Aspergillus and Fusarium Corneal Infections Are Regulated by Th17 Cells and IL-17−Producing Neutrophils

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 3, 2014, doi:10.4049/jimmunol.1302235 The Journal of Immunology

Aspergillus and Fusarium Corneal Infections Are Regulated by Th17 Cells and IL-17–Producing Neutrophils

Patricia R. Taylor, Sixto M. Leal, Jr., Yan Sun, and Eric Pearlman

Fusarium and Aspergillus species of mold are major causes of corneal infections in the United States and worldwide, resulting in severe and blindness. As there is evidence for T cell responses to these pathogenic fungi in infected individuals, we examined the role of IL-17A (IL-17) and IFN-g in murine models of fungal . We found that C57BL/6 mice given intratracheal or s.c. immunization of conidia prior to corneal infection exhibited enhanced fungal killing and lower compared with unimmunized mice. Protective immunity was associated with temporal recruitment of IL-17–producing neutrophils and Th17 and Th1 cells and dependent on production of IL-17 but not IFN-g. Protection was also impaired in neutrophil-depleted and Rag22/2 mice. Together, the results of these studies identify an essential role for IL-17–producing neutrophils and Th17 cells in regulating the growth of fungal hyphae and the severity of corneal disease. The Journal of Immunology, 2014, 192: 000–000. Downloaded from spergillus and Fusarium are filamentous fungi that are ripheral corneal stroma, which then migrate to the infected area of the ubiquitous in the environment and can cause life-threatening (12–14). Given that neutrophils cannot ingest hyphae, their A systemic diseases. Potentially fatal pulmonary aspergillosis ability to inhibit hyphal growth is dependent on reactive oxygen can occur in patients given hematopoietic stem cell transplants and species (ROS) production and iron sequestration (15, 16). in patients with chronic pulmonary obstruction (1, 2). Pulmonary In addition to neutrophils, infected examined after pa- +

and systemic aspergillosis and fusariosis can occur in patients with tients had undergone transplant were found to contain CD3 and http://www.jimmunol.org/ immune suppression due to HIV/AIDS, hematopoietic stem cell CD4+ T cells and express IL-17A and IFN-g (17). Given that T cells transplants, organ transplants, and cancer therapy (3–5). These were likely sensitized following inhalation of fungal spores, experi- organisms are also the major cause of blinding corneal infections in ments described in the current study sensitized mice to Aspergillus immune-competent individuals following corneal injury by plant and Fusarium by intratracheal or s.c. injection of killed, swollen material containing fungal spores (conidia) (6–8). Fusarium was conidia prior to corneal infection with live conidia. We found that also found to be the causative organism in an outbreak of contact Th17 and Th1 cells are recruited to the corneal stroma and that IL-17, –associated keratitis in the United States, Europe, and Singapore, but not IFN-g, is required to kill the fungi. We also demonstrate that with .300 cases of corneal infections in a 1-y period in the United neutrophils are recruited prior to T cells, that a subpopulation of

States alone (9–11). Once in the corneal stroma, the conidia ger- neutrophils also produce IL -17, and that optimal protective im- by guest on October 2, 2021 minate, and hyphae spread through the tissue and can penetrate into munity requires T cells and IL-17–producing neutrophils. the anterior chamber. Both the hyphae and the ensuing cellular infiltrate cause severe corneal opacification, visual impairment, Materials and Methods and blindness. Source of mice Using mouse models of cornea infection, we reported that All animals were treated in accordance with the guidelines provided in the dormant conidia in the cornea stroma do not recruit neutrophils or Association for Research in Vision and statement for the induce keratitis, as the external hydrophobin protein layer is not Use of Animals in Ophthalmic and Vision Research and approved by Case recognized by the immune system (12). However, following germi- Western Reserve University Institutional Animal Care and Use Committee. Female C57BL/6 mice (6–12 wk old) and Rag22/2 mice were purchased nation, conidia express b-glucan and a-mannan, which activate the from The Jackson Laboratory (Bar Harbor, ME). Il172/2 mice were kindly C-type lectins dectin-1 and dectin-2, respectively, on resident corneal provided by Dr. Yoichiro Iwakura (University of Tokyo). macrophages. IL-1b and CXC chemokines are then produced, which recruit neutrophils from peripheral, limbal blood vessels to the pe- Aspergillus and Fusarium strains Aspergillus fumigatus strain Af-BP is a clinical isolate from a fungal keratitis patient treated at Bascom Palmer Eye Institute (Miami, FL) provided by Department of Ophthalmology and Visual Sciences, Case Western Reserve Univer- Dr. Darlene Miller and used in our earlier studies (13). Fusarium oxysporum sity, Cleveland, OH 44106 strain 8996 is a fungal keratitis clinical isolate from a patient treated at the Received for publication August 21, 2013. Accepted for publication January 22, Cole Eye Institute, Cleveland Clinic Foundation (Cleveland, OH), which 2014. we also used previously in mouse models of keratitis (14, 18). The RFP- expressing strain of A. fumigatus (Af-dsRed) has a gpdA promoter, con- This work was supported by National Institutes of Health Grants F32EY022278 (to P.R.T.), F31EY019841 (to S.M.L.), RO1 EY018612 (to E.P.), P30 EY011373 (to E.P.), stitutively expresses monomeric dsRed protein, and was generated in the Research to Prevent Blindness Foundation, and the Ohio Lions Eye Research collaboration with Dr. Michelle Momany at the University of Georgia Foundation (to E.P.). (Athens, GA) (13). GFP-expressing F. oxysporum were obtained from Dr. Seogchan Kang (Pennsylvania State University). Address correspondence and reprint requests to Dr. Eric Pearlman, Department of Ophthalmology and Visual Sciences, Case Western Reserve University, 10900 Euclid Subcutaneous and intratracheal immunization with heat-killed, Avenue, Cleveland, OH 44106. E-mail address: [email protected] swollen conidia The online version of this article contains supplemental material. Abbreviations used in this article: AspHE, Aspergillus hyphal extract; FusHE, A. fumigatus and F. oxysporum were harvested from plate-grown cultures, Fusarium hyphal extract; qPCR, quantitative PCR; ROS, reactive oxygen species. and conidia were obtained by passing the culture suspension through sterile PBS-soaked cotton gauze positioned at the tip of a 10-ml syringe as Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 described (13). Conidia were incubated 6 h in Sabouraud dextrose broth to

