INFECTION AND IMMUNITY, Sept. 2010, p. 3871–3882
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 78, No. 9
Conidia but Not Yeast Cells of the Fungal Pathogen
Histoplasma capsulatum Trigger a Type I Interferon Innate
Immune Response in Murine Macrophages?†
Diane O. Inglis,1Charlotte A. Berkes,1Davina R. Hocking Murray,1and Anita Sil1,2*
Department of Microbiology and Immunology,1Howard Hughes Medical Institute,2University of
California—San Francisco, San Francisco, California 94143-0414
Received 2 March 2010/Returned for modification 12 March 2010/Accepted 29 June 2010
Histoplasma capsulatum is the most common cause of fungal respiratory infections and can lead to progres-
sive disseminated infections, particularly in immunocompromised patients. Infection occurs upon inhalation
of the aerosolized spores, known as conidia. Once inside the host, conidia are phagocytosed by alveolar
macrophages. The conidia subsequently germinate and produce a budding yeast-like form that colonizes host
macrophages and can disseminate throughout host organs and tissues. Even though conidia are the predom-
inant infectious particle for H. capsulatum and are the first cell type encountered by the host during infection,
very little is known at a molecular level about conidia or about their interaction with cells of the host immune
system. We examined the interaction between conidia and host cells in a murine bone-marrow-derived mac-
rophage model of infection. We used whole-genome expression profiling and quantitative reverse transcription-
PCR (qRT-PCR) to monitor the macrophage signaling pathways that are modulated during infection with
conidia. Our analysis revealed that type I interferon (IFN)-responsive genes and the beta type I IFN (IFN-?)
were induced in macrophages during infection with H. capsulatum conidia but not H. capsulatum yeast cells.
Further analysis revealed that the type I IFN signature induced in macrophages in response to conidia is
independent of Toll-like receptor (TLR) signaling and the cytosolic RNA sensor MAVS but is dependent on the
transcription factor interferon regulatory factor 3 (IRF3). Interestingly, H. capsulatum growth was restricted
in mice lacking the type I IFN receptor, indicating that an intact host type I IFN response is required for full
virulence of H. capsulatum in mice.
Studying the interaction between macrophages and intracel-
lular pathogens has provided fundamental information about
the innate immune response to microbial challenge. Macro-
phages use a number of different receptors to recognize and
phagocytose microbes, resulting in the activation of a variety of
antimicrobial effector mechanisms (1, 2, 6, 7, 36, 51, 72, 79, 83).
Intracellular pathogens have evolved to modulate some innate
immune mechanisms and replicate within the phagosome or
cytosol of the host cell. While our understanding of the mac-
rophage response to bacterial intracellular pathogens has ad-
vanced in recent years, our knowledge of the host response to
fungal intracellular pathogens is still limited. Transcriptional
profiling of host cells has been used as a comprehensive
method to reveal host pathways that are activated in response
to infection (34, 38, 46, 50, 56). This work describes the mac-
rophage transcriptional response to the infectious form of the
fungal pathogen Histoplasma capsulatum.
H. capsulatum, the etiologic agent of histoplasmosis, is a
primary fungal pathogen that infects healthy as well as immu-
nocompromised individuals (14). Approximately 500,000 infec-
tions are thought to occur every year in the United States alone
(23, 48, 86, 88). Immunocompromised individuals tend to de-
velop progressive, disseminated disease that can be fatal. H.
capsulatum is endemic in the Ohio River Valley through the
midwestern United States into Texas and is a leading pathogen
affecting both AIDS patients in the Midwest (76) as well as
individuals taking tumor necrosis factor alpha (TNF-?) antag-
onists (20, 21, 32, 73).
H. capsulatum is a dimorphic fungus that is adapted to grow
either in the soil or in a mammalian host. In the soil, it grows
in a hyphal (or filamentous) form. The hyphae generate two
types of vegetative spores, macroconidia (8 to 25 ?m) and
microconidia (2 to 6 ?m), which are distinguished mainly on
the basis of size (64). After inhalation, conidia are taken up by
macrophages and other phagocytic cells (13, 23, 88). Once
inside the host, conidia germinate and give rise to yeast cells,
which evade phagocytic killing and multiply within alveolar
macrophages (AvMs). Yeast cells use phagocytic cells as vehi-
cles to spread to multiple organs of the reticuloendothelial
system (such as the spleen, liver, lymph nodes, and bone mar-
row) and to other organs in patients with disseminated disease
(19, 23, 36, 58). Whereas the yeast form is the parasitic form of
the organism, conidia are thought to be the infectious particle
of H. capsulatum. Thus, studying the interaction of conidia
with immune cells sheds light on the initial stages of infection.
Recognition of H. capsulatum conidia or yeast cells by host
cells and the resultant downstream signaling events are just
beginning to be investigated. A number of germ line-encoded
receptors (e.g., membrane-bound Toll-like receptors, or TLRs,
and cytosolic NOD-like receptors, or NLRs) have been iden-
tified as critical for recognition of microbes by immune cells
* Corresponding author. Mailing address: Howard Hughes Medical
Institute, Department of Microbiology and Immunology, University of
California—San Francisco, San Francisco, CA 94143-0414. Phone:
(415) 502-1805. Fax: (415) 476-8201. E-mail: email@example.com.
† Supplemental material for this article may be found at http://iai
?Published ahead of print on 6 July 2010.
(35, 39, 62). In the case of fungi, the main surface-expressed
pattern recognition receptors (PRRs) involved in detection of
these organisms are TLR2 and TLR4; the mannose receptor
(MR); Dectin-1, which recognizes the major fungal cell wall
carbohydrate ?-glucan; Dectin-2; and DC-SIGN (9–11, 57, 65,
80, 87). As of yet, the roles of these and other PRRs in the host
response to H. capsulatum are largely unexplored, although it
is known that ?-glucan present in the yeast cell wall is shielded
from recognition by Dectin-1 by the presence of an outer layer
of ?-(1–3)-glucan in particular H. capsulatum strains (65).
In contrast to H. capsulatum, much is known about the host
response to a variety of other types of pathogenic agents. A
critical host response to viral infection is the induction of type
I interferons (IFNs), a family of cytokines (including beta IFN
[IFN-?] and multiple IFN-? molecules) that signal through the
type I IFN receptor (IFNAR). Type I IFN production is initi-
ated via phosphorylation and activation of the IFN regulatory
factor 3 (IRF3) and IRF7 transcription factors, which then
activate expression of type I IFNs. A secondary response is
stimulated when secreted IFN-? signals in an autocrine- and
paracrine-type manner through the type I IFN receptor, IFNAR
(composed of the IFNAR1 and IFNAR2 subunits), which in
turn leads to induction of a large set of type I IFN response
genes through activation of the JAK/STAT pathway (for re-
views, see references 17 and 18). Type I IFNs directly induce
critical antiviral effectors and influence the function of NK
cells and CD8?T cells in antiviral defense. In recent years, it
has been observed that bacterial and parasitic infections also
induce a type I IFN signature in host cells, but signaling of
these cytokines through IFNAR can benefit either the host or
the pathogen (5, 12, 33, 61, 75, 85). The ability of fungi to
trigger a type I IFN response is largely unknown and only
beginning to be explored (8).
In this study, we used transcriptional profiling to investigate
the macrophage response to infection with H. capsulatum
conidia. Surprisingly, murine bone marrow-derived macro-
phages (BMDMs) induced a classic type I interferon (IFN)
transcriptional signature in response to infection with H. cap-
sulatum conidia, but not in response to infection with isogenic
yeast cells. We showed that the transcription factor IRF3,
which is required for previously characterized type I interferon
responses to other stimuli, is required for the induction of
IFN-? transcript in BMDMs in response to conidia, whereas
the TLR adaptors MyD88 and TRIF and the cytosolic RNA-
sensing adaptor MAVS are not. Interestingly, induction of the
interferon-responsive gene Ifi205 was observed during infec-
tion of alveolar macrophages with conidia but not yeast cells,
again suggesting that these two H. capsulatum cell types can
elicit a different host response. Finally, mice lacking IFNAR1
restricted the growth of H. capsulatum in the lungs and spleen
compared to that in wild-type (WT) controls, indicating that
type I IFN signaling in response to H. capsulatum benefits the
pathogen rather than the host.
MATERIALS AND METHODS
Cell culture and bone marrow-derived macrophage infections. For bone mar-
row collection, 8-week-old wild-type C57BL/6 mice were obtained from Charles
River Laboratories. Macrophages were differentiated from the bone marrow
from femurs of 8-week-old mice for 6 days in bone marrow-derived macrophage
medium (BMM) containing Dulbecco’s modified Eagle’s medium (DMEM)-
H21, 20% fetal calf serum, 10% colony-stimulating factor (CSF) from 3T3 cells,
2 mM glutamine, 1 mM sodium pyruvate, and penicillin-streptomycin (Pen/
Strep) at 37°C in 5% CO2. Femurs from 8-week-old myd88?/?trif?/?double-
knockout mice in the C57BL/6 background were obtained from the laboratory of
G. Barton, University of California—Berkeley, and differentiated as described
above. For all bone marrow-derived macrophage experiments, cells were grown
in the same medium. Bone marrow-derived macrophages from mavs knockout
(?/?) and mavs heterozygous (?/?) littermate controls were obtained from the
laboratory of R. Vance, University of California—Berkeley. Bone marrow-de-
rived macrophages from irf3?/?mice were obtained from the laboratory of J.
