Microbiology Papers in Press. Published February 20, 2015 as doi:10.1099/mic.0.000059
Microbiology Human neutrophils produce extracellular traps against Paracoccidioides brasiliensis --Manuscript Draft-Manuscript Number:
MIC-D-14-00233R1
Full Title:
Human neutrophils produce extracellular traps against Paracoccidioides brasiliensis
Short Title:
P. brasiliensis induces NETs
Article Type:
Standard
Section/Category:
Host-microbe interaction
Corresponding Author:
Angel Gonzalez Universidad de Antioquia Medellin, COLOMBIA
First Author:
Susana P Mejia
Order of Authors:
Susana P Mejia Luz E Cano Juan A Lopez Orville Hernandez Angel Gonzalez
Abstract:
Neutrophils play an important role as effector cells and contribute to the resistance of the host against microbial pathogens. Neutrophils are able to produce extracellular traps (NETs) in response to medically important fungi including Aspergillus spp., Candida albicans and Cryptococcus gattii. However, NET production in response to Paracoccidioides brasiliensis has yet to be studied. We have demonstrated that human neutrophils produce NETs against both conidia and yeasts of P. brasiliensis. Although the NADPH oxidase inhibitor DPI did not alter NET production against conidia, it partially suppressed NET formation against P. brasiliensis yeasts. Cytochalasin-D or IFN-γ did not affect the production of NETs against the fungus. Additionally, a mutant strain of P. brasiliensis with reduced expression of an alternative oxidase (AOX) induced significantly higher levels of NETs in comparison with the wild type strain. Finally, CFU quantification of P. brasiliensis showed no significant differences when neutrophils were treated with DPI, DNaseI or cytochalasin D when compared with untreated cells. These data establish that NET formation by human neutrophils appear to be either dependent or independent of ROS production correlating to the fungal morphotype used for stimulation. However, this mechanism was ineffective in killing the fungus.
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Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document): Mejia et al NETs 2014 R3.doc
1
1
Human neutrophils produce extracellular traps against Paracoccidioides brasiliensis
2
Susana P. Mejía1,2, Luz E. Cano1,2, Juan A. López3, Orville Hernandez4,
3
& Ángel González3*
4 5
Running title: P. brasiliensis induces NETs
6 7
1
8
(CIB), Carrera 72A No. 78B 141, Medellín, Colombia.
9
2
Medical and Experimental Mycology Group, Corporación para Investigaciones Biológicas
School of Microbiology, Universidad de Antioquia, Calle 70 No. 52-51, Medellín,
10
Colombia.
11
3
12
Universidad de Antioquia, Calle 70 No 52-51, Medellín, Colombia.
13
4
14
Carrera 72A No. 78B 141, Medellín, Colombia.
15
*Corresponding author. Universidad de Antioquia, School of Microbiology, Calle 70 No
16
52-51, Medellín, Colombia. Phone: 574-2195483. E-mail address:
17
[email protected]
Basic and Applied Microbiology Research Group (MICROBA), School of Microbiology,
Cellular and Molecular Biology Unit, Corporación para Investigaciones Biológicas (CIB),
18 19
Abstract word counts: 194
20
Main text word counts: 5101
21
Figures number: 4
22 23
Keywords: Neutrophil, NETs, Paracoccidioides brasiliensis, ROS.
2 1
Abstract
2
Neutrophils play an important role as effector cells and contribute to the resistance of the
3
host against microbial pathogens. Neutrophils are able to produce extracellular traps
4
(NETs) in response to medically important fungi including Aspergillus spp., Candida
5
albicans and Cryptococcus gattii. However, NET production in response to
6
Paracoccidioides brasiliensis has yet to be studied. We have demonstrated that human
7
neutrophils produce NETs against both conidia and yeasts of P. brasiliensis. Although the
8
NADPH oxidase inhibitor DPI did not alter NET production against conidia, it partially
9
suppressed NET formation against P. brasiliensis yeasts. Cytochalasin-D or IFN-γ did not
10
affect the production of NETs against the fungus. Additionally, a mutant strain of P.
11
brasiliensis with reduced expression of an alternative oxidase (AOX) induced significantly
12
higher levels of NETs in comparison with the wild type strain. Finally, CFU quantification
13
of P. brasiliensis showed no significant differences when neutrophils were treated with
14
DPI, DNaseI or cytochalasin D when compared with untreated cells. These data establish
15
that NET formation by human neutrophils appear to be either dependent or independent of
16
ROS production correlating to the fungal morphotype used for stimulation. However, this
17
mechanism was ineffective in killing the fungus.
18 19 20 21 22 23 24
3 1
INTRODUCTION
2
The fungus Paracoccidioides brasiliensis is a dimorphic and pathogenic species that causes
3
paracoccidioidomycosis (PCM), the most frequent systemic mycosis in Latin America.
4
This mycosis is predominantly found in South America, especially in Brazil, Colombia,
5
Venezuela and Argentina (Brummer et al., 1993; Negroni, 1993; Colombo et al., 2011).
