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
1. Milner JD, Sandler NG, Douek DC. Th17 cells,
Job’s syndrome and HIV: opportunities for bac-
terial and fungal infections. Curr Opin HIV AIDS.
2. Antachopoulos C. Invasive fungal infections in
congenital immunodeficiencies. Clin Microbiol
3. Thomas PA. Fungal infections of the cornea. Eye.
4. Gower EW, et al. Trends in fungal keratitis in the
United States, 2001 to 2007. Ophthalmology. 2010;
5. Liesegang TJ, Forster RK. Spectrum of micro-
bial keratitis in South Florida. Am J Ophthalmol.
6. Rosa RH Jr, Miller D, Alfonso EC. The changing
spectrum of fungal keratitis in south Florida. Oph-
7. Xie L, Zhong W, Shi W, Sun S. Spectrum of fun-
gal keratitis in north China. Ophthalmology.
8. Bharathi MJ, Ramakrishnan R, Meenakshi R, Pad-
mavathy S, Shivakumar C, Srinivasan M. Microbial
keratitis in South India: influence of risk factors,
climate, and geographical variation. Ophthalmic Epi-
9. Bhartiya P, Daniell M, Constantinou M, Islam FM,
Taylor hour Fungal keratitis in Melbourne. Clin
Experiment Ophthalmol. 2007;35(2):124–130.
10. Dunlop AA, et al. Suppurative corneal ulceration
in Bangladesh. A study of 142 cases examining the
microbiological diagnosis, clinical and epidemio-
logical features of bacterial and fungal keratitis.
Aust N Z J Ophthalmol. 1994;22(2):105–110.
11. Saha R, Das S. Mycological profile of infec-
tious keratitis from Delhi. Indian J Med Res. 2006;
12. Perez-Balbuena AL, Vanzzini-Rosano V, Valadez-
Virgen Jde J, Campos-Moller X. Fusarium keratitis
in Mexico. Cornea. 2009;28(6):626–630.
13. Chang DC, et al. Multistate outbreak of Fusarium
keratitis associated with use of a contact lens solution.
14. Srinivasan M. Fungal keratitis. Curr Opin Ophthal-
15. Prajna NV, et al. Comparison of natamycin and
voriconazole for the treatment of fungal keratitis.
Arch Ophthalmol. 2010;128(6):672–678.
16. Pham CT. Neutrophil serine proteases: specific reg-
ulators of inflammation. Nat Rev Immunol. 2006;
17. Nauseef WM. How human neutrophils kill and
degrade microbes: an integrated view. Immunol Rev.
18. Karthikeyan RS, et al. Expression of innate and
adaptive immune mediators in human corneal tis-
sue infected with Aspergillus or fusarium. J Infect
19. Leal SM Jr, Cowden S, Hsia YC, Ghannoum MA,
Momany M, Pearlman E. Distinct roles for Dec-
tin-1 and TLR4 in the pathogenesis of Aspergillus
fumigatus keratitis. PLoS Pathog. 2011;6:e1000976.
20. Tarabishy AB, et al. MyD88 regulation of Fusarium
keratitis is dependent on TLR4 and IL-1R1 but not
TLR2. J Immunol. 2008;181(1):593–600.
21. Winterbourn CC. Reconciling the chemistry and
biology of reactive oxygen species. Nat Chem Biol.
22. Segal BH, Romani LR. Invasive aspergillosis in
chronic granulomatous disease. Med Mycol. 2009;
23. Morgenstern DE, Gifford MA, Li LL, Doerschuk
CM, Dinauer MC. Absence of respiratory burst in
X-linked chronic granulomatous disease mice leads
to abnormalities in both host defense and inflam-
matory response to Aspergillus fumigatus. J Exp
24. Pollock JD, et al. Mouse model of X-linked chronic
granulomatous disease, an inherited defect in
phagocyte superoxide production. Nat Genet. 1995;
25. Philippe B, et al. Killing of Aspergillus fumigatus by
alveolar macrophages is mediated by reactive oxidant
intermediates. Infect Immun. 2003;71(6):3034–3042.
26. Rex JH, Bennett JE, Gallin JI, Malech HL, Melnick
DA. Normal and deficient neutrophils can coop-
erate to damage Aspergillus fumigatus hyphae.
J Infect Dis. 1990;162(2):523–528.
27. Henriet SS, et al. Human leukocytes kill Aspergillus
nidulans by reactive oxygen species-independent
mechanisms. Infect Immun. 2011;79(2):767–773.
28. Faust N, Varas F, Kelly LM, Heck S, Graf T. Inser-
tion of enhanced green fluorescent protein into
the lysozyme gene creates mice with green fluores-
cent granulocytes and macrophages. Blood. 2000;
29. Viola A, Luster AD. Chemokines and their recep-
tors: drug targets in immunity and inflammation.
Annu Rev Pharmacol Toxicol. 2008;48:171–197.
30. Wilson RW, et al. Gene targeting yields a CD18-
mutant mouse for study of inflammation. J Immunol.
31. Lambeth JD. NOX enzymes and the biology of reac-
tive oxygen. Nat Rev Immunol. 2004;4(3):181–189.
