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© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e‐mail: [email protected].
Dectin-1 Induces M1 Macrophages and Prominent Expansion of
CD8+IL-17+ Cells in Pulmonary Paracoccidiodomycosis
Flávio V. Loures1, Eliseu F. Araújo1, Claudia Feriotti1, Silvia B. Bazan1, Tânia A.
Costa1, Gordon D. Brown2 and Vera L. G. Calich1
1Departamento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São
Paulo, São Paulo, SP, Brazil
2Aberdeen Fungal Group, Section of Immunity and Infection, Institute of Medical
Sciences, University of Aberdeen, Aberdeen, UK
Corresponding Author: Vera L. G. Calich, Departamento de Imunologia, Instituto
de Ciências Biomédicas da Universidade de São Paulo, Av. Prof. Lineu Prestes 1730,
CEP 05508-900, São Paulo, SP, Brazil. Phone: 55-11- 30917397. Fax: 55-11-30917224.
E-mail: [email protected]
Journal of Infectious Diseases Advance Access published March 5, 2014 at U
niversidade de São Paulo on M
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Abstract
Dectin-1, the innate immune receptor that recognizes -glucan, plays an important role
in immunity against fungal pathogens. Paracoccidioides brasiliensis, the etiological
agent of paracoccidioidomycosis, has a sugar-rich cell wall mainly composed of
mannans and glucans. This fact motivated us to use dectin-1-sufficient and deficient
mice to investigate the role of -glucan recognition in the immunity against pulmonary
paracoccidioidomycosis. Initially, we verified that P. brasiliensis infection reinforced
the tendency of dectin-1 deficient macrophages to express an M2 phenotype. This
prevalent anti-inflammatory activity of dectin1-/- macrophages resulted in impaired
fungicidal ability, low nitric oxide production and elevated synthesis of IL-10.
Compared with dectin-1-sufficient mice, the fungal infection of dectin-1-/- mice was
more severe and resulted in enhanced tissue pathology and mortality rates. The absence
of dectin-1 has also impaired the production of Th1/Th2/Th17 cytokines and the
activation and migration of T cells to the site of infection. Remarkably, dectin-1
deficiency increased the expansion of regulatory T cells and reduced the differentiation
of T cells to the IL-17+ phenotype, impairing the migration of IL-17+CD8+ T cells and
PMN neutrophils to infected tissues. In conclusion, dectin-1 exerts an important
protective role in pulmonary paracoccidioidomycosis by controlling the innate and
adaptive phases of anti-fungal immunity.
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Introduction
Among several families of pattern recognition receptors (PRRs), the family of
C-type lectin receptors has emerged as a major sensor of pathogens by recognizing
carbohydrate moieties on pathogens [1, 2]. The most studied C-type lectin receptor is
dectin- 1, which contains an extracellular C-type lectin domain and an intracellular
immunoreceptor tyrosine-based activation motif (ITAM)-like motif that initiates cell
signaling and activation following interaction with several fungal pathogens [3-5].
Dectin-1 recognizes β-1-3-glucans present on the cell wall of medically important fungi
[3, 4]. Following ligation of β-glucans to dectin-1, a large number of cellular events
such as phagocytosis, activation of signaling pathways, generation of reaction oxygen
species and release of cytokines occur [4, 6]. These events can directly affect the quality
and quantity of the adaptive immune response. Several studies have shown that mouse
and human DCs that have taken up antigens via dectin-1 present antigenic peptides to
both CD4+ and CD8+ T cells, resulting in potent Ag-specific CD4 and CD8+ T cell
responses [7-11]. In addition, the cellular activation mediated by this receptor can drive
host adaptive immunity to a prevalent Th17 response [12-17].
Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by
the dimorphic fungus Paracoccidioides brasiliensis and constitutes the most prevalent
deep mycosis in Latin America. In previous studies, our group showed that TLR2
deficiency leads to increased Th17 immunity, which was associated with diminished
expansion of regulatory T cells (Treg) and increased lung pathology due to unrestrained
inflammatory reactions. In addition, a more severe P. brasiliensis infection associated
with increased production of Th17 cytokines, enhanced proinflammatory immunity and
impaired expansion of regulatory T cells was shown to be influenced by TLR4
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expression [18-21]. However, the role of dectin-1 in PCM was not investigated
thoroughly. Nevertheless, a recent report indicated that dectin-1 was involved in the
internalization of P. brasiliensis by human monocytes and neutrophils, suggesting that it
has a function in immunity against P. brasiliensis infection [22]. Herein, we found that
dectin-1 receptor controls the innate and adaptive phases of the immune response in P.
brasiliensis-infected mice. The absence of dectin-1 expression induces a preferential
differentiation of macrophages to an anti-inflammatory, M2-like phenotype that results
in decreased fungicidal ability and nitric oxide (NO) production. Compared to wild type
(WT) mice, the fungal infection and tissue pathology is more severe in dectin-1-/- mice,
leading to increased mortality rates. Altogether, our studies demonstrate for the first
time the crucial role of dectin-1 in the protective immunity developed by P. brasiliensis-
infected hosts.
Materials and Methods
Fungus
P. brasiliensis Pb18, a highly virulent isolate [23] was maintained by weekly
sub-cultivation in semisolid culture medium at 37o C. Washed yeast cells were adjusted
to 20 × 106 cells/mL (for in vivo infection) and 4 × 104 cells/mL (for in vitro infection).
Viability was determined with Janus Green (Merck) and was always higher than 85%.
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Mice and intratracheal infection
Eight to 12-week old male C57BL/6 Clec7a-/- (Dectin1 -/-) and wild type (WT)
mice were obtained from the pathogen free Isogenic Breeding Unit of the Institute of
Biomedical Sciences, University of São Paulo. All animal procedures were approved by
the Ethics Committee on Animal Experiments of our Institute (Proc.76/04/CEEA). Mice
were anesthetized and submitted to intratracheal (i.t.) infection with 1x106 yeast cells as
previously described [20, 24].