Cox, University of California—San Francisco (UCSF).
Bone marrow-derived macrophages were seeded at 7 ? 105cells/well in 6-well
dishes or at 2 ? 105cells/well in 24-well dishes in BMM. After 16 to 20 h of
growth, macrophages were infected with conidia or yeast cells resuspended in
DMEM or phosphate-buffered saline (PBS). Conidia or yeast cells were centri-
fuged onto macrophages and incubated at 37°C in 5% CO2for the times indi-
cated. For quantitative reverse transcription-PCR (qRT-PCR), macrophages
were infected at a multiplicity of infection (MOI) of 10 for the times indicated
and then were washed twice in prewarmed BMM prior to collection in RNeasy
minikit cell lysis reagent (Qiagen). For microarray time course experiments,
macrophages were washed 1 h postinfection with prewarmed medium and then
collected at the indicated time points in cell lysis reagent.
Mice. ifnar1?/?mice that were back-crossed for at least 8 generations to the
C57BL/6 background were obtained from the laboratory of J. Cox (74). Age- and
sex-matched WT (C57BL/6) mice for infections were purchased from Charles
River Laboratories. All mice were handled according to protocols approved by
the UCSF Institutional Animal Care and Use Committee.
Histoplasma growth and conidia purification. Histoplasma strains were
thawed from frozen stocks as yeast cells onto Histoplasma-macrophage medium
(HMM) at 37°C with 5% CO2and passaged up to 3 times on plates. For infection
of macrophages, yeast cells were grown to early log phase in HMM and washed
and resuspended in warm PBS. Clumps of cells were pelleted by centrifugation
of 50 ml of culture at 50 g for 5 min in a conical tube. The top 10 ml, which was
enriched for single cells, doublets, and triplets, was collected, counted on an
improved-Neubauer-phase hemacytometer, and diluted in PBS for infection.
Conidia from the G217B, G184AR, and G184AS strains were obtained by
plating approximately 3 ? 107yeast cells on 15-cm petri plates containing
synthetic medium 1 (3) or on Bird agar (http://www.fgsc.net/fgn51/fgn51metz
.html) supplemented with cysteine-HCl and penicillin-streptomycin (Pen/Strep)
as indicated. The G186AR strain, which grows poorly on synthetic media, was
grown on Sabouraud dextrose agar to produce conidia. Plates were sealed in
parafilm and cultured at room temperature for 4 to 12 weeks in a biosafety level
3 facility: G184AR and G184AS strains required 10 weeks to produce reasonable
numbers of conidia, whereas G217B and G186AR routinely produced adequate
yields of conidia within 4 to 5 weeks of incubation. Conidia were harvested by
flooding the plates with PBS and dislodging the conidia with a bent glass rod.
Mycelial fragments were removed from the conidial suspension by filtration
through sterile glass wool. Conidia were pelleted by centrifugation at 2,000 ? g,
at 4°C for 10 min, washed, resuspended in PBS or PBS with Pen/Strep, and
stored at 4°C until use. Conidia were heat killed by incubation in PBS at 95°C for
20 min. Conidial viability was confirmed by plating serial dilutions on brain heart
infusion (BHI) agar with 10% sheep blood, 0.05% cysteine-HCl, and 10 ?g/ml
gentamicin and incubating for at least 10 days at 30°C.
RNA preparation. Macrophage RNA was purified using a RNeasy minikit and
Qiashredder columns (Qiagen) according to the manufacturer’s instructions with
the following modification. Macrophage lysates were centrifuged for 5 min at
14,000 rpm to pellet any yeast cells or conidia prior to loading onto Qiashredder
colums. For qRT-PCR analysis, RNA was treated with RQ1 RNase-free DNase
I (Promega) for 20 min at room temperature. RQ1 stop solution was added, and
reaction mixtures were incubated at 65°C for 15 min to inactivate the DNase I
Microarray analysis. Total RNA was amplified to generate anti-sense RNA
(aRNA) using the amino allyl MessageAmp II aRNA kit (Ambion). Each sample
was labeled with Cy5 and competitively hybridized to a reference sample con-
sisting of a pool of experimental samples labeled with Cy3. The aRNAs were
fragmented with RNA fragmentation reagent (Ambion) according to the man-
ufacturer’s instructions prior to hybridizing the samples to microarrays. Microar-
rays were printed at the UCSF Center for Advanced Technology, using the
MEEBO (Mouse Exonic Evidence-Based Oligonucleotide) 70-mer oligonucleo-
tide set (Illumina; for more details, see http://alizadehlab.stanford.edu/). Mi-
croarrays were scanned using Gene Pix Pro 6.0 software on an Axon 4000B
scanner (Molecular Devices). Grids were generated for each array with Gene Pix
6.0 (Molecular Devices), and the data were uploaded to the NOMAD database
3872INGLIS ET AL.INFECT. IMMUN.
(http://ucsf-nomad.sourceforge.net/) for quality control and normalization. Sig-
nificantly induced genes were determined using the MeV implementation of
SAM (Significance Analysis of Microarrays) with a false discovery rate of less
than 5%. Information linked to each unique Oligo ID can be accessed at http:
//meebo.ucsf.edu:8080/meebo/meeboInfo.jsp?oligoid ? (insert Oligo ID here).
The data were organized for presentation with XCluster (http://genome-www5
.stanford.edu/download/) and Java Treeview software (22, 70).
qRT-PCR analysis. For qRT-PCR, 1 ?g of DNase I-treated total macrophage
RNA was reverse transcribed with Affinity Script multitemperature reverse
transcriptase (Stratagene) and 500 ng oligo(dT19V) (Integrated DNA Technol-
ogies, San Diego, CA) for 2 h. cDNA was diluted 3- to 4-fold with pyrogen-free
water. Two microliters of diluted cDNA was used in each 25-?l reaction. Reac-
tions were run on an Mx3000P machine (Stratagene), and MxPro software
(Stratagene) was used to determine threshold and threshold cycle (CT) values.
qRT-PCR data were normalized to hypoxanthine phosphoribosyltransferase 1
(HPRT1) expression using the Pfaffl method (63). The IFN-? expression shown
is relative to that of the mock-infected control unless otherwise indicated. Data
are representative of at least 3 (in many cases 4 or more) independent experi-
ments. Error bars represent the standard error of the mean for replicate qRT-
PCRs. The primers used in this study were IFNb-F (CTGGAGCAGCTGAAT
GGAAAG), IFNb-R (CTTGAAGTCCGCCCTGTAGGT), mHPRT1-F (AGG
TTGCAAGCTTGCTGGT), and mHPRT1-R (TGAAGTACTCATTATAGTC
Quantitation of phagocytosis and cell staining. For immunostaining, macro-
phages were seeded in 24-well dishes on 12-mm coverslips and infected as
described above. After 2 h, macrophages were washed twice with BMM to
remove unbound conidia or yeast cells. Coverslips were fixed in phosphate-
buffered saline (PBS) with 3.7% formaldehyde for 5 min and then washed in PBS
and stored at 4°C until stained. Extracellular conidia or yeast cells were detected
with 1:300 anti-Histoplasma mold or 1:500 anti-Histoplasma yeast cell antibodies
(a kind gift of Joseph Wheat, Miravista Labs) in PBS with 1% bovine serum
albumin (BSA) for 30 min at room temperature. Goat anti-rabbit Alexa 594
(Molecular Probes) secondary antibody was used at 1:500, concanavalin A-flu-
orescein isothiocyanate (FITC) (Invitrogen) was used at 1:400 to stain macro-
phages, and 10 ?g/ml calcofluor (fluorescence brightener 28; Sigma-Aldrich) was
used to stain all fungal cells (4, 47). Fluorescence images were obtained on a
Zeiss Axiovert 200 inverted microscope using Axiovision 4.4 software. Red,
green, and blue fluorescent channels were merged with Adobe Photoshop. Cy-
tochalasin D was resuspended in dimethyl sulfoxide (DMSO) and used at a 5 ?M
final concentration. At least 100 macrophages were evaluated per condition. To
visualize germination during macrophage infection, coverslips seeded with conid-
ium-infected macrophages were fixed at 24 h postinfection (hpi) in PBS with
3.7% formaldehyde for 5 min before staining with periodic acid-Schiff (PAS)
Isolation and infection of AvMs. Bronchoalveolar lavage (BAL) was per-
formed to obtain AvMs from the lungs of 8- to 12-week-old female C57BL/6
mice. Briefly, mice were sacrificed by cervical dislocation and lungs were flushed
with a total of 20 ml BAL solution (5 mM EDTA in PBS without Ca2?and
Mg2?). Cells were pelleted and resuspended in AKT solution (150 mM NH4Cl,
10 mM KHCO3, 0.1 mM EDTA in double-distilled water [ddH2O], pH 7.2 to 7.4)
to lyse contaminating red blood cells. The remaining cells were pelleted and
resuspended in high-glucose DMEM (UCSF Cell Culture Facility) supple-
mented with 10% fetal bovine serum (HyClone; Thermo Fisher [www.hyclone
.com]), penicillin, and streptomycin (UCSF Cell Culture Facility). AvMs were
seeded at a density of 6.0 ? 105cells/well in 24-well plates and allowed to settle
overnight prior to infection with G217B yeast cells or conidia at an MOI of 10.