32. Andrews T, Sullivan KE. Infections in patients with
inherited defects in phagocytic function. Clin Micro-
biol Rev. 2003;16(4):597–621.
33. El-Benna J, Dang PM, Gougerot-Pocidalo MA.
Priming of the neutrophil NADPH oxidase activa-
tion: role of p47phox phosphorylation and NOX2
mobilization to the plasma membrane. Semin
34. Hohl TM, et al. Aspergillus fumigatus triggers
inflammatory responses by stage-specific beta-
glucan display. PLoS Pathog. 2005;1(3):e30.
35. van Bruggen R, et al. Complement receptor 3, not
Dectin-1, is the major receptor on human neutro-
phils for beta-glucan-bearing particles. Mol Immunol.
36. Lessing F, et al. The Aspergillus fumigatus tran-
scriptional regulator AfYap1 represents the major
regulator for defense against reactive oxygen inter-
mediates but is dispensable for pathogenicity in an
intranasal mouse infection model. Eukaryot Cell.
37. Lambou K, Lamarre C, Beau R, Dufour N, Latge
JP. Functional analysis of the superoxide dismutase
family in Aspergillus fumigatus. Mol Microbiol. 2010;
38. Paris S, et al. Catalases of Aspergillus fumigatus.
Infect Immun. 2003;71(6):3551–3562.
39. Fallon JP, Reeves EP, Kavanagh K. Inhibition of
neutrophil function following exposure to the
Aspergillus fumigatus toxin fumagillin. J Med
Microbiol. 2010;59(pt 6):625–633.
40. Tsunawaki S, Yoshida LS, Nishida S, Kobayashi T,
Shimoyama T. Fungal metabolite gliotoxin inhibits
assembly of the human respiratory burst NADPH
oxidase. Infect Immun. 2004;72(6):3373–3382.
41. Perrin RM, et al. Transcriptional regulation of
chemical diversity in Aspergillus fumigatus by
LaeA. PLoS Pathog. 2007;3(4):e50.
42. Qiao J, et al. Afyap1, encoding a bZip transcription-
al factor of Aspergillus fumigatus, contributes to
oxidative stress response but is not essential to the
virulence of this pathogen in mice immunosup-
pressed by cyclophosphamide and triamcinolone.
Med Mycol. 2008;46(8):773–782.
43. Wood ZA, Schroder E, Robin Harris J, Poole LB.
Structure, mechanism and regulation of peroxire-
doxins. Trends Biochem Sci. 2003;28(1):32–40.
44. Koharyova M, Kolarova M. Oxidative stress and
thioredoxin system. Gen Physiol Biophys. 2008;
45. Glaser AG, Menz G, Kirsch AI, Zeller S, Crameri R,
Rhyner C. Auto- and cross-reactivity to thioredoxin
allergens in allergic bronchopulmonary aspergillosis.
46. Ramanathan RK, et al. A phase I trial of PX-12,
a small-molecule inhibitor of thioredoxin-1,
administered as a 72-hour infusion every 21 days
in patients with advanced cancers refractory to
standard therapy [published online ahead of print
August 24, 2011]. Invest New Drugs. doi:10.1007/
47. Ramanathan RK, et al. A Phase I pharmacoki-
netic and pharmacodynamic study of PX-12, a
novel inhibitor of thioredoxin-1, in patients with
advanced solid tumors. Clin Cancer Res. 2007;
48. Welsh SJ, Williams RR, Birmingham A, Newman
DJ, Kirkpatrick DL, Powis G. The thioredoxin
redox inhibitors 1-methylpropyl 2-imidazolyl
disulfide and pleurotin inhibit hypoxia-induced
factor 1alpha and vascular endothelial growth fac-
tor formation. Mol Cancer Ther. 2003;2(3):235–243.
49. Ramanathan RK, et al. A randomized phase II
study of PX-12, an inhibitor of thioredoxin in
patients with advanced cancer of the pancreas
following progression after a gemcitabine-con-
taining combination. Cancer Chemother Pharmacol.
50. Chauhan N, Latge JP, Calderone R. Signalling and
oxidant adaptation in Candida albicans and Aspergil-
lus fumigatus. Nat Rev Microbiol. 2006;4(6):435–444.
51. Brown GD, Herre J, Williams DL, Willment JA,
Marshall AS, Gordon S. Dectin-1 mediates the
biological effects of beta-glucans. J Exp Med. 2003;
52. Vetvicka V, Thornton BP, Ross GD. Soluble beta-
glucan polysaccharide binding to the lectin site of
neutrophil or natural killer cell complement recep-
tor type 3 (CD11b/CD18) generates a primed state
of the receptor capable of mediating cytotoxicity
of iC3b-opsonized target cells. J Clin Invest. 1996;
53. Lavigne LM, Albina JE, Reichner JS. Beta-glucan
is a fungal determinant for adhesion-dependent
human neutrophil functions. J Immunol. 2006;
54. Boyle KB, et al. Class IA phosphoinositide 3-kinase
beta and delta regulate neutrophil oxidase activa-
tion in response to Aspergillus fumigatus hyphae.
