2482 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 7 July 2012
Fungal antioxidant pathways promote survival
against neutrophils during infection
Sixto M. Leal Jr.,1,2 Chairut Vareechon,1,2 Susan Cowden,3 Brian A. Cobb,2
Jean-Paul Latgé,4 Michelle Momany,3 and Eric Pearlman1,2
1Department of Ophthalmology and Visual Sciences and 2Department of Pathology, Case Western Reserve University, Cleveland, Ohio, USA.
3Department of Plant Biology, University of Georgia, Athens, Georgia, USA. 4Laboratoire des Aspergillus, Institut Pasteur, Paris, France.
Filamentous fungi are a common cause of blindness and visual impairment worldwide. Using both murine
model systems and in vitro human neutrophils, we found that NADPH oxidase produced by neutrophils was
essential to control the growth of Aspergillus and Fusarium fungi in the cornea. We demonstrated that neutro-
phil oxidant production and antifungal activity are dependent on CD18, but not on the β-glucan receptor dec-
tin-1. We used mutant A. fumigatus strains to show that the reactive oxygen species–sensing transcription factor
Yap1, superoxide dismutases, and the Yap1-regulated thioredoxin antioxidant pathway are each required for
protection against neutrophil-mediated oxidation of hyphae as well as optimal survival of fungal hyphae in
vivo. We also demonstrated that thioredoxin inhibition using the anticancer drug PX-12 increased the sensitiv-
ity of fungal hyphae to both H2O2- and neutrophil-mediated killing in vitro. Additionally, topical application
of PX-12 significantly enhanced neutrophil-mediated fungal killing in infected mouse corneas. Cumulatively,
our data reveal critical host oxidative and fungal anti-oxidative mediators that regulate hyphal survival dur-
ing infection. Further, these findings also indicate that targeting fungal anti-oxidative defenses via PX-12 may
represent an efficacious strategy for treating fungal infections.
Pathogenic fungi, such as Aspergillus and Fusarium species, can
cause lethal pulmonary and systemic disease in immune-sup-
pressed individuals, including those with HIV infection (1, 2).
These organisms are also a major cause of infectious blindness and
corneal ulcers in immunocompetent individuals, and in contrast
to individuals with systemic and pulmonary fungal infections,
there is no indication that fungal keratitis patients are other than
fully immunocompetent (3, 4). In the hot and humid southeast-
ern United States, fungal infections of the cornea account for up
to 35% of all corneal ulcers (5, 6). Globally, fungal infections of
the cornea account for up to 65% of corneal ulcers, with estimates
of 80,000 total cases and 10,000 cornea transplants per year due
to fungal infections in India alone (7–12). Other risk factors for
disease in the USA, Britain, and Europe include contact lens wear,
as illustrated by a 2005–2006 fungal keratitis outbreak associated
with a lens care product (13). A. flavus, A. fumigatus, F. solani, and
F. oxysporum are the main etiologic agents of fungal keratitis (14).
These organisms are prevalent in vegetative matter and suspended
in air, and are inoculated into the corneal stroma via traumatic
injury associated with agricultural work (14). Current treatment
with topical antimycotics is often ineffective, with up to 60% of
cases requiring corneal transplantation (3, 14, 15).
Neutrophils are the predominant cell type infiltrating fungus-
infected lungs and corneas, and contribute to tissue destruction
by release of proteolytic enzymes and reactive oxygen and nitrogen
species (13–17). Our recent studies characterizing fungus-infect-
ed human corneas in India showed that neutrophils constitute
greater than 90% of cellular infiltrates in corneal ulcers in patients
infected for less than 7 days and more than 70% total infiltrate at
later stages of infection (18). Similarly, neutrophils are the first
cells recruited to the corneal stroma in murine models of Aspergil-
lus and Fusarium keratitis (19, 20), indicating that neutrophils are
the main effector cells required for killing fungal hyphae. A role for
neutrophils in control of fungal infection is also suggested by the
increased incidence of systemic and pulmonary fungal infections
in patients with neutropenia (2).
Neutrophils produce NADPH oxidase (NOX), which catalyzes
the conversion of molecular O2 to superoxide anion (O2–) with the
release of ROS and protons into the extracellular space (17, 21).