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302235 2 IL-17 AND FUNGAL KERATITIS allow germination and expression of b-glucan, which initiates the host synthesis system (Invitrogen), and quantitative PCR (qPCR) was performed response (13). Conidia suspensions were centrifuged, diluted in PBS to using SYBR Green (Applied Biosystems, Carlsbad, CA) as the detecting 3 3 108 conidia/100 ml, and then killed by boiling for 5 min. probe (Il17a gene NM_010552 primers: 59-TCAGCGTGTCCAAACACTG Mice were immunized with heat-killed swollen conidia by the intra- AG-39 and 39-CGCCAAGGGAGTTAAAGACTT-59). qPCR products were tracheal or s.c. routes. For airborne sensitization, mice were given a single also detected by agarose gel electrophoresis and compared with Actb (which transtracheal instillation 10 d prior to corneal infection. The trachea was encodes the b-actin gene) as the loading control. surgically exposed, and 10 ml33 107 A. fumigatus spores suspended in PBS was inoculated through the tracheal wall into the lumen as described IL-17 and IFN-g neutralization in vivo for a model of sarcoid (19). For s.c. immunization, 3 3 108 heat-killed m swollen conidia in 100 ml PBS was injected into the base of the tail 10 and Mice were given a single injection of 25 g mouse anti–IL-17A (R&D m 3 4 g 3 d prior to corneal infection. Systems) or 200 g(2 10 neutralizing units) anti–IFN- (R&D Sys- tems) in 5 ml PBS into the conjunctival sac 3 h prior to corneal infection. Aspergillus and Fusarium hyphal extracts Abs diffuse into the corneal stroma and have neutralizing activity, which we confirmed by cytokine analysis of infected corneas. Aspergillus and Fusarium conidia were added to Sabouraud broth in a shaking incubator at 37˚C (Aspergillus) or 34˚C (Fusarium) and incubated In vivo neutrophil depletion 18 h to generate hyphae. After harvesting by passing through an aspiration m filter, hyphae were then frozen in a bath of liquid nitrogen, pulverized To deplete neutrophils systemically, 200 g rat anti-mouse NIMP-R14 in m using a mortar and pestle, and passed through a 30-mm filter. Protein 200 l PBS (prepared in-house) or rat IgG (control) was injected into the concentration was adjusted to 1 mg/ml protein and stored at 220˚C. i.p. cavity. After 24 h, the number of bone marrow neutrophils was ex- amined by flow cytometry. Mouse model of Aspergillus and Fusarium keratitis In vitro chemokine production Conidia were harvested from A. fumigatus and F. oxysporum cultures as described above and adjusted to a final concentration of 5 3 104 conidia/ml The MK/T-1 murine corneal fibroblast cell line was maintained in DMEM- Downloaded from m in PBS. Mice were anesthetized by i.p. injection of tribromoethanol, and low glucose media containing 10% FBS and 50 g/ml hygromycin the corneal epithelium was abraded using a 30-gauge needle. A 33-gauge (Invitrogen) at 37˚C as described (20, 21), and cells were harvested when Hamilton syringe was inserted into the abrasion, and 2 ml13 105 live 70% confluent. Bone marrow–derived macrophages were obtained from naive C57BL/6 mice as described (12). Briefly, total bone marrow cells conidia in PBS were injected into the corneal stroma as described (13, 14). were collected from the femurs and tibias of mice, incubated in erythrocyte Mice were photographed daily using a stereomicroscope, and corneal opacity was visualized in the intact cornea using a high-resolution stereo lysis buffer (eBioscience), and cultured in a 6-ml bacteriologic-grade Petri dish containing Macrophage Growth Media (DMEM with L-glutamine,