As a positive control for induction of Ifi205, AvMs were treated for 2 h with 100
ng/ml lipopolysaccharide (LPS) (Sigma). Four hours postinfection, AvM total
RNA samples were isolated using the RNeasy minikit (Qiagen) and 250 ng total
RNA was reverse transcribed using the Affinityscript quantitative PCR (qPCR)
cDNA synthesis kit (Agilent). qRT-PCR analysis of Ifi205 expression in AvMs
was performed using the same methods as for analysis of IFN-? expression in
BMDMs, with the exception that reactions were carried out using SYBR green
qPCR master mix (Applied Biosystems). The primer sequences are CATCTTC
GGCTTCATCTAAC for Ifi205 fwd and ACATGGAAATACTGGCTCAC for
Mouse infections. Mice were anesthetized with isoflurane and infected intra-
nasally with 2 ? 106conidial CFU (from the G217B strain) or 2 ? 104yeast CFU
(from the G217B strain) in a volume of 25 to 40 ?l PBS. Since germination of
conidia must occur before they give rise to actively dividing yeast cells, and
because 100% of the conidia do not germinate, different numbers of infectious
particles for conidia and yeast cells were selected to allow a similar progression
of fungal burden and disease in both cases. At the indicated time points, mice
were euthanized using CO2inhalation followed by cervical dislocation. Lungs
and spleens were homogenized in Hank’s medium supplemented with 10 ?g/ml
gentamicin with disposable 15-ml conical homogenizers. Dilution series were
plated on brain-heart infusion (BHI) agar with 10% sheep blood, 0.05% cysteine,
and 10 ?g/ml gentamicin at 30°C for 10 to 14 days before enumeration of CFU.
P values were calculated using the Mann-Whitney rank sum test.
For histopathological analysis of infected tissues, age- and sex-matched WT
and ifnar?/?mice of the C57BL/6 background were infected intranasally with a
suspension of 2 ? 106G217B conidial CFU in sterile PBS. Mice were weighed
and monitored for symptoms at regular intervals. At the indicated time points,
two mice per strain were euthanized as described above. Postmortem, the tra-
chea was cannulated and the lungs were inflated in situ with 0.7 ml of 10%
formalin–PBS. The lungs were removed and fixed in 10% formalin–PBS before
serial dehydration and paraffin embedding. Five-micrometer parasagittal sec-
tions were taken at 100-?m intervals from the right lungs. At each level, sections
were stained for hematoxylin and eosin (H&E) or periodic acid-Schiff (PAS)
(Sigma-Aldrich). Sections were analyzed using light microscopy and photo-
graphed using a Leica DM1000 with a Leica DFC290 color camera. Images of
sections were montaged using Photoshop CS3 (Adobe Systems, Inc.) for addi-
tional comparative analysis of inflammatory regions.
Microarray data accession number. The GEO accession number for the mi-
croarray data is GSE20022.
Phagocytosis of conidia and infection of murine bone-mar-
row derived macrophages. Although numerous studies have
documented the interaction between macrophages and His-
toplasma yeast cells (59, 60), there has been only limited anal-
ysis of infection of macrophages with conidia. We generated
conidia from the virulent laboratory strain G217B, which has
been studied extensively in the yeast form. G217B yeast cells
were induced to form filaments and sporulate by incubation on
Materials and Methods) at room temperature. Under these con-
duced, with the remaining cells in the preparation being macro-
condia. To determine whether G217B microconidia (hereafter
referred to as conidia) were efficiently ingested by BMDMs, we
infected macrophages with conidia or yeast cells (which are
known to be efficiently phagocytosed by macrophages) at a
multiplicity of infection (MOI) of 3 or 5. After a 2-h incubation
period, we used polyclonal antibodies and calcofluor white to
detect Histoplasma yeast cells and conidia (see Materials and
Methods). Only external Histoplasma cells were accessible to
the antibodies, whereas both external and internal fungal cells
were accessible to calcofluor white (47), which binds to chitin
in the fungal cell wall (Fig. 1A). Quantitation of the staining
revealed that conidia and yeast cells were phagocytosed by
wild-type macrophages with similar efficiencies (85.8% of yeast
cells and 86.4% of conidia associated with macrophages were
internalized). Germination of conidia to give rise to yeast cells
was observed approximately 16 to 24 h postinfection (hpi) by
staining the infected macrophages with periodic acid-Schiff
base (PAS) (Fig. 1B). Ultimately, infection of macrophages
with conidia resulted in lysis of the macrophage monolayer, as
is observed for infection of BMDMs with H. capsulatum yeast
cells (data not shown).
Macrophages infected with conidia express type I interferon
response genes. To identify host signaling pathways induced
specifically in response to infection by conidia, we used Mouse
Exonic Evidence-Based Oligonucleotide (MEEBO) microar-
rays (Illumina) to determine the transcriptional profile of mu-
rine BMDMs infected with G217B conidia or yeast cells. Mac-
VOL. 78, 2010HISTOPLASMA-INFECTED MACROPHAGE TYPE I IFN RESPONSE3873
rophages were infected at an MOI of 5, and RNA was
harvested at 0, 3, 6, and 9 hpi. As expected, we found that
infection with both conidia and yeast cells resulted in induction
of general inflammatory response genes, including chemokines
and cytokines (C. A. Berkes et al., unpublished data). How-
ever, a group of 74 genes were significantly induced only in
macrophages infected with conidia (Fig. 2; see Table S1 in
supplemental material for the gene list). Many of these genes
are known to be induced by type I IFNs, suggesting that mac-
rophages were producing type I IFNs specifically in response to
infection with H. capsulatum conidia. Induction of type I IFN
response genes during infection of macrophages with conidia is
interesting because previous reports of type I IFN responses to
fungal infection are limited, although signaling through IF-
NAR1 has been shown to play a critical role in host survival
during infection with the fungal pathogen Cryptococcus neofor-
To test whether the type I IFN signaling pathway is required
for the transcriptional response of macrophages to conidia, we
infected macrophages deficient in the type I IFN receptor
(ifnar1?/?macrophages) with conidia and examined the result-
ant transcriptional response. Cells lacking the type I IFN re-
ceptor are capable of primary induction of type I IFNs but are
deficient in the secondary response that amplifies the primary
signal and results in the expression of downstream genes (37,
81). ifnar1?/?macrophages were unable to mount a wild-type
transcriptional response to H. capsulatum conidia (Fig. 2),
strongly suggesting that the production of type I IFNs and
subsequent signaling through IFNAR are required for the
transcriptional response to conidia.
H. capsulatum conidia trigger the induction of IFN-? tran-
script in macrophages. To confirm our transcriptional profiling
data, we used qRT-PCR as a sensitive assay to detect IFN-?
expression in infected macrophages. WT macrophages were
infected with G217B conidia at an MOI of 10, and RNA was
harvested at multiple time points between 1 and 6 hpi. Maxi-
mal (12-fold) induction of IFN-? occurred between 3 and 4 hpi
and declined by 6 hpi (Fig. 3A). Over the course of multiple
experiments, we routinely observed that infection with G217B
conidia at an MOI of 10 resulted in a range of IFN-? induction
that was largely dependent on the age of the conidia—i.e.,
conidia purified from plates incubated for a longer period (e.g.,
10 weeks) stimulated higher levels of IFN-? message than
conidia purified from plates incubated for shorter periods (e.g.,
4 weeks). We were not able to detect IFN-? protein production
by enzyme-linked immunosorbent assay (ELISA) (data not
shown), although the dependence of the host transcriptional
signature on IFNAR (Fig. 2) strongly suggests that type I IFN
proteins are produced and signal through IFNAR during in-
FIG. 1. Conidia are internalized by macrophages and germinate
intracellularly to give rise to yeast cells. (A) Differential interference
contrast (DIC) image (left) and immunofluorescence staining (right)
of macrophages infected with conidia of the G217B strain. Both inter-
nal and external conidia are stained in blue with calcofluor, whereas
only external conidia are stained in red with anti-Histoplasma antibod-
ies. Macrophages are stained green using concanavalin A-FITC.
(B) Periodic acid-Schiff (PAS) staining of conidia that have germinated
and are producing yeast cells within macrophages 24 hpi. With PAS,
conidia typically stain a darker magenta color than yeast cells. Three
representative yeast cells are indicated with arrowheads, and two rep-
resentative conidia are indicated with arrows.