Phagocytic, fungicidal, and flow cytometric assays of macrophages
Thioglycollate-induced peritoneal macrophages were isolated by adherence to
culture plates (1x106 cells/well) and cultivated overnight with medium (DMEM, Sigma)
containing 10% fetal calf serum, 100 U/mL penicillin and 100 뼈g/mL streptomycin in
the presence or absence of recombinant IFN-뼈 (20 ng/mL, BD-Pharmingen). For
phagocytic assays, macrophages were infected with heat-inactivated FITC-labeled P.
brasiliensis yeasts at a macrophage:yeast ratio of 1:1 for 4 h at 37°C in 5% CO2. Fungi
adhesion/ingestion was measured by flow cytometry using detached macrophages
labeled with APC anti-F4/80 antibodies as previously described [25, 26]. For fungicidal
assays, macrophages were infected with P.brasiliensis yeasts in a macrophage:yeast
ratio of 25:1 and cocultivated for 2 h at 37o C in 5% CO2 [27]. The monolayers were
then washed and incubated for 48h. Plates were centrifuged (400xg, 10 min, 4o C) and
supernatants stored at –70o C. The wells were washed with distilled water and 100 뼈l
of cell homogenates were assayed for the presence of viable yeasts. For TLR2 and
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TLR4 expression, macrophages were detached from plastic with fresh cold medium and
a rubber cell scraper on ice and then labeled with anti-F4/80, TLR2 and TLR4 labeled
(eBioscience) for 20 min at 4o C. The samples were analyzed by flow cytometry
(FACSCalibur, BD-Pharmingen).
CFU assays, mortality rates and histological analysis
The numbers of viable microorganisms in cell cultures and infected organs were
determined by counting the number of colony forming units (CFU) as previously
described [28]. The numbers (log10) of viable P. brasiliensis per gram of tissue (in vivo)
or per mL of cell homogenate (in vitro) were determined, and expressed as the means
SEM. Mortality studies were done with groups of 10-12 mice inoculated i.t. with 1x 106
yeast cells. Deaths were registered daily and experiments were repeated twice. For
histological examinations, the left lung of infected mouse was removed and fixed in
10% formalin. Five m sections were stained by the hematoxilin-eosin (H&E) for an
analysis of the lesions and silver stained (Grocott stain) for fungal evaluation.
Morphometrical analysis was performed using a Nikon DXM 1200c camera and Nikon
NIS AR 2.30 software. The area of lesions was measured (in µm2) in 10 microscopic
fields per slide in 5 mice per group. Results are expressed as the mean ± SEM of total
area of lesions for each mouse.
Measurement of cytokines and NO
Cytokines levels were measured by capture ELISA (eBiosciences) according to
the manufacturer's protocol. NO production was quantified by a standard Griess reaction
[29]. All determinations were performed in triplicate and expressed as µM NO.
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Assessment of leukocyte subpopulations
After 2 and 8 weeks of infection, lungs were digested enzymatically for 30
minutes with collagenase (1 mg/mL) in culture medium (Sigma). Total lung leukocyte
numbers were assessed in the presence of trypan blue and viability was always >85%.
For differential leukocyte counts, samples of lung cell suspensions were cytospun
(Shandon Cytospin) onto glass slides and stained by the Diff-Quik blood stain (Baxter
Scientific). A total of 200 to 400 cells were counted from each sample. For flow
cytometry, lung leukocytes were resuspended at 106 cells/mL in staining buffer. Anti-
CD44, CD25, CD62L, CD69, CD4 and CD8 monoclonal antibodies (eBiosciences)
were used. Cells were fixed with 1% paraformaldehyde (Sigma) and analyzed in a
FACSCANTO flow cytometer (BD Pharmingen). For PCR experiments, macrophages
were isolated from lung leukocytes suspensions by positive selection using anti-CD11b-
coated magnetic beads (Miltenyi Biotec).
Quantitative Real-Time PCR
Total RNA from in vitro infected macrophages and lung CD11b+ cells obtained
at week 2 after infection was extracted using Trizol (Invitrogen) reagent, reverse
transcribed, and cDNA amplified. Real-time PCR was performed using TaqMan
universal master mix. First-strand cDNAs were synthesized from 2 µg RNA using the
High Capacity RNA-to-cDNA kit (Applied Biosystems). Real-time polymerase chain
reaction (RT-PCR) was performed using the TaqMan real-time PCR assay (Applied) for
the following molecules: TLR2, TLR4, suppressor of cytokine signaling-3 (SOCS3),
suppressor of cytokine signaling-1 (SOCS1), arginase 1 (ARG1), NO-synthase 2
(NOS2), found in inflammatory zone protein (FIZZ1), and chitinase-3 like 3 (Chi3l3 or
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Ym1). Analysis was performed with the ABI PRISM 7000 sequence detection system
(Applied). All values were normalized to GAPDH, and the relative gene expression was
calculated using the Pfaffl method [30].
Intracellular staining
Lung leukocytes were stimulated for 6h in complete medium in the presence of
50 ng/mL phorbol 12-myristate 13-acetate, 500 ng/mL ionomycin (Sigma-Aldrich) and
monensin (3 mM, eBioscience). After surface staining for CD4, CD8, CD25, and CD69
some cultures were fixed, permeabilized, and stained by anti-IFN-γ, IL-4 and anti-IL17
antibodies (eBioscience). The expression of cell surface markers, as well as the
intracellular expression of IL-4, IFN-γ and IL-17 in lung infiltrating leucocytes, were
analyzed in a FACSCANTO flow cytometer (BD Pharmingen) and the FlowJo software
(Tree Star).
Statistical analysis
Data are expressed as the mean ± SEM. Differences between groups were
analyzed by Student's t test or analysis of variance (ANOVA) followed by the Tukey
test. Differences between survival times were determined with the LogRank test. Data
were analyzed using GraphPad Prism 5.01 software for Windows. Error bars represent
± SEM; p values ≤ 0.05 were considered significant.