fluorescence MZFLIII microscope (Leica Microsystems) and Spot RT Slider http://www.jimmunol.org/ KE camera (Diagnostic Instruments). All images were captured using Na-pyruvate, HEPES, penicillin/streptomycin, 10% FBS, and 30% L929 SpotCam software (RT Slider KE; Diagnostic Instruments). Corneal opacity cell-conditioned media) at 37˚C. Growth medium was replaced on day 5, and adherent cells were isolated on day 7. MK/T-1 cells and macrophages was quantified using Metamorph software as described previously (13, 14) were then cultured with media alone or media containing 100 mg/ml (Supplemental Fig. 2). Aspergillus hyphal extract (AspHE), 10 ng/ml recombinant mouse IL-17A Quantification of Aspergillus and Fusarium in infected corneas (R&D Systems), or both for 3 h at 37˚C. Supernatants were collected, and chemokines were analyzed by ELISA. Metamorph was also used to quantify total fungal mass in mice infected with the Af-dsRed and GFP expressing fungi as described (13). For as- Statistical analysis sessment of fungal viability, whole eyes were homogenized under sterile Statistical analysis was performed for each experiment using an unpaired conditions in 1 ml PBS using the Mixer Mill MM300 (Retsch; Qiagen, by guest on October 2, 2021 t test or Tukey one-way ANOVA analysis (Prism; GraphPad Software). A Valencia, CA) at 33 Hz for 4 min. Subsequently, serial log dilutions were p value ,0.05 was considered significant. performed and plated onto bacteriologic grade Sabouraud dextrose agar plates (BD Biosciences). A. fumigatus was cultured at 37˚C, and F. oxysporum was cultured at 34˚C. The number of CFUs was determined by direct counting. Results Cytokine quantification by ELISA Protective immunity in Fusarium and Aspergillus keratitis is associated with elevated local and systemic IL-17 and IFN-g For cytokine production, corneas were dissected, placed in 0.5 ml reagent diluent, and homogenized using a Retsch MM 300 ball miller at 33 Hz for As IL-17A and IFN-g are elevated in corneas of Fusarium- and 4 min (Qiagen). Soluble protein extracts were then diluted, and cytokine Aspergillus-infected patients (17), we determined if these cytokines production was measured by ELISA according to the manufacturer’s are generated in immunized mice. C57BL/6 mice were immunized directions (R&D Systems, Minneapolis, MN). with heat-killed, swollen conidia by the intratracheal or s.c. routes, Flow cytometry infected intrastromally with live Fusarium and Aspergillus conidia, and cytokines were examined. Corneas were dissected, incubated for 1 h at 37˚C in 80 U collagenase (C0130; Sigma-Aldrich), and washed in FACS buffer (PBS plus 1% FBS Splenocytes from immunized and unimmunized mice were in- plus 0.5% Na azide). Spleens were removed, and a single-cell suspension cubated 3 h with soluble AspHE or hyphal extract from Fusarium was generated and incubated in erythrocyte lysis buffer (eBioscience) for (FusHE), and cytokines were measured by ELISA. As shown in 5 min at 37˚C. Cells were incubated with anti-mouse CD16/32 Ab (Fc Fig. 1A, both cytokines were elevated in FusHE- or AspHE- block, clone 93; eBioscience), washed, and incubated with anti-mouse NIMP-R14 Ab or anti-mouse CD4 Ab (eBioscience.) For intracellular stimulated splenocytes from C57BL/6 mice immunized by either staining, cells from 10 corneas were pooled and incubated in fixation buffer the intratracheal or s.c. routes, but not from unimmunized mice. (eBioscience) and resuspended in permeabilization buffer (eBioscience). Th17 and Th1 cells were also detected in the spleens of immu- Cells were then incubated in Fc block, stained with anti-mouse IL-17A or nized mice 13 d after the initial immunization (Supplemental Fig. 1). IFN-g–conjugated (eBioscience) Abs, and analyzed using an Accuri C6 These cells also expressed cell-surface CD44 (Supplemental Fig. 1), flow cytometer (BD Biosciences). Cell populations were not gated, so that all cells were analyzed. Gates for positive Ab responses were determined which, together with cytokine production, is consistent with a pe- based on isotype controls. To isolate neutrophils from infected corneas, we ripheral memory phenotype (22). To examine cytokine production used a FACSAria III cell sorter (BD Biosciences), which yielded .98% during corneal infection, corneas were dissected and homoge- + pure NIMP-R14 neutrophils (which was also confirmed by Wrights- nized, and IL-17A and IFN-g in the soluble fractions were mea- Giemsa staining, not shown). sured by ELISA. Fig. 1B shows that production of IL-17A was Quantitative PCR maximal at 24 and 48 h, but was lower after 72 h. In contrast, IFN-g Neutrophil RNAwas extracted using the RNAeasy mini kit according to was elevated in immunized mice at 48 h and further increased at the manufacturer’s directions (Qiagen). Samples with an OD260/280 ratio 72 h postinfection. Neither cytokine was detected in infected of 2.0 were used to generate cDNA using the SuperScript First Strand corneas from unimmunized mice. The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 1. IL-17A (IL-17) and IFN-g production in Aspergillus and Fusarium keratitis. (A) IL-17 and IFN-g production by splenocytes recovered from C57BL/6 mice 10 d after s.c. (SC IMM) or intratracheal (IT IMM) injection of heat-killed, swollen Aspergillus fumigatus (Asp) or Fusarium oxysporum (Fus) conidia. IL-17 and IFN-g in splenocyte supernatants were quantified by ELISA after 3 h stimulation with AspHE or FusHE. (B) Aspergillus- and Fusarium-infected corneas were dissected and homogenized, and total IL-17 and IFN-g was measured. (C) Representative infected corneas in unimmunized and immunized mice; original magnification 320. (D and E) Percent and total corneal opacity quantified by image analysis. (F) Viable fungi in homogenized eyes quantified by CFUs. (G) Representative corneas showing dsRed-expressing A. fumigatus and GFP-expressing F. oxysporum 24 h postinfection. (H and I); original magnification 320. Quantification of total RFP and GFP as a measure of total fungal mass in infected corneas. (J) Total NIMP-R14+ neutrophils in collagenase-digested corneas. (A, B, and J) Mean 6 SD of five mice per group. (D–F, H, and I) Data points represent individual corneas. Data are representative of three experiments with similar results. *p , 0.01, by Student t test. UNIM, splenocytes or corneal lysates from naive, unimmunized mice.

