Interactions between TLR2, TLR4, and mannose receptors with gp43 from Paracoccidioides...

as in other systemic mycosis conditions, an effi cient

immune response depends on the interplay between innate

and adaptive host defenses in combination with fungal

pathogenic mechanisms. Although the need for innate

immunity in resisting fungal infection is well-recognized

[4], the molecular mechanisms underlying the recognition

of P. brasiliensis by innate immune cells remains poorly

defi ned [5].

Activation of monocytes and macrophages is one of the

fi rst events in the innate immune response to fungal infec-

tions. This activation is induced by the recognition of cell

surface components of the foreign microorganisms. Patho-

gen recognition is mediated by a series of germline encoded

pattern recognition receptors (PRRs) that are either soluble

or membrane-bound. These PRRs recognize conserved

Received 24 September 2010; Received in fi nal revised form 22 December

2010; Accepted 20 February 2011

Correspondence: Maria Terezinha S. Pera ç oli, Departamento de Micro-

biologia e Imunologia, Instituto de Bioci ê ncias, UNESP, CEP 18618-

970, Botucatu, S ã o Paulo, Brasil. Tel.: � 55 015 14 3811 6058; fax: � 55

015 14 3815-3744. E-mail: [email protected]

Interactions between TLR2, TLR4, and mannose receptors

with gp43 from Paracoccidioides brasiliensis induce

cytokine production by human monocytes

ERIKA NAKAIRA-TAKAHAGI * , MARJORIE A. GOLIM † , CAMILA F. BANNWART * , ROSANA PUCCIA ‡ &

MARIA T. S. PERA Ç OLI *

* Departamento de Microbiologia e Imunologia , Instituto de Bioci ê ncias , Universidade Estadual Paulista , UNESP , Botucatu , S ã o

Paulo , † Hemocentro , Faculdade de Medicina , Universidade Estadual Paulista , UNESP , Botucatu , S ã o Paulo , and ‡ Departamento

de Microbiologia , Imunologia e Parasitologia , Universidade Federal de S ã o Paulo , UNIFESP , S ã o Paulo , Brasil

The glycoprotein gp43 is an immunodominant antigen secreted by Paracoccidioides brasiliensis , the agent of paracoccidioidomycosis. The present study evaluated whether gp43 can interact with toll-like (TLR2, TLR4) and mannose (MR) receptors on the surface of human monocytes, and how that affects their expression and cytokine pro-duction. Monocytes were incubated with or without monoclonal antibodies anti-TLR2, anti-TLR4, or anti-MR, individually or in combination, prior to the addition of gp43. The gp43 binding to monocyte surface, as well as expression of TLR2, TLR4, and MRs were analyzed by fl ow cytometry, while production of TNF- α and IL-10 was monitored by ELISA. The results suggested that gp43 binds to TLR2, TLR4, and MR receptors, with TLR2 and MR having the strongest effect. All three receptors infl uenced the production of IL-10, while TNF- α production was associated with expression of TLR4 and MR. The modulatory effect of gp43 was demonstrated by high levels of TLR4 expression associated with increased production of TNF- α after 4 h of culture. Alternatively, high levels of TLR2 expression, and elevated production of IL-10, were detected after 18 h. We showed that interaction between gp43 and monocytes may affect the innate immune response by modulating the expression of the pattern recognition receptors TLR2, TLR4 and MR, as well as production of pro- and anti-infl ammatory cytokines.

Keywords Paracoccidioides brasiliensis , gp43 , monocytes , Toll-like receptors, mannose receptor, TNF- α , IL-10

Introduction

Paracoccidioides brasiliensis is a thermal dimorphic fun-

gus that causes paracoccidioidomycosis, the most preva-

lent systemic mycosis currently diagnosed in Latin

America [1,2]. Clinical manifestations of the disease are

those of a chronic granulomatous disease, with involve-

ment of the lung, reticuloendothelial system, mucocutane-

ous areas, and other organs [3]. In paracoccidioidomycosis,

© 2011 ISHAM DOI: 10.3109/13693786.2011.565485

Medical Mycology October 2011, 49, 694–703

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© 2011 ISHAM, Medical Mycology, 49, 694–703