6
P. brasiliensis is characterized by its ability to switch from the mycelial to the yeast
7
morphotype; the former occurs at temperatures of 18-24oC and is found in the external
8
environment while the latter grows at 36oC in culture or the host (Kanetsuna & Carbonell,
9
1970; Kanetsuna et al., 1972; Brummer et al., 1993; Tomazett et al., 2010). Infection is
10
caused after inhalation of P. brasiliensis conidia, small propagules produced by the mycelia
11
being able to reach the lungs. In 90% of clinically active cases, PCM produces a chronic
12
systemic and progressive disease, which progresses slowly and can take months or even
13
years to develop, while the remaining 10% of patients develop an acute form (Londero &
14
Melo, 1983; Brummer et al., 1993; Restrepo et al., 2008; Marques, 2013; Restrepo &
15
Tobon, 2010).
16
In experimental animal models, it has been shown that an acute inflammatory process takes
17
place during the early stages of the infection with involvement of phagocytic cells, mainly
18
neutrophils and macrophages (Gonzalez et al., 2003; Gonzalez et al., 2008; Lopera et al.,
19
2011). This initial inflammatory response is associated with a decrease in the fungal burden
20
in the lungs of infected mice (Pina et al., 2006; Gonzalez et al., 2008).
21
It has been demonstrated in vitro that human neutrophils are involved in resistance to P.
22
brasiliensis infection. These cells can ingest P. brasiliensis yeasts through phagocytosis but
23
when these are too large they form an extracellular vacuole before killing the fungus (Dias
24
et al., 2004). High activities of NADPH oxidase, endogenous peroxidase and acid
4 1
phosphatase have also been demonstrated during the fungal phagocytosis process (Dias et
2
al., 2004). Nonetheless, although P. brasiliensis is phagocytosed and the respiratory burst is
3
activated, these mechanisms do not seem to be sufficient to kill the fungus (Kurita et al.,
4
1999a, b ; Dias et al., 2008).
5
Other studies have shown that cytokines such as interleukin-15 (IL-15), IL-8, interferon-γ
6
(IFN-γ), granulocyte macrophage colony-stimulating factor (GM-CSF) and tumor necrosis
7
factor-α (TNF-α), increase the fungicidal/fungistatic effect of these cells ( Kurita et al.,
8
1999a; Kurita et al., 2000; Rodrigues et al., 2007; Tavian et al., 2008; Acorci et al., 2009).
9
Interestingly, the antifungal activity of IFN-γ, TNF-α and GM-CSF is exerted in a dose-
10
dependent manner and this response is correlated with production of reactive oxygen
11
species (ROS) (Rodrigues et al., 2007).
12
In the last decade, a new neutrophil defence strategy has been described, namely neutrophil
13
extracellular traps (NETs) (Brinkmann et al., 2004). These structures are produced after
14
cell activation and are composed of decondensed chromatin complexed with different
15
cytoplasmatic proteins, such as histones as well as over 30 different neutrophil proteins
16
(Brinkmann et al., 2004; Fuchs et al., 2007; Medina, 2009; Papayannopoulos &
17
Zychlinsky, 2009;). NETs can be produced by NADPH oxidase activation-dependent or
18
independent mechanisms and subsequent ROS production (Brinkmann et al., 2004;
19
Pilsczek et al., 2010). These network structures can capture microbes, degrade their
20
virulence factors and finally eliminate the pathogens (Brinkmann et al., 2004; Fuchs et al.,
21
2007).
22
An increasing number of bacteria, fungi, viruses and protozoan parasites have been shown
23
to induce NETs (Brinkmann et al., 2004; Guimaraes-Costa et al., 2009; Urban et al., 2009;
24
Saitoh et al., 2012; Jenne et al., 2013) but to date only three fungal species (C. albicans,
5 1
Aspergillus spp. and Cryptococcus gattii) have been reported to activate their production
2
(Urban et al., 2006; Bruns et al., 2010; Springer et al., 2010; Bianchi et al., 2011). NET
3
production is induced after recognition of β-glucan present on the cell wall of C. albicans,
4
through interaction with the complement receptor 3 (CR3) located on the surface of the
5
phagocytic cells (Byrd et al., 2013). NETs play an important role against C. albicans and A.
6
fumigatus hyphae. These structures are not phagocytosed due to their large size and NET
7
formation may be a good strategy to arrest fungal spread (Urban et al., 2009; Bianchi et al.,
8
2011). Although C. gattii induces NET production, these structures do not kill the fungus.
9
It is noteworthy that these capsulated yeasts produce extracellular fibrils, which are
10
associated with its virulence and the inhibition of neutrophil-mediated killing (Springer et
11
al., 2010).
12
Interestingly, the induction of NETs by P. brasiliensis has yet to be described. Here we
13
report that human neutrophils can produce NETs after interaction with both morphotypes of
14
P. brasiliensis (conidia and yeast cells). The production of these structures appears to be
15
either dependent or independent of ROS production correlating to the fungal morphotype
16
used for stimulation. However, this mechanism was ineffective in killing the fungus.
17 18
MATERIALS AND METHODS
19
Strains and media
20
A strain of Paracoccidioides brasiliensis (ATCC-60855; Manassas, VA, USA) originally
21
isolated from a Colombian patient and characterized by its abundant conidia production
22
was used in this study. The fungus was maintained at 18°C by passage every two weeks on
23
solid synthetic McVeigh-Morton modified (SMVM) medium (Restrepo and Jimenez 1980).