Corneal opacity in Fusarium- and Aspergillus-infected mice is mice with a dsRed-expressing A. fumigatus–orF. oxysporum– shown in Fig. 1C. As we reported earlier (13, 14), naive C57BL/6 expressing GFP strain and quantified fungal mass by image mice receiving live Fusarium or Aspergillus conidia develop analysis as before (13, 16). Aspergillus and Fusarium hyphae were pronounced corneal opacification over 72 h. In marked contrast, present throughout the corneas of unimmunized mice at 24 h infected corneas of mice immunized by either the intratracheal or postinfection (Fig. 1G). In contrast, there was significantly less s.c. routes had less corneal disease. Total and percent corneal RFP Aspergillus and GFP Fusarium fungal mass in the corneas of opacification were quantified by image analysis of infected cor- immunized mice (Fig. 1G–I). To determine if the enhanced fungal neas as described previously (13) (Supplemental Fig. 2). There killing was due to increased numbers of infiltrating neutrophils, was significantly less total and percent corneal opacity at each infected corneas were collagenase digested, cells were incubated time point in infected mice that had been immunized by either with NIMP-R14, and the total number of neutrophils in each route compared with unimmunized mice, with no differences be- cornea was assessed by flow cytometry. However, as shown in tween intratracheal and s.c. immunization (Fig. 1D, 1E). Consistent Fig. 1J, there was no significant difference in the number of with less severe corneal disease, significantly lower CFU values infiltrating neutrophils recovered from corneas of unimmunized were observed in immunized compared with unimmunized mice at compared with immunized mice infected with either Aspergillus 48and72h,butnot24h(Fig.1F). or Fusarium. We and others reported that measuring CFUs in filamentous Together, these data show that intratracheal or s.c. immunization fungi does not necessarily reflect total fungal mass as a single CFU results in enhanced fungal clearance and elevated local and sys- can represent hyphae of different lengths (13, 23). We infected temic IL-17A and IFN-g, but not increased neutrophil infiltration. 4 IL-17 AND FUNGAL KERATITIS

