Medical Mycology, 2020, 0, 1–9

doi: 10.1093/mmy/myaa006
Advance Access Publication Date: 0 2020
Original Article

Original Article

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Histoplasma capsulatum chemotypes I and II induce IL-8 secretion
in lung epithelial cells in distinct manners
Cristiane Alcantara1 , Bruna Rocha Almeida 1
, Bianca Carla Silva
Campitelli Barros 1 , Cristina Mary Orikaza 1
, Marcos Sergio Toledo 2

and Erika Suzuki 1,∗

1
Department of Microbiology, Immunology, and Parasitology, Escola Paulista de Medicina - Universidade Federal
de São Paulo, São Paulo – SP, Brazil and 2 Department of Biochemistry, Escola Paulista de Medicina - Universidade
Federal de São Paulo, São Paulo – SP, Brazil
∗
To whom correspondence should be addressed. Erika Suzuki, PhD, Universidade Federal de São Paulo, Escola Paulista de Medicina,
Rua Botucatu 862, Ed. Antonio C. M. Paiva - 6 andar, São Paulo - SP - Brazil 04023-062. E-mail: erika.suzuki@unifesp.br
Received 24 November 2019; Revised 30 January 2020; Accepted 5 February 2020; Editorial Decision 3 February 2020

Abstract
The cell wall is one of the most important structures of pathogenic fungi, enabling initial interaction with
the host and consequent modulation of immunological responses. Over the years, some researchers have
shown that cell wall components of Histoplasma capsulatum vary among fungal isolates, and one of the
major differences is the presence or absence of α-(1,3)-glucan, classifying wild-type fungi as chemotypes II
or I, respectively. The present work shows that an isolate of H. capsulatum chemotype I induced lower levels
of interleukin (IL)-8 secretion by the lung epithelial cell line A549, when compared to chemotype II yeasts.
Thus, we expected that the absence of α-glucan in spontaneous variant yeasts, which were isolated from
chemotype II cultures, would modify IL-8 secretion by A549 cells, but surprisingly, these fungi promoted
similar levels of IL-8 secretion as their wild-type counterpart. Furthermore, when using a specific inhibitor
for Syk activation, we observed that this inhibitor reduced IL-8 levels in A549 cell cultures infected with wild
type chemotype I fungi. This inhibitor failed to reduce this cytokine levels in A549 cell cultures infected with
chemotype II and their spontaneous variant yeasts, which also do not present α-glucan on their surface. The
importance of SFKs and PKC δ in this event was also analyzed. Our results show that different isolates of
H. capsulatum modulate distinct cell signaling pathways to promote cytokine secretion in host epithelial
cells, emphasizing the existence of various mechanisms for Histoplasma pathogenicity.

Key words: Histoplasma capsulatum, chemotype, epithelial cell, cytokine, cell signaling.

Introduction In the 1970s, different groups observed that the composition

of the yeast cell wall may vary among H. capsulatum isolates
Histoplasma capsulatum is a thermally dimorphic fungus that
and classified these fungi according to the absence (chemotype I)
causes histoplasmosis, a systemic fungal disease, mostly endemic
or presence (chemotype II) of α-(1,3)-glucan in the cell wall.4–6
in parts of the USA and Latin America.1 This fungus infects the
In the 1990s, Eissenberg et al.7 verified that while chemotype I
host by inhalation of mycelium microconidia and, once in the
isolates were able to be internalized by hamster tracheal epithe-
lungs, differentiates into the yeast form. For this process to occur,
lial cells, chemotype II yeasts rarely infected these cells. Despite
H. capsulatum modulates the expression of several molecules,
this fact, both chemotypes were able to destroy monolayers of
some of which may act as virulence factors or are important
a macrophage-like cell line. Conversely, variants of chemotype
for remodeling the fungal cell wall. Therefore, H. capsulatum
II that did not present α-(1,3)-glucan infected the tracheal
can subvert the host immune system and establish a successful
epithelial cells, but lost the capability to kill macrophages.7
infection in the host.2,3

