Toll-like receptors and fungal infections: the role of TLR2, TLR4 and MyD88 in paracoccidioidomycosis.

Abstract

The aim of this minireview is to present a concise view of the most important pattern recognition receptors used by the innate immune system to sense and control pathogen growth into host tissues. A brief review of the role of Toll-like receptors (TLRs) in fungal infections followed by some recent results on the function of TLR4, TLR2 and the MyD88 adaptor molecule in the pathogenesis of paracoccidioidomycosis are presented.

Full text

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M I N I R E V I E W
Toll-likereceptorsandfungalinfections:theroleofTLR2,TLR4and
MyD88inparacoccidioidomycosis
Vera L. G. Calich, Adriana Pina, Ma´ ıra Felonato, Simone Bernardino, Tania A. Costa & Fl´ avio V. Loures
Departamento de Imunologia, Instituto de Cie ˆncias Biom´ edicas, Universidade de Sa ˜o Paulo, Sa ˜o Paulo, SP, Brazil
Correspondence: Vera L. G. Calich,
Departamento de Imunologia, Instituto de
Cie ˆncias Biom´ edicas da Universidade de Sa ˜o
Paulo, Av. Prof. Lineu Prestes 1730,
CEP 05508-900, Sa ˜o Paulo, SP, Brazil.
Tel.: 155 11 30917397; fax: 155 11
30917224; e-mail: vlcalich@icb.usp.br
Received 8 November 2007; accepted 17
December 2007.
First published online 1 April 2008.
DOI:10.1111/j.1574-695X.2008.00378.x
Editor: Willem van Leeuwen
Keywords
toll-like receptors; fungal pathogens;
paracoccidioidomycosis; TLR2; TLR4; MyD88.
Abstract
The aim of this minireview is to present a concise view of the most important
pattern recognition receptors used by the innate immune system to sense and
control pathogen growth into host tissues. A brief review of the role of Toll-like
receptors (TLRs) in fungal infections followed by some recent results on the
function of TLR4, TLR2 and the MyD88 adaptor molecule in the pathogenesis of
paracoccidioidomycosis are presented.
Innate immunity and pattern
recognition receptors
Cells of the innate immune system constantly sense the
presence of invading microorganisms using several kind of
conserved, transmembrane or intracytoplasmatic receptors
called pattern recognition receptors (PRRs). These receptors
recognize conserved molecular structures shared by groups
of microorganisms and known as pathogen-associated mo-
lecular patterns (PAMPs). Because PAMPs are produced by
pathogens but not by the host cells, their recognition by
PRRs allows for self–nonself discrimination (Janeway &
Medzhitov, 2002). The most important types of PRR are
the Toll-like receptors (TLRs), the non-TLRs such as in-
tracellularnucleotide-binding
(NOD)-like proteins and the C-type lectin receptors (CLRs)
(Gordon, 2002; Brown & Gordon, 2003; Inohara & Nunez,
2003; Akira et al., 2006). The interaction between PAMPs
and PRRs leads to the activation of cells of the innate
immune system and subsequent production of mediators
that are used to eliminate the invading pathogen and to
control the adaptative immune responses. TLRs are crucial
for many aspects of microbial elimination, including re-
oligomerization domain
cruitment of phagocytes to the site of infection, microbial
killing andactivation of dendriticcells (DCs), whichbecome
immunogenic and endowed with a unique ability to induce
full activation of T cells (Reis e Sousa, 2004). Interestingly,
recent findings indicate that direct recognition of PAMPs by
DCsis critical for priming appropriate T-cell responses. TLR
signaling resulted in activated DCs that primed an effective
T helper 1 (Th1) or Th2 response, whereas indirect activa-
tion by inflammatory mediators alone (proinflammatory
cytokines) induced DCs that, although supporting expan-
sion of CD41T-cell clones, did not promote Th1 or Th2
effector differentiation (Sp¨ orri & Reis e Sousa, 2005).