Individuals with inherited defects in NOX such as in chronic
granulomatous disease (CGD) exhibit an increased incidence of
bacterial and fungal infections, supporting the concept that the
specific expression of NOX by neutrophils is required for killing
of fungi (22). However, even though it is the hyphal stage of these
organisms that is invasive, most studies on CGD patients and
transgenic mice with mutations in NOX genes have focused only
on the role of NOX in killing conidia (23–26). Infected human cor-
neas and lungs exhibit primarily hyphal stages of fungal growth,
and conidia are rarely detected. Given that hyphae are significant-
ly larger in size, and are not readily phagocytosed, they are likely
killed through distinct mechanisms not required for anti-conidial
defenses, and a recent study suggests that NOX is not required to
control the growth of all filamentous fungi (27).
In the current study, we examined the role of ROS in killing
Aspergillus and Fusarium hyphae by human neutrophils and in a
murine model of fungal keratitis. We show that hyphae activate
neutrophil NOX through CD18 and that NOX activation is essen-
tial for killing hyphae. In addition, utilizing mutant A. fumigatus
strains, we show that the ROS-sensing transcription factor Yap1,
the ROS-detoxifying enzyme superoxide dismutase, and the Yap1-
regulated thioredoxin antioxidant pathway, but not catalases or
fungal secondary metabolites such as gliotoxin are required for
resistance to oxidation by neutrophils. Last, using pharmacologic
inhibitors of thioredoxin, we provide proof of concept that tar-
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J Clin Invest. 2012;122(7):2482–2498. doi:10.1172/JCI63239.
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 7 July 2012
geting fungal anti-oxidative stress responses can enhance fungal
clearance from infected tissues and may represent a new avenue
for treatment of fungal infections.
Neutrophils have an essential role in regulating fungal growth in the cornea.
To examine the role of neutrophils in fungal keratitis, we used two
complementary approaches: systemic depletion of neutrophils from
immune-competent C57BL/6 mice and adoptive transfer of neutro-
phils into Cxcr2–/– and Cd18–/– mice. In the first approach, neutro-
phils were depleted from transgenic C57BL/6 mice expressing eGFP
downstream of the promoter LysM (LysM-eGFP mice; ref. 28) by i.p.
injection of a neutrophil-specific monoclonal antibody (NIMPR-14),
while control mice were given rat isotype antibody. Injection of
400 μg NIMP antibody on day 1 resulted in significantly decreased
neutrophils in peripheral blood smears at 0, 24, and 48 hours after
infection (S.M. Leal Jr., unpublished observations). After 24 hours,
corneas were infected with conidia (40,000 in 2 μl) isolated from
the A. fumigatus strain Af-dsRed, which constitutively expresses the
red fluorescent protein dsRed under control of the glyceraldehyde
dehydrogenase promoter. Subsequently, RFP+ fungal growth and
eGFP+ neutrophil infiltration were assessed in live corneas. We also
examined the effect of neutrophils on corneal opacity.
Figure 1, A and B, show-significantly increased eGFP+ neutrophils
at 24 and 48 hours after infection in control, isotype-treated mice,
but not in neutrophil-depleted (NIMP) mice, thus confirming neu-
trophil depletion in NIMP-treated mice. Conversely, Figure 1, A and
C, shows significantly decreased dsRed-expressing fungal hyphae at
24 and 48 hours in isotype controls compared with NIMP-treated
mice, which is consistent with increased CFU in NIMP-treated mice
Neutrophil depletion enhances fungal growth during corneal infection. (A) Transgenic C57BL/6 mice with neutrophil-specific eGFP expression
downstream of the lysozyme promotor (LysM) were depleted of neutrophils (Neuts) with neutrophil-specific NIMPR-14 antibody (i.p.) and infected
with 40,000 Af-dsRed conidia. Eyes were imaged at 24 and 48 hours after infection for neutrophil infiltration (eGFP), fungal growth (dsRed), and
corneal opacity (BF). In addition, PASH stains were performed on 5-μm sections of corneas at 48 hours after infection. (B) MetaMorph software
was used to quantify neutrophil infiltration (eGFP emission) and (C) fungal dsRed expression. (D) At 4 and 48 hours after infection, eyes were
homogenized and plated on SDA plates and CFU quantified by direct counting. MetaMorph software was utilized to quantify (E) corneal opacity
area and (F) total corneal opacity (described in detail in Supplemental Figure 1). Three independent experiments (n = 5) were performed. *P < 0.05.
Original magnification, ×20 (eye images); ×400 (histology).
2484 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 7 July 2012
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 7 July 2012
(Figure 1D). We also found significantly lower corneal opacity in
neutrophil-depleted mice as measured by both area of opacity and
total corneal opacity at 24 hours but not 48 hours after infection.
