Fusarium and Aspergillus species of mold are major causes of corneal infections in the United States and worldwide, resulting in severe visual impairment 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-γ in murine models of fungal keratitis. We found that C57BL/6 mice given intratracheal or s.c. immunization of conidia prior to corneal infection exhibited enhanced fungal killing and lower corneal opacity 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-γ. Protection was also impaired in neutrophil-depleted and Rag2−/− 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.
Aspergillus and Fusarium are filamentous fungi that are ubiquitous in the environment and can cause life-threatening systemic diseases. Potentially fatal pulmonary aspergillosis can occur in patients given hematopoietic stem cell transplants and in patients with chronic pulmonary obstruction (1, 2). Pulmonary and systemic aspergillosis and fusariosis can occur in patients with immune suppression due to HIV/AIDS, hematopoietic stem cell transplants, organ transplants, and cancer therapy (3–5). These organisms are also the major cause of blinding corneal infections in immune-competent individuals following corneal injury by plant material containing fungal spores (conidia) (6–8). Fusarium was also found to be the causative organism in an outbreak of contact lens–associated keratitis in the United States, Europe, and Singapore, with >300 cases of corneal infections in a 1-y period in the United States alone (9–11). Once in the corneal stroma, the conidia germinate, and hyphae spread through the tissue and can penetrate into the anterior chamber. Both the hyphae and the ensuing cellular infiltrate cause severe corneal opacification, visual impairment, and blindness.
Using mouse models of cornea infection, we reported that dormant conidia in the cornea stroma do not recruit neutrophils or induce keratitis, as the external hydrophobin protein layer is not recognized by the immune system (12). However, following germination, conidia express β-glucan and α-mannan, which activate the C-type lectins dectin-1 and dectin-2, respectively, on resident corneal macrophages. IL-1β and CXC chemokines are then produced, which recruit neutrophils from peripheral, limbal blood vessels to the peripheral corneal stroma, which then migrate to the infected area of the cornea (12–14). Given that neutrophils cannot ingest hyphae, their ability to inhibit hyphal growth is dependent on reactive oxygen species (ROS) production and iron sequestration (15, 16).
In addition to neutrophils, infected corneas examined after patients had undergone transplant were found to contain CD3+ and CD4+ T cells and express IL-17A and IFN-γ (17). Given that T cells were likely sensitized following inhalation of fungal spores, experiments described in the current study sensitized mice to Aspergillus and Fusarium by intratracheal or s.c. injection of killed, swollen conidia prior to corneal infection with live conidia. We found that Th17 and Th1 cells are recruited to the corneal stroma and that IL-17, but not IFN-γ, is required to kill the fungi. We also demonstrate that neutrophils are recruited prior to T cells, that a subpopulation of neutrophils also produce IL -17, and that optimal protective immunity requires T cells and IL-17–producing neutrophils.
Materials and Methods
Source of mice
All animals were treated in accordance with the guidelines provided in the Association for Research in Vision and Ophthalmology statement for the Use of Animals in Ophthalmic and Vision Research and approved by Case Western Reserve University Institutional Animal Care and Use Committee. Female C57BL/6 mice (6–12 wk old) and Rag2−/− mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Il17−/− mice were kindly provided by Dr. Yoichiro Iwakura (University of Tokyo).
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 Dr. Darlene Miller and used in our earlier studies (13). Fusarium oxysporum strain 8996 is a fungal keratitis clinical isolate from a patient treated at the Cole Eye Institute, Cleveland Clinic Foundation (Cleveland, OH), which we also used previously in mouse models of keratitis (14, 18). The RFP-expressing strain of A. fumigatus (Af-dsRed) has a gpdA promoter, constitutively expresses monomeric dsRed protein, and was generated in collaboration with Dr. Michelle Momany at the University of Georgia (Athens, GA) (13). GFP-expressing F. oxysporum were obtained from Dr. Seogchan Kang (Pennsylvania State University).