Temporal recruitment of Th17 and Th1 cells to although there was a distinct population of NIMP-R14+ cells in Aspergillus- and Fusarium-infected corneas unimmunized, infected corneas, these were IL-17A negative; in Given that CD4 cells are present and IL-17A and IFN-g are contrast, 16.7–19.6% total corneal cells from immunized, infected + + ∼ expressed in Aspergillus- and Fusarium-infected human corneas mice were NIMP-R14 /IL-17A , which represents 46% of the total NIMP-R14+ cells. At this time point, all of the IL-17A–pro- (17), we examined Th17 and Th1 cell infiltration in Aspergillus- + 2 + ducing cells in infected corneas were NIMPR-14 (Fig. 3A), with and Fusarium-infected corneas. Although there is a CD4 IL-17 + population 24 h postinfection, Th17 (CD4+, IL-17+) cells were not no detectable NK1.1 cells or gd T cells (Supplemental Fig. 3). detected at this time, but were present 48 and 72 h postinfection, There was no significant difference between immunized and unim- where they represented ∼85 and ∼35%, respectively of total CD4 munized mice in either the total number of neutrophils in infected cells in the cornea (Fig. 2A). In contrast, Th1 (CD4+, IFN-g+) corneas (Fig. 3B), or in total CXCL1 and CXCL2 (Fig. 3C). CXCL1 cells were not detected until 72 h postinfection (Fig. 2A). Al- and CXCL2 were produced by AspHE and/or rIL-17–stimulated though there were fewer Th17 cells at 72 compared with 48 h, the macrophages, whereas corneal fibroblasts produced only CXCL1 mean fluorescence intensity of IL-17 appeared higher at the later (Fig. 3D). No chemokines were detected in unstimulated cells. time point. CD4 cells were not detected in corneas of unimmu- To examine if neutrophils in infected corneas also express Il17a nized mice at any time (data not shown). transcripts, cells from infected corneas were tagged with NIMP- To determine if the apparent biphasic recruitment of Th17 and R14, isolated by fluorescence cell sorting, and Il17a transcripts Th1 cells in infected corneas is related to chemokine production, were identified by qPCR. Fig. 3E shows a highly purified pop- corneas were dissected and homogenized 6, 24, and 48 h postin- ulation of cell-sorted neutrophils from immunized and unimmu- nized mice; however, Il17a gene expression was only detected in fection, and T cell chemokines were measured by ELISA. Fig. 2B Downloaded from shows elevated production of CCL20 and CCL22, which are neutrophils from immunized, infected mice (Fig. 3F). Similarly, specific for Th17 cells, at 24 h postinfection. Conversely, CXCL9 total IL-17A protein was detected in cell lysates of flow-sorted and CXCL10, which recruit Th1 cells, were elevated 48 h post- corneal neutrophils from immunized, but not unimmunized, mice infection. T cell chemokines were not detected in unimmunized, (Fig. 3G). infected mice at any time point. To determine the source of T cell These findings demonstrate that neutrophils are an early source chemokines, mouse corneal fibroblasts and bone marrow–derived of IL-17A in fungal infected corneas of immunized mice. http://www.jimmunol.org/ macrophages were stimulated in vitro with AspHE and/or recombi- IL-17 but not IFN-g regulates protective immunity in fungal nant mouse IL-17 for 3 h, and supernatants were assayed for T cell keratitis recruiting chemokines by ELISA. Fig. 2C shows IL-17–stimulated mouse corneal fibroblasts selectively produced CXCL10, CCL20, To determine the relative contribution of IL-17A and IFN-g to the and CCL22, whereas IL-17–stimulated macrophages produced protective immune response in fungal keratitis, C57BL/6 mice CXCL9 and CXCL10; however, AspHE did not induce production were immunized s.c., and neutralizing Abs to IL-17A or IFN-g of these chemokines in either cell type. were injected into the 2 h prior to infection with live Aspergillus or Fusarium conidia. We found that infected corneas IL-17–producing neutrophils are recruited to infected corneas

from mice given neutralizing Abs had no detectable IL-17A by guest on October 2, 2021 prior to T cells (Fig. 4A). Further, anti–IL-17A–treated C57BL/6 mice exhibited As neutrophils are recruited to the cornea within hours of infection significantly elevated corneal opacity, similar to unimmunized with Aspergillus or Fusarium (12–14), we next ascertained if the mice (Fig. 4B, Supplemental Fig. 4A). CFUs were also signifi- CD42, IL-17+ cells in corneas of immunized mice 24 h postin- cantly higher in mice receiving anti–IL-17A compared with mice fection were neutrophils. Total cells from infected corneas were given control IgG (Fig. 4C). incubated with the Ly6G-specific NIMP-R14 Ab, and intracellular As a second approach to determine the role of IL-17 in fun- IL-17A was assessed by flow cytometry. As shown in Fig. 3A, gal keratitis, Il17a2/2 mice were immunized and infected with

FIGURE 2. Recruitment of Th1 and Th17 cells to infected corneas. (A) IL-17– (left panel) and IFN-g–expressing (right panel) CD4+ cells in A. fumigatus– and F. oxysporum–infected corneas from immunized C57BL/6 mice; corneas were examined 24, 48, and 72 h postinfection. (B) CCL20 and CCL22 (Th17-recruiting chemokines) and CXCL9 and CXCL10 (Th1-recruiting chemokines) in A. fumigatus–infected corneas of unimmunized (UNIM) and immunized (IMM) mice. (C) T cell chemokines in culture supernatants of murine corneal fibroblasts and bone marrow–derived macrophages stimulated for 3 h with AspHE, recombinant mouse IL-17, or both. Data are representative of two experiments [mean and SD in (B) and (C)]. ND, not detected. The Journal of Immunology 5 Downloaded from

FIGURE 3. Recruitment of IL-17–producing neutrophils to infected corneas. (A) Intracellular IL-17 in NIMP-R14+ neutrophils 24 h after Aspergillus and Fusarium corneal infection of unimmunized (UNIM) and immunized (IMM) C57BL/6 mice. (B) Total number of NIMP-R14+ neutrophils per cornea quantified by flow cytometry. Data points represent individual corneas. (C) CXCL1 and CXCL2 production in infected corneas. (D) Chemokine production by corneal fibroblasts and bone marrow macrophages after 3-h stimulation with AspHE, recombinant mouse IL-17, or both. (E) Cell-sorted NIMP-R14+

neutrophils 24 h after A. fumigatus corneal infection of unimmunized and immunized C57BL/6 mice. Il17a gene expression (F) and total IL-17 protein (G) http://www.jimmunol.org/ in cell lysates from cell-sorted neutrophils from infected corneas 24 h after Aspergillus infection. Actb (which encodes b-actin) serves as a loading control. Data are representative of two experiments with similar results, with five mice per group [(C, D, G) mean and SD]. *p , 0.001 (unpaired Student t test).

