ARTICLE
Auteur(s) : Celia Murciano, Alberto Yánez, M Luisa Gil,
Daniel Gozalbo
Departament de Microbiologia i Ecologia, Facultat de Farmàcia,
Universitat de València, Avgda. Vicent Andrés Estellés s/n, 46100
Burjassot, Spain
Resistance to candidiasis requires the coordinated action of both
innate and adaptive host immune responses [1, 2]. Recognition of
pathogen-associated molecular patterns (PAMPs) of invading fungi by
the innate immune system through pathogen recognition receptors
(PRRs) is the first step towards activating a rapid immunological
response and ensuring survival after infection; phagocytes can kill
the pathogen via intracellular and extracellular mechanisms,
macrophage activation releasing several key mediators, including
proinflammatory cytokines such as TNF-α, which are important for
protecting the host against disseminated candidiasis [1-3].
Antifungal CD4+ T helper 1 (Th1)-mediated responses play a central
role in anti-C. albicans defences, providing control of fungal
infectivity through production of IFN-γ. This cytokine is required
for optimal activation of phagocytes and suppresses the induction
of the Th2 response, which is associated with the susceptibility of
mice to systemic C. albicans infection and is characterized by
the production of anti-inflammatory cytokines, such as IL-10 and
IL-4; however, the pro-inflammatory (Th1) host response needs to be
counterbalanced through Th2 and Treg cells to ensure an optimal,
protective Th1 response [1].Toll-like receptors (TLRs) constitute a
family of PRRs that mediate recognition of microbes through PAMPs,
induce subsequent inflammatory responses and also regulate the
adaptive responses [3-5]. Our group has shown that TLR2 is
essential for murine resistance to invasive candidiasis, and
triggers production of proinflammatory cytokines, whereas TLR4
appears not to have a relevant role in these events [6-8]. Other
authors have described a role for TLR2 and TLR4 in murine
resistance to infection and in the in vitro cytokine production in
response to C. albicans cells [9-11]. TLR2, identified as the
receptor for the C. albicans cell wall-associated PAMP
phospholipomannan, triggers the production of proinflammatory
cytokines through the activation of the nuclear factor kappa B [12,
13]. In addition, recognition by TLR4 of O-linked mannosyl residues
present in the C. albicans cell wall mannoproteins has been
shown to mediate cytokine (TNF-α) induction in murine macrophages,
whereas TLR2 has a minor role through recognition of β-glucan by
the dectin-1/TLR2 receptor complex [14]. In addition, controversial
hypotheses have been proposed concerning the role of
C. albicans recognition by TLR2 and TLR4 in host protection
[8, 9, 15, 16], and two distinct models have been proposed.
According to one of these models, TLR2 basically recognizes the
C. albicans hyphae and confers susceptibility to infection
through induction of IL-10 and Treg cells that results in a Th2
response, whereas the yeast form is recognized by both TLR2 and
TLR4, leading to a Th1 protective response [11, 15]. In the other
model, proposed by our group, both yeasts and hyphae signal through
TLR2, and differences in the immune response to both fungal
morphologies may involve interaction with other PRRs, such dectin-1
[8, 16]. These inconsistencies point out the complexity of the
fungus-host interaction, which may be influenced by a wide range of
host- and fungal-related parameters. Therefore, in vitro assays of
cytokine production in response to C. albicans can be
influenced by the nature of the stimuli used. In this work, we have
studied the in vitro production of the Th1-cytokines TNF-α and
IFN-γ, key molecules involved in the host protective response to
C. albicans, in murine cells from wild type, TLR2-/- and
TLR4-/- knockout mice, challenged with viable and non-viable
(heat-killed, paraformaldehyde-fixed, and antimycotic-treated)
C. albicans cells in order to reveal possible differences in
the induced host response due to alterations of the fungal cell
surface that may affect exposure of PAMPs due to inactivating
treatments.