FIG. 2. Heat map of type I IFN response genes induced by mac-
rophages infected with Histoplasma conidia but not yeast. C57BL/6
(WT) macrophages were subjected to either mock infection, infection
with H. capsulatum yeast cells, or infection with two independent
preparations of H. capsulatum conidia (Con 1 and Con 2). WT and
ifnar1?/?macrophages were mock infected (data not shown) or in-
fected with a third preparation of G217B conidia (Con 3). At 3, 6, and
9 hpi, macrophages were subjected to gene expression profiling. Genes
with statistically significant induction in two independent wild-type
macrophage infection experiments relative to the mock infection are
shown. Yellow indicates gene upregulation, blue indicates downregu-
lation, and black indicates no change. The color bar indicates the log2
3874 INGLIS ET AL.INFECT. IMMUN.
fection of bone-marrow derived macrophages with His-
To determine whether induction of IFN-? transcript by His-
toplasma is an active process that requires viable spores, we
compared the IFN-? responses of WT macrophages infected
with live or heat-killed G217B conidia (Fig. 3B). Whereas
infection with G217B yeast cells failed to induce IFN-?, infec-
tion with heat-killed conidia induced intermediate levels of
IFN-? transcript compared to infection with live conidia. Thus,
induction of IFN-? does not fully depend on conidial viability
and at least partially reflects a heat-resistant property of
Conidia from evolutionarily diverged Histoplasma strains
trigger induction of IFN-? transcript in macrophages. Molec-
ular studies of H. capsulatum biology and pathogenesis have
largely taken place in three distinct strains: the North Ameri-
can clinical isolate G217B and the Latin American clinical
isolates G186AR and G184AR (“R” indicates that the yeast
form of the organism has a rough colony morphology). These
strains were originally classified on the basis of the polysaccha-
ride composition of their cell walls (16, 67–69), which is a
microbial property that could influence the host immune re-
sponse. G217B yeast cells lack ?-(1,3)-glucan in their cell wall,
whereas the cell walls of G186AR and G184AR yeast cells are
rich in ?-(1,3)-glucan. Variants of G186AR and G184AR that
lack ?-(1,3)-glucan (the so-called “smooth” G186AS and
G184AS strains) are avirulent (44, 45), whereas G217B is vir-
ulent despite its lack of ?-(1,3)-glucan. Recent molecular phy-
logeny studies confirmed that G217B is in a phylogenetic clade
that is significantly diverged from the G186AR and G184AR
lineages (40). To determine whether the IFN-? induction by
conidia was a property restricted to the G217B strain or
whether spores and yeast cells from other strains could induce
IFN-?, we attempted to generate conidia from the G186AR,
G186AS, G184AR, and G184AS strains. Like many strains
that have undergone extensive laboratory passaging, our stock
of the G186AS strain failed to produce conidia (data not
shown). However, we were able to produce conidia from
G184AR, G184AS, and G186AR yeast cells, as described in
Materials and Methods. All of these strains, including G217B,
were plated simultaneously and grown for approximately 10
weeks at room temperature. Macrophages were infected with
G217B, G184AR, G186AR, or G184AS conidia, and qRT-
PCR was used to detect IFN-? induction 4 h after infection
(Fig. 3C). Infection with G217B conidia resulted in approxi-
mately 25-fold induction of IFN-?, but infection with G186AR
conidia failed to induce significant levels of IFN-?. Interest-
ingly, whereas G184AR conidia induced modest levels of
IFN-? (7.5-fold), infection with G184AS conidia resulted in a
150-fold induction of IFN-? transcript. These data suggest that
the unknown microbial property that triggers production of
IFN-? by host cells is enhanced in the smooth G184AS strain
and is masked in the rough G184AR and G186AR strains,
although the molecular basis of this difference is unknown. To
determine if ?-(1,3)-glucan modulates type I IFN production,
we attempted to generate conidia from the G186A ags1? strain
(66), which is smooth because these cells produce no ?-(1,3)-
glucan due to a deletion in the ?-(1,3)-glucan synthase. How-
ever, like many laboratory strains, the ags1? strain failed to
sporulate (data not shown). No yeast cells from any strains
tested, including G217B, G184AR, G184AS, G186AR, and
G186AS, were capable of inducing appreciable levels of IFN-?
FIG. 3. Expression level of IFN-? in conidium- and yeast-infected macrophages. (A) qRT-PCR analysis to determine fold induction of IFN-?
was performed on macrophage samples at various time points after infection with G217B conidia at an MOI of 10. (B) qRT-PCR analysis to
determine fold induction of IFN-? at 3 hpi in macrophages infected with live or heat-killed (HK) conidia or with live yeast cells at an MOI of 10.
(C and D) Macrophage lysates were subjected to qRT-PCR to detect relative induction of IFN-? after mock infection or infection with G217B,
G184AR, G184AS, G186AR, or G186AS conidia (C) or yeast cells (D) at an MOI of 10. ND, not determined.
VOL. 78, 2010HISTOPLASMA-INFECTED MACROPHAGE TYPE I IFN RESPONSE 3875
transcript in macrophages (Fig. 3D), again suggesting that pro-
duction of IFN-? is a specific characteristic of infection with H.
capsulatum conidia but not their isogenic yeast cells.
The type I IFN response of BMDMs is independent of
MyD88 and TRIF signaling and the adaptor protein MAVS
but dependent on IRF3. Canonical production of type I IFNs
by macrophages during infection occurs in response to signal-
ing through host Toll-like receptors (TLRs) or a cytosolic
nucleic acid detection pathway (42, 77). The induction of
IFN-? through either of these pathways is dependent on the
transcription factor IRF3. We observed that IFN-? induction
during infection with conidia was completely dependent on
IRF3 (Fig. 4A), indicating that production of IFN-? transcript
during infection with conidia is likely to occur via known path-
To determine whether host TLR signaling was required for
the type I IFN response to conidia, we utilized macrophages
from mice lacking TLR adaptor molecules MyD88 and TRIF.
myd88?/?trif?/?macrophages, which are deficient in TLR
signaling, were fully capable of inducing IFN-? in response to
infection with G217B conidia (Fig. 4B), suggesting that TLR
signaling is not required for IFN-? production by macrophages
in response to Histoplasma conidia.
Cytosolic detection of microbial nucleic acids by host cells
also results in production of IFN-?. Sensing of RNA by the
cytosolic RNA receptors RIG-I and MDA5 requires the innate
immune signaling adaptor MAVS, which is required for type I
IFN production in response to viral infection (25, 43, 52, 71,
91). Levels of induction of IFN-? transcript by infection with
conidia in mavs?/?and mavs?/?littermate control macro-
phages were comparable (Fig. 4C), indicating that cytosolic
detection of conidial RNA is unlikely to be responsible for
production of IFN-? by host cells. It is currently unknown
whether cytosolic sensing of conidial DNA contributes to the
type I IFN response.
Phagocytosis is required for IFN-? induction in conidium-
infected macrophages. Since TLR signaling is dispensable for
IFN-? production in response to conidial infection, our data
suggested that cytosolic sensing of a conidial molecule(s) might
be required for production of IFN-? by host macrophages. If
so, it is likely that phagocytosis of conidia would be necessary
to trigger a type I IFN response in macrophages. Macrophages
were pretreated with either DMSO (control) or 5 ?M actin
polymerization inhibitor cytochalasin D (15, 24), infected with
G217B conidia, and then subjected to staining as described in
Materials and Methods to determine internalization of fungal
cells. Cytochalasin-treated macrophages were still associated
with conidia but were unable to phagocytose them (Table 1).
In contrast to DMSO-treated control cells, cytochalasin-
treated macrophages showed a 25-fold reduction in production
of IFN-? by qRT-PCR when infected with G217B, suggesting
that phagocytosis of conidia is required for the type I response
(Table 1). Cytochalasin-treated macrophages exposed to LPS
were capable of inducing IFN-?, indicating that the cytocha-
lasin treatment did not generally inhibit IFN-? expression in
these cells (data not shown).
Alveolar macrophages induce an interferon-responsive gene
in response to infection with conidia but not yeast cells. By
probing the transcriptional profile of bone marrow-derived
macrophages during infection with conidia or yeast cells, we
were able to uncover differential responses elicited in host cells
by these two fungal cell types. To perform an initial investiga-
tion to determine whether conidia and yeast cells might elicit
different responses in a lung macrophage, we isolated alveolar
macrophages (AvMs) from 30 mice by BAL. Macrophages
were infected with either conidia or yeast cells, and host RNA
was harvested at 4 hpi to examine early transcriptional re-
sponses. No detectable IFN-? transcript was observed by qRT-
PCR during infection of AvMs with either conidia or yeast cells
(data not shown). However, we were able to detect a repro-
ducible 6-fold induction of interferon-responsive gene Ifi205
(53) in AvMs infected with conidia but not yeast cells (Fig. 5);
Ifi205 was also induced by BMDMs in response to conidia but
not yeast cells (see Table S1 in the supplemental material).
This experiment supports the idea that conidia and yeast cells
could provoke different transcriptional responses in host cells
Signaling through the type I IFN receptor IFNAR1 contrib-
utes to the pathogenesis of H. capsulatum during host infec-
tion. The observation that infection with H. capsulatum conidia
FIG. 4. The type I IFN response to conidia is dependent on IRF3 and independent of MyD88, TRIF, and MAVS. qRT-PCR was used to
determine fold IFN-? induction in irf3?/?macrophages (A), myd88?/?trif?/?macrophages (B), and mavs?/?and mavs?/?macrophages
(C) infected with G217B conidia at an MOI of 10.