Corneal opacity at 48 hours was likely due to fungus-mediated tis-
sue pathology (image analysis methods described in Supplemental
Figure 1; supplemental material available online with this article;
doi:10.1172/JCI63239DS1) (Figure 1, E and F). Figure 1A also
shows a representative PAS-hematoxylin (PASH)–stained cornea
section of a 48-hour-infected LysM-eGFP mouse with infiltrating
cells in the stroma and anterior chamber, and few intact fungal
hyphae. In contrast, the corneas of neutrophil-depleted mice exhib-
ited reduced cellular infiltrates and prominent fungal hyphae in
both the corneal stroma and the anterior chamber.
As a second approach, we utilized two mouse strains with known
defects in neutrophil infiltration during infection and adoptively
transferred WT naive bone marrow–derived neutrophils (BMNs)
into these mice to study the specific role of neutrophils in killing
fungi during corneal infection. Cxcr2–/– neutrophils are unable to
recognize and respond to ELR+ CXC chemokines, whereas Cd18–/–
neutrophils are unable to bind to ICAM-1 on limbal vessel vascular
endothelial cells (29, 30).
C57BL/6, Cxcr2+/–, and Cxcr2–/– corneas were infected with A.
fumigatus conidia as described above. Figure 2A shows fungal
dsRed expression and corneal opacity in C57BL/6 and Cxcr2+/–
heterozygous mice, which both increase at 48 hours after infection.
In contrast, infected Cxcr2–/– mice had significantly increased fun-
gal dsRed expression (Figure 2B) and lower corneal opacity scores
(Figure 2, C and D) at 24 and 48 hours compared with C57BL/6
mice. However, Cxcr2–/– mice given C57BL/6 syngeneic neutrophils
i.v. had significantly lower fungal dsRed values (Figure 2B) and
higher corneal opacity (Figure 2, C and D) compared with Cxcr2–/–
mice not receiving neutrophils, indicating that neutrophils con-
tribute to both fungal killing and corneal opacity. Corneal sections
from these mice showed intense cellular infiltration, neutrophil
recruitment, and minimal intact fungal hyphae at 48 hours after
infection in C57BL/6 mice and Cxcr2+/– mice, whereas Cxcr2–/– cor-
neas exhibited minimal cellular infiltrates, but abundant hyphae in
the stroma and anterior chamber (Figure 2A). Following adoptive
transfer of C57BL/6 neutrophils into Cxcr2–/– mice, neutrophils
were detected in the corneal stroma, and fungal growth in the cor-
nea was lower than in the absence of neutrophils (Figure 2A).
Similarly, C57BL/6 neutrophils transferred to Cd18–/– mice during
infection conferred a protective response with increased cell migra-
tion to the cornea and lower fungal load. Figure 2, E and F, shows
that fungal dsRed expression at 48 hours was significantly increased
in Cd18–/– mice compared with C57BL/6 mice. However, Cd18–/– mice
given syngeneic BMNs from transgenic C57BL/6 LysM-eGFP mice
exhibited significantly decreased fungal dsRed expression (Figure 2F)
along with significantly increased eGFP+ neutrophil infiltration
(Figure 2G) and increased corneal opacity (Figure 2, H and I).
Together, these findings demonstrate that both CXCR2 and
CD18 regulate neutrophil recruitment to fungus-infected corneas
and that neutrophils have an essential role in controlling fungal
growth at this site.
Neutrophil NOX activity is required for control of fungal growth during
corneal infection. NOX is an enzyme complex required for reduction
of molecular O2 to O2– (31). Superoxide is the limiting reagent in
subsequent reactions, leading to the transient synthesis of ROS
with greater oxidative and fungal killing potential (21), and indi-
viduals with CGD due to impaired NOX function are unable to
control microbial infections and often succumb to filamentous
fungal infections in the lung (2, 32).