Materials and methods
Mice and yeast strains
TLR2-/- and TLR4-/- knockout mice (against a C57BL/6 genetic
background) were kindly provided by Dr. S. Akira (Osaka University,
Osaka, Japan) [17] and wild type C57BL/6 mice were obtained from
Harlan Ibérica (Barcelona, Spain). Eight-to-ten-week-old mice were
used for all experimental assays. All assays involving mice were
approved by the Institutional Animal Care and Use Committee. The
low virulence, non-germinative C. albicans PCA2 strain and the
high virulence, C. albicans ATCC 26555 strain were used in
this study [18, 19].
Preparation of fungal stimuli for in vitro assays
Yeasts and hyphae (germ tube-bearing yeast cells) from the high
virulence C. albicans ATCC 26555 were obtained following
incubation of starved yeast cells for 3 h at 28°C (budding
yeast) or 37°C (germ-tubes) in a minimal synthetic medium, as
previously described [19]. Cells were collected by centrifugation
and washed with phosphate-buffered saline (PBS) prior to
inactivating treatments: (1) heat-killing was performed by
incubating the cells (20 x 106 cells/mL in PBS) at 100
°C for 1 h, as reported elsewhere [6, 7]; (2) fixed cells were
obtained by incubating the cells (20 x 106
cells/mL) in 4% paraformaldehyde (fixation buffer, eBioscience, San
Diego, CA, USA) as previously described [20], followed by extensive
washing with PBS to remove the fixing agent, and (3)
antimycotic-treated cells we obtained by incubating the cells (20
x 106 cells/mL in PBS) in the presence of 3 μg/mL
amphotericin B (Gifco, Barcelona, Spain) for 72 h at room
temperature, followed by extensive washing to remove the
antimycotic, as previously described [20]. After treatments,
inactivation of cells was checked by the absence of growth
following incubation of samples (2 x 106 cells) on
Sabouraud-dextrose agar plates for 48 h at 28°C.
C. albicans PCA2 and ATCC 26555 yeast cells, collected from
exponentially growing cultures in liquid YPD medium (1% yeast
extract, 2% peptone, 2% glucose) at 28°C, were used as viable
fungal stimuli: PCA2 cells as a yeast stimuli, since this low
virulence, non-germinative strain is unable to develop hyphal forms
[18], and ATCC 26555 cells as hyphal stimuli, as these cells
exhibit the hyphal pattern of growth when incubated at 37°C in
complete culture medium for murine cells (RPMI 1640 medium
supplemented with 5% heat-inactivated FBS and 1%
penicillin-streptomycin, Gifco, Barcelona, Spain) in a 5%
CO2 atmosphere. All procedures were performed under
conditions designed to minimize endotoxin contamination as
described elsewhere [6, 7, 20-22].
Isolation of mouse peritoneal macrophages and in vitro
production of TNF-α
Resident peritoneal macrophages were obtained as previously
described [6, 7]. Macrophages (2.3 x 105 cells in 200 μL
of complete medium per well, in a 96-well tissue culture plate)
were challenged for 24 h with Escherichia coli O111: B4 LPS
(250 ng/mL, Sigma, Spain), zymosan (7.5 x 106
particles/mL, Molecular Probes, Invitrogen, Spain) and equivalent
amounts of killed budding yeast or hyphal (germ-tube) cells (150 μg
dry weight/mL), or for 6 h with viable fungal cells (250 000
cells per well) either of PCA2 or ATCC 26555 strains (see above).
Culture supernatants were tested using a commercial ELISA kit for
TNF-α (eBioscience, San Diego, CA, USA). Assays in the absence of
exogenous stimuli were performed as negative controls to check
background activation. Student’s two tailed t-test was used to
compare cytokine production by TLR2-/- and TLR4-/- cells to control
C57BL/6 cells; data are expressed as mean ± SD and significance was
accepted at *p < 0.05.