TABLE 1. Cytochalasin D treatment-inhibited internalization of
conidia and induction of type I IFNs
Mean ? SD
% of conidia
Avg ? SD
G217B conidia ? DMSO
G217B conidia ? 5 ?M
68.5 ? 2.0
8.6 ? 0.5
31.2 ? 2.7
1.23 ? 1.5
3876INGLIS ET AL.INFECT. IMMUN.
triggered a type I IFN signature in bone marrow-derived mac-
rophages raises the possibility that type I IFNs could influence
the outcome of H. capsulatum infection in the mouse, although
the production of type I IFNs in vivo and the cell types that
produce them have not been investigated. For other patho-
gens, examination of the outcome of infection in the ifnar1?/?
mice, which are deficient in the secondary response that results
in robust expression of interferon-dependent genes (37, 81),
has been used as an initial query to shed light on the role of
type I IFN signaling during infection. Interestingly, in response
to infection with bacterial pathogens, this type of approach has
been used to show that host type I IFN signaling confers either
resistance or susceptibility, depending on the bacterial patho-
gen in question (5, 61, 74). To determine whether type I IFN
signaling contributes to the outcome of H. capsulatum infec-
tion, we subjected WT and ifnar1?/?mice to an intranasal
infection with 2 ? 106CFU of G217B conidia. Lungs and
spleens from infected animals were harvested for enumeration
of CFU at 5, 10, and 14 days postinfection (dpi). Whereas the
level of fungal burden was not significantly different between
the WT and mutant mouse strains at 5 and 10 dpi (based on a
P value of ?0.05), the fungal burden was reproducibly lower in
the ifnar1?/?mice in both the lungs (Fig. 6) and spleen (data
not shown) by 14 dpi. These data indicate that signaling
through the type I IFN receptor is required for full virulence of
Since we observed decreased fungal burden in ifnar1?/?
mice at later time points in infection when conidia have ger-
minated to give rise to yeast cells, we were interested to know
if infection of wild-type and ifnar1?/?mutant mice with His-
toplasma yeast cells would give a comparable difference in
fungal burden. We observed that during mouse infections with
H. capsulatum yeasts, the fungal burden was significantly lower
in the lungs of ifnar1?/?mice at 14 dpi (Fig. 6). These data
indicate that signaling through the type I IFN receptor is re-
quired for maximal disease burden during Histoplasma infec-
Histological examination of lung sections from WT and mu-
tant mice infected with Histoplasma conidia revealed signifi-
cant differences in the inflammatory infiltrate (Fig. 7). Infected
lungs of both WT and ifnar1?/?mice had a similar pattern of
inflammation centered around the bronchioles (Fig. 7A, B, E,
and F); however, the lungs of WT mice contained a denser
inflammatory infiltrate as well as larger foci of inflammation.
Additionally, there were differences in the compositions of the
inflammatory infiltrate between the two infected mouse strains
(Fig. 7C and D). In WT lungs at 5 dpi, the infiltrate consisted
largely of granulocytes and lymphocytes with numerous eosin-
ophils. In contrast, at the same time point, the ifnar1?/?infil-
trate was largely composed of macrophages, with only a minor
lymphocytic component. Giant cells, which presumably result
from coalescence of infected macrophages, were observed in
nearly all the inflammatory foci of WT lungs (Fig. 7C and 8),
but were not found in the ifnar1?/?lungs (Fig. 7D). By 14 dpi,
the extent of inflammation had decreased relative to 5 dpi, but
was still higher in wild-type mice than in ifnar1?/?mice (Fig.
7E, F, G, and H). The uninfected lung sections from WT and
ifnar1?/?mice did not look appreciably different (data not
shown). Taken together with the CFU data (Fig. 6), these
experiments indicate that signaling through the type I IFN
receptor is required for the normal extent and character of the
inflammatory response to Histoplasma as well as maximal fun-
gal burden in host tissues during Histoplasma infection.
H. capsulatum is an environmental fungus that is able to
colonize a number of mammalian species via inhalation of
infectious spores (conidia). As a primary pathogen, H. capsu-
FIG. 5. Induction of Ifi205 in AvMs infected with G217B conidia.
qRT-PCR analysis to determine fold induction of Ifi205 was per-
formed on AvM samples 4 h after infection with G217B conidia or
yeast cells at an MOI of 10. Fold changes are calculated relative to the
level in the mock-infected control. Error bars represent the standard
error of the mean for replicate qRT-PCRs. The data shown include
two (yeast cell A and B) or three (conidia A, B, and C) biological
FIG. 6. Host type I IFN signaling is required for maximal fungal
burden in host tissues following infection with H. capsulatum. WT and
ifnar1?/?mice were subjected to intranasal infection with G217B
conidia or yeast cells. Two representative conidial infections and one
representative yeast cell infection are shown. Lungs from infected
animals were harvested at the indicated hours (h) or days (d) postin-
fection and assessed for CFU. Horizontal bars indicate mean log10
CFU values. Significant P values (P ? 0.05) for WT versus ifnar1?/?
comparisons are indicated on the figure.
VOL. 78, 2010HISTOPLASMA-INFECTED MACROPHAGE TYPE I IFN RESPONSE3877
FIG. 7. Wild-type mice have a more extensive inflammatory response to H. capsulatum conidia than ifnar1?/?mice. Shown are hematoxylin-
and-eosin-stained lung sections from mice infected with G217B conidia. Panels A and B are low-power images of representative inflammatory foci
at 5 dpi in WT (A) or ifnar?/?(B) mice. WT inflammatory foci are larger and more densely packed with immune cells. Scale bar, 200 ?m. Panels
C and D are high-power views of boxed regions from panels A and B. WT infiltrate contains many neutrophils, macrophages and eosinophils, with
giant cells (GC) also present. Scale bar, 20 ?m. (E and F) Low-power images of representative inflammatory foci at 14 dpi in WT (E) or ifnar?/?
(F) mice. Again, WT mouse inflammatory foci are larger and more densely packed than those of ifnar?/?mice. Scale bar, 200 ?m. Panels G and
H are high-power views of boxed regions from panels E and F. The WT shows more densely organized macrophage and lymphocytic inflammation.
Scale bar, 20 ?m.
3878INGLIS ET AL.INFECT. IMMUN.
latum causes significant morbidity among healthy individuals
(14), but little is understood about the host response to this
intracellular fungus. This study represents the first examination
of the macrophage transcriptional profile in response to H.
capsulatum infectious particles. We found that infection of
macrophages with conidia results in induction of IFN-? tran-
script, as well as induction of a classic type I IFN secondary
response signature. These data are one of the first demonstra-
tions of type I IFN induction in macrophages in response to an
infection with fungal cells. Even more interesting is that induc-
tion of a type I IFN signature by macrophages in response to
H. capsulatum occurred only in response to conidia; the yeast
form of the organism, which is produced within the host as
conidia germinate, was unable to stimulate this response, even
at an MOI of 10 (data not shown). Similarly, a more limited
examination of the alveolar macrophage response revealed
that infection with conidia but not yeast induced the interfer-
on-responsive gene Ifi205. Since conidia represent the most
common infectious particle, they are likely to be the initial H.
capsulatum cell encountered by host macrophages. These data
suggest that in a natural infection, conidia could trigger early
differential immune responses that influence the progression
of H. capsulatum infection.
Type I IFN induction is elicited either in response to acti-
vation of TLRs or in response to cytosolic receptors (77). Since
induction of IFN-? in response to conidia is independent of
TLR signaling, it is likely that a cytosolic response pathway
might be engaged by an unknown conidial component. Al-
though H. capsulatum yeast cells are known to remain in the
phagosome of macrophages during infection, the subcellular
location of H. capsulatum conidia has not been investigated. Of
note, some pathogens can trigger cytosolic signaling pathways
despite being confined to the phagosome (55): for example, the
bacterial pathogen Mycobacterium tuberculosis is able to access
cytosolic signaling pathways to stimulate IFN-? despite its lo-
calization in the phagosome of macrophages (74).
It is unclear which feature of conidia is recognized by host
macrophages, although we did observe that the unknown in-
ducing factor was partially resistant to heat treatment. The
host sensors required for the response are also unknown. Type
I IFN production is triggered by signaling through cytosolic
receptors that recognize nucleic acids, including DNA, RNA,
cyclic-di-GMP, and cyclic-di-AMP (41, 55, 78, 89, 92). We have
shown that induction of type I IFNs in response to conidia is
independent of the adaptor MAVS, which is required for rec-
ognition of pathogen RNA by the RNA helicases RIG-I and
MDA5. Thus, in contrast to the bacterial pathogen Legionella
pneumophila (54), it seems unlikely that pathogen RNA con-
tributes to the induction of the type I IFN response to H.
capsulatum conidia. The role of conidial DNA in the induction
of the type I IFN response has not been tested, and DNA
remains a viable candidate ligand that could be sensed by host
receptors. In this model, some unknown aspect of conidial but
not yeast cell biology would allow fungal DNA to access the
cytosol. In the case of the bacterial pathogen Listeria monocy-
togenes, introduction of bacterial genomic DNA into the cy-
tosol of macrophages is sufficient to induce IFN-?, but this
transcriptional response is enhanced by co-delivery of muramyl
dipeptide, a constituent of the bacterial cell wall peptidoglycan
(46). These data suggest that recognition of multiple ligands by
different cytosolic receptors can contribute to induction of type
I IFNs during infection with a pathogen. Notably, in the cases
of L. monocytogenes, M. tuberculosis, and several other well-
studied pathogens, the host receptors required for the type I
response are unknown (55). The identification of these host
molecules, as well as those that participate in the response to
Histoplasma conidia, will shed light on common and distinct
host pathways that are utilized to sense and respond to a
diversity of pathogens.
The magnitude of induction of IFN-? by H. capsulatum
conidia varied with respect to age and strain background.
“Older” spores were more likely to induce higher levels of
IFN-?, suggesting that these spores might accumulate higher
levels of the inducing factor or activity that is recognized by the
host. We also examined the ability of conidia from several
evolutionarily diverged H. capsulatum strains to induce IFN-?.
Whereas the North American G217B conidia induced inter-
mediate levels of IFN-?, the “rough” Latin American G184AR
strain induced only modest levels of IFN-?, and G186AR
conidia did not appear to induce any. Interestingly, conidia
from the “smooth” variant of G184AR, termed G184AS, in-
duced high levels of IFN-?. (We were unable to produce
conidia from the G186AS strain to test whether enhanced
IFN-? production is a common property of smooth strains.)