Thus far, 13 TLRs have been described that recognize a
wide variety of pathogen structures including triacyl lipo-
peptides (TLR1 in association with TLR2), lipoteichoic acid
and lipoproteins of Gram-positive bacteria (TLR2), double-
stranded RNA (TLR3), lipopolysaccharide of Gram-nega-
tive bacteria (TLR4), bacterial flagellin (TLR5), diacyl
lipopeptides (TLR6 in association with TLR2), single-
stranded RNA (TLR7) and nonmethylated CpG of bacterial
DNA (TLR9) (Takeda & Akira, 2005). Intracellular NOD
proteins sense the presence of intracellular muropeptides
(Inohara & Nunez, 2003). Upon ligand binding, innate
FEMS Immunol Med Microbiol 53 (2008) 1–7
c ?2008 Federation of European Microbiological Societies
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immune receptors engage intracellular signaling pathways
that result in the activation of conserved transcription
factors for cell activation. One key transcription factor used
by TLR-mediated innate immunity is NFkB. Almost all
TLRs signal via MyD88, an adaptor protein, for NFkB
activation, subsequent inflammatory cytokine production
and control of adaptative immunity (Takeda & Akira, 2005;
Akira et al., 2006). Besides their well-described role in the
immunorecognition of conserved pathogen motifs, TLRs
can be used to enhance microbial pathogenicity. Thus,
Yersinia pestis was shown to evade the host’s immune system
by TLR2 activation and subsequent production of IL-10,
one important macrophage deactivating cytokine (Sing
et al., 2002). Furthermore, studies with whole pathogens or
purified components have shown distinct patterns of TLR
usage: TLR2 and TLR4 control in vivo Brucella abortus
infection whereas only TLR4 is activated by the purified
bacterial lipopolysaccharide (Campos et al., 2004).
TLR-activated DCs can induce the differentiation of
distinct T-cell-mediated effector mechanisms. Lipopolysac-
charide-stimulated DCs produce low levels of IL-10 but high
levels ofIL-12 andtumor necrosis factor-a (TNF-a),favoring
Th1 immunity. In contrast, peptidoglycan-activated DCs
secrete low levels of IL-12 associated with prevalent produc-
tion of IL-10, resulting in prevalent Th2 immunity (Qi et al.,
2003). Interestingly, the recognition of microbial products by
TLRswasfoundtoblockthesuppressiveeffectofTregulatory
(Treg) cells on pathogen-specific adaptative immune re-
sponse and this effect was partially due to the synthesis of
IL-6 by TLR-stimulated DCs (Pasare & Medzhitov, 2003).
Dendritic cell-specific intercelullar adhesion molecule-3-
grabbing nonintegrin (DC-SIGN), mannose receptor (MR)
and dectin-1 are C-type lectins that recognize glycoproteins
and carbohydrates in pathogen cells. This interaction con-
trols phagocytosis, microbicidal activity and signaling pro-
cesses that direct cell adhesion and migration. As fungal cell
walls are carbohydrate-rich structures, these PRRs are
directly involved in the host’s innate immunity to these
pathogens. DC-SIGN as well as MRs are primarily activated
by IL-4 and associated with the Th2 pattern of the immune
response (Brown & Gordon, 2003; Cambi et al., 2005;
Koppel et al., 2005). Dectin-1 activation by Candida albicans
or curdlan, a dectin-1-specific b-glucan, was recently shown
to induce the preferential secretion of tumor growth factor
(TGF)-b and IL-6 and the subsequent activation of Th17
lymphocytes. This T-cell subset secretes IL-17, induces
chemokine secretion at sites of infection, causes recruitment
of neutrophils and is important in defense against extra-
cellular pathogens, including Candida albicans (Leibund-
gut-Landmann et al., 2007; Palm & Medzhitov, 2007).
Upon ligand binding, innate immunoreceptors engage
intracellular signaling pathways that converge to the activa-
tion of conserved transcription factors and subsequent cell
activation. Almost all TLRs signal via MyD88 for NFkB
activation, resulting in Th1 immunity associated with the
prevalent IL-12 secretion. TLR2 activation, however, induces
high levels of IL-10, and expansion of regulatory T cells
(Treg) which further control T effector lymphocytes (Gor-
don, 2002; Akira et al., 2006; Sutmuller et al., 2006).