Interaction of gp43, TLR2, TLR4 and MR on human monocytes 695

microbial structures, including bacterial lipopolysaccha-

rides, or fungal β -glucan, both of which are known as patho-

gen associated molecular patterns (PAMPs) [6 – 8]. These

PAMPs decorate the surface of the fungus, and can include

β -glucans, chitin, and mannoproteins [7,9]. Over the past

couple of years, a growing number of opsonic and non-

opsonic PRRs that recognize fungal PAMPs have been iden-

tifi ed. Of particular interest are those of the Toll-like

receptor (TLR) and C-type lectin families, which appear to

have central roles in antifungal immunity [10]. The TLR

family of proteins, fi rst described in Drosophila , possess

extracellular leucine-rich repeat regions, which are involved

in microbial recognition, and an intracellular Toll/interleu-

kin-1 region (TIR) domain, which is necessary for signaling.

The TLR have only been examined for a limited number of

fungi and some ligands of these PRRs such as mannan and

phospholipomannan, have been described [7,10].

The involvement of TLRs in fungi recognition and

resistance of mammalian hosts has been reported for

Candida albicans , Aspergillus fumigatus , and Cryptococcus neoformans [4,11,12], and more recently for P. brasiliensis

[13]. Moreover, the results of previous studies have sug-

gested that purifi ed pathogenic fungal cell wall compo-

nents can be used to identify primary PRR receptors and

characterize host cell signaling pathways that facilitate the

recognition of fungal PAMPs [5,14].

MR (CD206) is a group VI C-type lectin receptor (CLR)

that is composed of eight extracellular C-type lectin-like

domains (CTLDs), a fi bronectin type II repeat domain, a

cysteine-rich domain, and a short cytoplasmic tail. This

receptor is widely expressed on peritoneal [9] and alveolar

macrophages [15], as well as on human monocytes. MR

recognizes oligosaccharides that terminate in mannose,

fucose, and N-acetylglucosamine [14]. It has been impli-

cated in the processes of endocytosis and phagocytosis,

and to induce the production of pro- and anti-infl ammatory

cytokines following interactions with fungi [16,17]. MR

also interacts with other canonical PRRs to mediate intra-

cellular signaling [18]. For example, stimulation via the

TLR family of proteins results in the initiation of signaling

cascades that culminate in activation of nuclear factor kB

(NFkB) and mitogen-activated protein kinases. This pro-

cess facilities the transcription of genes that regulate the

adaptative immune response, including those for many

cytokines and chemokines [19].

The gp43 glycoprotein is the main antigenic component

secreted by P. brasiliensis , which is recognized by most

sera from patients with active paracoccidioidomycosis

[20,21]. Accordingly, gp43 can be used to monitor patients

receiving treatment with antifungals [22]. Structurally,

gp43 contains only one N -linked high mannose chain that

has been fully characterized [23]. Gp43 may also represent

a virulence factor due to its adhesive properties, and has

been shown to modulate P. brasiliensis infection in a ham-

ster intratesticular model [24]. Gp43 elicits cellular immu-

nity and has successfully been used as a protective antigen

in experimentally infected mice [25]. Additional studies

have shown that gp43 can inhibit macrophage functions

such as phagocytosis, fungal intracellular killing, as well

as the production of nitric oxide (NO) and hydrogen per-

oxide (H 2 O 2 ) [26,27]. These activities induced by gp43

may facilitate the infection of host tissue by the fungus,

particularly in the initial phase of the infection [28] since

phagocytosis of P. brasiliensis may be mediated by MRs

on the surface of macrophage [26,27]. Until now, gp43 of

P. brasiliensis has been recognized as an important fungal

component, primarily acting as an immunodominant anti-

gen [28]. Since studies on the binding of gp43 to human

monocytes have not previously been investigated, the goal

of the present study was to determine whether the immu-

nodominant antigen of P. brasiliensis , gp43, can interact

with TLR2, TLR4, and MR on the surface of human mono-

cytes, and whether expression of these receptors and

cytokine production is affected by these interactions.

Materials and methods

Healthy blood donors

Twenty healthy individuals, 25 – 50 years of age (mean:

34.5 � 8.4), were recruited from the University Hospital,

Botucatu Medical School, S ã o Paulo State University,

Brazil, for donation of blood. This study was approved by

the Ethics Committee of the Botucatu Medical School, and

informed consent was obtained from all blood donors.