Although the molecular differences between the rough and
smooth variants have not been characterized, it is known that
the cell walls of the yeast form of the rough and smooth strains
are fundamentally different: the rough yeast strains express the
cell wall carbohydrate ?-(1,3)-glucan, whereas the smooth
strains do not. ?-(1,3)-Glucan is thought to be specific to yeast
cells, so unless ?-(1,3)-glucan has a previously unsuspected
role in conidial biology, it is likely that some other undeter-
mined property of the G184AS smooth variant is contributing
to the increased induction of IFN-?. In either case, the rough
conidia either fail to accumulate the inducing factor or shield
that factor from recognition by host cells.
FIG. 8. Giant cell formation in conidium-infected lungs of wild-
type mice. Shown is high magnification of a giant cell containing H.
capsulatum macroconidia (black arrows), microconidia (white arrow-
heads), and yeast cells (black arrowheads) observed in the lungs of
conidium-infected WT mouse strains at 5 dpi. Conidial forms could
represent ungerminated cells or conidial remnants that persist after
germination. Scale bar, 20 ?m.
VOL. 78, 2010HISTOPLASMA-INFECTED MACROPHAGE TYPE I IFN RESPONSE3879
During a natural infection, conidia are inhaled by the host,
undergo germination, and produce yeast cells that colonize the
host for the remainder of the infection. We observed that only
H. capsulatum conidia, and not yeast cells, were able to induce
IFN-? transcript in bone marrow-derived macrophages. Alveo-
lar macrophages assayed at a single time point postinfection
induced expression of Ifi205, an interferon-responsive gene
(53), in response to conidia but not yeast cells, which also
suggests that these host cells might respond differentially to
various fungal cell types. Even though we observed induction
of an interferon-responsive gene at 4 hpi, we did not observe
induction of IFN-? in alveolar macrophages at 4 hpi in re-
sponse to either conidia or yeast cells, which suggests that
induction of Ifi205 could be dependent on production of IFN-?
species or that the chosen time point was not optimal for
detection of IFN-? transcript. Of note, Ifi205 expression can be
activated in response to either type I or type II interferons (53),
so it is also formally possible, although unexpected, that type II
interferons could trigger Ifi205 expression in AvMs infected
with conidia. Regardless, these data are consistent with the
model that conidia and yeast cells trigger nonequivalent re-
sponses in macrophages. Most studies of Histoplasma-host in-
teraction have utilized yeast cells, which are an excellent model
for macrophage-fungus interactions that occur after germina-
tion of conidia. Our data highlight the value of examining the
interaction of host cells with conidia, which, although techni-
cally challenging, sheds light on the initial stages of a natural
infection. Fungal pathogens are notorious for adopting differ-
ent morphologies in response to distinct environmental stim-
uli, and there is precedent for a host response that is tailored
to individual morphological states. For example, it has been
suggested that distinct morphological forms of the fungal
pathogen Candida albicans are differentially recognized by
TLR4 and by Dectin-1 (26, 82). Certainly the conidial and
yeast forms of H. capsulatum have notable differences that
could easily influence the host response: for example, electron
microscopy clearly reveals that two morphological forms dis-
play fundamental differences in the structures of their cell walls
(27–31). Furthermore, we have observed that conidia and yeast
cells are molecularly distinct; approximately 300 transcripts
accumulate preferentially in conidia as compared to yeast cells
(D. O. Inglis, M. Voorhies, and A. Sil, unpublished data).
H. capsulatum yeast cells may lack the ability to induce
IFN-? in macrophages, or they may actively suppress induction
of this pathway in host cells. Even though yeast cells are
thought to suppress other types of innate immune responses
during infection (49), preliminary coinfection experiments of
WT macrophages with conidia and yeast cells did not reveal a
clear ability of yeast cells to inhibit the induction of IFN-?
(data not shown). Macrophages infected with heat-killed yeast
cells also failed to induce IFN-? (data not shown), indicating
that yeast cells are unlikely to be actively suppressing the type
I IFN response of macrophages.
By comparing fungal burdens in WT and ifnar1-deficient
mice, we determined that type I IFN signaling does not protect
the host from H. capsulatum-associated disease. In fact, type I
IFN signaling promotes maximal fungal burden in lungs and
spleens at later time points during infection, regardless of
whether mice were infected with conidia or yeast cells. At
present, the identity of the host cells (e.g., macrophages versus
plasmacytoid dendritic cells [pDCs]) that produce type I IFNs
during in vivo infection and the kinetics of type I IFN produc-
tion are unknown. Presumably, some host cells, such as pDCs,
or even alveolar macrophages at different times in infection,
might produce type I IFNs in response to both yeast cells and
conidia, suggesting that bone marrow-derived macrophages,
although a useful model for assessing host-pathogen signaling,
do not reflect the full complexity of in vivo interactions. Given
the myriad roles of type I IFNs in the host, the possible effects
of induction of IFN-? cells infected with H. capsulatum could
include modulation of (i) downstream cytokine production, (ii)
apoptosis of infected macrophages, or (iii) specific aspects of
the adaptive immune response to H. capsulatum. Interestingly,
it was previously observed that chronic infection of macro-
phages or mice with lymphocytic choriomeningitis virus
(LCMV) clone 13, which induces type I IFNs, caused sensiti-
zation of the host to H. capsulatum infection (84, 90). Although
the possible interpretations of these data are complex, they are
consistent with the model that increased levels of type I IFNs
correlate with increased sensitivity to H. capsulatum infection.
Of note, type I IFN signaling has been shown to play both
protective and sensitizing roles in response to bacterial infec-
tion (17, 55). Our data are reminiscent of the observation that
organs lacking IFNAR1 are more restrictive for bacterial
growth during infection with L. monocytogenes and M. tuber-
culosis (5, 61, 74). In the case of the fungal pathogen Crypto-
coccus neoformans (8), ifnar1?/?mice displayed a higher fun-
gal burden in the lungs and brain, as well a dramatic decrease
in survival. Disruption of IFNAR1 also results in increased
sensitivity to infection with the fungal pathogen Candida albi-
cans (K. Kuchler, personal communication). Thus, analogous
to what is observed for bacterial pathogens, it may be that type
I IFN signaling may play protective or sensitizing roles during
fungal infections, depending on the distinct strategies used by
individual pathogens to promote disease.
We are grateful to Daniel Portnoy, Denise Monack, Jeffery Cox,
Joseph DeRisi, Russell Vance, Charlie Kim, Paolo Manzanillo, Greg
Barton, Jonathan Jones, and members of the Sil laboratory for useful
discussion as this work progressed. We thank Sil laboratory members
and Denise Monack for comments on the manuscript. We thank the
laboratories of Greg Barton, Jeffery Cox, Joseph DeRisi, and Russell
Vance for mutant mice and/or BMDMs. We appreciate the assistance
of Margaret Mayes, Research Morphology Core Facility, Department
of Pathology, UCSF, for preparation of the tissue sections and Kirk
Jones for histopathological analysis. We thank M. Paige Nittler, Katie
Hermens, Sajeev Batra, and the Bay Area PO1 Group for the produc-
tion of MEEBO arrays. We are grateful to Joseph Wheat for providing
polyclonal antibodies that recognize H. capsulatum.
This work was supported by an Irvington Institute for Immunology
Postdoctoral fellowship to D.O.I., UCSF Immunology training grant
(T32 AI07334) support to C.A.B., Microbial Pathogenesis and Host
Defense Training Grant (NIH T32 A1060537) support to D.O.I. and
C.A.B., NIH (R01AI066224 and PO1AI063302) and an HHMI Early
Career Scientist Award to A.S., and the Sandler Program in Basic
Sciences and a Howard Hughes Medical Institute Biomedical Re-
search Support Program grant (5300246) to the UCSF School of Med-
1. Aderem, A. 2003. Phagocytosis and the inflammatory response. J. Infect. Dis.
2. Akira, S., S. Uematsu, and O. Takeuchi. 2006. Pathogen recognition and
innate immunity. Cell 124:783–801.
3880INGLIS ET AL.INFECT. IMMUN.
3. Anderson, K. L., and S. Marcus. 1968. Sporulation characteristics of His-
toplasma capsulatum. Mycopathol. Mycol. Appl. 36:179–187.
4. Andreas, S., S. Heindl, C. Wattky, K. Moller, and R. Ruchel. 2000. Diagnosis
of pulmonary aspergillosis using optical brighteners. Eur. Respir. J. 15:407–
5. Auerbuch, V., D. G. Brockstedt, N. Meyer-Morse, M. O’Riordan, and D. A.
Portnoy. 2004. Mice lacking the type I interferon receptor are resistant to
Listeria monocytogenes. J. Exp. Med. 200:527–533.
6. Beutler, B., K. Hoebe, X. Du, and R. J. Ulevitch. 2003. How we detect
microbes and respond to them: the Toll-like receptors and their transducers.
J. Leukoc. Biol. 74:479–485.
7. Beutler, B., Z. Jiang, P. Georgel, K. Crozat, B. Croker, S. Rutschmann, X.
Du, and K. Hoebe. 2006. Genetic analysis of host resistance: Toll-like recep-
tor signaling and immunity at large. Annu. Rev. Immunol. 24:353–389.
8. Biondo, C., A. Midiri, M. Gambuzza, E. Gerace, M. Falduto, R. Galbo, A.
Bellantoni, C. Beninati, G. Teti, T. Leanderson, and G. Mancuso. 2008.