Interestingly, recent work has shown that the preferential
activation of dectin-1 by the selective agonist curdlan, and
possibly dectin-2 by Candida albicans hyphae, is mediated by
the caspase recruitment domain (CARD9) adaptor protein,
resulting in increased IL-23 secretion and preferential induc-
tion of Th17 cells (Gross et al., 2006; Leibundgut-Landmann
et al., 2007; Palm & Medzhitov, 2007). The fungal morpho-
type is also recognized by different PRRs and this fact was
suggested to be exploited as an escape mechanism by fungal
cells. For instance, Candida hyphae are recognized only by
TLR2, inducing prevalent secretion of anti-inflammatory
cytokines, whereas Candida blastoconidia interact with
TLR4, Dectin-1 and TLR2, resulting in a complex pattern of
cell activation (Romani, 2004; Netea et al., 2006).
In summary, PRR activation orchestrates the develop-
ment of innate and adaptative immune responses, which are
necessary for protection against infection, reinfection or
containment of chronic infections. However, if activation of
innate immune receptors is excessive, high levels of proin-
flammatory mediators [IFN-g, TNF-a, nitric oxide (NO)]
are secreted and can exert a deleterious effect to the host.
Septic shock induced by lipopolysaccharide and TLR4
activation by Gram-negative bacteria is a good example of
inadequate activation of immunity, which results in severe
host pathology.
Recognition of fungi by TLR
Netea et al. (2002) were the first to describe the use of TLRs
by a fungal pathogen, Candida albicans. C3H/HeJ mice,
which express a defective TLR4 gene, present an increased
susceptibility to disseminated candidiasis and impaired
recruitment of neutrophils to the site of infection when
compared with normal, C3H/HeN mice. In addition, the
chemokines keratinocyte-derived chemokine (KC) and
macrophage inflammatory protein (MIP-2) were shown to
be released in lower amounts by TLR4-defective macro-
phages. Following this pioneer work, other groups reported
their studies on the role of TLRs and the MyD88 adaptor
protein in Candida albicans infections. The main biological
effects observed in diverse experimental approaches are
summarized in Table 1. As can be seen, some discrepant
findings were obtained with TLR2- and TLR4-deficient
hosts (Netea et al., 2002, 2004; Villam´ on et al., 2004b;
Murciano et al., 2006), although MyD88 deficiency appears
consistently to lead to impaired protection or phagocyte–
fungus interaction. This adaptor protein was shown to be
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involved in the induction of protective immune responses
by DCs (Bellocchio et al., 2004a), as well as in the phagocy-
tosis, killing and synthesis of cytokines by Candida-infected
cells (Marr et al., 2003; Villam´ on et al., 2004a).
TLR2 usage was shown to have protective or detrimental
effects in models of Candida albicans infection; the conflict-
ing results, however, could be attributed to the use of
different experimental protocols (Netea et al., 2004;
Villam´ on et al., 2004a,b), but brought important contribu-
tions to the understanding of immunopathology of infec-
tious processes. The deleterious effect of TLR2 signaling
during infection was associated with increased synthesis of
IL-10 and an enhanced survival of CD41CD251Treg cells,
resulting in deficient T-cell immunity and impaired fungal
clearance (Netea et al., 2004).
As observed with Candida albicans, the role of TLR in
Cryptococcus neoformans infection needs to be further
explored (Table 2). Glucuronoxylomannan, the major com-
ponent of the polysaccharide capsule of Cryptococcus neofor-
mans, is shed from the fungus and circulates in blood and
cerebrospinal fluid of infected hosts. This polysaccharide
was reported to activate cells transfected with CD14 and
TLR4 but this interaction results in incomplete activation
of cells, and no secretion of TNF-a (Shoham et al., 2001).
In vivo, MyD88 and TLR2 but not TLR4 were shown to be
required to induce protection against Cryptococcus neofor-
mans infection (Yauch et al., 2004; Biondo et al., 2005). A
more recent report, however, suggests that TLR2 and TLR4
do not or only marginally contribute to the host response to
this pathogen (Nakamura et al., 2006).