C The Author(s) 2020. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. 1
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2 Medical Mycology, 2020, Vol. 00, No. 00
Later, among other reports describing the differences of host
response to both chemotypes, Sepúlveda et al.8 verified that mice
infected with high doses of chemotype I yeasts, when compared
to chemotype II, led to drastic weight loss, higher fungal burden
in the lungs, longer infection duration, and delayed resolution
of inflammation.8
Pulmonary epithelial cells may recognize pathogens by in-
teracting with pathogen-associated molecular patterns (PAMPs)
through pattern recognition receptors (PRRs), such as Toll-like
receptors (TLRs) and Dectin-1. This interaction leads to the re-
cruitment of several adapter proteins and the initiation of sig-
naling pathways, which may culminate in the secretion of cy-
tokines and the modulation of the host immune system.9,10 In
fact, over the last years, our group has verified that Paracoccid-
ioides brasiliensis yeasts, for example, promote interleukin (IL)-6
and IL-8 secretion by the human lung epithelial cell line A549
in a protein kinase C δ (PKC δ)-, ERK 1/2- and p38 MAPK-
dependent manner.11,12 We verified that H. capsulatum yeasts
also induce IL-6 and IL-8 secretion in A549 cells by activating
Src-family kinases (SFKs).13
Activation of SFKs is essential for immune responses against
some infections. These kinases may phosphorylate receptors,
such as Dectin-1 and TLR4, creating docking sites for recruit-
ment of the spleen-tyrosine kinase (Syk). In turn, Syk starts a
cell signaling pathway that may culminate with activation of the
transcription factor nuclear factor-κB (NF-κB) and expression
of cytokines.14,15
During the last decade, there is increasing evidence that lung
epithelial cells are able to influence the host immune responses
against infections.16 Thus, the aim of this work is to study if
different chemotypes of H. capsulatum promote IL-8 secretion
similarly in A549 epithelial cell cultures. The activation and in-
volvement of SFKs, Syk and PKC δ in this cytokine secretion
were also analyzed during A549 cell-H. capsulatum infection.
Methods
Histoplasma capsulatum cultures
H. capsulatum isolates 268 and G217B are part of the fungal
collection of Escola Paulista de Medicina - Universidade Federal
de São Paulo, Brazil, and were kindly provided by Dr. Zoilo
Pires de Camargo. Isolate 268 belongs to a new Latin American
clade according to recent phylogenetic studies (Dr. Anderson M.
Rodrigues and Dr. Zoilo P. de Camargo, personal commu-
nication). These isolates were cultivated as yeasts in liquid
Histoplasma macrophage medium (HMM), a synthetic culture
medium consisting of Ham’s F-12 nutrient mixture (Sigma-
Aldrich Corporation/Merck KGaA, Darmstadt, Germany)
supplemented with 18.2 g/l glucose, 1 g/l sodium glutamate,
84 mg/l cysteine, and 5.96 g/l HEPES.17 Yeasts were cultivated
in an orbital shaker incubator (Innova 42 New Brunswick Sci.,
NJ, USA) at 37◦ C, 120 rpm, until late exponential growth phase
(48 to 72 hours).
Klimpel and Goldman’s protocol for enrichment of
α-(1,3)-glucan-negative yeasts
By using Klimpel and Goldman’s protocol,8,17 we obtained
a spontaneous variant from isolate 268, which, at the end of
this process, did not present α-(1,3)-glucan. First, yeasts of H.
capsulatum isolate 268 were cultivated in liquid HMM until late
exponential growth phase. Next, the culture was centrifuged
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for 1 minute at 600 × g and the supernatant containing
individual fungal cells was collected. The concentration of
yeasts in liquid HMM was adjusted to 1 × 106 yeasts/ml,
and cultures were maintained in an orbital shaker incubator
at 37◦ C, 120 rpm. This procedure was repeated nine times
every 48 hours. Yeasts were then plated in solid HMM (HMM
containing 0.6% agarose and 25 μM FeSO4 ) and incubated
at 37◦ C. After 3 days, the morphology of fungal colonies was
analyzed and smooth-textured colonies (named 268S) were
selected as possible yeasts that did not contain α-(1,3)-glucan.
The presence or absence of this polysaccharide was evalu-
ated by immunofluorescence or flow cytometry as described
below.
Epithelial cell culture
The human lung adenocarcinoma epithelial cell line A549 was
cultured in ‘complete DMEM’ that consisted of DMEM (Dul-
becco’s Modified Eagle’s Medium) supplemented with 10% fetal
bovine serum (FBS, Vitrocell, SP, Brazil), 10 mM HEPES, 100
U/ml penicillin, and 100 μg/ml streptomycin at 37◦ C, 5% CO2 .
Epithelial cell interaction with H. capsulatum
A549 cells were cultivated in 6-well or 24-well plates (9 × 104
or 2.4 × 104 cells per well, respectively) and after 72 hours,
these cells were maintained overnight in FBS-free DMEM. For
some experiments, prior to fungal infection A549 cells were
incubated for 2 hours in serum-free DMEM containing 10 μM
PP2 (inhibitor of SFK activation - Sigma-Aldrich Corpora-
tion/Merck KGaA, Darmstadt, Germany), 2 μM PRT062607
(inhibitor of Syk activation - Selleck Chemicals, Houston,
TX, USA), 10 μM PI-PKC δ (a peptide inhibitor of PKC δ
activation – Biomatik, USA/Canada), 0.015% DMSO (used as
vehicle for PP2 and PRT062607), or 0.01% NH4 OH (used as
vehicle for PI-PKC δ).
The preparation of H. capsulatum yeasts (268, 268S, and
G217B) for A549 cell infection involved washing the fungi
three times with FBS-free DMEM. After the last centrifuga-
tion, fungal pellets were weighted and resuspended in FBS-free
DMEM so that the relationship of pellet weight/volume of cul-
ture medium was the same among all three fungi (268, 268S,
and G217B). Then, fungal suspensions were forced five times
through a 23-gauge needle to disperse aggregated yeasts. Next,
aliquots of fungi were fixed with 4% formaldehyde and counted
in a Neubauer chamber. Finally, the same amount of yeasts was
Alcantara et al.
added to A549 cell cultures. Multiplicity of infection (MOI) and
incubation time periods are described in figure legends.
By MTT [‘3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide’] assays, A549 cell viability was analyzed, and
more than 95% of the cells remained viable in all experiments
(data not shown).
Measurement of IL-8 levels in epithelial cell cultures
infected with H. capsulatum
After an overnight infection of A549 cells with H. capsulatum
yeasts, cell culture supernatants were collected and centrifuged at
1300 × g for removal of yeasts and cell debris. IL-8 levels were
determined in these supernatants by using the IL-8 DuoSet
ELISA kits, following the manufacturer’s instructions (R&D
Systems Incorporation, Minneapolis, MN, USA).