Subcutaneous and intratracheal immunization with heat-killed, swollen conidia
A. fumigatus and F. oxysporum were harvested from plate-grown cultures, 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 described (13). Conidia were incubated 6 h in Sabouraud dextrose broth to allow germination and expression of β-glucan, which initiates the host response (13). Conidia suspensions were centrifuged, diluted in PBS to 3 × 108 conidia/100 μl, and then killed by boiling for 5 min.
Mice were immunized with heat-killed swollen conidia by the intratracheal or s.c. routes. For airborne sensitization, mice were given a single transtracheal instillation 10 d prior to corneal infection. The trachea was surgically exposed, and 10 μl 3 × 107 A. fumigatus spores suspended in PBS was inoculated through the tracheal wall into the lumen as described for a model of sarcoid (19). For s.c. immunization, 3 × 108 heat-killed swollen conidia in 100 μl PBS was injected into the base of the tail 10 and 3 d prior to corneal infection.
Aspergillus and Fusarium hyphal extracts
Aspergillus and Fusarium conidia were added to Sabouraud broth in a shaking incubator at 37°C (Aspergillus) or 34°C (Fusarium) and incubated 18 h to generate hyphae. After harvesting by passing through an aspiration filter, hyphae were then frozen in a bath of liquid nitrogen, pulverized using a mortar and pestle, and passed through a 30-μm filter. Protein concentration was adjusted to 1 mg/ml protein and stored at −20°C.
Mouse model of Aspergillus and Fusarium keratitis
Conidia were harvested from A. fumigatus and F. oxysporum cultures as described above and adjusted to a final concentration of 5 × 104 conidia/μl in PBS. Mice were anesthetized by i.p. injection of tribromoethanol, and the corneal epithelium was abraded using a 30-gauge needle. A 33-gauge Hamilton syringe was inserted into the abrasion, and 2 μl 1 × 105 live conidia in PBS were injected into the corneal stroma as described (13, 14). Mice were photographed daily using a stereomicroscope, and corneal opacity was visualized in the intact cornea using a high-resolution stereo fluorescence MZFLIII microscope (Leica Microsystems) and Spot RT Slider KE camera (Diagnostic Instruments). All images were captured using SpotCam software (RT Slider KE; Diagnostic Instruments). Corneal opacity was quantified using Metamorph software as described previously (13, 14) (Supplemental Fig. 2).
Quantification of Aspergillus and Fusarium in infected corneas
Metamorph was also used to quantify total fungal mass in mice infected with the Af-dsRed and GFP expressing fungi as described (13). For assessment of fungal viability, whole eyes were homogenized under sterile conditions in 1 ml PBS using the Mixer Mill MM300 (Retsch; Qiagen, Valencia, CA) at 33 Hz for 4 min. Subsequently, serial log dilutions were 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.
Cytokine quantification by ELISA
+ neutrophils (which was also confirmed by Wrights-Giemsa staining, not shown).
Il17a gene NM_010552 primers: 5′-TCAGCGTGTCCAAACACTGAG-3′ and 3′-CGCCAAGGGAGTTAAAGACTT-5′). qPCR products were also detected by agarose gel electrophoresis and compared with Actb (which encodes the β-actin gene) as the loading control.
IL-17 and IFN-γ neutralization in vivo
In vivo neutrophil depletion
To deplete neutrophils systemically, 200 μg rat anti-mouse NIMP-R14 in 200 μl PBS (prepared in-house) or rat IgG (control) was injected into the i.p. cavity. After 24 h, the number of bone marrow neutrophils was examined by flow cytometry.
In vitro chemokine production
20, 21), and cells were harvested when 70% confluent. Bone marrow–derived macrophages were obtained from naive C57BL/6 mice as described (12l-glutamine, Na-pyruvate, HEPES, penicillin/streptomycin, 10% FBS, and 30% L929 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 were then cultured with media alone or media containing 100 μg/ml Aspergillus
Statistical analysis was performed for each experiment using an unpaired t test or Tukey one-way ANOVA analysis (Prism; GraphPad Software). A p value <0.05 was considered significant.