TLR2-, TLR4- and MyD88-dependent activation of host
cells were shown to play a role in cytokine secretion,
polymorphonuclearneutrophil (PMN)activation and
susceptibility to infection by another opportunistic fungal
pathogen, Aspergillus fumigatus (Wang et al., 2001; Marr
et al., 2003; Meier et al., 2003; Netea et al., 2003; Bellocchio
et al., 2004a,b; Braedel et al., 2004; Dubordeau et al., 2006).
As described for Candida albicans, the germination from
conidia to hyphae was proposed as an escape mechanism of
A. fumigatus as conidia cells are recognized by TLR4 and
TLR2, resulting in the production of proinflammatory cyto-
kines, while hyphae stimulate production of IL-10 using a
TLR2-dependent mechanism (Netea et al., 2003). Although
some experimental approaches have revealed the important
roleofMyD88adaptorproteinincellsignalingandprotective
responses (Mambula et al., 2002; Bellocchio et al., 2004a),
other reports claimed that MyD88 signaling and activation of
NFkB are not important for fungal clearance (Marr et al.,
2003; Dubordeau et al., 2006) (Table 3).
As a whole, several reports have illustrated the use of
different TLRs by a single fungal species, resulting in diverse
biological activities. Studies with purified components of
fungal cell walls revealed the major PRR and signaling
pathways used by host cells to recognize fungal PAMPs;
however, this picture is less clear when whole pathogens are
used to infect normal or PRR-deficient hosts. The final
activation, although influenced by the missing receptor, is
mediated by the remaining PRRs, which can compensate or
not the deficient receptor.
PRR and Paracoccidioides brasiliensis
infection
Paracoccidioides brasiliensis, the causative agent of human
paracoccidioidomycosis, is primarily a respiratory pathogen,
infecting the host through inhalation of airborne spores.
Table 1. The role of TLRs and MyD88 adaptor protein in some experimental models of Candida albicans infection
PRR deficiencyBiological effect Reference
TLR4High susceptibility; normal PMN and macrophage killing activity; normal TNF-a, low KC, MIP-2
and impaired PMN influx
No increased susceptibility to disseminated infection. TLR4-deficient mice mount Th1 immunity
High resistance; TLR2 induces IL-10, Treg cells and suppressed immunity
High susceptibility; low TNF-a, MIP-2; decreased PMN influx; no effect phagocytosis and NO
production
Hyphae: impaired phagocytosis, killing and cytokine secretion
High susceptibility, impaired production of cytokine, low type-1 CD41and CD81Tcells
MyD88: high susceptibility. TLR signaling: depends on morphotypes, route of infection.
Th1 response: DC-MyD-dependent
Netea et al. (2002)
TLR4
TLR2
TLR2
Murciano et al. (2006)
Netea et al. (2004)
Villam´ on et al. (2004a)
MyD88
MyD88
MyD88, TLR4,
TLR2, TLR9
Marr et al. (2003)
Villam´ on et al. (2004b)
Bellocchio et al. (2004a)
Table 2. The role of TLRs and MyD88 adaptor protein in some experimental models of Cryptococcus neoformans infection
PRR deficiencyBiological effectReference
TLR2, TLR4, CD14
TLR-2, TLR4, MyD88, CD14
TLR-2, TLR4, MyD88
TLR2 and TLR4
Glucoronoxylomannan stimulates cells via CD14 and TLR4; no TNF-a synthesis
MyD88 has a major role in protection; CD14 and TLR2, minor roles; TLR4, no effect
MyD88 and TLR2 have a major role in protection; TLR4 not important
Not important to protection
Shoham et al. (2001)
Yauch et al. (2004)
Biondo et al. (2005)
Nakamura et al. (2006)
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The great majority of infected subjects develop an asympto-
matic pulmonary infection, although some individuals
present clinical manifestations, which give rise to the
localized (benign) or disseminated (severe) forms of the
disease. Clinical and experimental evidence indicates that
cell-mediated immunity plays a significant role in host
defense against P. brasiliensis infection, whereas high levels
of specific antibodies are associated with the most severe
forms of the disease (Borges-Walmsley et al., 2002; Calich
et al., 2008). Our laboratory developed a murine pulmonary
model of infection in which A/Sn mice developed a chronic
benign,pulmonary-restricted
whereas B10.A mice developed a progressive disseminated
disease. The main immunological characteristics of this
model are described elsewhere (Calich & Blotta, 2005).