Isolation of splenocytes and in vitro production of IFN-γ
Splenocytes from mice intravenously (i.v.) infected with
C. albicans PCA2 cells (400 000 cells/mouse) were obtained at
day 3 post-infection as described elsewhere [7, 21, 22].
Splenocytes were resuspended (107 cells/mL) in 1 mL
of complete medium per well in a 24-well tissue culture plate, in
the presence of 2.5 μg/mL amphotericin B to avoid fungal growth,
and challenged for 48 h with LPS (500 ng/mL), zymosan (7.5 x
106 particles/mL) and killed C. albicans ATCC 26555
yeast cells (30 μg dry weight/mL) in a 5% CO2
atmosphere. Culture supernatants were tested using a commercial
ELISA kit for IFN-γ (eBioscience, San Diego, CA, USA). Assays in
the absence of exogenous stimuli were used as negative controls to
check background activation. Student’s two tailed t-test was used
to compare cytokine production by TLR2-/- and TLR4-/- cells to
control C57BL/6 cells; data are expressed as mean ± SD and
significance was accepted at *p < 0.05 and **p < 0.01.
Results
In vitro production of TNF-α by macrophages in response to
C. albicans stimuli
TNF-α production by macrophages in response to fungal stimuli was
triggered through TLR2 in all cases (figure 1). TLR2-/-
macrophages showed a significant reduction in TNF-α production
(65-78% inhibition) in response to all three types of killed,
budding yeast cells, as well as in response to killed, germ-tube
cells (70-88% inhibition), whereas no reduction was observed in
TLR4-/- macrophages, as compared to wild type cells (figure 1). The
expected results were obtained with control stimuli: (1) the TNF-α
production in response to LPS (a pure agonist of TLR4) showed a 73%
reduction in TLR4-/- cells (the retained response suggests the
presence of some contamination in the LPS preparation), and (2)
TNF-α production in response to zymosan (a TLR2 agonist) showed a
70% reduction in TLR2-/- cells. Therefore, the different
inactivating cell treatments used did not result in significant
changes in the macrophage response against C. albicans
mediated through TLR2 and TLR4. To compare these results with the
response to viable fungal cells, we performed in vitro assays using
viable cells of C. albicans PCA2 and ATCC 26555 strains as
stimuli, and a shorter incubation period to avoid fungal overgrowth
(figure 2).
Results showed that after six hours of the challenge, there were
detectable levels of TNF-α, and again TLR2-/- macrophages showed an
impaired production of TNF-α in response to both yeast (PCA2; 79%
inhibition) and hyphae (ATCC 26555; 75% inhibition) as compared
with wild type macrophages, whereas no significant differences were
found between wild type and TLR4-/- cells (figure 2). Control
stimuli (zymosan, and heat-killed C. albicans cells) produced
the expected results according to the results shown in figure 1. This
observation indicates that killed and viable cells share common
PAMPs that are able to trigger TNF-α production by murine
macrophages in response to C. albicans, through a
TLR2-dependent signalling. Moreover, zymosan as well as inactivated
and viable fungal stimuli still retained TNF-α-inducing capacity
(roughly 20-30%) in TLR2-/- macrophages, which indicates that other
receptors also participate in triggering TNF-α production upon
recognition of these stimuli.
In vitro production of IFN-γ by splenocytes in response to
C. albicans stimuli
To assess the antifungal production of IFN-γ upon primary
infection, animals were infected i.v. with a low dose of the
non-germinative, low virulence C. albicans PCA2 strain. This
infection does not produce any mortality or any significant
symptoms of disease in mice (wild type, TLR2-/- and TLR4-/-) and
induces substantial Th1 acquired protection to reinfection with a
high virulence C. albicans strain [7, 21, 22]. Therefore, this
is a suitable experimental approach for inducing the presence, in
the spleen, of IFN-γ-producing cells that are immunoresponsive to
C. albicans. The production IFN-γ was assessed in splenocytes
obtained three days after i.v. infection of mice with the
C. albicans PCA2 strain. These splenocytes were challenged in
vitro with all three types of killed yeast stimuli obtained from
C. albicans ATCC 26555 strain (figure 3). As
expected, IFN-γ production in response to LPS was strongly
inhibited in TLR4-/- mice (98% inhibition) as well as in response
to zymosan in TLR2-/- mice (76% inhibition). Production of IFN-γ
was significantly inhibited (80-90% inhibition) in response to all
three, non-viable, fungal stimuli in TLR2-/- splenocytes, whereas
wild type and TLR4-/- cells showed similar levels of IFN-γ
production.