Gp43 from P. brasiliensis

Native gp43 was isolated from P. brasiliensis isolate B-339

(Pb339) and purifi ed from culture supernatants using affi n-

ity chromatography. Columns of Affi -Gel 10 bound to

Mab17c, an anti-gp43 monoclonal antibody that recognizes

all gp43 isoforms, was used as previously described [29].

Isolation of peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PBMC) were isolated

from heparinized (50 U/ml heparin) venous blood using His-

topaque density-gradient centrifugation [density (d) � 1.077]

(Sigma-Aldrich, Inc., St Louis, MO, USA). Briefl y, 5 ml of

heparinized blood was added to an equal volume of RPMI

1640 tissue culture medium (Sigma-Aldrich) containing

2 mM L-glutamine, 10% heat-inactivated fetal calf serum

(FCS), 20 mM HEPES, and 40 μ g/ml gentamicin (complete

medium). Samples were added to 5 ml of Histopaque and

centrifuged at 400 g for 30 min at room temperature. The

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696 Nakaira-Takahagi et al .

without 0.5 μ g/ml anti-TLR2 (TLR2.1), anti-TLR4

(HTA125), or anti-MR (15-2) MAbs from Biolegend (San

Diego, CA, USA) individually or in combination for recep-

tor blockade. After 60 min at room temperature, cells were

washed in PBS/1% FCS and incubated with 10 ng biotin-

labeled-gp43 for 30 min on ice. After washing the cells in

PBS/1% FCS, monocytes were incubated with streptavi-

din-FITC (SouthernBiotech, Birmingham, AL, USA) and

PE/Cy7-labeled anti-CD14 (M5E2, 0.5 μ g) for 30 min on

ice. After a third wash with PBS/1% FCS, cells were fi xed

with 4% paraformaldehyde (PFA) and analyzed using a

FACSCalibur fl ow cytometer and CellQuest software (Bec-

ton Dickinson, Franklin Lakes, NJ, USA). Background

staining was determined by analyzing cells incubated with

0.5 μ g FITC-labeled control isotype antibodies for 30 min

at room temperature in the dark. Ten thousand monocyte

events, defi ned as cells with respective side scatter (SSC)

and CD14 staining characteristics were acquired in the list

mode fi le for each sample, and the distribution of each

gp43 � population was determined. Results are expressed

as the mean fl uorescence intensity (MFI) values of the

gated positive events.

Flow cytometry analysis of TLR2, TLR4, and MR expression

in monocytes

Expression of TLR2, TLR4, and MR by human mono-

cytes was assessed using a FACSCalibur fl ow cytometer

and CellQuest software (Becton Dickinson, Franklin

Lakes, NJ, USA). Peripheral blood mononuclear cells

(PBMCs) containing 5 � 10 5 monocytes/ml from healthy

subjects were incubated for 4 h or 18 h at 37 ° C and 5%

CO 2 with complete medium in the presence or absence of

gp43 (10 ng/ml) or LPS (1 μ g/ml). Cells were washed and

incubated with the following MAbs, according to the

manufacturer ’ s instructions: 0.5 μ g of PE/Cy7-labeled

anti-CD14 (M5E2), 0.5 μ g of PE-labeled anti-TLR2

(TL2.1), 0.5 μ g of FITC-labeled anti-TLR4 (HTA125), or

0.5 μ g of FITC-labeled anti-MR (15-2). All of these anti-

bodies were purchased from Biolegend (San Diego, CA,

USA). Stained cells were incubated for 30 min in the dark

at room temperature, then washed and fi xed with 2% PFA

in PBS. Background staining was determined from cells

incubated with 0.5 μ g FITC-, PE-, or PE/Cy7-labeled con-

trol isotype antibodies, for 30 min at room temperature in

the dark. Cell samples were washed twice with PBS, then

analyzed by fl ow cytometry. Ten thousand monocyte

events, defi ned as cells with respective side scatter (SSC)

and CD14 staining characteristics, were acquired in the

list mode fi le from each sample, and corresponding levels

of fl uorescence for TLR2, TLR4, and MR were obtained

for the gated CD14 � cells. Results are expressed as the

MFI value of the gated events.

interface layer of the PBMCs was then carefully aspirated

and washed twice with phosphate buffer saline (0.1 M, pH

7.2) containing 0.05 mM ethylenediaminetetraacetic acid

(PBS-EDTA), and once with complete medium at 300 g for

10 min. Cell viability, as determined by 0.2% trypan blue

dye exclusion, was � 95% in all experiments. In addition,

monocytes were counted using neutral red (0.02%) staining,

and were resuspended at a concentration of 1 � 10 6 mono-

cytes/ml in complete medium.