IFN-alpha/beta signaling is required for polarization of cytokine responses
toward a protective type 1 pattern during experimental cryptococcosis. J. Im-
9. Bourgeois, C., O. Majer, I. E. Frohner, L. Tierney, and K. Kuchler. 20 June
2010, posting date. Fungal attacks on mammalian hosts: pathogen elimina-
tion requires sensing and tasting. Curr. Opin. Microbiol. [Epub ahead of
10. Brown, G. D. 2006. Dectin-1: a signalling non-TLR pattern-recognition re-
ceptor. Nat. Rev. Immunol. 6:33–43.
11. Brown, G. D. 2006. Macrophage receptors and innate immunity: insights
from dectin-1. Novartis Found. Symp. 279:114–126, 216–219.
12. Bukholm, G., B. P. Berdal, C. Haug, and M. Degre. 1984. Mouse fibroblast
interferon modifies Salmonella typhimurium infection in infant mice. Infect.
13. Bullock, W. E. 1993. Interactions between human phagocytic cells and His-
toplasma capsulatum. Arch. Med. Res. 24:219–223.
14. Chu, J. H., C. Feudtner, K. Heydon, T. J. Walsh, and T. E. Zaoutis. 2006.
Hospitalizations for endemic mycoses: a population-based national study.
Clin. Infect. Dis. 42:822–825.
15. Cooper, J. A. 1987. Effects of cytochalasin and phalloidin on actin. J. Cell
16. Davis, T. E., Jr., J. E. Domer, and Y. T. Li. 1977. Cell wall studies of
Histoplasma capsulatum and Blastomyces dermatitidis using autologous and
heterologous enzymes. Infect. Immun. 15:978–987.
17. Decker, T., M. Muller, and S. Stockinger. 2005. The yin and yang of type I
interferon activity in bacterial infection. Nat. Rev. Immunol. 5:675–687.
18. Decker, T., S. Stockinger, M. Karaghiosoff, M. Muller, and P. Kovarik. 2002.
IFNs and STATs in innate immunity to microorganisms. J. Clin. Invest.
19. Deepe, G. S., Jr. 2000. Immune response to early and late Histoplasma
capsulatum infections. Curr. Opin. Microbiol. 3:359–362.
20. Deepe, G. S., Jr. 2005. Modulation of infection with Histoplasma capsulatum
by inhibition of tumor necrosis factor-alpha activity. Clin. Infect. Dis.
21. Deepe, G. S., Jr. 2007. Tumor necrosis factor-alpha and host resistance to the
pathogenic fungus, Histoplasma capsulatum. J. Investig. Dermatol. Symp.
22. Eisen, M. B., P. T. Spellman, P. O. Brown, and D. Botstein. 1998. Cluster
analysis and display of genome-wide expression patterns. Proc. Natl. Acad.
Sci. U. S. A. 95:14863–14868.
23. Eissenberg, L. G., and W. E. Goldman. 1991. Histoplasma variation and
adaptive strategies for parasitism: new perspectives on histoplasmosis. Clin.
Microbiol. Rev. 4:411–421.
24. Elliott, J. A., and W. C. Winn, Jr. 1986. Treatment of alveolar macrophages
with cytochalasin D inhibits uptake and subsequent growth of Legionella
pneumophila. Infect. Immun. 51:31–36.
25. Evans, J. D., and C. Seeger. 2006. Cardif: a protein central to innate immu-
nity is inactivated by the HCV NS3 serine protease. Hepatology 43:615–617.
26. Gantner, B. N., R. M. Simmons, and D. M. Underhill. 2005. Dectin-1 me-
diates macrophage recognition of Candida albicans yeast but not filaments.
EMBO J. 24:1277–1286.
27. Garrison, R. G., and K. S. Boyd. 1978. Electron microscopy of yeastlike cell
development from the microconidium of Histoplasma capsulatum. J. Bacte-
28. Garrison, R. G., and J. W. Lane. 1973. Scanning-beam electron microscopy
of the conidia of the brown and albino filamentous varieties of Histoplasma
capsulatum. Mycopathol. Mycol. Appl. 49:185–191.
29. Garrison, R. G., and J. W. Lane. 1971. Yeastlike to mycelial phase transfor-
mation of Histoplasma capsulatum as observed by scanning electron micros-
copy. Mycopathol. Mycol. Appl. 43:183–193.
30. Garrison, R. G., J. W. Lane, and M. F. Field. 1970. Ultrastructural changes
during the yeastlike to mycelial-phase conversion of Blastomyces dermatitidis
and Histoplasma capsulatum. J. Bacteriol. 101:628–635.
31. Garrison, R. G., J. W. Lane, and D. R. Johnson. 1971. Electron microscopy
of the transitional conversion cell of Histoplasma capsulatum. Mycopathol.
Mycol. Appl. 44:121–129.
32. Giles, J. T., and J. M. Bathon. 2004. Serious infections associated with
anticytokine therapies in the rheumatic diseases. J. Intensive Care Med.
33. Gold, J. A., Y. Hoshino, S. Hoshino, M. B. Jones, A. Nolan, and M. D.
Weiden. 2004. Exogenous gamma and alpha/beta interferon rescues human
macrophages from cell death induced by Bacillus anthracis. Infect. Immun.
34. Henry, T., A. Brotcke, D. S. Weiss, L. J. Thompson, and D. M. Monack. 2007.
Type I interferon signaling is required for activation of the inflammasome
during Francisella infection. J. Exp. Med. 204:987–994.
35. Hoebe, K., Z. Jiang, K. Tabeta, X. Du, P. Georgel, K. Crozat, and B. Beutler.
2006. Genetic analysis of innate immunity. Adv. Immunol. 91:175–226.
36. Huffnagle, G. B., and G. S. Deepe. 2003. Innate and adaptive determinants of
host susceptibility to medically important fungi. Curr. Opin. Microbiol.
37. Hwang, S. Y., P. J. Hertzog, K. A. Holland, S. H. Sumarsono, M. J. Tymms,
J. A. Hamilton, G. Whitty, I. Bertoncello, and I. Kola. 1995. A null mutation
in the gene encoding a type I interferon receptor component eliminates
antiproliferative and antiviral responses to interferons alpha and beta and
alters macrophage responses. Proc. Natl. Acad. Sci. U. S. A. 92:11284–11288.
38. Jenner, R. G., and R. A. Young. 2005. Insights into host responses against
pathogens from transcriptional profiling. Nat. Rev. Microbiol. 3:281–294.
39. Kabelitz, D., and R. Medzhitov. 2007. Innate immunity–cross-talk with adap-
tive immunity through pattern recognition receptors and cytokines. Curr.
Opin. Immunol. 19:1–3.
40. Kasuga, T., J. W. Taylor, and T. J. White. 1999. Phylogenetic relationships of
varieties and geographical groups of the human pathogenic fungus His-
toplasma capsulatum Darling. J. Clin. Microbiol. 37:653–663.
41. Kato, H., O. Takeuchi, S. Sato, M. Yoneyama, M. Yamamoto, K. Matsui, S.
Uematsu, A. Jung, T. Kawai, K. J. Ishii, O. Yamaguchi, K. Otsu, T. Tsu-
jimura, C. S. Koh, C. Reis e Sousa, Y. Matsuura, T. Fujita, and S. Akira.
2006. Differential roles of MDA5 and RIG-I helicases in the recognition of
RNA viruses. Nature 441:101–105.
42. Kawai, T., and S. Akira. 2006. TLR signaling. Cell Death Differ. 13:816–825.
43. Kawai, T., K. Takahashi, S. Sato, C. Coban, H. Kumar, H. Kato, K. J. Ishii,
O. Takeuchi, and S. Akira. 2005. IPS-1, an adaptor triggering RIG-I- and
Mda5-mediated type I interferon induction. Nat. Immunol. 6:981–988.
44. Klimpel, K. R., and W. E. Goldman. 1988. Cell walls from avirulent variants
of Histoplasma capsulatum lack alpha-(1,3)-glucan. Infect. Immun. 56:2997–
45. Klimpel, K. R., and W. E. Goldman. 1987. Isolation and characterization of
spontaneous avirulent variants of Histoplasma capsulatum. Infect. Immun.
46. Leber, J. H., G. T. Crimmins, S. Raghavan, M. P. Meyer, J. S. Cox, and D. A.
Portnoy. 2008. Distinct TLR- and NLR-mediated transcriptional responses
to an intracellular pathogen. PLoS Pathog. 4:e6.
47. Luther, K., M. Rohde, J. Heesemann, and F. Ebel. 2006. Quantification of
phagocytosis of Aspergillus conidia by macrophages using a novel antibody-
independent assay. J. Microbiol. Methods 66:170–173.
48. Marques, S. A., A. M. Robles, A. M. Tortorano, M. A. Tuculet, R. Negroni,
and R. P. Mendes. 2000. Mycoses associated with AIDS in the Third World.
Med. Mycol. 38(Suppl. 1):269–279.
49. Marth, T., and B. L. Kelsall. 1997. Regulation of interleukin-12 by comple-
ment receptor 3 signaling. J. Exp. Med. 185:1987–1995.
50. McCaffrey, R. L., P. Fawcett, M. O’Riordan, K. D. Lee, E. A. Havell, P. O.
Brown, and D. A. Portnoy. 2004. A specific gene expression program trig-
gered by Gram-positive bacteria in the cytosol. Proc. Natl. Acad. Sci. U. S. A.
51. Medzhitov, R., and C. Janeway, Jr. 2000. Innate immune recognition: mech-
anisms and pathways. Immunol. Rev. 173:89–97.