24
Conidia production and purification were assessed as described previously (Restrepo et al.,
6 1
1986; Del P. Jimenez et al., 2004). The numbers and viability of conidia were evaluated by
2
staining with Janus green (Invitrogen, CA, USA). Conidia suspensions with viabilities
3
higher than 90% were used throughout the experiments.
4
Yeast morphotypes were maintained by weekly subcultures in solid Sabouraud medium
5
(Becton Dickinson, NJ, USA) supplemented with 0.01% thiamine (Becton Dickinson, NJ,
6
USA), 0.014% asparagine (SIGMA-Aldrich, St. Louis, MO, USA), 100 U penicillin and
7
100 μg streptomycin (SIGMA) per mL at 36°C with 5% CO2. Unless otherwise indicated,
8
yeast cells were grown in broth Sabouraud medium supplemented with 0.01% thiamine and
9
0.014% asparagine plus 100 U penicillin and 100 μg streptomycin per mL at 36ºC with
10
aeration on a mechanical shaker, and were routinely collected during their exponential
11
growth phase (at 72h). Fungal growth was collected in phosphate-buffered saline solution
12
(PBS), and passed twelve times through a 5 mL 21G syringe with a 1 ½” needle to
13
eliminate clumped fungal cells. The fungal suspension was centrifuged at 1500g and 4°C
14
for 5 min. Viability and numbers of yeasts were determined by staining with Janus green
15
(Invitrogen, CA, USA). The fungal unit was considered to be equivalent to a single cell or
16
mother cell with 2-7 daughter cells. Fungal cells were counted in a haemocytometer and re-
17
suspended in RPMI 1640 culture medium (GIBCO Laboratories, Grand Island, NY, USA)
18
to obtain the desired number.
19
Moreover, a mutant strain of P. brasiliensis (PbAOX) (Ruiz et al., 2011) with modified
20
expression of an alternative oxidase (AOX) gene, involved in the detoxification or
21
reduction of reactive oxygen species (ROS) (Ruiz et al., 2011), was also employed in the
22
present investigation using antisense RNA technology.
23
A Candida albicans strain (ATCC 90028) was used as a positive control for NET
24
production by neutrophils, (Urban et al., 2006; Byrd et al., 2013)]. This strain was
7 1
maintained by three days of sub-culturing in solid Sabouraud medium at 35°C with 5%
2
CO2, and harvested during its exponential growth phase (48h). Fungal mass was isolated by
3
scraping the medium surface and re-suspending this material in RPMI 1640 medium.
4
Viability and numbers of yeasts were determined by staining with Janus green and counting
5
in a haemocytometer.
6 7
Reagents
8
Recombinant human IFN-γ, phorbol myristate acetate (PMA), diphenyleneiodonium
9
chloride (DPI), cytochalasin D, lipopolysaccharides (LPS) and luminol were purchased
10
from SIGMA-Aldrich (St. Louis, MO, USA). DNaseI was provided by Mo-BIO
11
Laboratories Inc. (CA, USA). SYTOX green was obtained from Molecular Probes
12
(Invitrogen, CA, USA).
13 14
Isolation of human neutrophils
15
Blood samples were obtained from healthy human volunteers by following the guidelines
16
of resolution 8430 of 1993 of the Colombian Ministry of Health. Blood was collected in
17
EDTA-containing Vacutainer tubes (BD Biosciences, San Jose, CA). Polymorphprep
18
[(density 1.113 g/mL; 460 mOsm); Axis Shield, Oslo, Norway] was used for blood gradient
19
centrifugation at 550 g. Neutrophils were collected and washed with HBSS buffer (GIBCO
20
Laboratories, Grand Island, NY, USA) and diluted in RPMI 1640 medium. The number and
21
viability of cells were evaluated by trypan blue exclusion (Invitrogen, CA, USA).
22
Neutrophil cells with viability higher than 95% were used.
23 24
8 1
NET Visualization
2
For staining techniques, we used 12 mm glass coverslips (Fisherbrand, United Kingdom)
3
in 24-well non-treated culture plates (Brand, Germany). As a first step, neutrophils were
4
adhered onto the glass coverslip, 2x105 PMNs/wells were seeded and the preparations
5
incubated for 30min at 37°C. They were then inoculated with 4x104 yeasts of P.
6
brasiliensis in 250 µL of RPMI 1640 medium and incubated for 3 h at 37 °C. In some wells
7
PMA (25nM) was added to stimulate neutrophils. Furthermore, in some experiments
8
100U/mL of DNaseI were added at the same time as the stimulus in order to degrade any
9
DNA that had been released. Then, monolayers were fixed by adding 4% paraformaldehyde
10
for 10 min at room temperature. Additionally, cells were permeabilized using 0.5% triton
11
X-100 for 1 min. NETs were stained with a rabbit anti-human elastase AB21595 (Abcam,
12
Cambridge, MA, USA) 0.6g in a 10% PBS-BSA solution (GIBCO Laboratories, Grand
13
Island, NY, USA); finally, an Alexa 488-conjugated goat anti-rabbit IgG (Invitrogen, CA,
14
USA) 0.3 g in PBS was used as the secondary antibody. Additionally, DNA was stained
15
with 1 µg/ml of propidium iodide (Invitrogen, CA, USA) and the fungus with 100 µg/ml of
16
calcofluor white (Fluka-SIGMA-Aldrich, Buchs, Switzerland) following the manufacture’s
17
instructions. NETs were visualized using an Axio Vert A.1 inverted fluorescent microscope
18
(Carl Zeiss, Jena, Germany) and the images captured using an Axiocam IC camera (Carl
19
Zeiss).