J Immunol. 2011;186(5):2978–2989.
55. Abram CL, Lowell CA. The ins and outs of leuko-
cyte integrin signaling. Annu Rev Immunol. 2009;
56. Leal SM Jr, Cowden S, Hsia YC, Ghannoum MA,
Momany M, Pearlman E. Distinct roles for Dec-
tin-1 and TLR4 in the pathogenesis of Aspergillus
fumigatus keratitis. PLoS Pathog. 2010;6:e1000976.
57. Werner JL, et al. Requisite role for the dectin-1
beta-glucan receptor in pulmonary defense
against Aspergillus fumigatus. J Immunol. 2009;
58. Goodridge HS, et al. Activation of the innate immune
receptor Dectin-1 upon formation of a ‘phagocytic
synapse’. Nature. 2011;472(7344):471–475.
59. Vedder NB, Harlan JM. Increased surface expres-
sion of CD11b/CD18 (Mac-1) is not required for
stimulated neutrophil adherence to cultured endo-
thelium. J Clin Invest. 1988;81(3):676–682.
60. Amulic B, Cazalet C, Hayes GL, Metzler KD, Zych-
linsky A. Neutrophil function: from mechanisms
to disease. Annu Rev Immunol. 2012;ƒ30:459–489.
61. Goodridge HS, Wolf AJ, Underhill DM. Beta-
glucan recognition by the innate immune system.
Immunol Rev. 2009;230(1):38–50.
62. Mocsai A, Ruland J, Tybulewicz VL. The SYK tyro-
sine kinase: a crucial player in diverse biological
functions. Nat Rev Immunol. 2010;10(6):387–402.
63. Chai LY, et al. Aspergillus fumigatus cell wall com-
ponents differentially modulate host TLR2 and
TLR4 responses. Microbes Infect. 2011;13(2):151–159.
2498 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 7 July 2012
64. Netea MG, et al. Immune sensing of Candida albi-
cans requires cooperative recognition of mannans
and glucans by lectin and Toll-like receptors. J Clin
65. Chen GY, Nunez G. Sterile inflammation: sensing
and reacting to damage. Nat Rev Immunol. 2010;
66. Chun KH, Seong SY. CD14 but not MD2 transmit
signals from DAMP. Int Immunopharmacol. 2010;
67. Dickinson BC, Chang CJ. Chemistry and biology
of reactive oxygen species in signaling or stress
responses. Nat Chem Biol. 2011;7(8):504–511.
68. Aratani Y, et al. Relative contributions of myelo-
peroxidase and NADPH-oxidase to the early host
defense against pulmonary infections with Candi-
da albicans and Aspergillus fumigatus. Med Mycol.
69. Elahi S, Pang G, Ashman RB, Clancy R. Nitric
oxide-enhanced resistance to oral candidiasis.
70. de Jesus-Berrios M, Liu L, Nussbaum JC, Cox GM,
Stamler JS, Heitman J. Enzymes that counteract
nitrosative stress promote fungal virulence. Curr
71. Bahn YS, Sundstrom P. CAP1, an adenylate cyclase-
associated protein gene, regulates bud-hypha tran-
sitions, filamentous growth, and cyclic AMP levels
and is required for virulence of Candida albicans.
J Bacteriol. 2001;183(10):3211–3223.
72. Sugui JA, et al. Genes differentially expressed in
conidia and hyphae of Aspergillus fumigatus
upon exposure to human neutrophils. PLoS One.
73. Thon M, Al-Abdallah Q, Hortschansky P, Brakhage
AA. The thioredoxin system of the filamentous fun-
gus Aspergillus nidulans: impact on development
and oxidative stress response. J Biol Chem. 2007;
74. Spikes S, et al. Gliotoxin production in Aspergillus
fumigatus contributes to host-specific differences
in virulence. J Infect Dis. 2008;197(3):479–486.
75. Belkacemi L, Barton RC, Hopwood V, Evans EG.
Determination of optimum growth conditions for
gliotoxin production by Aspergillus fumigatus and
development of a novel method for gliotoxin detec-
tion. Med Mycol. 1999;37(4):227–233.
76. Chapman RW, Phillips JE, Hipkin RW, Curran AK,
Lundell D, Fine JS. CXCR2 antagonists for the treat-
ment of pulmonary disease. Pharmacol Ther. 2009;
77. Sugui JA, et al. Gliotoxin is a virulence factor of
Aspergillus fumigatus: gliP deletion attenuates
virulence in mice immunosuppressed with hydro-
cortisone. Eukaryot Cell. 2007;6(9):1562–1569.
78. Bok JW, et al. GliZ, a transcriptional regulator
of gliotoxin biosynthesis, contributes to Asper-
gillus fumigatus virulence. Infect Immun. 2006;
79. Kale SP, Milde L, Trapp MK, Frisvad JC, Keller NP,
Bok JW. Requirement of LaeA for secondary metabo-
lism and sclerotial production in Aspergillus flavus.
Fungal Genet Biol. 2008;45(10):1422–1429.