Production of monocyte culture supernatants

Monocyte suspensions (1 � 10 6 /ml) were distributed into

24-well fl at-bottomed plates (1 ml/well) (Nunc, Life Tech.

Inc., MD, USA) and incubated for 2 h at 37 ° C in humidifi ed

5% CO 2 . Non-adherent cells were removed by aspiration

and each well was rinsed twice with complete medium.

These preparations were � 90% pure for monocytes as

determined by morphologic examination and staining for

non-specifi c esterase [30]. To evaluate cytokine production,

monocytes were incubated with or without monoclonal anti-

bodies (MAbs) raised against TLR2 (TLR2.1), TLR4

(HTA125), or MR (15-2) at 0.5 μ g/ml for 60 min at room

temperature . All of these antibodies were purchased from

Biolegend (San Diego, CA, USA). Following incubation,

monocytes were washed and treated with complete medium

with or without gp43 (10 ng/ml) or lipopolysaccharide (LPS)

from Escherichia coli O55B5 (Sigma-Aldrich) (1 μ g/ml) for

4 h and 18 h at 37 ° C with 5% CO 2 . Culture supernatants

were harvested and stored at � 80 ° C until assayed.

Detection of cytokines

Concentrations of TNF- α and IL-10 in cell-free superna-

tants obtained from monocyte cultures treated with gp43

or LPS were detected using Quantikine ELISA kits (R&D

Systems, Minneapolis, MN, USA) according to the manu-

facturer ’ s protocol. Assay sensitivity limits were 10 pg/ml

for TNF- α and 7.5 pg/ml for IL-10.

Preparation of biotin-labeled gp43

Purifi ed native gp43 at 100 ng/ml in PBS was incubated

for 2 h on ice with 20 μ l EZ-link sulpho-NHS-biotin

reagent solution (Pierce, Rockford, IL, USA), prepared

according to the manufacturer ’ s instructions. Unlabeled

biotin was removed using a dialysis chamber (Pierce) incu-

bated in PBS overnight.

Flow cytometry to monitor gp43 binding to monocytes

To evaluate gp43 binding to TLR2, TLR4, and MR, 5 � 10 5

monocytes/ml in complete medium were incubated with or

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Statistical analysis

Data are expressed as the mean � standard error (SEM).

Differences between groups were analyzed using analysis

of variance (ANOVA) followed by the Tukey test using

INSTAT 3.05 software (GraphPad, San Diego, CA, USA).

A P value � 0.05 was considered signifi cant [31].

Results

Binding of gp43 to TLR2, TLR4, and MR expressed

by monocytes

Binding of gp43 to monocytes was evaluated using the

MFI detected in CD14 � cell populations. Prior to fl uores-

cence detection, monocytes were incubated with different

concentrations of biotin-labeled gp43 (i.e., 1, 5, 10, or

20 ng/ml) for various intervals (i.e., 10, 30, and 60 min).

At 10 ng/ml gp43, and a 30 min incubation period, the

highest levels of MFI were detected (data not shown). The

viability of monocytes cultured in the presence or absence

of gp43 was also determined and was found to be � 95%

for all samples analyzed. This would indicate that any

observed reduction in gp43 binding was not due to cell

death. When cells were cultured with gp43 in the absence

of anti-TLR2, -TLR4, or -MR antibodies, an increase in

gp43 located on the surface of monocytes was detected

after 30 min. In contrast, when monocytes were incubated

with anti-TLR2, -TLR4, and/or -MR MAbs, individually

or in combination, a signifi cant decrease in MFI was

observed compared to monocyte cultures that were not

pretreated with MAbs (Fig. 1). The highest inhibition of

MFI was associated with monocytes treated with all three

receptor blocking antibodies (76.7%), while treatment with

anti-MR antibodies alone showed a similar decrease in

MFI (71.1%). In contrast, only a slight decrease in MFI

values was observed following treatment of monocytes

with anti-TLR4 antibodies (29.6%). These results suggest

that TLR2, TLR4, and MRs have a role in the gp43 binding

to monocytes, especially TLR2 and MR.