Indirect immunofluorescence and flow cytometry
H. capsulatum yeasts were washed three times with PBS (10 mM
sodium phosphate buffer, pH 7.2, 150 mM NaCl), fixed with
4% formaldehyde in PBS for 15 minutes, and incubated with
5% bovine serum albumin (BSA) in PBS. After 1 hour, yeasts
were incubated with anti-α-(1,3)-glucan antibody (MOPC
104E - Sigma-Aldrich Corporation/Merck KGaA, Darmstadt,
Germany) for 2 hours and then with the secondary antibody
anti-mouse immunoglobulin conjugated to AlexaFluor R
488
(Thermo Fisher Scientific, Rockford, IL, USA) for 1 hour. Before
each step, yeasts were washed with PBS three times. All these
steps were performed at room temperature.
For indirect immunofluorescence analysis, after the secondary
antibody step, yeasts were incubated for 10 minutes with PBS
containing 1% BSA and 1 μg/ml calcofluor (Sigma-Aldrich
Corporation/Merck KGaA, Darmstadt, Germany). Next, yeasts
were washed twice with PBS, resuspended in SlowFadeTM
(Thermo Fisher Scientific, Rockford, IL, USA), and 10 μl were
placed on glass slides and covered with coverslips. Fluores-
cence was analyzed in a fluorescence microscope Olympus BX51
(Japan).
For flow cytometry analysis, after the secondary antibody
step, the fungal suspension was applied to the flow cytometer
FACS Canto II (Becton Dickinson Immunocytometry Systems,
San Jose, CA, USA) and 10 000 events were analyzed. Besides
all caution to prevent yeast clumping (described in ‘Epithelial
cell interaction with H. capsulatum’), the flow cytometry gating
strategy included a first dot plot gate of FSC-A versus FSC-
H (area vs height) to exclude eventual clumps in the analysis,
and then the fungal population was gated to further analyze α-
(1,3)-glucan intensity by histogram. Moreover, registered events
that were out of the population gate (considering the size and
cellular complexity) were not analyzed. The collected data were
analyzed by using the software BDFACSDiva (Becton Dickinson
3
Immunocytometry Systems, San Jose, CA, USA) and Flowing
Software (Perttu Terho, Turku, Finland).
SDS-PAGE and Western blot
Activation of SFKs, Syk, or PKC δ in A549 epithelial cells was
analyzed after H. capsulatum infection by Western blot. In these
experiments, the MOI was 10 yeasts per A549 cell. For protein
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extraction, A549 epithelial cells were washed with PBS contain-
ing 1 mM sodium vanadate (PBS/SV) and collected with a cell
scraper. Then, cells were resuspended with M-PER R
protein
extraction buffer (Thermo Fisher Scientific, Rockford, IL, USA)
containing 1 mM sodium vanadate and a mixture of protease in-
R hibitors (1 μM leupeptin, 125 μg/ml aprotinin, and 500 μg/ml
AEBSF - Sigma-Aldrich Corporation/Merck KGaA, Darmstadt,
Germany). Cell lysis was performed by alternating vigorous vor-
texing (10 seconds) and placing the samples on ice for 5 minutes.
After six cycles, samples were centrifuged at 13 000 × g for
10 minutes and protein quantification of the supernatant was
performed with Micro BCA Protein Assay Kit (Thermo Fisher
Scientific, Rockford, IL, USA), according to the manufacturer’s
instructions.
Next, samples were solubilized in sample buffer (Tris-HCl
73.5 mM, pH 6.8, 40 mM SDS, 280 mM glycerol, and 0.03%
bromophenol blue). Same amount of protein of each sample
was subjected to SDS-PAGE. Subsequently, proteins were trans-
ferred to PVDF membranes (Sigma-Aldrich Corporation/Merck
KGaA, Darmstadt, Germany), which were blocked with 5%
milk solution in TBST (200 mM Tris-HCl buffer, pH 8.0 con-
taining 0.1% Tween-20) for 1 hour at 25◦ C. Then, membranes
were incubated with the following primary antibodies: anti- P-
SFK (Tyr416 ), P-Syk (Tyr525/526 ), P-PKC δ (Thr505 ) (Cell Sig-
naling Technology - Beverly, MA, USA), or β-actin (Sigma-
Aldrich Corporation/Merck KGaA, Darmstadt, Germany). After
overnight incubation at 4◦ C, membranes were incubated with
HRP (‘horseradish peroxidase’)-conjugated secondary antibod-
ies diluted in 1% BSA in TBS for 1 hour at 25◦ C. After each step,
PVDF membranes were washed with TBST containing 1 mM
sodium vanadate and, to detect antibody-positive bands, mem-
branes were incubated with SuperSignal West Pico Chemilumi-
nescent Substrate R
reagent (Thermo Fisher Scientific, Rockford,
IL, USA). Proteins recognized by the antibodies were detected
using a UVITEC system (Alliance 4.7, Cambridge, UK), and
quantification of these proteins was performed by densitometric
analyses using Scion Image (Scion Co., USA).
Results
IL-8 secretion by A549 cells during interaction with
different isolates of H. capsulatum
Since our group has recently described that isolate 496 of
H. capsulatum induced cytokine secretion by the human lung
4 Medical Mycology, 2020, Vol. 00, No. 00
Figure 1. IL-8 levels in A549 cell cultures during infection with H. capsulatum,
isolates 268 or G217B. A549 epithelial cells were incubated with H. capsula-
r
tum yeasts, isolates 268 () or G217B ( ), at different MOIs. After 16 h, culture
supernatants were collected and IL-8 levels were determined by ELISA. Val-
ues represent the mean of triplicates ± standard deviation (SD). *, P < 0.01
compared to A549 cells infected with H. capsulatum, isolate G217B. Similar
results were obtained in two other independent experiments.
epithelial cell line A549,13 we analyzed whether other isolates of
this fungal species also promote IL-8 secretion by these epithelial
cells. Figure 1 shows that H. capsulatum isolates 268 and G217B
increased IL-8 levels in A549 cell cultures in a MOI-dependent
manner. Furthermore, 268 yeasts induced IL-8 secretion by A549
cells up to twofold higher than G217B. Therefore, despite the
fact that H. capsulatum yeasts promote IL-8 secretion by A549
cells, this cytokine levels may vary depending on the isolate used
for the infection of epithelial cell cultures.
Presence of α-(1,3)-glucan in different isolates of H.
capsulatum yeasts
One stark difference among the H. capsulatum isolates is the
presence (or absence) of α-(1,3)-glucan in the yeast cell wall.8,18
Some groups have already described that this polysaccharide is
absent in isolate G217B.8,18 On the other hand, isolate 268 of
H. capsulatum was obtained from the fungal collection of Escola
Paulista de Medicina – Universidade Federal de São Paulo, and
there was no information about the presence of α-(1,3)-glucan
in this fungus. Thus, the occurrence of this polysaccharide was
evaluated in both isolates 268 and G217B. For this, we ana-
lyzed macroscopic colony features and microscopic morphology,
since yeasts that have α-(1,3)-glucan form large clumps in liquid
medium and rough colonies on solid medium. Conversely, when