20 21
Determination of NET formation (area of NETs)
22
The area of NETs (%) was determined with ImageJ 1.48 v software (National Institutes of
23
Health, Bethesda, MD). Forty images were captured with a 20x objective and analysed
9 1
randomly from different regions of each coverslip; individual cell and NETs areas were
2
framed with the free hand selection (yellow line) and the inner region was measured, the
3
sum of these areas was taken as the total area. With this data, the area of NETs was
4
calculated with Microsoft Excel 2010.
5 6
Quantification of extracellular DNA
7
Co-cultures of P. brasiliensis at the concentrations described above with 2x105 freshly
8
isolated human neutrophils were carried out in 200 µl RPMI 1640 medium using black 96-
9
well culture plates (Greiner, Germany) for 3 h at 37 °C. In some wells PMA (25 nM), LPS
10
(3 µg/mL) or C. albicans (4x104 yeasts) was added to stimulate neutrophils.
11
In order to inhibit NET formation, neutrophil cultures were pre-incubated with 10 µg/mL
12
cytochalasin D (an inhibitor of phagocytosis) and 16 µM DPI (an inhibitor of NADPH
13
oxidase) for 30 min at 37 ºC, after which P. brasiliensis yeasts or conidia were added. In
14
addition, in some experiments 100 U/mL of DNaseI were added at the same time as the
15
stimulus in order to degrade any DNA that had been released. Some neutrophils were also
16
pre-treated with IFN-γ (100 UI) 1h before the second stimuli were added. Finally, 5 μL of
17
SYTOX green (a fluorescence high-affinity nucleic acid stain) at a concentration of 5 μM
18
were added to the wells and the fluorescence measured (excitation 480 nm and emission
19
530 nm) in a spectrofluorometer (Spectra Max Gemini- Molecular Devices, Silicon Valley,
20
CA, USA). To quantify the amount of extracellular DNA, total DNA from 2x10 5
21
neutrophils was obtained using a commercial extraction kit (Mini kit quiamp DNA,
22
QUIAGEN); this DNA was taken as 100 % of total and used to calculate the extracellular
23
DNA released by cells.
24
10 1
ROS production by human neutrophils
2
Activation of neutrophils upon stimulation with P. brasiliensis yeast (wild type-Pb60855-,
3
yeasts carrying the empty vector -PbEv60855- or a mutant strain with diminished
4
expression of AOX –PbAOX-) was determined by measuring reactive oxygen species
5
(ROS) production. Quantification of ROS was performed using a luminol-enhanced
6
chemiluminescence method. Briefly, freshly isolated human neutrophils were seeded on
7
white 96- well culture plates (Greiner, Germany) at 2x105 cells/well in PBS with glucose
8
and luminol (50 µM, Sigma-Aldrich). Cells were incubated for 10 min at 37 ºC and
9
infected with 4x104 P. brasiliensis yeasts. As positive control, neutrophils were stimulated
10
with 25 nM PMA (Sigma-Aldrich). NADPH oxidase activity inhibited prior incubation (20
11
min) with 10 μM DPI (Sigma-Aldrich). Unstimulated neutrophils were included in each
12
experiment as negative control. An additional negative control consisting of P. brasiliensis
13
yeasts alone was also included, and the luminescence of this control was subtracted from
14
co-cultures. Finally, the chemiluminescence was measured every 2 min for 2 h in a
15
Varioskan Flash Multimode Reader (Thermo Scientific, Waltham, MA, USA). The area
16
under curve was calculated using Excel 2010.
17 18
Killing assay
19
In order to determine the efficiency of NETs to kill P. brasiliensis, a colony-forming unit
20
(CFU) assay was performed. Co-incubation of fresh human neutrophils with P. brasiliensis
21
yeasts (Pb60855 and PbAOX strains) in 96-well culture plates (Brand, Germany) was
22
carried out as described previously. In additional experiments, some wells were treated with
23
16 µM DPI, 100 U/mL DNaseI and 10 µg/mL of cytochalasin D. After 3h of incubation at
24
37 °C, wells were aspirated and washed with PBS. Dilutions of 1:10 and 1:100 were made
11 1
and 100 µL of each dilution was plated onto BHI agar supplemented with 4% (vol/vol)
2
horse serum, 500 µM EDTA and 1% glucose (SIGMA-Aldrich, St. Louis, MO, USA).
3
Cultures were incubated at 36 °C with 5 % CO2. CFUs were counted after three days of
4
culturing and thereafter until no further increase in colony numbers could be observed.