Involvement of TLR2, TLR4, and MR in the production

of IL-10 and TNF- α

To evaluate whether TLR2, TLR4, or MR infl uence the

production of IL-10 and TNF- α , human monocytes were

blocked by incubation with MAbs specifi c for these

receptors 60 min prior to the addition of gp43. IL-10 and

TNF- α production were subsequently assayed after 4 h

and 18 h. To eliminate the possibility that trace amounts

of LPS could be present in the preparations of purifi ed

gp43, leading to unspecifi c results, separate monocyte

cultures were treated with polymixin B (PMX-B). The

addition of PMX-B to monocyte cultures signifi cantly

decreased TNF- α production by LPS-stimulated cells,

while there was no effect on the cytokine release after

gp43 stimulation (data not shown). IL-10 production

detected at 18 h after gp43 stimulation was observed to

be signifi cantly higher than the levels of IL-10 detected

at 4 h in cultures that were not pretreated with MAbs, yet

were stimulated with gp43. However, when MAbs to

TLR2, TLR4, or MR were added individually, or in com-

bination, IL-10 production was inhibited. Lower levels of

IL-10 were also associated with signifi cantly lower

cytokine levels compared to monocyte cultures that were

not treated with MAbs (Fig. 2A). Overall, these results

suggest that IL-10 production by monocytes may be

dependent on the binding of gp43 to TLR2, TLR4, and

MR on the cell surface.

For TNF- α , signifi cantly higher levels were detected at

the 4-h timepoint compared to the 18-h timepoint follow-

ing stimulation with gp43 (10 ng/ml), or compared to con-

trol cultures. When monocytes were pretreated with TLR2

MAbs, TNF- α production was not inhibited, and levels of

TNF- α were similar to those of control cultures stimulated

with gp43. In contrast, preincubation of monocytes with

MAbs to TLR4, or MR, prior to the addition of gp43

resulted in lower levels of TNF- α production at both the

4 h and 18 h Furthermore, down-regulation of TNF- α pro-

duction in cultures treated with MAbs to TLR2 was only

observed when MAbs specifi c for TLR4 and MR were also

included. Overall, the lowest levels of TNF- α production

were detected in cultures pretreated with MAbs specifi c

for TLR4 and MR, suggesting that these receptors play a

role in gp43-mediated stimulation of TNF- α production

(Fig. 2B).

Gp43 modulates the expression of TLR2, TLR4, and MR

on monocytes

Monocytes were cultured in the absence or presence of

gp43 (10 ng/ml) for 4 h and 18 h at 37 ° C. In these assays,

the former served as negative control, while addition of

LPS (1 μ g/ml) served as a positive control. In Fig. 3A – C,

the expression profi les of MR, TLR2, and TLR4 in mono-

cyte populations are shown. A higher percentage of MR �

monocytes was detected in cultures stimulated with gp43

or LPS, compared with control monocyte cultures or non-

stimulated cultures, at both the 4 h and 18 h. In addition,

when TLR2 � , TLR4� , and MR� cell populations were

compared, less than 2% of monocytes constitutively

expressed MR prior to stimulation with LPS and gp43.

However, following stimulation with LPS and gp43,

the percentage of MR� monocytes increased three- and

fi ve-fold, respectively (Fig. 3A). In contrast, greater

than 90% of CD14� cells expressed TLR2 and TLR 4

(Fig. 3B – C).

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Analysis of MFI profi les showed that the treatment of

monocytes with LPS or gp43 did not increase the levels

of MR expression at either the 4 h or 18 h timepoints

(Fig. 4A). In contrast, a signifi cant increase in TLR2

expression was detected at the same timepoints following

stimulation with LPS compared with control, non-

stimulated cultures. Although culturing monocytes with

gp43 was found to enhance TLR2 expression after 4 h,

Fig. 1 Binding of gp43 to human monocytes. Monocytes (5 � 10 5 /ml) were incubated in the absence (without blockade), or presence of anti-TLR2,

anti-TLR4, and anti-MR MAbs individually, or in combination, for 60 min at room temperature. Afterwards, cells were treated with biotin-labeled gp43

(10 ng/ml) and with streptavidin-FITC for 30 min on ice, then analyzed by fl ow cytometry. (A) Representative dot-plots of gated CD14 � cells that have

bound to gp43; (B) MFI of gp43 � /CD14� cells expressed as the mean � SEM of experiments from 20 healthy individuals. Percentage inhibition of

gp43 binding is also represented. (C) Representative histograms of the distribution of gp43 � /CD14 � cells prior to and following incubation of monocytes

with anti-MR MAbs. * P � 0.05, when compared to all other samples; � P � 0.05, when compared to incubation with anti-TLR4 MAbs.