Discussion
The results reported in this work indicate that TLR2 is a major PRR
involved in triggering production of proinflammatory and
Th1-cytokines, such as TNF-α and IFN-γ, by murine macrophages and
splenocytes, respectively, in response to C. albicans stimuli,
but do not support a significant role of TLR4 in C. albicans
recognition, according to our previous observations [6, 7]. In
addition, fungal cell, surface-associated ligands (PAMPs) for TLRs
appear to be well conserved in viable and all three types of
non-viable fungal stimuli assayed (heat-killed, formaldehyde-fixed
and antimycotic-treated cells), regardless of the inactivating
treatment, which does not affect significantly the exposure of
fungal ligands for TLR2 and TLR4. However, it should be noted that
Wheeler and Fink [23] have reported that some inactivating fungal
treatments increase β-glucan exposure and lead to high levels of
TNF-α elicitation, probably through the dectin-1/TLR2 receptor
complex. It has been demonstrated that dectin-1, a phagocytic
receptor for the fungal cell wall polymer β-glucan, collaborates
with TLR2 in eliciting the inflammatory response to yeast, and that
dectin-1 mediates macrophage recognition of C. albicans yeasts
but not hyphae, as hyphal cells do not expose β-glucan at the cell
surface, thus suggesting that failure of hyphae to activate
dectin-1 may contribute to an impaired Th1 response [24, 25].
Moreover, dectin-1 can also trigger signalling through a
TLR2-independent pathway leading to cytokine production [26, 27].
Although it can not be ruled out that, under the experimental
conditions used, differences in the detected levels of cytokine
production in response to all three types of inactivated fungal
stimuli, may involve modifications of glucan exposure, our results
suggest that glucan exposure is not significantly affected by the
inactivating treatments, as reduction of cytokine production in
TLR2-/- cells was similar is response to viable and all three types
of inactivated fungal stimuli. Moreover, it has been found that
macrophages sense differently C. albicans and S. cerevisiae
through a mechanism involving both TLR2 and galectin-3, which
probably associate for the binding of ligands expressing β-1,2
mannosides specific to the C. albicans cell wall surface [28].
These observations further support the model proposed by our group
of the role of TLRs in host response to C. albicans [6-8, 16],
indicating that TLR2 is the major PRR involved in the induction of
proinflammatory cytokines through a MyD88-dependent signalling
pathway in response to both yeasts and hyphae. However, since
zymosan and fungal stimuli still retained some cytokine-inducing
capacity in the absence of TLR2, obviously other receptors (such as
dectin-1 and others) may be involved in this process. Furthermore,
the differences in the macrophage secretory response to
C. albicans yeasts and hyphae [8, 11] may involve PRRs other
than TLR2, such as dectin-1 and galectin-3, that may recognize
different PAMPs exposed on the yeast and/or hypha cell surface and
collaborate with TLR2.
Acknowledgements
This work was supported by grants PI03/0647, PI04/1472 (Ministerio
de Sanidad y Consumo, Spain) and ACOMP06/51 and ACOMP06/43
(Generalitat Valenciana). We thank Dr. S. Akira (Osaka University,
Japan) for providing TLR2 and TLR4 knockout mice. C. M. and A.Y.
are the recipients of a fellowship from the Ministerio de Educación
y Ciencia (Spain) and from the University of Valencia,
respectively.
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