Cybb–/– mice lack the gene encoding the NOX subunit gp91phox
and thus do not express a functional NOX complex. These mice are
also more susceptible to Aspergillus lung infections (23). To deter-
mine the role of NOX in fungal keratitis, we infected Cybb–/– mice
with Af-dsRed, and ROS levels in the corneas were measured at
48 hours after infection after intrastromal injection of carboxy-
fluorescein diacetate (CFDA), which emits green fluorescence
upon oxidation. Fungal dsRed expression and corneal opacifica-
tion were measured by image analysis as described above. Figure 3,
A and B, shows CFDA activity in C57BL/6 and Cybb–/– corneas at
48 hours after infection; however, total fluorescence was signifi-
cantly lower in Cybb–/– corneas (there was no fluorescence in naive
corneas injected with CFDA; S.M. Leal Jr., unpublished observa-
tion). Conversely, fungal dsRed expression and CFU (Figure 3, C
and D) were elevated in Cybb–/– compared with C57BL/6 corneas,
indicating impaired fungal clearance in Cybb–/– corneas. Interesting-
ly, Cybb–/– corneas had more severe disease than in C57BL/6 mice,
with significantly higher total corneal opacity scores (Figure 3,
E and F), which correlated with increased neutrophil infiltra-
tion and formation of microabscesses and the presence of intact
fungal hyphae (Figure 3G). As there is no defect in the ability of
Cybb–/– neutrophils to migrate to the cornea, it is likely that these
findings represent “frustrated” neutrophils that are unable to kill
hyphae but can still recruit neutrophils to this site. Very similar
results were found when mice were infected with other pathogenic
Aspergillus and Fusarium species that cause keratitis (Supplemental
Figures 2–4), indicating that ROS has a more general role in inhib-
iting growth of filamentous fungi.
To ascertain directly whether the impaired fungal killing in
Cybb–/– mice is due to NOX that is specifically produced by neutro-
phils, we infected Cd18–/– mice with Af-dsRed as described above
and injected BMNs from C57BL/6 or Cybb–/– mice i.v. 2 hours after
infection. Corneas were imaged 24 hours after infection. Figure 3,
H and I, shows that fungal dsRed expression was significantly
reduced following adoptive transfer of C57BL/6 neutrophils,
whereas mice given Cybb–/– neutrophils had the same fungal dsRed
expression as mice not receiving neutrophils. These data clearly
demonstrate that NOX-dependent ROS production by neutro-
phils is essential for inhibiting fungal growth in the cornea.
Neutrophil adoptive transfer restricts fungal growth during corneal
infection. (A) C57BL/6, Cxcr2+/–, and Cxcr2–/– mice were infected with
30,000 Af-dsRed conidia. At 24 hours after infection, one group of fun-
gus-infected Cxcr2–/– mice were injected i.v. with 4 × 106 BMNs from
C57BL/6 (B6) mice. At 24 hours after infection, corneas were imaged
for cellular infiltration, fungal growth, and corneal opacity. At this time
point, mice were euthanized, eyes were fixed in formalin, and 5-μm cor-
neal sections were PASH stained. (B) MetaMorph software was used
to quantify fungal dsRed expression, (C) corneal opacity area, and (D)
total corneal opacity in infected corneas. (E) Similar to Cxcr2–/– mice,
C57BL/6 and Cd18–/– mice were infected with Af-dsRed conidia, and at
24 hours after infection one group of infected Cd18–/– mice were given
4 million adoptively transferred BMNs isolated from a LysM-eGFP
mouse (eGFP+ Neuts) and eyes were imaged at 48 hours. (F) Fungal
dsRed expression, (G) eGFP+ neutrophil infiltration, (H) corneal opacity
area, and (I) total corneal opacity were quantified using MetaMorph soft-
ware. Three independent experiments (n = 5) were performed. *P < 0.05.
Original magnification, ×20 (eye images); ×400 (histology).
2486 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 7 July 2012
Neutrophil NOX is required for control of A. fumigatus fungal growth during corneal infection. (A) C57BL/6 mice and Cybb–/– mice were infected
with 40,000 A. fumigatus strain Af-dsRed conidia. Eyes were imaged at 48 hours after infection for ROS-mediated CFDA dye oxidation, fungal
dsRed expression, and corneal opacity. (B) CFDA dye oxidation, (C) fungal dsRed expression, (D) CFU, (E) corneal opacity area, and (F) total
corneal opacity were quantified after infection. (G) 5-μm sections of 48-hour-infected fungal corneas were stained with PASH or neutrophil-specific
NIMP antibody. (H) Cd18–/– mice were infected with A. fumigatus strain Af-dsRed conidia. At 2 hours after infection, one set of mice were left
untreated, the second set received an i.v. injection of 4 × 106 BMNs isolated from C57BL/6 mice, and a third set received the same number of
neutrophils isolated from Cybb–/– mice. (I) At 24 hours after infection, eyes were imaged and fungal dsRed expression quantified using MetaMorph
software. Three independent experiments (n = 5) were performed. *P < 0.05. Original magnification, ×20 (eye images); ×400 (histology).