5 6
Statistical analysis
7
All statistical analysis was performed using Graph Pad Prism software, version 5.0 (Graph
8
Pad software, La Jolla, CA, USA). Normal distribution was determined using the ANOVA
9
and verified by Kolmogorov-Smirnov normality test. Differences between groups were
10
analyzed using Student’s t-test or Mann-Whitney test according to the Gaussian distribution
11
of data. A P value of
Microbiology Human neutrophils produce extracellular traps against Paracoccidioides brasiliensis --Manuscript Draft-Manuscript Number:
MIC-D-14-00233R1
Full Title:
Human neutrophils produce extracellular traps against Paracoccidioides brasiliensis
Short Title:
P. brasiliensis induces NETs
Article Type:
Standard
Section/Category:
Host-microbe interaction
Corresponding Author:
Angel Gonzalez Universidad de Antioquia Medellin, COLOMBIA
First Author:
Susana P Mejia
Order of Authors:
Susana P Mejia Luz E Cano Juan A Lopez Orville Hernandez Angel Gonzalez
Abstract:
Neutrophils play an important role as effector cells and contribute to the resistance of the host against microbial pathogens. Neutrophils are able to produce extracellular traps (NETs) in response to medically important fungi including Aspergillus spp., Candida albicans and Cryptococcus gattii. However, NET production in response to Paracoccidioides brasiliensis has yet to be studied. We have demonstrated that human neutrophils produce NETs against both conidia and yeasts of P. brasiliensis. Although the NADPH oxidase inhibitor DPI did not alter NET production against conidia, it partially suppressed NET formation against P. brasiliensis yeasts. Cytochalasin-D or IFN-γ did not affect the production of NETs against the fungus. Additionally, a mutant strain of P. brasiliensis with reduced expression of an alternative oxidase (AOX) induced significantly higher levels of NETs in comparison with the wild type strain. Finally, CFU quantification of P. brasiliensis showed no significant differences when neutrophils were treated with DPI, DNaseI or cytochalasin D when compared with untreated cells. These data establish that NET formation by human neutrophils appear to be either dependent or independent of ROS production correlating to the fungal morphotype used for stimulation. However, this mechanism was ineffective in killing the fungus.
Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation
Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document): Mejia et al NETs 2014 R3.doc
1
1
Human neutrophils produce extracellular traps against Paracoccidioides brasiliensis
2
Susana P. Mejía1,2, Luz E. Cano1,2, Juan A. López3, Orville Hernandez4,
3
& Ángel González3*
4 5
Running title: P. brasiliensis induces NETs
6 7
1
8
(CIB), Carrera 72A No. 78B 141, Medellín, Colombia.
9
2
Medical and Experimental Mycology Group, Corporación para Investigaciones Biológicas
School of Microbiology, Universidad de Antioquia, Calle 70 No. 52-51, Medellín,
10
Colombia.
11
3
12
Universidad de Antioquia, Calle 70 No 52-51, Medellín, Colombia.
13
4
14
Carrera 72A No. 78B 141, Medellín, Colombia.
15
*Corresponding author. Universidad de Antioquia, School of Microbiology, Calle 70 No
16
52-51, Medellín, Colombia. Phone: 574-2195483. E-mail address:
17
[email protected]
Basic and Applied Microbiology Research Group (MICROBA), School of Microbiology,
Cellular and Molecular Biology Unit, Corporación para Investigaciones Biológicas (CIB),
18 19
Abstract word counts: 194
20
Main text word counts: 5101
21
Figures number: 4
22 23
Keywords: Neutrophil, NETs, Paracoccidioides brasiliensis, ROS.
2 1
Abstract
2
Neutrophils play an important role as effector cells and contribute to the resistance of the
3
host against microbial pathogens. Neutrophils are able to produce extracellular traps
4
(NETs) in response to medically important fungi including Aspergillus spp., Candida
5
albicans and Cryptococcus gattii. However, NET production in response to
6
Paracoccidioides brasiliensis has yet to be studied. We have demonstrated that human
7
neutrophils produce NETs against both conidia and yeasts of P. brasiliensis. Although the
8
NADPH oxidase inhibitor DPI did not alter NET production against conidia, it partially
9
suppressed NET formation against P. brasiliensis yeasts. Cytochalasin-D or IFN-γ did not
10
affect the production of NETs against the fungus. Additionally, a mutant strain of P.
11
brasiliensis with reduced expression of an alternative oxidase (AOX) induced significantly
12
higher levels of NETs in comparison with the wild type strain. Finally, CFU quantification
13
of P. brasiliensis showed no significant differences when neutrophils were treated with
14
DPI, DNaseI or cytochalasin D when compared with untreated cells. These data establish
15
that NET formation by human neutrophils appear to be either dependent or independent of
16
ROS production correlating to the fungal morphotype used for stimulation. However, this
17
mechanism was ineffective in killing the fungus.
18 19 20 21 22 23 24
3 1
INTRODUCTION
2
The fungus Paracoccidioides brasiliensis is a dimorphic and pathogenic species that causes
3
paracoccidioidomycosis (PCM), the most frequent systemic mycosis in Latin America.
4
This mycosis is predominantly found in South America, especially in Brazil, Colombia,
5
Venezuela and Argentina (Brummer et al., 1993; Negroni, 1993; Colombo et al., 2011).