52. Meylan, E., J. Curran, K. Hofmann, D. Moradpour, M. Binder, R. Barten-
schlager, and J. Tschopp. 2005. Cardif is an adaptor protein in the RIG-I
antiviral pathway and is targeted by hepatitis C virus. Nature 437:1167–1172.
53. Mondini, M., S. Costa, S. Sponza, F. Gugliesi, M. Gariglio, and S. Landolfo.
2010. The interferon-inducible HIN-200 gene family in apoptosis and in-
flammation: implication for autoimmunity. Autoimmunity 43:226–231.
54. Monroe, K. M., S. M. McWhirter, and R. E. Vance. 2009. Identification of
host cytosolic sensors and bacterial factors regulating the type I interferon
response to Legionella pneumophila. PLoS Pathog. 5:e1000665.
55. Monroe, K. M., S. M. McWhirter, and R. E. Vance. 2010. Induction of type
I interferons by bacteria. Cell. Microbiol. 12:881–890.
56. Nau, G. J., J. F. Richmond, A. Schlesinger, E. G. Jennings, E. S. Lander, and
R. A. Young. 2002. Human macrophage activation programs induced by
bacterial pathogens. Proc. Natl. Acad. Sci. U. S. A. 99:1503–1508.
57. Netea, M. G., G. Ferwerda, C. A. van der Graaf, J. W. Van der Meer, and
B. J. Kullberg. 2006. Recognition of fungal pathogens by toll-like receptors.
Curr. Pharm. Des. 12:4195–4201.
58. Newman, S. L. 1999. Macrophages in host defense against Histoplasma
capsulatum. Trends Microbiol. 7:67–71.
59. Newman, S. L., C. Bucher, J. Rhodes, and W. E. Bullock. 1990. Phagocytosis
of Histoplasma capsulatum yeasts and microconidia by human cultured mac-
VOL. 78, 2010HISTOPLASMA-INFECTED MACROPHAGE TYPE I IFN RESPONSE3881
rophages and alveolar macrophages. Cellular cytoskeleton requirement for Download full-text
attachment and ingestion. J. Clin. Invest. 85:223–230.
60. Newman, S. L., and W. E. Bullock. 1994. Interaction of Histoplasma capsu-
latum yeasts and conidia with human and animal macrophages. Immunol.
61. O’Connell, R. M., S. K. Saha, S. A. Vaidya, K. W. Bruhn, G. A. Miranda, B.
Zarnegar, A. K. Perry, B. O. Nguyen, T. F. Lane, T. Taniguchi, J. F. Miller,
and G. Cheng. 2004. Type I interferon production enhances susceptibility to
Listeria monocytogenes infection. J. Exp. Med. 200:437–445.
62. Petrilli, V., C. Dostert, D. A. Muruve, and J. Tschopp. 2007. The inflamma-
some: a danger sensing complex triggering innate immunity. Curr. Opin.
63. Pfaffl, M. W. 2001. A new mathematical model for relative quantification in
real-time RT-PCR. Nucleic Acids Res. 29:e45.
64. Pine, L. 1960. Morphological and physiological characteristics of His-
toplasma capsulatum, p. 40–75. In H. C. Sweany (ed.), Histoplasmosis.
Thomas, Springfield, IL.
65. Rappleye, C. A., L. G. Eissenberg, and W. E. Goldman. 2007. Histoplasma
capsulatum alpha-(1,3)-glucan blocks innate immune recognition by the
beta-glucan receptor. Proc. Natl. Acad. Sci. U. S. A. 104:1366–1370.
66. Rappleye, C. A., J. T. Engle, and W. E. Goldman. 2004. RNA interference in
Histoplasma capsulatum demonstrates a role for alpha-(1,3)-glucan in viru-
lence. Mol. Microbiol. 53:153–165.
67. Reiss, E. 1977. Serial enzymatic hydrolysis of cell walls of two serotypes of
yeast-form Histoplasma capsulatum with alpha(1 leads to 3)-glucanase,
beta(1 leads to 3)-glucanase, pronase, and chitinase. Infect. Immun. 16:181–
68. Reiss, E., H. Hutchinson, L. Pine, D. W. Ziegler, and L. Kaufman. 1977.
Solid-phase competitive-binding radioimmunoassay for detecting antibody
to the M antigen of histoplasmin. J. Clin. Microbiol. 6:598–604.
69. Reiss, E., S. E. Miller, W. Kaplan, and L. Kaufman. 1977. Antigenic, chem-
ical, and structural properties of cell walls of Histoplasma capsulatum yeast-
form chemotypes 1 and 2 after serial enzymatic hydrolysis. Infect. Immun.
70. Saldanha, A. J. 2004. Java Treeview—extensible visualization of microarray
data. Bioinformatics 20:3246–3248.
71. Seth, R. B., L. Sun, C. K. Ea, and Z. J. Chen. 2005. Identification and
characterization of MAVS, a mitochondrial antiviral signaling protein that
activates NF-kappaB and IRF 3. Cell 122:669–682.
72. Sieling, P. A., and R. L. Modlin. 2002. Toll-like receptors: mammalian “taste
receptors” for a smorgasbord of microbial invaders. Curr. Opin. Microbiol.
73. Smith, J. A., and C. A. Kauffman. 2009. Endemic fungal infections in patients
receiving tumour necrosis factor-alpha inhibitor therapy. Drugs 69:1403–
74. Stanley, S. A., J. E. Johndrow, P. Manzanillo, and J. S. Cox. 2007. The type
I IFN response to infection with Mycobacterium tuberculosis requires ESX-
1-mediated secretion and contributes to pathogenesis. J. Immunol. 178:
75. Stanley, S. A., S. Raghavan, W. W. Hwang, and J. S. Cox. 2003. Acute
infection and macrophage subversion by Mycobacterium tuberculosis re-
quire a specialized secretion system. Proc. Natl. Acad. Sci. U. S. A. 100:
76. Sternberg, S. 1994. The emerging fungal threat. Science 266:1632–1634.
77. Stetson, D. B., and R. Medzhitov. 2006. Type I interferons in host defense.
78. Takaoka, A., Z. Wang, M. K. Choi, H. Yanai, H. Negishi, T. Ban, Y. Lu, M.
Miyagishi, T. Kodama, K. Honda, Y. Ohba, and T. Taniguchi. 2007. DAI
(DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune
response. Nature 448:501–505.
79. Takeda, K., and S. Akira. 2003. Toll receptors and pathogen resistance. Cell.
80. Taylor, P. R., L. Martinez-Pomares, M. Stacey, H. H. Lin, G. D. Brown, and
S. Gordon. 2005. Macrophage receptors and immune recognition. Annu.
Rev. Immunol. 23:901–944.
81. Vadiveloo, P. K., G. Vairo, P. Hertzog, I. Kola, and J. A. Hamilton. 2000.
Role of type I interferons during macrophage activation by lipopolysaccha-
ride. Cytokine 12:1639–1646.
82. van der Graaf, C. A., M. G. Netea, I. Verschueren, J. W. van der Meer, and
B. J. Kullberg. 2005. Differential cytokine production and Toll-like receptor
signaling pathways by Candida albicans blastoconidia and hyphae. Infect.
83. Vieira, O. V., R. J. Botelho, and S. Grinstein. 2002. Phagosome maturation:
aging gracefully. Biochem. J. 366:689–704.
84. Villarete, L., R. de Fries, S. Kolhekar, D. Howard, R. Ahmed, and B. Wu-
Hsieh. 1995. Impaired responsiveness to gamma interferon of macrophages
infected with lymphocytic choriomeningitis virus clone 13: susceptibility to
histoplasmosis. Infect. Immun. 63:1468–1472.
85. Weigent, D. A., T. L. Huff, J. W. Peterson, G. J. Stanton, and S. Baron. 1986.
Role of interferon in streptococcal infection in the mouse. Microb. Pathog.
86. Wheat, L. J., and C. A. Kauffman. 2003. Histoplasmosis. Infect. Dis. Clin.
North Am. 17:1–19, vii.
87. Willment, J. A., and G. D. Brown. 2008. C-type lectin receptors in antifungal
immunity. Trends Microbiol. 16:27–32.
88. Woods, J. P. 2003. Knocking on the right door and making a comfortable
home: Histoplasma capsulatum intracellular pathogenesis. Curr. Opin. Mi-
89. Woodward, J. J., A. T. Iavarone, and D. A. Portnoy. 2010. c-di-AMP secreted
by intracellular Listeria monocytogenes activates a host type I interferon
response. Science 328:1703–1705.
90. Wu-Hsieh, B. A., J. K. Whitmire, R. de Fries, J. S. Lin, M. Matloubian, and
R. Ahmed. 2001. Distinct CD8 T cell functions mediate susceptibility to
histoplasmosis during chronic viral infection. J. Immunol. 167:4566–4573.
91. Xu, L. G., Y. Y. Wang, K. J. Han, L. Y. Li, Z. Zhai, and H. B. Shu. 2005.
VISA is an adapter protein required for virus-triggered IFN-beta signaling.
Mol. Cell 19:727–740.
92. Yoneyama, M., M. Kikuchi, T. Natsukawa, N. Shinobu, T. Imaizumi, M.
Miyagishi, K. Taira, S. Akira, and T. Fujita. 2004. The RNA helicase RIG-I
has an essential function in double-stranded RNA-induced innate antiviral
responses. Nat. Immunol. 5:730–737.
Editor: G. S. Deepe, Jr.
3882 INGLIS ET AL.INFECT. IMMUN.