human
cells is unknown.
The p65 guanylate-binding proteins (GBPs) are
another family of enzymes induced by IFN that participate in immunity against T.gondii150. Both humans and
mice express GBPs (seven GBPs are in the human genome
and 13GBPs are found in the mouse genome)151, and it
is thought that these proteins participate in T.gondiispecific immune responses by cooperating with IRGs in
IFNmediated parasite killing144,152154.

Conclusions and perspectives


The identification of the key recognition and effector
mechanisms that are required for resistance to T.gondii
not only explains the basic principles of immunity to the
parasite but also raises a question about the evolution
of host defences to protozoan parasites. Although TLRs
are generally highly conserved innate immune sensors,
the TLR11 family members TLR11, TLR12 and TLR13
(which is a sensor for bacterial RNA) are exceptions and
are missing in various animal species155. All three genes
are functional in mice and in multiple other species, but
TLR11 is the product of a pseudogene in humans and
the genes for both TLR12 and TLR13 are absent from
the human genome155. Even more marked species variation has been established for the major effector molecules downstream of IFN. As discussed above, more
than 21 functional effector genes of the IRG family are
not present in the human genome despite their crucial
role in parasite elimination in mice123. Thus, there is a
great variation between mouse and human responses
to T.gondii from recognition to the effector phases. It
is not known why humans lack all functional TLR11
family members and it would be beneficial to identify
the driving force behind this species variation. This is of
special interest because TLR11 mediates the recognition
of multiple other apicomplexan parasites that represent
a major threat to humans and animals. The analysis of
profilin proteins isolated from different apicomplexan
parasites revealed that TLR11 functions as a principal

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REVIEWS
recognition receptor for these pathogens, with the
exception of Plasmodium spp., which cause malaria6,7.
Malaria profilin avoids recognition by TLR11 through
poorly characterized mutations 6. As apicomplexan

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Acknowledgements

F.Y. gratefully thanks colleagues and the members of his laboratory for the many discussions that contributed to this
manuscript. Work in F.Y.s laboratory is supported by the US
National Institutes of Health (AI085263) and the Burroughs
Wellcome Fund.

Competing interests statement

The author declares no competing interests.

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