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Results
Absence of dectin-1 leads to decreased phagocytic and fungicidal abilities and NO
production by P. brasiliensis-infected macrophages.
Macrophages from dectin1-/- and WT mice were infected with P. brasiliensis
yeasts. Phagocytosis was determined by flow cytometry using fluorescent labeled yeasts
and macrophages. As shown in Figure 1A, dectin-1−/− macrophages showed a decreased
frequency of ingested/adhered yeast cells. In addition, CFU assays demonstrated an
increased number of viable yeasts recovered from dectin-1−/− macrophages (Figure 1B).
The levels of NO and cytokines were determined in cell supernatants of CFU assays and
diminished levels of NO (Figure 1C) and IL-6 (Figure 1D) were produced by dectin-1-/-
macrophages. Furthermore, deficient macrophages produced increased levels of MCP-1
and IL-10, an important macrophage-deactivating cytokine (Figure 1D).
Dectin-1 controls the expression of TLRs in vitro
To further explore the influence of dectin-1 in the recognition of P. brasiliensis
yeasts, the expression of other pathogen recognition receptors (PRRs) previously known
to be involved in the interaction of this fungal pathogen with macrophages [18-20] was
also evaluated. As seen in Figure 2A and B, P. brasiliensis-infected dectin-1-/- mice
presented decreased percentage of TLR4+ and a higher percentage of TLR2+
macrophages when compared with WT mice. To confirm the opposite expression of
TLR2 and TLR4 molecules in macrophages, the levels of mRNA to TLR2 and TLR4
were measured. As depicted in Figure 2C and D, the mRNA levels of TLR4 and TLR2
confirm the opposite expression of these receptors by WT and dectin-1-/- macrophages.
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The absence of dectin-1 determines a prevalent differentiation of macrophages to
an M2-like phenotype
M1 macrophages are associated with NO production and enhanced microbicidal
activity, whereas M2 macrophages promote healing and tissue repair but show impaired
microbicidal activity [31]. The diverse behavior of dectin-1-/- and WT macrophages led
us to suppose that the expression of dectin-1 was inducing a prevalent inflammatory or
M1-like differentiation in WT macrophages but its absence was stimulating a
predominant anti-inflammatory of M2-like differentiation. As can be seen in Figure 2E,
macrophages from uninfected WT mice showed a prevalent expression of M1-
polaryzed cells (SOCS3), whereas dectin-1-/- macrophages expressed elevated levels of
M2-associated markers (Ym1, ARG1, and SOCS1). P. brasiliensis infection induced
enhanced levels of mRNA but was not able to alter the prevalent M1 and M2 behavior
of dectin-1-sufficient and -deficient macrophages, respectively. Importantly, P.
brasiliensis infection enhanced the differences of M1 (NOS2 and SOCS3) and M2
(YM1, ARG1, SOCS1 and FIZZ-1) markers between WT and dectin-1-deficient
macrophages. Therefore, P. brasiliensis infection appears to intensify the differentiation
of dectin-1-/- macrophages to an M2 phenotype.
Absence of dectin-1 receptor increases mortality rates associated with increased
fungal loads and tissue pathology
The severity of fungal infection was assessed at early and late periods of the
disease. Pulmonary fungal burdens were increased in dectin-1-/- mice at 48h as well as at
2 and 8 weeks after infection (Figure 3A, B, and C). At week 8, CFUs were also
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increased in the livers and spleens of the deficient mice (Figure 3C). In addition, an
increased number of non- organized lesions containing high numbers of fungal cells and
intense tissue destruction were observed in dectin-1-/- mice (Figure 3D-K). Pulmonary
lesions in dectin-1-/- mice replaced large part of normal tissue, and were composed of
confluent necrotic lesions containing many budding yeasts (Figure 3G), surrounded by
inflammatory cells (Figure 3E). The lesions in the lungs of WT mice occupied a small
area and were composed of organized granulomas of small sizes (Figure 3D, F). The
hepatic lesions of WT were composed of scarce and isolated granulomas whereas in
dectin-1-/- mice a high number of granulomas with elevated number of yeasts occupied a
large area of the organ (Figure 3H-K). Compared with WT mice, the less organized and
more frequent pulmonary and hepatic lesions of dectin-1-/- mice resulted in increased
areas of damaged tissue (Figure 3L). To assess the influence of dectin-1 deficiency in
the disease outcome, mortality of infected mice was registered daily. As shown in
Figure 3M, at the 137th day of infection all dectin-1-/- mice were dead. In the same
period, 5 out of 12 WT mice were still alive and apparently healthy.
Dectin-1 controls the recruitment of PMN cells to the lungs
To better characterize the influence of dectin-1 expression in the inflammatory
reaction induced by P. brasiliensis infection, leukocyte recruitment to the lungs of P.
brasiliensis-infected dectin-1-/- and WT mice was studied. A reduced number of total
leukocytes and a decreased frequency and numbers of polymorphonuclear cells (PMN)
were observed in the lungs of dectin-1-/- mice. No important differences were noted in
the numbers of macrophages and lymphocytes (Figure 4A and B).
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Absence of dectin-1 results in reduced levels of cytokines
By eight weeks after infection, reduced levels of Th1 (IL-12, TNF-α, IFN-γ),
Th2 (IL-4 and IL-10), Th17 cytokines (TGF-β, IL-6, IL-17 and IL-23) and MCP-1
chemokine were detected in the lungs of dectin-1-/- mice (Figure 4B). In addition,
reduced levels of IL-1β, the cytokine produced upon inflammasomes activation, were
also detected in the lungs of dectin-1-/- mice. No major differences were found in the
liver, but IL-12 was found in reduced levels in dectin-1-/- mice. When splenic cytokines
were measured, decreased levels of IFN-γ, IL-10, TGF-β, IL-17 and IL-1β were
detected in dectin-1-/- mice (Figure S1).