Protective immunity in Fusarium and Aspergillus keratitis is associated with elevated local and systemic IL-17 and IFN-γ
As IL-17A and IFN-γ are elevated in corneas of Fusarium- and Aspergillus-infected patients (17), we determined if these cytokines are generated in immunized mice. C57BL/6 mice were immunized with heat-killed, swollen conidia by the intratracheal or s.c. routes, infected intrastromally with live Fusarium and Aspergillus conidia, and cytokines were examined.
Splenocytes from immunized and unimmunized mice were incubated 3 h with soluble AspHE or hyphal extract from Fusarium (FusHE), and cytokines were measured by ELISA. As shown in Fig. 1A, both cytokines were elevated in FusHE- or AspHE-stimulated splenocytes from C57BL/6 mice immunized by either the intratracheal or s.c. routes, but not from unimmunized mice. Th17 and Th1 cells were also detected in the spleens of immunized mice 13 d after the initial immunization (Supplemental Fig. 1). These cells also expressed cell-surface CD44 (Supplemental Fig. 1), which, together with cytokine production, is consistent with a peripheral memory phenotype (22). To examine cytokine production during corneal infection, corneas were dissected and homogenized, and IL-17A and IFN-γ in the soluble fractions were measured by ELISA. Fig. 1B shows that production of IL-17A was maximal at 24 and 48 h, but was lower after 72 h. In contrast, IFN-γ was elevated in immunized mice at 48 h and further increased at 72 h postinfection. Neither cytokine was detected in infected corneas from unimmunized mice.
Corneal opacity in Fusarium- and Aspergillus-infected mice is shown in Fig. 1C. As we reported earlier (13, 14), naive C57BL/6 mice receiving live Fusarium or Aspergillus conidia develop pronounced corneal opacification over 72 h. In marked contrast, infected corneas of mice immunized by either the intratracheal or s.c. routes had less corneal disease. Total and percent corneal opacification were quantified by image analysis of infected corneas as described previously (13) (Supplemental Fig. 2). There was significantly less total and percent corneal opacity at each time point in infected mice that had been immunized by either route compared with unimmunized mice, with no differences between intratracheal and s.c. immunization (Fig. 1D, 1E). Consistent with less severe corneal disease, significantly lower CFU values were observed in immunized compared with unimmunized mice at 48 and 72 h, but not 24 h (Fig. 1F).
We and others reported that measuring CFUs in filamentous fungi does not necessarily reflect total fungal mass as a single CFU can represent hyphae of different lengths (13, 23). We infected mice with a dsRed-expressing A. fumigatus– or F. oxysporum–expressing GFP strain and quantified fungal mass by image analysis as before (13, 16). Aspergillus and Fusarium hyphae were present throughout the corneas of unimmunized mice at 24 h postinfection (Fig. 1G). In contrast, there was significantly less RFP Aspergillus and GFP Fusarium fungal mass in the corneas of immunized mice (Fig. 1G–I). To determine if the enhanced fungal killing was due to increased numbers of infiltrating neutrophils, infected corneas were collagenase digested, cells were incubated with NIMP-R14, and the total number of neutrophils in each cornea was assessed by flow cytometry. However, as shown in Fig. 1J, there was no significant difference in the number of infiltrating neutrophils recovered from corneas of unimmunized compared with immunized mice infected with either Aspergillus or Fusarium.
Together, these data show that intratracheal or s.c. immunization results in enhanced fungal clearance and elevated local and systemic IL-17A and IFN-γ, but not increased neutrophil infiltration.