α-(1,3)-glucan is absent, yeasts grow as single cells or form very
small clumps in liquid media, and on solid medium, these fungi
form smooth colonies.8,17
As expected, Figure 2B, D shows that isolate G217B presented
smooth colonies on solid medium and only up to three yeasts
formed a clump. In contrast, we observed rough colonies of iso-
late 268 in solid medium and large clumps in liquid medium,
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Figure 2. Presence of α-(1,3)-glucan in H. capsulatum, isolates 268 and G217B.
(A) and (B): Macroscopic aspects of fungal colonies cultured in solid medium.
(C) and (D): Phase contrast of H. capsulatum yeasts cultured in liquid medium.
(E) and (F): Cell wall staining with calcofluor. (G) and (H): Indirect immunofluo-
rescence using anti-α-(1,3)-glucan antibody. Bar = 10 μm. (I) and (J): Analysis
of the presence of α-(1,3)-glucan, which was assessed by flow cytometry. Black
lines represent yeasts incubated with the secondary antibody only. Blue and
red filled areas correspond respectively to isolates 268 and G217B that were
incubated with anti-α-(1,3)-glucan antibody. Bars and values represent the
percentage of α-(1,3)-glucan positive yeasts. These data are representative of
four independent experiments. (A), (C), (E), (G), and (I): H. capsulatum, isolate
268. (B), (D), (F), (H), and (J): H. capsulatum, isolate G217B. This Figure is
reproduced in color in the online version of Medical Mycology.
suggesting that α-(1,3)-glucan is present in this H. capsulatum
isolate (Fig. 2A, C). In fact, by indirect immunofluorescence and
flow cytometry, we verified that most of the isolate 268 yeast
population was positive when using anti-α-(1,3)-glucan antibod-
ies (Fig. 2G, I), while no reactivity was observed in isolate G217B
yeasts (Fig. 2H, J). Taken together, these results corroborate the
morphological analyses that α-(1,3)-glucan is present in isolate
268 yeasts and absent in isolate G217B.
One way to obtain in vitro H. capsulatum yeasts that lack
α-(1,3)-glucan is submitting yeast cultures to the Klimpel and
Goldman’s protocol.8,17 For this, we used yeasts of the iso-
late 268 that originally presented this polysaccharide in the
cell wall, as shown in Figure 2. As presented in Figure 3, the
Alcantara et al.
Figure 3. Presence of α-(1,3)-glucan in H. capsulatum, isolates 268 and spon-
taneous variant 268S. (A) and (B): Macroscopic aspects of H. capsulatum
colonies cultured in solid medium. (C) and (D): Phase contrast of H. capsula-
tum cultured in liquid medium. (E) and (F): Cell wall staining with calcofluor.
(G) and (H): Indirect immunofluorescence using anti-α-(1,3)-glucan antibody.
Bar = 10 μm. (I) and (J): Analysis of the presence of α-(1,3)-glucan, which was
assessed by flow cytometry. Black lines represent yeasts incubated with the
secondary antibody only. Dark blue (I) and light blue (J) filled areas represent,
respectively, 268 and 268S yeasts incubated with anti-α-(1,3)-glucan antibody.
Bars and values represent the percentage of α-(1,3)-glucan positive yeasts.
(A), (C), (E), (G), and (I): H. capsulatum, isolate 268. (B), (D), (F), (H), and (J):
H. capsulatum, variant 268S. (K): A549 cells were incubated with 268 or 268S
yeasts (MOI 10:1). After overnight incubation, culture supernatants were col-
lected, and IL-8 levels were determined by ELISA. Values represent the mean
of triplicates ± SD and are representative of four independent experiments. ∗ ,
P <0.01 when compared to C (uninfected cells). This Figure is reproduced in
color in the online version of Medical Mycology.
5
Klimpel and Goldman’s protocol17 allowed the selection of
fungi (named 268S) that were morphologically similar to iso-
late G217B (Fig. 2), that is, smooth colonies on solid medium
and small yeast clumps in liquid medium. Furthermore, analy-
ses by indirect immunofluorescence and flow cytometry showed
that 268S yeasts were negative when using anti-α-(1,3)-glucan
antibodies (Fig. 3H, J).
Next, IL-8 was measured in A549 cell cultures infected with
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yeasts of isolate 268 or the spontaneous variant 268S, and we
observed that this cytokine secretion increased 4.2- and 4.9-fold
over basal levels, respectively. Therefore, a comparison between
A549 cell cultures infected with 268 or 268S shows similar IL-8
levels (Fig. 3K), indicating that the absence of α-(1,3)-glucan in
the cell wall of 268S did not modify A549 cell response towards
IL-8 secretion.
Activation of SFKs, Syk, and PKC δ in A549 cells
during interaction with H. capsulatum
Recently, our group demonstrated that different fungi induce
IL-8 secretion by epithelial cells in SFK- or PKC δ- dependent
manner.11,13 In the present work, we verified by Western blot
that the two isolates of H. capsulatum (268 and G217B) and the
spontaneous variant (268S) promoted SFK activation in A549
cells, reaching a peak at 3 hours of infection (Fig. 4A).
As SFKs may cause tyrosine phosphorylation of some recep-
tors such as Dectin-1, generating docking sites for Syk, which
undergoes to activation,15 we also analyzed the phosphorylation
of Syk at Tyr525/526 by Western blot (Fig. 4B). By densitometric
analysis and comparing to basal levels, we verified that the high-
est levels of Syk phosphorylation occurred when A549 cells were
incubated with isolate G217B (5.7-fold, 30 minutes of infection)
or with the variant 268S (5.9-fold, 1 hour). Isolate 268 yeasts
were able to increase Syk activation but only up to 2.6-fold over
basal levels. Relative values for Syk phosphorylation, determined
by densitometric analysis of the bands obtained by Western blot,
were: (i) for Control: blot on the left side = 0.13, blot on the right
side = 0.14; (ii) for 268 isolate: 5 min = 0.22, 15 min = 0.28,
30 min = 0.28, 1 h = 0.30, 3 h = 0.37; 5 h = 0.35; (iii) for
G217B isolate: 5 min = 0.09, 15 min = 0.40, 30 min = 0.77,
1 h = 0.47, 3 h = 0.38; 5 h = 0.42; and (iv) for 268S isolate:
5 min = 0.07, 15 min = 0.34, 30 min = 0.63, 1 h = 0.85,
3 h = 0.51; 5 h = 0.82.
Regarding PKC δ, an activation increase was observed when
A549 cells were incubated with 268, G217B, or 268S (Fig. 4C).
A maximum activation peak occurred at 5 hours when these
epithelial cells were infected with the isolate 268 (Fig. 4C), and by
densitometric analysis (data not shown), we verified an increase
by 6.0-fold over basal levels. Regarding infection of A549 cells
with isolate G217B and the variant 268S, we verified that these
fungi promoted an increase of PKC δ activation by 4.1- and
3.4-fold over basal levels, respectively.
6 Medical Mycology, 2020, Vol. 00, No. 00