Aspergillus or Fusarium conidia. Il17a2/2 mice exhibited signifi- Depletion of neutrophils ablates protective immunity in fungal cantly elevated corneal opacity (Fig. 4D, Supplemental Fig. 4B), keratitis similar to the effect of IL-17A–neutralizing Ab. CFU were also 2/2 To determine the role of neutrophils in fungal keratitis in immu- significantly elevated in infected Il17a mice on either a C57BL/6 nized mice, neutrophils were depleted by i.p. injection of 200 mg or BALB/c background (Fig. 4E, 4F). NIMP-R14 after immunization and 24 h prior to corneal infection.

In contrast, although IFN-g was not detected in infected corneas We found that NIMP-R14–treated mice had no detectable neu- by guest on October 2, 2021 following a subconjunctival injection of anti–IFN-g (Fig. 4G), trophils in the bone marrow (Fig. 6A); further, IL-17A production there was no significant difference in corneal opacity between mice in Fusarium-orAspergillus-infected corneas was undetectable in given neutralizing IFN-g and those given control IgG (Fig. 4H, neutrophil-depleted compared with mice given control IgG (Fig. Supplemental Fig. 4C, 4D). There was also no significant difference 6B), consistent with neutrophils being the predominant source of in CFU at 48 or 72 h after Aspergillus or Fusarium infection (Fig. IL-17A at this time point. Further, CFU were significantly higher 4I). Taken together, these data show that IL-17A but not IFN-g is in neutrophil-depleted mice compared with mice given control required for the protective immune response in fungal keratitis. IgG (Fig. 6C), and the percent and total corneal opacity was sig- Immunized Rag22/2 mice exhibit intermediate protective nificantly higher (Fig. 6D, Supplemental Fig. 4E). immunity in fungal keratitis These data clearly demonstrate that in immunized mice, neu- trophils are the major source of IL-17A 24 h after corneal infection To examine the role of T cells in IL-17–dependent protective 2 2 with Fusarium or Aspergillus, and that neutrophils have an es- immunity, Rag2 / mice, which have no functional T or B cells, sential role in limiting fungal growth and corneal disease. were immunized s.c. and infected intrastromally with A. fumigatus. At 48 h postinfection, Th17 cells (CD4+IL-17+)werepresentin C57BL/6, but not in Rag22/2, corneas; however, there was no sig- Discussion nificant difference in the number of IL-17A+ neutrophils in infected Filamentous molds, which are the major worldwide cause of fun- corneas of Rag22/2 compared with C57BL/6 mice (Fig. 5A). gal keratitis, are ubiquitous in the environment, and conidia are There were significantly less CFUs in immunized C57BL/6 and present in the air we breathe. The report that Aspergillus-specific Rag22/2 mice compared with unimmunized counterparts, indi- TcellIFN-g responses can be detected in healthy individuals de- cating enhanced killing activity in the absence of T cells (Fig. 5B); monstrates that we can respond to airborne spores in the absence of however, protection was only partial, as CFUs recovered from disease manifestations (24). Based on this finding and on reports immunized, infected Rag22/2 corneas were significantly higher that the concentration of spores in the air can reach very high levels than immunized C57BL/6. Total and percent corneal opacity were in some environments (25, 26), we examined the possibility that also significantly higher in immunized, infected Rag22/2 com- T cell responses contribute to the outcome of fungal keratitis in pared with C57BL/6 mice, which is consistent with the impaired regions with high airborne spore counts. As the prevalence of fun- fungal clearance (Fig. 5C, 5D). gal keratitis is very high in rural India and China, and the incidence Collectively, these findings demonstrate that immunized, increases further during harvest seasons (6, 7, 27), the study by infected Rag22/2 mice have an intermediate phenotype between Karthikeyan and colleagues (17) showed evidence of T cell in- unimmunized and immunized C57BL/6 mice and also indicate volvement in infected corneas from patients in southern India who that Th17 cells have a significant role in fungal keratitis. had undergone . CD3+ and CD4+ cells were 6 IL-17 AND FUNGAL KERATITIS Downloaded from http://www.jimmunol.org/