Although the importance of innate immunity in
resistance to fungal infection is well recognized (Roeder
et al., 2004; Romani, 2004), the molecular mechanisms
underlying recognition of P. brasiliensis by innate immune
cells are not well known (Calich & Blotta, 2005; Calich
et al., 2008). C3b receptors (CR3, CD11b/CD18) are mem-
brane integrins that recognize iC3b of the complement
system, several b-glucans and other cell-wall components
expressing high mannose content (Brown & Gordon,
2003). We were the first to demonstrate that P. brasiliensis
interaction with peritoneal macrophages was enhanced
by iC3b opsonization of yeast cells (Calich et al., 1979).
Studying murine macrophages, Jimenez etal.(2006) verified
that CR3 and MR were involved in the phagocytosis
of P. brasiliensis spores (conidia). In addition, gp43, the
immunodominant antigen of P. brasiliensis, was shown to
bind to MR and to inhibit the phagocytic and fungicidal
ability of peritoneal macrophages from resistant and suscep-
tible mice (Popi et al., 2002). This finding led the authors to
postulate the expression or secretion of gp43 as an escape
mechanism of fungal cells.
paracoccidioidomycosis
TLR4 and P. brasiliensis infection
Comparative studies of in vivo susceptibility of different
mouse strains to P. brasiliensis intraperitoneal infection led
us to verify that TLR4-deficient (C3H/HeJ) mice were more
resistant than TLR4 normal (C3H/HePas) animals (Calich
et al., 1985). Recent findings from our laboratory (F.V.
Loures, unpublished data) demonstrated that, compared
with the normal strain, macrophages from TLR4-deficient
mice had a lower phagocytic ability, which appears to
influencethe decreased numberof viable P.brasiliensis yeasts
recovered after cocultivation for a 72h period. Deficient
macrophages secrete lower levels of NO, IL-12 and mono-
cyte chemoattractant protein-1 (MCP-1) but produced
equivalent amounts of TNF-a. In contrast, IL-10 was
synthesized in higher amounts by TLR4-deficient macro-
phages. Consistent with in vitro results, 96h after
in vivo pulmonary infection, TLR4-deficient mice presented
decreased fungal loads in the lungs associated with lower
levels of NO and proinflammatory cytokines [IL-12, and
granulocyte macrophage colony-stimulating factor (GM-
CSF)]. Similar results were observed at week 11 after
infection of TLR4-mutant mice: decreased CFU counts
associated with low IL-12 levels but high IFN-g secretion.
Paralleling its mild infection, the deficient strain secreted
low levels of IgG1, IgG2b and IgM P. brasiliensis-specific
isotypes (F.V. Loures, unpublished data).