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Figure 4. Activation of SFKs, Syk, and PKC δ in A549 epithelial cells infected with H. capsulatum yeasts. A549 cells were incubated for the indicated time
periods with H. capsulatum yeasts, isolates 268 or G217B, or the spontaneous variant 268S. MOI was 10:1. Next, A549 cells were collected, lysed, and subjected
to SDS-PAGE. Activation of SFKs (A), Syk (B) and PKC δ (C) was analyzed using specific antibodies that recognize specific phosphorylated sites that indicate
activation of these kinases. β-actin was used as protein loading control. Blots are representative of two independent experiments.

Involvement of SFKs, Syk, and PKC δ in IL-8 secretion
by A549 cells during interaction with H. capsulatum
As previously mentioned, our group demonstrated that isolate
496 of H. capsulatum promoted IL-8 secretion by A549 cells
in SFK-dependent manner.13 In this work, we verified by ELISA
that PP2 (an inhibitor for SFK activation) decreased IL-8 levels
by 59% and 37% in cultures of A549 cells infected with isolate
268 and 268S, respectively (Fig. 5A). Interestingly, PP2 pro-
moted only a small reduction of IL-8 levels (13%) in A549 cell
cultures infected with isolate G217B. These results indicate that
isolate 268 and its spontaneous variant promoted IL-8 secretion
in A549 cells in a SFK-dependent manner.
Next, we used PRT062607 to inhibit Syk in A549 cell cultures
infected with H. capsulatum. Figure 5B, shows that this inhibitor
decreased IL-8 levels by 77% in A549 cell cultures infected with
isolate G217B. On the other hand, we did not verify any effect
of PRT062607 in IL-8 levels in cultures of A549 cells infected
with 268 or 268S, indicating that only isolate G217B promoted
IL-8 secretion in a Syk-dependent manner.
By using the peptide PI-PKC δ that specifically inhibits PKC δ
activation, we verified that this inhibitor was able to reduce IL-8
levels by 75%, 91%, and 34% in A549 cell cultures infected with
268, G217B, and 268S (Fig. 5C), respectively, thus indicating
that PKC δ contributed to IL-8 secretion that was promoted by
these fungi in A549 cells.
The efficiency of all inhibitors to reduce their respective kinase
activation was analyzed. For this, A549 cells were preincubated
with PP2, PRT062607, or PI-PKC δ and then with fungi. By
Western blot, we verified that these inhibitors reduced the acti-
vation of SFKs, Syk, or PKC δ in A549 cells infected with 268,
G217B, or 268S (Fig. 5).
Syk is activated in a SFK-dependent manner and also
contributes to SFK activation
As Syk activation may be dependent on SFK activation, we in-
fected A549 cells with H. capsulatum yeasts from isolates 268,
G217B, or 268S in the presence of the SFK inhibitor PP2 and
analyzed Syk activation by Western blot. Figure 6A shows that
PP2 reduced Syk phosphorylation up to 75% in A549 cells in the
presence of fungi. In a similar assay, but using the Syk inhibitor
PRT062607 instead of PP2, we verified that PRT062607 pro-
moted a reduction of SFK activation by 56%, 51%, and 68% in
A549 cells incubated with 268, G217B, and 268S, respectively
(Fig. 6B). Together, these results indicate that SFKs contribute
to Syk activation and vice versa.
Discussion
According to the composition of the cell wall, yeasts of H. capsu-
latum may be classified as chemotypes I or II. In the present work,
the absence of α-(1,3)-glucan was demonstrated in the yeast cell
wall of the H. capsulatum isolate G217B, corroborating previ-
ous data obtained by other groups and classifying this fungus as
chemotype I.8,18 Concomitantly, we verified that the isolate 268
contains this polysaccharide, and consequently, is a chemotype
II. Next, it was observed that the capability to promote IL-8
secretion in the lung epithelial cell line A549 was different be-
tween H. capsulatum isolates G217B and 268 (chemotypes I and
Alcantara et al. 7