FIGURE 4. The role of IFN-g and IL-17 in fungal keratitis in immunized mice. (A–F) Effect of IL-17 neutralization or deletion on fungal keratitis. Immunized C57BL/6 mice were given a subconjunctival injection of neutralizing anti–IL-17 or isotype (IgG) control 1 d prior to Aspergillus or Fusarium corneal infection and examined 24 h postinfection. (A) IL-17 in homogenized corneas showing effective Ab neutralization. (B and C) Representative corneas from immunized mice given anti–IL-17 (aIL-17) or control Abs (IgG) 24 and 48 h postinfection (B) (quantification is shown in Supplemental Fig. 4). (C) CFU quantification 48 h postinfection. (D) Representative corneas from immunized C57BL/6 and Il17a2/2 mice at indicated time points after Aspergillus or Fusarium cornea infection. CFU quantification of corneas from immunized Il17a2/2 mice on C57BL/6 background with C57BL/6 by guest on October 2, 2021 controls (left) or Il17a2/2 mice on BALB/c background with BALB/c controls (right) 48 h after Aspergillus (E)orFusarium (F) infection. (G–I) IFN-g neutralization. (G) IFN-g in homogenized corneas from immunized mice showing effective Ab neutralization. (H) Representative corneas from immunized mice given anti– IFN-g (aIFNg) or control Abs (IgG). (I) CFUs in corneas of Aspergillus-(top panel)orFusarium-infected (bottom panel) corneas at indicated time points. (C, E, F, and I) Data points represent individual corneas. (A and G) Data are mean and SD of five mice per group. Data are rep- resentative of three repeat experiments with five mice per group. *p , 0.01 (unpaired Student t test). Ctrl, control; ND, not detected. detected in Aspergillus-andFusarium-infected corneas, which production of neutrophil chemokines CXCL1 and CXCL2 in both also express elevated IFN-g and IL-17A compared with uninfected groups of animals, which were produced by corneal fibroblasts and corneas, whereas IL-4 expression was low (17). Although non-CD4 macrophages. Neutrophils also produce CXC chemokines (29). cells can also produce IL-17 (28), these observations are consistent Although IFN-g was produced in infected corneas, there was no with the presence of Th1 and Th17 cells. effect of IFN-g neutralization on either CFU or corneal disease. In To examine the role of IL-17 and IFN-g in fungal keratitis, we contrast, neutralization or deletion of IL-17A ablated the protec- developed a murine model in which Th17 and Th1 cells are in- tive immune response, indicating an essential role for this cy- duced systemically by either intratracheal or s.c. immunization with tokine in regulating fungal growth. Depletion of neutrophils or heat-killed, swollen conidia. As with human corneas, CD4 cells, eliminating lymphoid cells (T cells and gd T cells) as a source of IL-17A and IFN-g were detected in Aspergillus-andFusarium- IL-17 by using Rag22/2 mice each impaired fungal killing, in- infected mouse corneas. However, although Th1 and Th17 cells dicating an essential role for both cell types. are generated systemically following immunization, Th17 cells are Individuals who have impaired IL-17 responses due to pro- recruited to infected corneas earlier than Th1 cells. These finding are duction of autoantibodies to IL-17 are more susceptible to fungal consistent with production of the Th17 chemokines CCL20 and infections, especially mucosal candidiasis (30, 31). Similarly, CCL22, which were detected prior to the Th1 chemokines CXCL9 mutations in STAT3, which is required for IL-17 production, result and CXCL10 and produced by corneal fibroblasts in response to in increased susceptibility to Candida infections (32, 33). Murine IL-17. We also found that CD4 cells are not the only source of models of oral candidiasis also show a requirement for IL-17, with IL-17 in fungal keratitis, as ∼50% total neutrophils recruited to the cell sources being Th17 and innate lymphoid cells (34–36). the cornea 24 h postinfection expressed intracellular IL-17A Th17 cells and neutrophils were also found to be a source of IL-17 and were the predominant source of IL-17A at this time point. in a model of pulmonary aspergillosis and systemic histoplasmosis The number of neutrophils in infected corneas of immunized mice (23, 37, 38), and IL-17–producing neutrophils were reported in was the same as unimmunized mice, indicating that immunization models of Bacillus anthracis and Yersinia pestis infection and does not affect neutrophil recruitment. This is likely due to elevated LPS-induced lung inflammation (39–41). In addition to microbial The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 5. The role of Th17 cells in protective immunity to fungal infection. (A) Intracellular IL-17 in CD4+ cells and NIMP-R14+ neutrophils from infected corneas of immunized C57BL/6 and Rag22/2 mice 24 and 48 h after Aspergillus infection. (B) Quantification of CFU in corneas from unim- munized (UNIM) and immunized (IMM) C57BL/6 and Rag22/2 mice 48 h after Aspergillus infection. (C) Representative corneas from Aspergillus- infected UNIM and IMM C57BL/6 and Rag22/2 mice 24 and 48 h postinfection. (D) Percent and total corneal opacity quantified 24 and 48 h after Aspergillus infection. (A) Representative plots are shown. (B and D) Data points represent individual corneas. Data are representative of three experiments, with 10 mice per group. *p , 0.01, **p , 0.001 (unpaired Student t test). infections, IL-17–producing neutrophils have been reported in that T cells are not required. IL-23 has been implicated in IL-17 by guest on October 2, 2021 murine models of kidney ischemia reperfusion, early-stage collagen- production by neutrophils (38, 46); however, our recent studies induced arthritis, and human psoriasis tissue and T cell lymphomas show that in addition to IL-23, IL-6 is required for IL-17A pro- (42–45). Taken together, these reports not only support our current duction and that these cytokines also induce expression of a func- findings, but also indicate that IL-17–producing neutrophils are tional IL-17R (47). involved in multiple infectious and inflammatory diseases. Al- IL-17 also mediates corneal infections with HSV, Pseudomonas though T cells reportedly contribute to IL-17 production by neu- aeruginosa, and Candida albicans (48–50). The latter study shows trophils in pulmonary aspergillosis (38), the current study clearly neutrophils and CD4 cells as a source of IL-17 during Candida shows IL-17–producing neutrophils in Rag22/2 mice, indicating infection (50), which is consistent with our findings; however,