Dectin-1 signaling increases the differentiation of CD4+ and CD8+ T cells
To determine the role of dectin-1 receptor in the acquired phase of immunity
against P. brasiliensis, the phenotype and activation of lung inflammatory cells were
analyzed. When lymphocytes were phenotyped (Figure 5A), a reduced number of
activated/effector CD4+CD25+ and CD4+CD44highCD62Llow T cells were observed in
the lungs of dectin-1-/- mice at week 2 of infection (Figure 5B). By week 8
postinfection, only CD4+CD25+ T cells appeared in reduced numbers in the lungs of
dectin-1-/- mice (Figure 5C). A significantly reduced recruitment of total, naïve and
activated/effector (CD8+, CD8+CD44lowCD62Lhigh, CD8+CD44highCD62Llow) T cells
was observed in the lungs of dectin-1-/- mice at week 2 of infection (Figure 5D).
However, at week 8 after infection only CD8+CD44highCD62Llow appeared in reduced
numbers in the lungs of dectin-1 -/- mice (Figure 5E).
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Absence of dectin-1 promotes down regulation of IL-17-producing CD8+
T cells associated with regulatory T cell expansion
To better clarify the importance of dectin-1 in the polarization of T cell
responses, the phenotype of IL-17, IFN-γ and IL-4-producing cells was defined in the
inflammatory infiltrates of lungs (Figure 6A). As shown in Figure 6B and C,
significantly diminished numbers of CD8+ IL-17+ T cells were detected in the lungs of
dectin-1-/- mice, but no differences in the numbers of CD4+ IL-17+ T cells were detected.
Similar numbers of IL-4 and IFN- producing cells were also observed. These findings
indicate that the absence of dectin-1 receptor induces an impaired migration of IL-
17+CD8+ T cells, also known as Tc17 cells, to the lungs of dectin-1-/- mice.
Additionally, increased frequency and numbers of CD4+CD25+FoxP3+ Treg cells were
observed in the lung inflammatory exudates of dectin-1-/- mice (Figure 6D e E).
Discussion
In response to fungal cell wall components, dectin-1 induces intracellular
signaling that promote the activation of transcription factors (NFkB and NFAT) which
control the production of cytokines, and chemokines as well as the release of reactive
oxygen intermediates and eicosanoids [32-35].
In this study, we verified that dectin-1-/- macrophages ingested decreased
numbers of yeasts, but allowed increased growth of P. brasiliensis compared to WT
cells. Dectin-1-/- macrophages also showed impaired NO production suggesting that
dectin-1 receptor participates in the recognition of P. brasiliensis and in the induction of
cellular mechanisms that control fungal growth. The higher levels of IL-10 produced by
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dectin-1-/- macrophages may have contributed to the ineffective activation of these cells.
Indeed, WT cells expressed elevated levels of SOCS3, indicating a tendency to an
inflammatory "M1-like" profile, whereas dectin-1-/- macrophages showed elevated
levels of Ym1, ARG1, FIZZ1, and SOCS1, typical markers of alternatively activated
macrophages, suggesting a prevalent "M2-like" differentiation. Importantly, P.
brasiliensis infection reinforced the expression of M2 markers by dectin-1-deficient
macrophages, possibly influencing the ineffective immunity developed by dectin-1-/-
mice. However, a recent report showed an association between M2 markers and dectin-
1 expression, indicating that not all fungal ligands behave similarly [36].
Although the expression of TLR4 and TLR2 (protein and mRNA) was not
affected by P. brasiliensis infection of WT macrophages, dectin-1-/- cells expressed
reduced levels of TLR4 and increased levels of TLR2. Interestingly, our previous
studies have shown the inhibitory and stimulatory effects of TLR2 and TLR4,
respectively, on Th17 immunity against P. brasiliensis infection [18, 20, 21]. Thus, it is
possible that the inhibited Th17 response of dectin-1-/- mice was influenced by the
increased expression of TLR2 and the lower expression of TLR4 detected in dectin-1-/-
macrophages.
Consistent with the in vitro data, the results of in vivo CFU assays showed a
more severe infection in dectin-1-/- mice than in WT mice. Importantly, at the chronic
phase, the increased fungal loads were concomitant with reduced levels of IL-1β, Th1,
Th2 and Th17 cytokines, indicating a major role for dectin-1 in the differentiation of all
T cell subsets.
Some studies have described the involvement of dectin-1 in the cleavage of pro-
IL-1β and pro-IL-18 into their active forms by caspase-1 or other inflammatory or
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pathogen-derived proteases. As an example, Hise et al. [37] demonstrated that the
interaction of Candida albicans with TLR2 and dectin-1 regulates the production of IL-
1β via the NLRP3 inflammasome caspase-1-dependent pathway. Using MyD88-/- mice,
we have previously demonstrated an association between disease severity and reduced
Th17 response and IL-1 production [21]. Thus, the decreased levels of IL-1 here
observed could have contributed to the suppressed T cell immunity developed
by dectin-1-/- mice.
The diminished synthesis of IL-1β, TGF-β, IL-6 and IL-23 was linked to the
defective CD8+17+ T cell responses developed by dectin-1-/- mice. Recent studies have
described the involvement of dectin-1 in the induction of Th17 immune response [38,
39]. Besides, the proliferation of antigen-specific CD8+ T cells and the in vivo cross
priming of cytotoxic T lymphocytes were reported to be mediated by dectin-1 signaling
[9]. Similar responses were observed in mice and humans infected by fungal pathogens
[40] and were here confirmed and expanded in pulmonary paracoccidioidomycosis.
Th17 immunity is generally associated with enhanced synthesis of CXC
chemokines and the induction of neutrophil chemotaxis to inflammatory sites [41, 42].
Here, the reduced neutrophil influx into the lungs of infected dectin-1-/- mice was
concurrent with decreased Th17 cytokine production. This finding is consistent with our
previous report showing that Th17 polarization in pulmonary PCM is associated with
PMN-rich inflammatory reactions [18, 20, 21].
The less organized and more severe lesions observed in the histopathology
study, the elevated fungal burdens, and the increased fungal dissemination to several
organs appear to have contributed to the increased mortality rates of dectin-1-/- mice.