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Figure 5. Effect of SFKs, Syk, and PKC δ inhibitors on IL-8 secretion by A549 cells infected with H. capsulatum yeasts. A549 cells were incubated in the presence
of vehicles (−) or inhibitors (+) of activation of (A) SFKs (PP2), (B) Syk (PRT062607 -PRT-), or (C) PKC δ (Peptide Inhibitor of PKC δ -PI-). After 2 h, H. capsulatum
yeasts (268, G217B, or 268S) were added to the A549 cell cultures (MOI 10:1). After 16 h, culture supernatants were collected and IL-8 levels were determined by
ELISA. IL-8 values represent the mean of triplicates ± SD. ∗, P < 0.01 when compared to control without the inhibitor and in the presence of vehicle (−) (graphs
on the left side of the figure). Activation of SFKs, Syk or PKC δ in the presence of inhibitors, or their respective vehicles, was analyzed by Western blot, using
antibodies that recognize specific phosphorylated sites that indicate activation of these kinases in A549 cells infected for 4 h with fungi (blots on the right side
of the figure). β-actin was used as a protein loading control. Similar results were obtained in two independent experiments.

II, respectively). This result agrees with the findings from other
researchers who demonstrated that yeasts of these chemotypes
promote distinct responses in the host.2
A variant named 268S was obtained after spontaneous loss of
α-(1,3)-glucan of the chemotype II 268 yeasts and both 268 and
268S fungi stimulated similar IL-8 levels in A549 cell cultures.
At first, this result was surprising, because we were expecting
alterations in the cytokine levels, since G217B, that also does
not present α-(1,3)-glucan, induced lower levels of IL-8 secre-
tion than 268 yeasts, which contain α-(1,3)-glucan. In addition,
other groups demonstrated differences in host cell response, such
as increase of macrophage survival after infection with α-(1,3)-
glucan-free chemotype II-derived variant yeasts.7,8
To study epithelial cell signaling differences that could reflect
the IL-8 secretion levels, promoted by 268, G217B, and 268S,
we first analyzed SFK activation, which is important for this
cytokine secretion promoted by H. capsulatum in A549 cells.13
In fact, we verified that these three fungi induced similar SFK
activation and inhibition of these kinases led to decrease of IL-8
secretion by A549 cells, indicating that these fungi promoted this
cytokine release in a SFK-dependent manner, especially 268 and
268S. Regarding PKC δ, all three fungi promoted activation of
this kinase in A549 cells with 268 and G217B yeasts leading to
the highest levels, and when using a specific inhibitor of PKC
δ, we verified a decrease in IL-8 secretion in epithelial cell-H.
capsulatum cultures. This IL-8 reduction was observed mainly
with 268 and G217B yeasts, coinciding with the result of PKC δ
activation.
Interesting differences in cell signaling mechanisms were ob-
served when Syk activation was analyzed in H. capsulatum in-
fected A549 epithelial cell cultures. The two fungi that lacked
α-(1,3)-glucan, 268S and G217B yeasts, induced the highest lev-
els of phosphorylated Syk. As α-(1,3)-glucan has been described
as an important cell wall component that conceals the underlying
8 Medical Mycology, 2020, Vol. 00, No. 00

A549 cell cultures infected with the spontaneous variant 268S.