6
P. brasiliensis is characterized by its ability to switch from the mycelial to the yeast
7
morphotype; the former occurs at temperatures of 18-24oC and is found in the external
8
environment while the latter grows at 36oC in culture or the host (Kanetsuna & Carbonell,
9
1970; Kanetsuna et al., 1972; Brummer et al., 1993; Tomazett et al., 2010). Infection is
10
caused after inhalation of P. brasiliensis conidia, small propagules produced by the mycelia
11
being able to reach the lungs. In 90% of clinically active cases, PCM produces a chronic
12
systemic and progressive disease, which progresses slowly and can take months or even
13
years to develop, while the remaining 10% of patients develop an acute form (Londero &
14
Melo, 1983; Brummer et al., 1993; Restrepo et al., 2008; Marques, 2013; Restrepo &
15
Tobon, 2010).
16
In experimental animal models, it has been shown that an acute inflammatory process takes
17
place during the early stages of the infection with involvement of phagocytic cells, mainly
18
neutrophils and macrophages (Gonzalez et al., 2003; Gonzalez et al., 2008; Lopera et al.,
19
2011). This initial inflammatory response is associated with a decrease in the fungal burden
20
in the lungs of infected mice (Pina et al., 2006; Gonzalez et al., 2008).
21
It has been demonstrated in vitro that human neutrophils are involved in resistance to P.
22
brasiliensis infection. These cells can ingest P. brasiliensis yeasts through phagocytosis but
23
when these are too large they form an extracellular vacuole before killing the fungus (Dias
24
et al., 2004). High activities of NADPH oxidase, endogenous peroxidase and acid
4 1
phosphatase have also been demonstrated during the fungal phagocytosis process (Dias et
2
al., 2004). Nonetheless, although P. brasiliensis is phagocytosed and the respiratory burst is
3
activated, these mechanisms do not seem to be sufficient to kill the fungus (Kurita et al.,
4
1999a, b ; Dias et al., 2008).
5
Other studies have shown that cytokines such as interleukin-15 (IL-15), IL-8, interferon-γ
6
(IFN-γ), granulocyte macrophage colony-stimulating factor (GM-CSF) and tumor necrosis
7
factor-α (TNF-α), increase the fungicidal/fungistatic effect of these cells ( Kurita et al.,
8
1999a; Kurita et al., 2000; Rodrigues et al., 2007; Tavian et al., 2008; Acorci et al., 2009).
9
Interestingly, the antifungal activity of IFN-γ, TNF-α and GM-CSF is exerted in a dose-
10
dependent manner and this response is correlated with production of reactive oxygen
11
species (ROS) (Rodrigues et al., 2007).
12
In the last decade, a new neutrophil defence strategy has been described, namely neutrophil
13
extracellular traps (NETs) (Brinkmann et al., 2004). These structures are produced after
14
cell activation and are composed of decondensed chromatin complexed with different
15
cytoplasmatic proteins, such as histones as well as over 30 different neutrophil proteins
16
(Brinkmann et al., 2004; Fuchs et al., 2007; Medina, 2009; Papayannopoulos &
17
Zychlinsky, 2009;). NETs can be produced by NADPH oxidase activation-dependent or
18
independent mechanisms and subsequent ROS production (Brinkmann et al., 2004;
19
Pilsczek et al., 2010). These network structures can capture microbes, degrade their
20
virulence factors and finally eliminate the pathogens (Brinkmann et al., 2004; Fuchs et al.,
21
2007).
22
An increasing number of bacteria, fungi, viruses and protozoan parasites have been shown
23
to induce NETs (Brinkmann et al., 2004; Guimaraes-Costa et al., 2009; Urban et al., 2009;
24
Saitoh et al., 2012; Jenne et al., 2013) but to date only three fungal species (C. albicans,
5 1
Aspergillus spp. and Cryptococcus gattii) have been reported to activate their production
2
(Urban et al., 2006; Bruns et al., 2010; Springer et al., 2010; Bianchi et al., 2011). NET
3
production is induced after recognition of β-glucan present on the cell wall of C. albicans,
4
through interaction with the complement receptor 3 (CR3) located on the surface of the
5
phagocytic cells (Byrd et al., 2013). NETs play an important role against C. albicans and A.
6
fumigatus hyphae. These structures are not phagocytosed due to their large size and NET
7
formation may be a good strategy to arrest fungal spread (Urban et al., 2009; Bianchi et al.,
8
2011). Although C. gattii induces NET production, these structures do not kill the fungus.
9
It is noteworthy that these capsulated yeasts produce extracellular fibrils, which are
10
associated with its virulence and the inhibition of neutrophil-mediated killing (Springer et
11
al., 2010).
12
Interestingly, the induction of NETs by P. brasiliensis has yet to be described. Here we
13
report that human neutrophils can produce NETs after interaction with both morphotypes of
14
P. brasiliensis (conidia and yeast cells). The production of these structures appears to be
15
either dependent or independent of ROS production correlating to the fungal morphotype
16
used for stimulation. However, this mechanism was ineffective in killing the fungus.
17 18
MATERIALS AND METHODS
19
Strains and media
20
A strain of Paracoccidioides brasiliensis (ATCC-60855; Manassas, VA, USA) originally
21
isolated from a Colombian patient and characterized by its abundant conidia production
22
was used in this study. The fungus was maintained at 18°C by passage every two weeks on
23
solid synthetic McVeigh-Morton modified (SMVM) medium (Restrepo and Jimenez 1980).