Importantly, this profile was associated with impaired activation of effector/memory
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CD4+ and CD8+ T cells concomitantly with increased number and frequency of
CD4+CD25+FoxP3+ Treg cells at the site of infection. Thus, in pulmonary PCM, the
dectin-1 seems to be involved in the modulation of adaptive immunity, and its
expression contributes to the development of efficient T cell immunity modulated by
moderate expansion of Treg cells.
Our laboratory characterized the role of CD4+ and CD8+ T cells in murine PCM
and demonstrated that, whatever the pattern of host susceptibility, in pulmonary PCM
the fungal loads are primarily controlled by CD8+ T cells [43, 44]. Several studies have
demonstrated the importance of dectin-1 in the activation of CD8+ T lymphocytes as
well as in their cytotoxic activity [9-11]. Human DCs activated with curdlan, act as
efficient APCs and induce prevalent Th17 and CD8+ T cell responses [10, 11]. Our data
on intracellular cytokines confirmed the prominent involvement of dectin-1 in the
development of CD8+ T cells and their polarization toward IL-17 production. A reduced
number of infiltrating CD8+IL-17+ T cells was detected at weeks 2 and 8 after infection
of dectin-1-/- mice indicating that in pulmonary PCM dectin-1 expression has a more
critical influence in the differentiation and migration of IL-17+CD8+ T cells than CD4+
T cells.
In conclusion, the absence of the dectin-1 appears to impair inflammatory innate
immunity as evidenced by the M2-like profile of macrophages, which present impaired
fungicidal ability. Moreover, dectin-1 deficiency suppresses the development of
protective adaptive immunity, as shown by the decreased production of Th1, Th2 and
Th17 cytokines, and diminished activation and migration of CD4+ and CD8+ T cells to
the site of infection. This defective innate and adaptive immunity of dectin-1-/- mice,
which was concomitant with increased Treg expansion, resulted in uncontrolled growth
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and dissemination of the fungal cells, which consistently compromises the survival of P.
brasiliensis-infected hosts.
Funding – This project was supported by Fundação de Amparo à Pesquisa do Estado
de São Paulo (FAPESP), Research Grant VLGC 2010/52275-5). FAPESP Postdoctoral
fellowships (FVL, SBB); CAPES Postdoctoral fellowship (CF).
Acknowledgements
We are grateful to Paulo Albee for his invaluable technical assistance.
Footnotes
Competing Interests: The authors declare that no competing interests exist.
Funding: This project was supported by Fundação de Amparo à Pesquisa do Estado
de São Paulo (FAPESP), Research Grant VLGC 2010/52275-5). FAPESP Postdoctoral
fellowships (FVL, SBB); CAPES Postdoctoral fellowship (CF).
Meetings: Previous data were presented at the Second International Gordon
Conference on Immunology of Fungal Infections, held in Galveston, Texas, USA, 2013,
and at the International Congress of Immunology, Milan, Italy, 2013.
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References
1. Brown GD. Innate antifungal immunity: the key role of phagocytes. Annu Rev
Immunol 2011; 29:1-21.
2. Hardison SE, Brown GD. C-type lectin receptors orchestrate antifungal immunity.
Nat Immunol 2012; 13:817-822.
3. Hohl TM, van Epps HL, Rivera A, Morgan LA, Chen PL, Feldmesser M, Pamer EG.
Aspergillus fumigatus triggers inflammatory responses by stage-specific beta-glucan
display. PLoS Pathog 2005; 1:e30.
4. Taylor PR, Tsoni SV, Willment JA, Dennehy KM, Rosas M, Findon H, Haynes, C.
Steele K, Botto M, Gordon S, Brown GD. Dectin-1 is required for beta-glucan
recognition and control of fungal infection. Nat Immunol 2007; 8:31-38.
5. Brown GD. Dectin-1: a signalling non-TLR pattern-recognition receptor. Nat Rev
Immunol 2006; 6:33-43.
6. Huang H, Ostroff GR, Lee CK, Agarwal S, Ram S, Rice PA, Specht CA, Levitz
SM. Relative contributions of dectin-1 and complement to immune responses to
particulate β-glucans. J Immunol 2012; 189:312-317.
7. Weck MM, Appel S, Werth D, Sinzger C, Bringmann A, Grünebach F, Brossar P.
hDectin-1 is involved in uptake and cross-presentation of cellular antigens. Blood 2008;
111:4264-4272.
at Universidade de SÃ
£o Paulo on March 7, 2014
http://jid.oxfordjournals.org/D
ownloaded from
Acce
pted M
anus
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19
8. Carter RW, Thompson C, Reid DM, Wong SY, Tough DF. Preferential induction of
CD4+ T cell responses through in vivo targeting of antigen to dendritic cell-associated
C-type lectin-1. J Immunol 2006; 177:2276-2284.
9. Leibundgut-Landmann,S, Osorio F, Brown GD, Reis e Sousa C. Stimulation of
dendritic cells via the dectin-1/Syk pathway allows priming of cytotoxic T-cell
responses. Blood 2008; 112:4971-4980.
10. Ni L, Gayet I, Zurawski S, Duluc D, Flamar AL, Li XH, O'Bar A, Clayton S,
Palucka AK, Zurawski G, Banchereau J, Oh S. Concomitant activation and antigen
uptake via human dectin-1 results in potent antigen-specific CD8+ T cell responses. J
Immunol 2010; 185:3504-3513.
11. Agrawal S, Gupta S, Agrawal A. Human dendritic cells activated via dectin-1 are
efficient at priming Th17, cytotoxic CD8 T and B cell responses. PLoS One 2010;
5:e13418.
12. Reid DM, Gow NA, Brown GD. Pattern recognition: recent insights from Dectin-1.
Curr Opin Immunol 2009; 21:30-37.