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signifi cant changes in MFI values were observed only

after 18 h of culture. This increase in TLR2 expression

was signifi cantly higher than the TLR2 levels of control

and gp43-stimulated cells for 4 h (Fig. 4B). TLR4 expres-

sion induced by gp43 was also signifi cantly higher than

control and LPS-stimulated cultures at the 4-h timepoint.

However, TLR4 expression in monocytes stimulated with

gp43 was signifi cantly lower than the 4 h value at the 18-h

timepoint, yet it did not show a statistical difference with

control cultures. These results indicate that treatment

with gp43 up-regulates TLR4 expression in monocytes

after 4 h, and up-regulates TLR2 expression after 18 h

(Fig. 4C). However, treatment with gp43 did not affect

MR expression (Fig. 4A).

Fig. 2 Role of TLR2, TLR4, and MRs in the production of IL-10 and TNF- α by human monocytes stimulated with gp43. Monocytes (5 � 10 5 /ml)

were incubated in the absence (without blockage) or presence of anti-TLR2, anti-TLR4, and anti-MR antibodies individually, or in combination as

indicated, for 60 min at room temperature. Cells were then stimulated with gp43 (10 ng/ml) and assayed after 4 h and 18 h of the incubation. Levels of

cytokines present in the supernatants were detected by ELISA. Results represent the mean � SEM of IL-10 (A) and TNF- α (B) concentrations determined

in monocytes from 20 healthy individuals. IL-10 production: * P � 0.01, when compared with all other samples and timepoints; � P � 0.01, vs. gp43 at

4 h. TNF- α production: * P � 0.05, when compared with control at 4 h; � P � 0.05, when compared with all other samples and timepoints.

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Discussion

Currently, the exact route of infection by P. brasiliensis in

humans is not well-characterized. However, it is known

that this fungus can infect tissue macrophages and mono-

cytes [32]. In recent studies of experimental infection by

yeast cells of P. brasiliensis , possible roles for TLR2,

TLR4, and dectin-1 receptors expressed by monocytes and

neutrophils were identifi ed [13]. Activation of these recep-

tors led to cell activation and an intense infl ammatory

response. In addition, a virulent strain of P. brasiliensis

has been shown to induce high levels of pro- and anti-

infl ammatory cytokines during human monocytes infec-

tion in vitro [33].

The present study examined whether the immunodomi-

nant antigen of P. brasiliensis , gp43, can interact with

TLR2, TLR4, and MR present on the surface of human

monocytes. The ability of gp43 to modulate the expression

of these receptors, as well as the production of pro- and

anti-infl ammatory cytokines, was also investigated. Both

up- and down-regulatory effects were observed. For exam-

ple, initial experiments investigated possible interactions

between gp43 and various PRRs. These results confi rmed

Fig. 3 MR, TLR2, and TLR4 expression on the surface of human

monocytes. Monocytes (5 � 10 5 /ml) were incubated in the absence

(control culture) or presence of either LPS (1 μ g/ml) or gp43 (10 ng/ml)

at 37 ° C. Expression of TLR2, TLR4, and MRs was analyzed after 4 h

and 18 h using fl ow cytometry. Results represent the mean � SEM

percentage of monocytes expressing MR (A), TLR2 (B) or TLR4 (C)

obtained from 20 healthy individuals. * P � 0.05, when compared with

controls.

Fig. 4 MFI for MR, TLR2, and TLR4 on human monocytes. Monocytes

(5 � 10 5 /ml) were incubated in the absence (control culture), or presence

of LPS (1 μ g/ml) or gp43 (10 ng/ml) at 37 ° C for 4 h and 18 h. Cell

samples were then analyzed by fl ow cytometry. Results represent the

mean � SEM of the MFI detected for MR (A), TLR2 (B), or TLR4 (C)

receptors expressed on monocytes obtained from 20 healthy individuals.