24
Conidia production and purification were assessed as described previously (Restrepo et al.,
6 1
1986; Del P. Jimenez et al., 2004). The numbers and viability of conidia were evaluated by
2
staining with Janus green (Invitrogen, CA, USA). Conidia suspensions with viabilities
3
higher than 90% were used throughout the experiments.
4
Yeast morphotypes were maintained by weekly subcultures in solid Sabouraud medium
5
(Becton Dickinson, NJ, USA) supplemented with 0.01% thiamine (Becton Dickinson, NJ,
6
USA), 0.014% asparagine (SIGMA-Aldrich, St. Louis, MO, USA), 100 U penicillin and
7
100 μg streptomycin (SIGMA) per mL at 36°C with 5% CO2. Unless otherwise indicated,
8
yeast cells were grown in broth Sabouraud medium supplemented with 0.01% thiamine and
9
0.014% asparagine plus 100 U penicillin and 100 μg streptomycin per mL at 36ºC with
10
aeration on a mechanical shaker, and were routinely collected during their exponential
11
growth phase (at 72h). Fungal growth was collected in phosphate-buffered saline solution
12
(PBS), and passed twelve times through a 5 mL 21G syringe with a 1 ½” needle to
13
eliminate clumped fungal cells. The fungal suspension was centrifuged at 1500g and 4°C
14
for 5 min. Viability and numbers of yeasts were determined by staining with Janus green
15
(Invitrogen, CA, USA). The fungal unit was considered to be equivalent to a single cell or
16
mother cell with 2-7 daughter cells. Fungal cells were counted in a haemocytometer and re-
17
suspended in RPMI 1640 culture medium (GIBCO Laboratories, Grand Island, NY, USA)
18
to obtain the desired number.
19
Moreover, a mutant strain of P. brasiliensis (PbAOX) (Ruiz et al., 2011) with modified
20
expression of an alternative oxidase (AOX) gene, involved in the detoxification or
21
reduction of reactive oxygen species (ROS) (Ruiz et al., 2011), was also employed in the
22
present investigation using antisense RNA technology.
23
A Candida albicans strain (ATCC 90028) was used as a positive control for NET
24
production by neutrophils, (Urban et al., 2006; Byrd et al., 2013)]. This strain was
7 1
maintained by three days of sub-culturing in solid Sabouraud medium at 35°C with 5%
2
CO2, and harvested during its exponential growth phase (48h). Fungal mass was isolated by
3
scraping the medium surface and re-suspending this material in RPMI 1640 medium.
4
Viability and numbers of yeasts were determined by staining with Janus green and counting
5
in a haemocytometer.
6 7
Reagents
8
Recombinant human IFN-γ, phorbol myristate acetate (PMA), diphenyleneiodonium
9
chloride (DPI), cytochalasin D, lipopolysaccharides (LPS) and luminol were purchased
10
from SIGMA-Aldrich (St. Louis, MO, USA). DNaseI was provided by Mo-BIO
11
Laboratories Inc. (CA, USA). SYTOX green was obtained from Molecular Probes
12
(Invitrogen, CA, USA).
13 14
Isolation of human neutrophils
15
Blood samples were obtained from healthy human volunteers by following the guidelines
16
of resolution 8430 of 1993 of the Colombian Ministry of Health. Blood was collected in
17
EDTA-containing Vacutainer tubes (BD Biosciences, San Jose, CA). Polymorphprep
18
[(density 1.113 g/mL; 460 mOsm); Axis Shield, Oslo, Norway] was used for blood gradient
19
centrifugation at 550 g. Neutrophils were collected and washed with HBSS buffer (GIBCO
20
Laboratories, Grand Island, NY, USA) and diluted in RPMI 1640 medium. The number and
21
viability of cells were evaluated by trypan blue exclusion (Invitrogen, CA, USA).
22
Neutrophil cells with viability higher than 95% were used.
23 24
8 1
NET Visualization
2
For staining techniques, we used 12 mm glass coverslips (Fisherbrand, United Kingdom)
3
in 24-well non-treated culture plates (Brand, Germany). As a first step, neutrophils were
4
adhered onto the glass coverslip, 2x105 PMNs/wells were seeded and the preparations
5
incubated for 30min at 37°C. They were then inoculated with 4x104 yeasts of P.
6
brasiliensis in 250 µL of RPMI 1640 medium and incubated for 3 h at 37 °C. In some wells
7
PMA (25nM) was added to stimulate neutrophils. Furthermore, in some experiments
8
100U/mL of DNaseI were added at the same time as the stimulus in order to degrade any
9
DNA that had been released. Then, monolayers were fixed by adding 4% paraformaldehyde
10
for 10 min at room temperature. Additionally, cells were permeabilized using 0.5% triton
11
X-100 for 1 min. NETs were stained with a rabbit anti-human elastase AB21595 (Abcam,
12
Cambridge, MA, USA) 0.6g in a 10% PBS-BSA solution (GIBCO Laboratories, Grand
13
Island, NY, USA); finally, an Alexa 488-conjugated goat anti-rabbit IgG (Invitrogen, CA,
14
USA) 0.3 g in PBS was used as the secondary antibody. Additionally, DNA was stained
15
with 1 µg/ml of propidium iodide (Invitrogen, CA, USA) and the fungus with 100 µg/ml of
16
calcofluor white (Fluka-SIGMA-Aldrich, Buchs, Switzerland) following the manufacture’s
17
instructions. NETs were visualized using an Axio Vert A.1 inverted fluorescent microscope
18
(Carl Zeiss, Jena, Germany) and the images captured using an Axiocam IC camera (Carl
19
Zeiss).