13. Chamilos G, Ganguly D, Lande R, Gregorio J, Meller S, Goldman WE, Gilliet M,
Kontoyiannis DP. Generation of IL-23 producing dendritic cells (DCs) by airborne
fungi regulates fungal pathogenicity via the induction of T(H)-17 responses. PLoS One
2010; 5:e12955.
at Universidade de SÃ
£o Paulo on March 7, 2014
http://jid.oxfordjournals.org/D
ownloaded from
Acce
pted M
anus
cript
20
14. Cheng SC, van de Veerdonk FL, Lenardon M, Stoffels M, Plantinga T, Smeekens
S, Rizzetto L, Mukaremera L, Preechasuth K, Cavalieri D, Kanneganti TD, van der
Meer JW, Kullberg BJ, Joosten LA, Gow NA, Netea MG. The dectin-
1/inflammasome pathway is responsible for the induction of protective T-helper 17
responses that discriminate between yeasts and hyphae of Candida albicans. J Leukoc
Biol 2011; 90:357-366.
15. Rivera A, Hohl TM, Collins N, Leiner I, Gallegos A, Saijo S, Coward JW, Iwakura
Y, Pamer EG. Dectin-1 diversifies Aspergillus fumigatus-specific T cell responses by
inhibiting T helper type 1 CD4 T cell differentiation. J Exp Med 2011; 208:369-381.
16. Carvalho A, Giovannini G, De Luca A, D'Angelo C, Casagrande A, Iannitti RG,
Ricci G, Cunha C, Romani L. Dectin-1 isoforms contribute to distinct Th1/Th17 cell
activation in mucosal candidiasis. Cell Mol Immunol 2012; 9:276-286.
17. Zenaro E, Donini M, Dusi S. Induction of Th1/Th17 immune response by
Mycobacterium tuberculosis: role of dectin-1, Mannose Receptor, and DC-SIGN. J
Leukoc Biol 2009; 86:1393-1401.
18. Loures FV, Pina A, Felonato M, Calich VL. TLR2 is a negative regulator of Th17
cells and tissue pathology in a pulmonary model of fungal infection. J Immunol 2009;
183:1279-1290.
at Universidade de SÃ
£o Paulo on March 7, 2014
http://jid.oxfordjournals.org/D
ownloaded from
Acce
pted M
anus
cript
21
19. Calich VL, Pina A, Felonato M, Bernardino S, Costa TA, Loures FV. Toll-like
receptors and fungal infections: the role of TLR2, TLR4 and MyD88 in
paracoccidioidomycosis. FEMS Immunol Med Microbiol 2008; 53:1-7.
20. Loures FV, Pina A, Felonato M, Araújo EF, Leite KR, Calich VL. TLR4 signaling
leads to a more severe fungal infection associated with enhanced proinflammatory
immunity and impaired expansion of regulatory T cells. Infect Immun 2010; 78:1078-
1088.
21. Loures FV, Pina A, Felonato M, Feriotti C, de Araújo EF, Calich VL. MyD88
signaling is required for efficient innate and adaptive immune responses to
Paracoccidioides brasiliensis infection. Infect Immun 2011; 79:2470-2480.
22. Bonfim CV, Mamoni RL, Blotta MH. TLR2, TLR4 and dectin-1 expression in
human monocytes and neutrophils stimulated by Paracoccidioides brasiliensis. Med
Mycol 2009; 47:722-733.
23. Kashino SS, Calich VL, Burger E, Singer-Vermes LM. In vivo and in vitro
characteristics of six Paracoccidioides brasiliensis strains. Mycopathol 1985; 92:173-
178.
24. Cano LE, Singer-Vermes LM, Vaz CA, Russo M, Calich VLG. Pulmonary
paracoccidioidomycosis in resistant and susceptible mice: relationship among
progression of infection, bronchoalveolar cell activation, cellular immune response, and
specific isotype patterns. Infect Immun 1995; 63:1777-1783.
at Universidade de SÃ
£o Paulo on March 7, 2014
http://jid.oxfordjournals.org/D
ownloaded from
Acce
pted M
anus
cript
22
25. Bernardino S, Pina A, Felonato M, Costa TA, Araújo EF, Feriotti C, Bazan
SB, Keller AC, Leite KR, Calich VL. TNF-α and CD8+ T cells mediate the beneficial
effects of nitric oxide synthase-2 deficiency in pulmonary paracoccidioidomycosis.
PLoS Negl Trop Dis 2013; 78:e2325.
26. Costa TA, Bazan SB, Feriotti C, Araújo EF, Bassi Ê, Loures FV, Calich VL. In
pulmonary paracoccidioidomycosis IL-10 deficiency leads to increased immunity and
regressive infection without enhancing tissue pathology. PLoS Negl Trop Dis 2013;
7:e2512.
27. Pina A, Bernardino S, Calich VL. Alveolar macrophages from susceptible mice are
more competent than those of resistant mice to control initial Paracoccidioides
brasiliensis infection. J Leukoc Biol 2008; 835:1088-1099.
28. Singer-Vermes LM, Caldeira CB, Burger E, Calich VLG. Experimental murine
paracoccidioidomycosis: relationship among dissemination of the infection, humoral
and cellular immune responses. Clin Exp Immunol 1992; 94:75-79.
29. Ding AH, Nathan CF, Stuehr DJ. Release of reactive nitrogen intermediates and
reactive oxygen intermediates from mouse peritoneal macrophages. J Immunol 1998;
141:2407-2412.
at Universidade de SÃ
£o Paulo on March 7, 2014
http://jid.oxfordjournals.org/D
ownloaded from
Acce
pted M
anus
cript
23
30. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-
PCR. Nucleic Acids Res 2001; 29:e45.
31. Gordon S, Martinez FO. Alternative activation of macrophages: mechanism and
functions. Immunity 2010; 32:593-604.
32. Netea MG, Brown GD, Kullberg BJ, Gow NA. An integrated model of the
recognition of Candida albicans by the innate immune system. Nat Rev Microbiol
2008; 6:67-78.
33. Xu S, Huo J, Lee KG, Kurosaki T, Lam KP. Phospholipase Cgamma2 is critical for
Dectin-1-mediated Ca2+ flux and cytokine production in dendritic cells. J Biol Chem
2009; 284:7038-7046.