TLR2 expression: * P � 0.05, vs. control at 4 h and 18 h; � P � 0.05 vs.

gp43 at 4 h. TLR4 expression: * P � 0.05) vs. control at 4 h and 18 h; � P � 0.05 vs. LPS at 4 h; 0 P � 0.05 vs. gp43 at 18 h.

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that TLR2, TLR4, and MRs are involved in the gp43 bind-

ing. The appearance of gp43 antigen onto the surface of

monocytes was observed to increase after 30 min of co-

culture, and this was signifi cantly inhibited when TLR2,

TLR4, and MR receptors were blocked with specifi c MAbs.

The highest inhibition of MFI was detected in monocytes

treated with anti-MR antibodies, or their association with

both anti-TLR2 and anti-TLR4 antibodies. These results

suggest, for the fi rst time, that TLR2 and MRs are the most

important receptors involved in the gp43 interaction with

human monocytes. However, the inability of the three

MAbs tested to completely inhibit gp43 binding may be

due to the importance of other unidentifi ed receptors

involved, such as dectin-1 and CD11/CD18 integrin, which

were not evaluated in the present study.

Binding of Penicilliun marneffei conidia to human

monocytes has been shown to be signifi cantly inhibited by

pretreating monocytes with MAbs against MR, TLR1,

TLR2, TLR6, CD14, CD11b, and CD18 [17]. These results

indicate that various PRRs on the surface of human mono-

cytes participate in the initial recognition of the fungus

[17]. Previously, MR was reported to serve as recognition

sites for pathogenic fungi, including C. albicans , Pneumo-cystis jiroveci , and P. marneffei yeasts [17,34], in addition

to P. brasiliensis [27,35,36]. Correspondingly, these data

supported the hypothesis that MR is a common phagocytic

receptor for a wide variety of fungal pathogens. Engage-

ment of MR has also been shown to induce the production

of pro-infl ammatory cytokines [16]. However, few studies

have characterized the interactions that occur between fun-

gal antigens and cells of the innate immune system. In

these studies, components of C. albicans , such as phospho-

lipomannan, were shown to be detected by TLR2 and

TLR6 [37], the glucoronoxilomanan of C. neoformans was

found to be recognized by TLR4 [38], and cryptococcal

mannoproteins required the recognition of terminal man-

nose groups by MR [39].

In contrast, receptors that induce cytokine production in

response to fungal pathogens have been well-characterized.