20 21
Determination of NET formation (area of NETs)
22
The area of NETs (%) was determined with ImageJ 1.48 v software (National Institutes of
23
Health, Bethesda, MD). Forty images were captured with a 20x objective and analysed
9 1
randomly from different regions of each coverslip; individual cell and NETs areas were
2
framed with the free hand selection (yellow line) and the inner region was measured, the
3
sum of these areas was taken as the total area. With this data, the area of NETs was
4
calculated with Microsoft Excel 2010.
5 6
Quantification of extracellular DNA
7
Co-cultures of P. brasiliensis at the concentrations described above with 2x105 freshly
8
isolated human neutrophils were carried out in 200 µl RPMI 1640 medium using black 96-
9
well culture plates (Greiner, Germany) for 3 h at 37 °C. In some wells PMA (25 nM), LPS
10
(3 µg/mL) or C. albicans (4x104 yeasts) was added to stimulate neutrophils.
11
In order to inhibit NET formation, neutrophil cultures were pre-incubated with 10 µg/mL
12
cytochalasin D (an inhibitor of phagocytosis) and 16 µM DPI (an inhibitor of NADPH
13
oxidase) for 30 min at 37 ºC, after which P. brasiliensis yeasts or conidia were added. In
14
addition, in some experiments 100 U/mL of DNaseI were added at the same time as the
15
stimulus in order to degrade any DNA that had been released. Some neutrophils were also
16
pre-treated with IFN-γ (100 UI) 1h before the second stimuli were added. Finally, 5 μL of
17
SYTOX green (a fluorescence high-affinity nucleic acid stain) at a concentration of 5 μM
18
were added to the wells and the fluorescence measured (excitation 480 nm and emission
19
530 nm) in a spectrofluorometer (Spectra Max Gemini- Molecular Devices, Silicon Valley,
20
CA, USA). To quantify the amount of extracellular DNA, total DNA from 2x10 5
21
neutrophils was obtained using a commercial extraction kit (Mini kit quiamp DNA,
22
QUIAGEN); this DNA was taken as 100 % of total and used to calculate the extracellular
23
DNA released by cells.
24
10 1
ROS production by human neutrophils
2
Activation of neutrophils upon stimulation with P. brasiliensis yeast (wild type-Pb60855-,
3
yeasts carrying the empty vector -PbEv60855- or a mutant strain with diminished
4
expression of AOX –PbAOX-) was determined by measuring reactive oxygen species
5
(ROS) production. Quantification of ROS was performed using a luminol-enhanced
6
chemiluminescence method. Briefly, freshly isolated human neutrophils were seeded on
7
white 96- well culture plates (Greiner, Germany) at 2x105 cells/well in PBS with glucose
8
and luminol (50 µM, Sigma-Aldrich). Cells were incubated for 10 min at 37 ºC and
9
infected with 4x104 P. brasiliensis yeasts. As positive control, neutrophils were stimulated
10
with 25 nM PMA (Sigma-Aldrich). NADPH oxidase activity inhibited prior incubation (20
11
min) with 10 μM DPI (Sigma-Aldrich). Unstimulated neutrophils were included in each
12
experiment as negative control. An additional negative control consisting of P. brasiliensis
13
yeasts alone was also included, and the luminescence of this control was subtracted from
14
co-cultures. Finally, the chemiluminescence was measured every 2 min for 2 h in a
15
Varioskan Flash Multimode Reader (Thermo Scientific, Waltham, MA, USA). The area
16
under curve was calculated using Excel 2010.
17 18
Killing assay
19
In order to determine the efficiency of NETs to kill P. brasiliensis, a colony-forming unit
20
(CFU) assay was performed. Co-incubation of fresh human neutrophils with P. brasiliensis
21
yeasts (Pb60855 and PbAOX strains) in 96-well culture plates (Brand, Germany) was
22
carried out as described previously. In additional experiments, some wells were treated with
23
16 µM DPI, 100 U/mL DNaseI and 10 µg/mL of cytochalasin D. After 3h of incubation at
24
37 °C, wells were aspirated and washed with PBS. Dilutions of 1:10 and 1:100 were made
11 1
and 100 µL of each dilution was plated onto BHI agar supplemented with 4% (vol/vol)
2
horse serum, 500 µM EDTA and 1% glucose (SIGMA-Aldrich, St. Louis, MO, USA).
3
Cultures were incubated at 36 °C with 5 % CO2. CFUs were counted after three days of
4
culturing and thereafter until no further increase in colony numbers could be observed.
5 6
Statistical analysis
7
All statistical analysis was performed using Graph Pad Prism software, version 5.0 (Graph
8
Pad software, La Jolla, CA, USA). Normal distribution was determined using the ANOVA
9
and verified by Kolmogorov-Smirnov normality test. Differences between groups were
10
analyzed using Student’s t-test or Mann-Whitney test according to the Gaussian distribution
11
of data. A P value of