Cytospin preparations of lung-infiltrating leukocytes at
week 2 of infection showed an increased number of PMN
neutrophils associated with decreased numbers of lympho-
cytes and monocytes. Diminished expression of CD251,
CD861and CD861IAk1cells were also observed by fluor-
escence-activated cell sorter (FACS) analysis of lung
lymphocytes. In addition, by week 2 of infection no differ-
ences in the lymphoproliferative activity of TLR4-normal
and TLR4-deficient spleen cells were detected. Although
Table 3. The role of TLRs and MyD88 adaptor protein in some experimental models of Aspergillus fumigatus infection
PRR deficiency Biological effectReference
TLR4, CD14, TLR2
TLR4, TLR2
TLR4 but not TLR2 recognizes hyphae
TLR4 and TLR2 recognize conidia and hyphae, and induce TNF-a and MIP-2;
impaired PMN influx
Fungal antigens recognized by TLR4 and TLR2; enhanced phagocytosis and
cytokine synthesis; activation and maturation of DCs
Individual TLRs activate human PMNs for specialized antifungal effector
functions
Conidia use TLR4 and TLR2 to induce proinflammatory cytokines; hyphae use
only TLR2 to produce IL-10
MyD88 and TLR2 required for optimal signaling responses of cells
TLR2, TLR4 and MyD88 signaling dispensable for fungal clearance
Normal phagocytosis and killing of conidia; normal cytokine secretion
MyD88: high susceptibility. TLR signaling: depends on morphotypes, route of
infection. Th1 response: DC-MyD-dependent
Wang et al. (2001)
Meier et al. (2003)
TLR4, TLR2Braedel et al. (2004)
TLR4, TLR2, TLR3, etcBellocchio et al. (2004b)
TLR4, TLR2Netea et al. (2003)
MyD88, TLR2, TLR4
MyD88, TLR2, TLR4
MyD88
MyD88 TLR4, TLR2, TLR9
Mambula et al. (2002)
Dubordeau et al. (2006)
Marr et al. (2003)
Bellocchio et al. (2004a)
FEMS Immunol Med Microbiol 53 (2008) 1–7
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differences in CFU counts and synthesis of some inflamma-
tory mediators occur in TLR4-deficient mice, such differ-
ences were not sufficient to alter their mean survival times
(Loures; F.V. Loures & V.L.G. Calich, unpublished data).
TLR2 and P. brasiliensis infection
We have also comparatively analyzed P. brasiliensis infection
of TLR2-normal (WT) and TLR2-gene knockout (KO) mice
in a C57BL/6 background (F.V. Loures & V.L.G. Calich,
unpublished data). In vitro infection of KO macrophages
resulted in lower phagocytic indexes, decreased recovery of
viable yeasts after 72h of cocultivation and low levels of NO
and MCP-1 in culture supernatants. Compared with WT
mice, 48h after infection KO mice presented diminished
pulmonary fungal loads andNO, but these findings werenot
associated with differences in the levels of pro- and anti-
inflammatory cytokines. Analysis of bronchoalveolar lavage
fluids obtained 72h after i.t. infection showed an increased
influx of neutrophils to the airspaces of TLR2 KO mice in
comparison with WT animals. Cytospin preparations of
lung-infiltrating leukocytes at weeks 2 and 4 of infection
showed a decreased proportion of macrophages but an
increased number of PMN neutrophils and lymphocytes. In
addition, a decreased number of IAk1macrophages and
CD41CD251T cells was detected in TLR2 KO mice.
Further studies are needed, however, to characterize this
T-cell subset more completely, which can exert effector or
regulatory functions. Indeed, in a previous report Netea
et al. (2004) showed that TLR2 KO mice are less susceptible
to Candida albicans infection due to the decreased presence
of regulatory T cells and a more efficient fungal-specific
immunity. At week 11 of infection TLR2-deficient and
normal mice presented similar humoral immunity but the
former strain presented increased fungal burden in the
lungs. Despite this difference, both mouse strains exhibited
equivalent mortality rates.
MyD88 adaptor molecule and
P. brasiliensis infection
When macrophages were in vitro infected with P. brasiliensis
yeasts for 72h, an increased number of viable fungi was
recovered from MyD88 KO macrophages in comparison
with normal cells. This diminished fungicidal ability paral-
leled a decreased synthesis of NO and IL-12. The early in
vivo infection reproduced the in vitro findings: higher fungal
loads were found in MyD88 KO mice associated with lower
levels of pulmonary NO and IL-12. This appears to indicate
that absence of MyD88 molecule causes profound effects in
cell activation, resulting in more severe infection. Mortality
studies confirmed the higher susceptibility of MyD88 KO
mice to P. brasiliensis infection, as their mean survival time
was significantly lower than that of WT controls (F.V. Loures
& V.L.G. Calich, unpublished data).