TLR4 has been shown to strongly stimulate pro-infl amma-

tory cytokines [40,41], whereas TLR2 is associated with

the release of IL-10 [42]. When human monocytes were

pretreated with MAbs specifi c for CD14 or TLR4, TNF- α

production was found to be inhibited following stimulation

with P. marneffei [17]. These results are consistent with

other studies where TLR4 and CD14 have been shown to

infl uence the production of TNF- α by monocytes and mac-

rophage activated by A. fumigatus [43,44]. An association

between TLR2 expression and IL-10 production in response

to pathogenic fungi, such as A. fumigatus and C. albicans ,

has also been described [42,45]. During an infection by

C. albicans , the deleterious effect of TLR2 signaling is

associated with increased production of IL-10 and the

development of T regulatory (CD4 � CD25 � ) cells, which

result in a defi cient cellular immune response and a reduced

ability to eliminate the infecting fungus [42]. Furthermore,

in work by Bonfi m et al . [13], a highly virulent strain of

P. brasiliensis (Pb18) was found to be predominantly asso-

ciated with induction of TNF- α , while a less virulent strain

of P. brasiliensis (Pb265) was preferentially recognized by

TLR2 and dectin-1 receptors. As a result, the latter involved

the production of IL-10, which may serve to induce a con-

trolled immune response benefi cial to the host. In contrast,

the results of the current study suggest that TLR2, TLR4,

and MRs may contribute to the expression of IL-10. How-

ever, only MR and TLR4 receptors may be important for

the production of TNF- α following binding to gp43. The

discrepancy between these results and those of Bonfi m

et al . [13] may be explained by the employment of gp43,

a purifi ed antigen of P. brasiliensis for binding studies,

while other studies have used whole yeast cells to stimulate

monocytes. According to Calich et al . [5], the use of puri-

fi ed components of fungal cells may elucidate the major

PRR and signaling pathways used by host cells to recog-

nize fungal PAMPs. Thus, it is possible that cross-talk

between TLRs and MR following binding of gp43 may

be necessary to mediate intracellular signaling for cyto-

kine production. Correspondingly, following binding of

Pneumocystis carini , MR has been shown to form func-

tional complexes with TLR2 receptors on the cell surface,

which facilitates signal transduction to induce cytokine

production [46]. Besides, the employment of ligand-

specifi c blocking antibodies to MR showed that this recep-

tor synergizes with TLR2 for maximum NF-kB activation

and proinfl ammatory cytokine production in response to

Pseudomonas aeruginosa infection [47].

The capacity of gp43 binding to modulate the expres-

sion of TLR2, TLR4, and MR on the surface of monocytes

was also investigated. In the presence of gp43, the percent-

age of MR � monocytes increased at both the 4-h and 18-h

timepoints, yet did not interfere with the percentage of

TLR2� and TLR4� cells. Furthermore, while TLR2 and

TLR4 were found to be constitutively expressed on over

90% of monocytes, less than 2% of monocytes were found

to express MR prior to stimulation with gp43. The latter

observation is consistent with a recent study by Netea

et al . [14], and work by Gazi and Martinez-Pomares [18],

demonstrating that under steady state conditions, 10 – 30%

of MR expression are found at the cell surface, while the

remaining 70% of MR are localized intracellularly [18].

The higher percentage of monocytes expressing TLR2 and

TLR4 prior to stimulation with gp43 may be explained by

the roles that TLR2 and TLR4 have in recognizing invad-

ing pathogens present in the circulation. However, gp43

was found to preferentially up-regulate TLR4 expression

and TNF- α production 4 h after stimulation with gp43,

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whereas high levels of TLR2 expression, and higher levels

of IL-10, were detected at the 18-h timepoint. These results

indicate that the binding of gp43 to TLRs can modulate

their expression on the cell surface, a phenomenon that

may be attributed to cell activation and an increased expres-

sion of cytokines by monocytes.

In the assays of MFI, treatment with gp43 primarily

resulted in an increase in the number of MR� cells, but it

has not modulated the levels of MR expression. These

results might be explained by the low percentage of MR�

cells initially present. However, it is possible that after

binding to MR, the resulting gp43/MR complex is internal-

ized, and its de novo synthesis and appearance on the

monocyte surface would appear to be down-regulated in

response to cell activation induced by pro-infl ammatory

cytokines produced during the activation of monocytes by

gp43. It has been reported that macrophages recycle MR

between the plasma membrane and the early endosomal

compartment, even in the absence of any ligand [18], or

after binding to terminal mannose residues on the surface

of Trypanosoma cruzi amastigotes. The latter results in

pathogen phagocytosis and intracellular multiplication

[48]. Activation of macrophage by interferon-gamma (INF-

γ ) has also been shown to down-regulate cell surface

expression of MR [49]. Correspondingly, adherence of

T. cruzi mediated by MR may select macrophages that have

not been activated by IFN- γ , and therefore, are permissive

to the intracellular reproduction of parasites [46] . Thus,

the binding of gp43 to MR present on the surface of non-

activated monocytes or macrophage may represent an eva-

sion mechanism of P. brasiliensis from a host ’ s immune

response, as previously suggested [28,32].

In conclusion, gp43 purifi ed from P. brasiliensis binds to

TLR2, TLR4, and MR on the surface of human monocytes,

and as a result, affects many functions of the innate immune

response. Specifi cally, activation of monocytes by gp43

modulates PRR expression, and induces the production of

pro- and anti-infl ammatory cytokines. In combination, these

effects, as well as the amount of gp43 produced by the fun-

gus and the susceptibility of the host, may contribute to the

establishment of a fungal infection, or its elimination.

Acknowledgements

This work was supported by the Funda ç ã o de Amparo à

Pesquisa do Estado de S ã o Paulo, Brazil (FAPESP – grants

no 2006/53366-9).

Declaration of interest : The authors report no confl icts of

interest. The authors alone are responsible for the content

and writing of the paper.

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Interactions between TLR2, TLR4, and mannose receptors with gp43 from Paracoccidioides...

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