The latter result and the lowest IL-8 level secretion induced in
this work by G217B suggested that Dectin-1 was not crucial for
this cytokine release in lung epithelial cells. We have analyzed
whether this receptor was essential for IL-8 secretion during
infection of A549 cells but, unfortunately, by using siRNA or
blocking antibodies towards Dectin-1, we have failed to obtain
reproducible results (data not shown). Moreover, β-glucan levels

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Figure 6. Effect of PP2 or PRT062607 on Syk or SFK activation during infection
of A549 cells with H. capsulatum yeasts. A549 cells were incubated for 2 h
in the presence of the vehicle (−), PP2 or PRT062607 (PRT) (+). Then, H.
capsulatum yeasts (268, G217B or 268S) were added to A549 cell cultures
(MOI 10:1). After 4 h, A549 cells were collected, lysed, and subjected to SDS-
PAGE. (A) Syk activation was analyzed by using antibodies that recognize
specific phosphorylated sites that indicate this kinase activation. (B) SFKs
activation was detected by using antibodies that recognize the activation of
these kinases. β-actin was used as a protein loading control. This result is
representative of two independent experiments.
β-glucan in the H. capsulatum chemotype II yeasts,19 one expla-
nation for 268S and G217B to promote higher activation of
Syk (than 268 isolate) may be attributable to recognition of the
exposed β-glucan by the host receptor Dectin-1.
However, when Syk inhibitor was used, only epithelial cell
cultures infected with G217B presented a decrease in IL-8 levels.
The same Syk inhibitor did not have any effect in IL-8 levels in
could also be low in these fungi due to the activity of H. capsula-
tum β-glucanases,20,21 which could lead to insufficient interac-
tion with the host Dectin-1 for IL-8 secretion by epithelial cells.
Quantification of β-glucan, measurement of β-glucanase activity
of these fungi, and the relationship of this polysaccharide toward
IL-8 secretion by A549 epithelial cells will be investigated in our
laboratory.
Taken all the results together, we demonstrated that one iso-
late of each chemotype (I and II) of H. capsulatum promoted IL-8
secretion by lung epithelial cells in different manners (Fig. 7). Be-
sides Dectin-1, other host receptors such as TLRs and integrins
may participate in IL-8 secretion by A549 cells.2,13 Regarding H.
capsulatum, in addition to β-glucans, other molecules may inter-
act with host epithelial receptors, such as mannans and the pro-
tein Yps3, which is expressed by some isolates of Histoplasma
and may be recognized by TLR2.22
H. capsulatum infects both immunocompetent and immuno-
compromised individuals. The severity of the disease depends
on the number of fungal particles inhaled and the host immune
response.2,23 Furthermore, this fungus presents an impressive