34. LeibundGut-Landmann S, Wüthrich M, Hohl TM. Immunity to fungi. Curr Opin
Immunol 2012; 24:449-458.
35. Hardison SE, Brown GD. C-type lectin receptors orchestrate antifungal immunity.
Nat Immunol 2012; 139:817-822.
36. Elcombe SE, Naqvi S, Van Den Bosch MW, MacKenzie KF, Cianfanelli F, Brown
GD, Arthur JS. Dectin-1 regulates IL-10 production via a MSK1/2 and CREB
dependent pathway and promotes the induction of regulatory macrophage markers.
PLoS One 2013; 8:e60086.
at Universidade de SÃ
£o Paulo on March 7, 2014
http://jid.oxfordjournals.org/D
ownloaded from
Acce
pted M
anus
cript
24
37. Hise AG, Tomalka J, Ganesan S, Patel K, Hall BA, Brown GD, Fitzgerald KA. An
essential role for the NLRP3 inflammasome in host defense against the human fungal
pathogen Candida albicans. Cell Host Microbe 2009; 5:487-497.
38. LeibundGut-Landmann S, Gross O, Robinson MJ, Osorio F, Slack EC, Tsoni SV,
Schweighoffer E, Tybulewicz V, Brown GD, Ruland J, Reis e Sousa C. Syk- and
CARD9-dependent coupling of innate immunity to the induction of T helper cells that
produce interleukin 17. Nat Immunol 2007; 8:630-638.
39. Osorio F, LeibundGut-Landmann S, Lochner M, Lahl K, Sparwasser T, Eberl G,
Reis e Sousa C. DC activated via dectin-1 convert Treg into IL-17 producers. Eur J
Immunol 2008; 38:3274-3281.
40. Acosta-Rodriguez EV, Rivino L, Geginat J, Jarrossay D, Gattorno M, Lanzavecchia
A, F Sallusto, Napolitani G. Surface phenotype and antigenic specificity of human
interleukin 17-producing T helper memory cells. Nat Immunol 2007; 8:639-646.
41. Miyamoto M, Prause O, Sjöstrand M, Laan M, Lötvall J, Linden A. Endogenous IL-
17 as a mediator of neutrophil recruitment caused by endotoxin exposure in mouse
airways. J Immunol 2003; 170:4665-4672.
42. Kolls JK, Linden A. Interleukin-17 family members and inflammation. Immunity
2004; 21:467-476.
at Universidade de SÃ
£o Paulo on March 7, 2014
http://jid.oxfordjournals.org/D
ownloaded from
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pted M
anus
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25
43. Chiarella AP, Arruda C, Pina A, Costa TA, Ferreira RC, Calich VLG. The relative
importance of CD4+ and CD8+T cells in immunity to pulmonary
paracoccidioidomycosis. Microbes Infect 2007; 9:1078-1088.
44. Arruda C, Kashino SS, Fazioli RA, Calich VL. A primary subcutaneous infection
with Paracoccidioides brasiliensis leads to immunoprotection or exacerbated disease
depending on the route of challenge. Microbes Infect 2007; 9:308-316.
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Figure Legends
Figure 1. Absence of dectin-1 impairs the phagocytic and fungicidal abilities of
macrophages and alters nitric oxide and cytokines production. (A) For phagocytic
assays, IFN-γ-primed and unprimed peritoneal macrophages from dectin-1-/- and WT
mice were infected with heat-inactivated FITC-labeled P. brasiliensis yeasts at a
macrophage yeast ratio of 1:1 for 4 h at 37°C in 5% CO2. Cell suspension were then
obtained, macrophages labeled with APC anti-F4/80 antibodies and fungi
adhesion/ingestion measured by flow cytometry. (B) For fungicidal assay IFN-γ-primed
and unprimed macrophages were infected with P. brasiliensis yeasts in a
macrophage:yeast ratio of 25:1 for 2h. Infected macrophages were then cultivated for 48
h at 37o C in 5% CO2. The monolayers were washed with distilled water to lyse
macrophages, and 100 l of cell homogenates were assayed for the presence of viable
yeasts by a CFU assay. Supernatants obtained from fungicidal assays were used to
determine the levels of nitrite (C) and cytokines (D). Data are the mean ± SEM of
quintuplicate samples from one experiment representative of 3 independent
determinations. *P < 0.05.
Figure 2. Dectin-1 controls the expression of TLRs receptors and determines the
differentiation of P. brasiliensis infected macrophages to an “M1-like” phenotype.
Normal and IFN-γ-primed macrophages from WT and dectin -/- mice were infected with
P. brasiliensis yeasts in a macrophage:yeast ratio of 25:1 and co cultivated for 48h. The
expression of TLR4 (A) and TLR2 (B) was then assayed by flow cytometry. The
acquisition and analysis gates were restricted to the F4/80+ labeled macrophage
population. For quantitative PCR analysis of TLR4 (C) and TLR2 (D) mRNA
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expression, macrophages from dectin-1-/- and WT mice were infected by viable P.
brasiliensis yeasts (1:25, fungus:macrophages ratio) for 2 h. After 12 h at 37o C in 5%
CO2, the total RNA from macrophage cultures was obtained, reverse transcribed, and
cDNA amplified. Real-time PCR was performed using TaqMan universal master mix.
Amplified products were normalized to the amount of GAPDH products from in vitro
cultivated macrophages. Quantitative PCR analysis of NO-synthase 2 (NOS2), SOCS1,
SOCS3, arginase 1 (ARG1), found in inflammatory zone protein (FIZZ1) and chitinase-
like lectin (Ym1) mRNA expression. (E) CD11b+ cells were isolated from total lung
leukocytes suspentions obtained from infected and uninfected WT and dectin-/- mice at
week 2 after infection with 1 × 106 P. brasiliensis yeasts by using anti-CD11b magnetic
beads. Total RNA was extracted using Trizol reagent, reverse transcribed, and cDNA
amplified. Real-time PCR was performed using TaqMan universal master mix.