Concluding remarks
An increasing number of reports document the primary
importance of innate immunity not only by providing the
first line of defense against invading pathogens but also by
controlling essential mechanisms that induce and regulate
adaptative immunity. Our results on the role of TLRs in
paracoccidioidomycosis suggest P. brasiliensis yeasts use
TLR2 and TLR4 to gain entry into macrophages and infect
mammalian hosts. Indeed, P. brasiliensis yeasts appear to be
recognized by TLR2 and TLR4, resulting in increased
phagocytic ability, NO secretion and fungal infection of
macrophages. These data appear to be paradoxical, but the
killing activity usually associated with NO secretion was not
able to reduce the fungal growth provided by the presence
of TLRs. Thus, interaction with TLRs could be considered a
pathogenicity mechanism of P. brasiliensis, which would use
host receptors of innate immunity (TLR2 and TLR4) to
infect cells and to guarantee its own multiplication.
The in vivo infection of TLR-deficient mice resulted in
decreased fungal burdens, again suggesting that TLRs are
used by P. brasiliensis yeasts to infect hosts. The opposite
result, however, was seen with MyD88-deficient macro-
phages and mice as more severe infections were observed
probably due to the intact fungal recognition mediated by
the expression of normal PRR but impaired ability of cell
activation resulting in diminished fungicidal ability.
As a whole, TLR deficiency caused less severe infections
associated with altered secretion of NO, cytokines and
chemokines, resulting in altered cellular influx to the site of
infection. In both PRR-deficient strains, the lung inflamma-
tory infiltrate was composed of a diminished number of
macrophages associated with an increased presence of PMN
neutrophils. The phagocytic and killing abilities of the latter
cells perhaps contribute to the decreased fungal inoculum
at the site of infection. Furthermore, the low level of MCP-1
was parallel to the decreased number of lung-infiltrating
monocytes.
An interesting observation was the decreased number of
CD41CD251T cells in the lungs of TLR2 KO mice,
although an equivalent situation was not observed in
TLR4-deficient animals.
Mortality studies have shown that TLR deficiency was not
able to change the late course of infection as no differences
were observed between TLR-deficient and TLR-normal
mice. Compensatory mechanisms appear to abolish the
immunological differences caused by PRR deficiencies. The
same was not true for MyD88 deficiency, which causes
higher mortality of infected mice.
FEMS Immunol Med Microbiol 53 (2008) 1–7
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Toll-like receptors and fungal infections

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In summary, our studies suggest that MyD88 deficiency
is more important than TLR2 or TLR4 deficiency and
P. brasiliensis yeasts appear to use TLRs as a virulence
mechanism, which facilitates the access of fungal cells into
murine macrophages. Despite their TLR-mediated activa-
tion, macrophages are not able to control fungal growth,
both in vitro and in vivo. However, the final balance between
fungal growth and activation of the immune system appears
to control disease outcome as the low fungal loads and
impaired immunity of TLR-deficient mice and the high
fungal burdens and enhanced immunity of normal-TLR
mice result in equivalent survival times. Furthermore, our
studies have also suggested that, besides TLR, other PRRs
play a role in the host immune response against by
P. brasiliensis infection.
In conclusion, studies aimed to characterize the role of
TLRs in fungal infections are firmly demonstrating their
important participation in the effector and regulatory
mechanisms of innate and adaptative immunity against
these pathogens. TLR2, TLR4 and the adaptor molecule
MyD88 appear to control the phagocytic rates, cell migra-
tion and activation, cytokine and chemokine secretion as
well as the expression of costimulatory molecules that affect
dendritic cell activation and their competence as antigen-
presenting cell (APC) to naı ¨ve T cells. The interaction
between TLR and other PRRs, in a synergistic or antagonis-
tic way with fungal agonists, can result in different effector
(Th1, Th2 and Th17) and regulatory responses (Treg),
which ultimately determine disease outcome. Despite the
important information in the current literature, additional
investigation is needed to characterize further the influence
of TLRs in the immunopathology of fungal infections.
Acknowledgements
We are grateful to Dr Shizuo Akira for providing TLR and
MyD88 KO mice. This work was supported by grants from
Fundac ¸a ˜o de Amparo a ` Pesquisa do Estado de Sa ˜o Paulo
(FAPESP) e Conselho Nacional de Pesquisas (CNPq).
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Toll-like receptors and fungal infections