Figure 7. Host epithelial cell kinases are distinctly modulated by different H. capsulatum chemotypes. Different isolates of H. capsulatum may activate different
receptors (TLRs, Dectin-1, Integrins α3 and α5, and others) on the epithelial cell membrane, resulting in the activation of distinct kinases, and consequently
promoting different levels of IL-8 secretion. Isolate 268 (chemotype II) and its spontaneous variant 268S induced higher levels of IL-8 secretion by epithelial cells
when compared to isolate G217B (chemotype I). Isolates 268 and G217B and the variant 268S induced the activation of SFKs, Syk and PKC δ in epithelial cells.
In addition, SFK activation is dependent on Syk and vice versa. SFKs participate in IL-8 secretion promoted by 268 and 268S. Syk is important for IL-8 secretion
induced by G217B. PKC δ participates in this cytokine secretion by A549 cells during contact with all fungi used in this work.
Alcantara et al.
genetic variability among its isolates that even led Sepúlveda
et al. (2017)24 to suggest new species of Histoplasma.24,25
Therefore, studies that correlate host response differences to
particular Histoplasma characteristics will aid understanding
this fungal pathogenicity.
Funding
This work was supported by Fundação de Amparo à Pesquisa do Estado de
São Paulo [FAPESP grant numbers 2015/25652-6, 2016/06285-5]; Con-
selho Nacional de Desenvolvimento Cientı́fico e Tecnológico [CNPq grant
numbers 130309/2017-6, 164126/2014-7, and 306163/2015-2]; and,
Coordenação de Aperfeiçoamento de Pessoal de Nı́vel Superior (CAPES).
Declaration of interest
The authors report no conflicts of interest. The authors alone are respon-
sible for the content and the writing of the paper.
References
1. Wheat LJ, Azar MM, Bahr NC et al. Histoplasmosis. Infect Dis Clin North Am.
2016; 30: 207–227.
2. Ray SC, Rappleye CA. Flying under the radar: Histoplasma capsulatum avoid-
ance of innate immune recognition. Semin Cell Dev Biol. 2019; 89: 91–98.
3. Gauthier GM. Fungal dimorphism and virulence: molecular mechanisms for tem-
perature adaptation, immune evasion, and in vivo survival. Mediators Inflamm.
2017; 2017: 8491383.
4. Domer JE. Monosaccharide and chitin content of cell walls of Histoplasma
capsulatum and Blastomyces dermatitidis. J Bacteriol. 1971; 107: 870–877.
5. Kanetsuna F, Carbonell LM, Gil F, Azuma I. Chemical and ultrastructural studies
on the cell walls of the yeastlike and mycelial forms of Histoplasma capsulatum.
Mycopathol Mycol Appl. 1974; 54: 1–13.
6. Reiss E. Serial enzymatic hydrolysis of cell walls of two serotypes of yeast-form
Histoplasma capsulatum with α(1,3)-glucanase, β(1,3)-glucanase, pronase, and
chitinase. Infect Immun. 1977; 16: 181–188.
7. Eissenberg LG, West JL, Woods JP, Goldman WE. Infection of P388D1
macrophages and respiratory epithelial cells by Histoplasma capsulatum: Se-
lection of avirulent variants and their potential role in persistent histoplasmosis.
Infect Immun. 1991; 59: 1639–1646.
8. Sepúlveda VE, Williams CL, Goldman WE. Comparison of phylogenetically
distinct Histoplasma strains reveals evolutionarily divergent virulence strategies.
MBio. 2014; 5: e01376–14.
9
9. Suzuki T, Chow C-W, Downey GP. Role of innate immune cells and their prod-
ucts in lung immunopathology. Int J Biochem Cell Biol. 2008; 40: 1348–1361.
10. Moyes DL, Runglall M, Murciano C et al. A biphasic innate immune MAPK
response discriminates between the yeast and hyphal forms of Candida albicans
in epithelial cells. Cell Host Microbe. 2010; 8: 225–235.
11. Alcantara C, Maza PK, Barros BCSC, Suzuki E. Role of protein kinase C in
cytokine secretion by lung epithelial cells during infection with Paracoccidioides
brasiliensis. Pathog Dis. 2015; 73: ftv045.
12. Maza PK, Oliveira P, Toledo MS et al. Paracoccidioides brasiliensis induces
secretion of IL-6 and IL-8 by lung epithelial cells. Modulation of host cytokine
Downloaded from https://academic.oup.com/mmy/advance-article-abstract/doi/10.1093/mmy/myaa006/5771418 by guest on 02 March 2020
levels by fungal proteases. Microbes Infect. 2012; 14: 1077–1085.
13. Maza PK, Suzuki E. Histoplasma capsulatum-induced cytokine secretion in lung
epithelial cells is dependent on host integrins, Src-family kinase activation, and
membrane raft recruitment. Front Microbiol. 2016; 7: 580.
14. Slomiany BL, Slomiany A. Syk: a new target for attenuation of Helicobacter
pylori-induced gastric mucosal inflammatory responses. Inflammopharmacol.
2019; 27: 203–211.
15. Wagener M, Hoving JC, Ndlovu H, Marakalala MJ. Dectin-1-Syk-CARD9 sig-
naling pathway in TB immunity. Front Immunol. 2018; 9: 225.
16. Leiva-Juárez MM, Kolls JK, Evans SE. Lung epithelial cells: therapeutically in-
ducible effectors of antimicrobial defense. Mucosal Immunol. 2018; 11: 21–34.
17. Klimpel KR, Goldman WE. Isolation and characterization of spontaneous avir-
ulent variants of Histoplasma capsulatum. Infect Immun. 1987; 55: 528–533.
18. Edwards JA, Alore EA, Rappleye CA. The yeast-phase virulence requirement for
α-glucan synthase differs among Histoplasma capsulatum chemotypes. Eukary-
otic Cell. 2011; 10: 87–97.
19. Rappleye CA, Eissenberg LG, Goldman WE. Histoplasma capsulatum α-(1,3)-
glucan blocks innate immune recognition by the β-glucan receptor. Proc Natl
Acad Sci. 2007; 104: 1366–1370.
20. Garfoot AL, Shen Q, Wüthrich M, Klein BS, Rappleye CA. The Eng1 β-glucanase
enhances Histoplasma virulence by reducing β-glucan exposure. MBio. 2016; 7:
e01388–15.
21. Garfoot AL, Dearing KL, VanSchoiack AD, Wysocki VH, Rappleye CA. Eng1
and Exg8 are the major β-glucanases secreted by the fungal pathogen Histo-
plasma capsulatum. J Biol Chem. 2017; 292: 4801–4810.
22. Garfoot AL, Rappleye CA. Histoplasma capsulatum surmounts obstacles to in-
tracellular pathogenesis. FEBS J. 2016; 283: 619–633.
23. Horwath MC, Fecher RA, Deepe GS. Histoplasma capsulatum, lung infection
and immunity. Future Microbiology. 2015; 10: 967–975.
24. Sepúlveda VE, Márquez R, Turissini DA, Goldman WE, Matute DR. Genome
sequences reveal cryptic speciation in the human pathogen Histoplasma capsu-
latum. MBio. 2017; 8: e01339–17.
25. Van Dyke MCC, Teixeira MM, Barker BM. Fantastic yeasts and where to find
them: the hidden diversity of dimorphic fungal pathogens. Curr Opin Microbiol.
2019; 52: 55–63.