FIGURE 6. Role of neutrophils in IL-17 production and Aspergillus and Fusarium keratitis. (A) Flow cytometry showing the absence of neutrophils in total bone marrow cells recovered from immunized C57BL/6 mice 48 h after i.p. injection of 200 mg NIMP-R14 (a-NEUT) or isotype IgG control (IgG). (B) Concentration of IL-17 in corneal lysates obtained from immunized C57BL/6 mice 24 h after Aspergillus (top panel)orFusarium (bottom panel) infection (data are mean and SD of five mice per group). (C) CFUs of Aspergillus-(top panel) and Fusarium-infected (bottom panel) corneas from im- munized, neutrophil-depleted (a NEUT) mice 48 h postinfection. Data points represent individual corneas. (D) Representative corneas from immunized, neutrophil-depleted (a-NEUT) C57BL/6 mice 24 and 48 h postinfection. Data are representative of two experiments with similar results, with five mice per group. *p , 0.05, **p , 0.001 (unpaired Student t test). 8 IL-17 AND FUNGAL KERATITIS these investigators reported increased fungal killing and less corneal 9. Chang, D. C., G. B. Grant, K. O’Donnell, K. A. Wannemuehler, J. Noble-Wang, C. Y. Rao, L. M. Jacobson, C. S. Crowell, R. S. Sneed, F. M. Lewis, et al; disease in the absence of CD4 cells or IL-17. The differences be- Fusarium Keratitis Investigation Team. 2006. Multistate outbreak of Fusarium tween their study and ours may relate to infection with Candida keratitis associated with use of a solution. JAMA 296: 953–963. compared with Aspergillus or Fusarium or to differences be- 10. Gaujoux, T., M. A. Chatel, C. Chaumeil, L. Laroche, and V. M. Borderie. 2008. 2/2 Outbreak of contact lens-related Fusarium keratitis in France. Cornea 27: 1018– tween the models, as they examined unimmunized IL-17A mice 1021. on a BALB/c background. However, although these investigators 11. Khor, W. B., T. Aung, S. M. Saw, T. Y. Wong, P. A. Tambyah, A. L. Tan, had reported differences between BALB/c and C57BL/6 mice (51), R. Beuerman, L. Lim, W. K. Chan, W. J. Heng, et al. 2006. An outbreak of 2/2 Fusarium keratitis associated with contact lens wear in Singapore. JAMA 295: we show in the current study that C57BL/6/IL-17A and BALB/ 2867–2873. 2 2 c/IL-17A / both exhibited impaired fungal clearance compared 12. Carrion, Sde. J., S. M. Leal, Jr., M. A. Ghannoum, V. Aimanianda, J. P. Latge´, with the parent strains. The difference between our results and theirs and E. Pearlman. 2013. The RodA hydrophobin on Aspergillus fumigatus spores masks dectin-1- and dectin-2-dependent responses and enhances fungal survival has therefore yet to be determined. in vivo. J. Immunol. 191: 2581–2588. A broadly accepted role for IL-17 in infection and autoimmunity 13. Leal, S. M., Jr., S. Cowden, Y. C. Hsia, M. A. Ghannoum, M. Momany, and is that IL-17 produced by Th17 and innate lymphoid cells such as E. Pearlman. 2010. Distinct roles for Dectin-1 and TLR4 in the pathogenesis of Aspergillus fumigatus keratitis. PLoS Pathog. 6: e1000976. NK T cells and gd T cells activate fibroblasts and epithelial cells, 14. Tarabishy, A. B., B. Aldabagh, Y. Sun, Y. Imamura, P. K. Mukherjee, J. H. Lass, which constitutively express the IL-17RA and IL-17RC subunits M. A. Ghannoum, and E. Pearlman. 2008. MyD88 regulation of Fusarium keratitis is dependent on TLR4 and IL-1R1 but not TLR2. J. Immunol. 181: 593– (52). IL-17A then induces production of proinflammatory and 600. chemotactic cytokines, which mediate neutrophil recruitment, 15. Leal, S. M., Jr., S. Roy, C. Vareechon, Sd. Carrion, H. Clark, M. S. Lopez- resulting in microbial killing and tissue damage. In fungal kera- Berges, A. Di Pietro, M. Schrettl, N. Beckmann, B. Redl, et al. 2013. Targeting iron acquisition blocks infection with the fungal pathogens Aspergillus fumigatus titis, IL-17 produced by neutrophils and Th17 cells likely activates and Fusarium oxysporum. [Published erratum appears in 2013 PLoS Pathog. 9: Downloaded from both these mechanisms; however, we reported that IL-17–pro- e1003436.] PLoS Pathog. 9: e1003436. ducing neutrophils also express functional IL-17 receptors and 16. Leal, S. M., Jr., C. Vareechon, S. Cowden, B. A. Cobb, J. P. Latge´, M. Momany, and E. Pearlman. 2012. Fungal antioxidant pathways promote survival against showed that IL-17–producing neutrophils generate more ROS and neutrophils during infection. J. Clin. Invest. 122: 2482–2498. have increased fungal killing activity than their IL-17–negative 17. Karthikeyan, R. S., S. M. Leal, Jr., N. V. Prajna, K. Dharmalingam, D. M. Geiser, counterparts (47). Therefore, IL-17 produced by neutrophils and E. Pearlman, and P. Lalitha. 2011. 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