Amplified products were normalized to the amount of GAPDH products from lungs.
Data represent the means ± SEM of at least 5 mice/group and are representative of two
independent experiments. (*P<0.05, **P<0.01 and ***P<0.001).
Figure 3. P. brasiliensis infected dectin-1-/- mice present increased mortality associated
with increased fungal loads and tissue pathology. (A-C) CFU counts from organs were
determined 48h (A), 2 weeks (B) and 8 weeks (C) after P. brasiliensis infection of WT
and dectin-/- mice. The bars represent means ± SEM of log10 CFU obtained from groups
of five to six mice. (D-K) Photomicrographs of lesions of WT (D-G) and dectin-1-/-
mice (H-K) at week 8 of infection with 1 x 106 P. brasiliensis yeasts. Compared with
dectin-1-/- mice (E, G), the pulmonary lesions of WT mice (D) were smaller and
composed of organized granulomas containing lower numbers of yeasts (F). The
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pulmonary lesions of dectin-1-/- mice were composed of confluent and unorganized
granulomas of various sizes (E) containing an elevated number of fungal cells (G). The
livers of WT (H, J) and dectin-1-/- (I, K) mice presented organized granulomas, which,
however, appeared in lower numbers and contained fewer yeasts (J) in WT than in
dectin-1-/- mice (K). H&E (D, F, H, J) and Groccot (E, G, I, K ) stained lesions (100x).
(L) Total area of lesions in the lungs and livers of mice (n=6) at week 8 after infection.
(M) Survival times of dectin-1-/- and WT mice after i.t. infection with 1 × 106 P.
brasiliensis yeast cells was determined in a period of 137 days. The results are
representative of two experiments with equivalent results. Data represent the means ±
SEM of at least 5 mice/group and are representative of three independent experiments.
(*P<0.05).
Figure 4. Absence of dectin-1 determines a sustained decreased recruitment of PMN
cells and reduced levels of Th1-, Th2- and Th17-associated cytokines in the lungs.
Dectin-1-/- and WT mice were inoculated i.t. with 1x106 P. brasiliensis yeast cells, and
at weeks 2 and 8 after infection lungs of both mouse strains (n=5–6) were excised,
washed in PBS, minced, and digested enzymatically. Lung cell suspensions were
centrifuged in the presence of 20% percoll to separate leukocytes from cell debris. Cell
suspensions were cytospun onto glass slides and stained by the Diff-Quik bloodstain.
(A) Number of total leukocytes. (B) Number and frequency of macrophages, PMN
neutrophils, and lymphocytes in the lung infiltrating leucocytes (LIL). (C) At week 8
after i.t. infection with 1x106 yeast cells of P. brasiliensis, lungs from dectin-1-/- and
WT mice were collected and disrupted in 5.0 mL of PBS and supernatants were
analyzed for cytokines and MCP-1 content by capture ELISA. The bars depict
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means ± SEM of cytokine or chemokine levels (5–6 animals per group). The results are
representative of three independent experiments.*P < 0.05.
Figure 5. Absence of dectin-1 determines decreased numbers of activated T
lymphocytes in the lungs. (A) Flow Cytometry gating strategy to effector/memory, and
naïve CD4+ T cells in lung infiltrating leukocytes (LIL). LIL lymphocytes were
identified on FSC and SSC analysis. Gated cells were measured for CD4 expression
fallowing by CD44 expression and cells expressing high and low levels of this molecule
were gated. Gated CD44high cells were then measured for the expression of low levels of
CD62L identifying the effector/memory CD4+CD44highCD62Llow subpopulation. Gated
CD44low cells were then measured for the expression of high levels of CD62L
identifying the naïve CD4+CD44lowCD62Lhigh subpopulation. (B-E) Characterization of
CD4+ (B, C) and CD8+ T cells (D, E) by flow cytometry in the lung infiltrating
leucocytes (LIL) from dectin-1-/- and WT mice inoculated i.t. with 1 x 106 P.
brasiliensis yeast cells. At weeks 2 (B, D) and 8 (C, E) after infection, lungs of both
mouse strains (n=5–6) were excised and digested enzymatically. Cell suspensions were
obtained and stained as described in Materials and Methods. The stained cells were
analyzed immediately on a FACS CANTO II equipment gating on lymphocytes as
judged from forward and side light scatters. One hundred thousands cells were counted
and the data expressed as absolute number of positive cells. Data are expressed as
means ± SEM and are representative of three independent experiments. *P < 0.05.
Figure 6. Absence of dectin-1 promotes down regulation of IL-17-producing CD8+ T
(Tc17) cells associated with regulatory T cell expansion. (A) Flow Cytometry gating
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strategy to detect cytokine positive CD4+ T cells in lung infiltrating leukocytes (LIL).
LIL lymphocytes were identified on FSC and SSC analysis. Gated cells were measured
for CD4 or CD8 expression. Gated cells from were then measured for IL-17
expression. The same procedure was used to identify IL-4 positive and IFN- positive
CD4+ and CD8+ T cells. (B-C) The presence of IL-17+, IFN-γ+ and IL-4+ CD4+ and
CD8+ T cells in the lung infiltrating leukocytes (LIL) was assessed by intracellular
cytokine staining by flow cytometry at week 2 (B) and 8 (C) after infection. Lung cells
were re-stimulated in vitro with PMA/ionomycin for 6h and subjected to intracellular
staining for IL-17, IL-4 and IFN-γ. The lymphocyte population was gated by the
forward/side scatters. (D) Flow Cytometry gating strategy to detect regulatory (Treg) in
lung infiltrating leukocytes (LIL). LIL lymphocytes were identified on FSC and SSC
analysis. Gated cells were measured for CD4 expression and then measured for CD25
expression. Gated CD4+CD25+ cells were measured for Foxp3 expression identifying
Treg cells (E). Results are from one experiment and are representative of three
independent experiments. Data are expressed as means ± SEM. * (P<0.05).
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