Temporal recruitment of Th17 and Th1 cells to Aspergillus- and Fusarium-infected corneas
Given that CD4 cells are present and IL-17A and IFN-γ are expressed in Aspergillus- and Fusarium-infected human corneas (17), we examined Th17 and Th1 cell infiltration in Aspergillus- and Fusarium-infected corneas. Although there is a CD4−IL-17+ population 24 h postinfection, Th17 (CD4+, IL-17+) cells were not detected at this time, but were present 48 and 72 h postinfection, where they represented ∼85 and ∼35%, respectively of total CD4 cells in the cornea (Fig. 2A). In contrast, Th1 (CD4+, IFN-γ+) cells were not detected until 72 h postinfection (Fig. 2A). Although there were fewer Th17 cells at 72 compared with 48 h, the mean fluorescence intensity of IL-17 appeared higher at the later time point. CD4 cells were not detected in corneas of unimmunized mice at any time (data not shown).
To determine if the apparent biphasic recruitment of Th17 and Th1 cells in infected corneas is related to chemokine production, corneas were dissected and homogenized 6, 24, and 48 h postinfection, and T cell chemokines were measured by ELISA. Fig. 2B shows elevated production of CCL20 and CCL22, which are specific for Th17 cells, at 24 h postinfection. Conversely, CXCL9 and CXCL10, which recruit Th1 cells, were elevated 48 h postinfection. T cell chemokines were not detected in unimmunized, infected mice at any time point. To determine the source of T cell chemokines, mouse corneal fibroblasts and bone marrow–derived macrophages were stimulated in vitro with AspHE and/or recombinant mouse IL-17 for 3 h, and supernatants were assayed for T cell recruiting chemokines by ELISA. Fig. 2C shows IL-17–stimulated mouse corneal fibroblasts selectively produced CXCL10, CCL20, and CCL22, whereas IL-17–stimulated macrophages produced CXCL9 and CXCL10; however, AspHE did not induce production of these chemokines in either cell type.
IL-17–producing neutrophils are recruited to infected corneas prior to T cells
As neutrophils are recruited to the cornea within hours of infection with Aspergillus or Fusarium (12–14), we next ascertained if the CD4−, IL-17+ cells in corneas of immunized mice 24 h postinfection were neutrophils. Total cells from infected corneas were incubated with the Ly6G-specific NIMP-R14 Ab, and intracellular IL-17A was assessed by flow cytometry. As shown in Fig. 3A, although there was a distinct population of NIMP-R14+ cells in unimmunized, infected corneas, these were IL-17A negative; in contrast, 16.7–19.6% total corneal cells from immunized, infected mice were NIMP-R14+/IL-17A+, which represents ∼46% of the total NIMP-R14+ cells. At this time point, all of the IL-17A–producing cells in infected corneas were NIMPR-14+ (Fig. 3A), with no detectable NK1.1+ cells or γδ T cells (Supplemental Fig. 3). There was no significant difference between immunized and unimmunized mice in either the total number of neutrophils in infected corneas (Fig. 3B), or in total CXCL1 and CXCL2 (Fig. 3C). CXCL1 and CXCL2 were produced by AspHE and/or rIL-17–stimulated macrophages, whereas corneal fibroblasts produced only CXCL1 (Fig. 3D). No chemokines were detected in unstimulated cells.
To examine if neutrophils in infected corneas also express Il17a transcripts, cells from infected corneas were tagged with NIMP-R14, isolated by fluorescence cell sorting, and Il17a transcripts were identified by qPCR. Fig. 3E shows a highly purified population of cell-sorted neutrophils from immunized and unimmunized mice; however, Il17a gene expression was only detected in neutrophils from immunized, infected mice (Fig. 3F). Similarly, total IL-17A protein was detected in cell lysates of flow-sorted corneal neutrophils from immunized, but not unimmunized, mice (Fig. 3G).
These findings demonstrate that neutrophils are an early source of IL-17A in fungal infected corneas of immunized mice.
IL-17 but not IFN-γ regulates protective immunity in fungal keratitis
To determine the relative contribution of IL-17A and IFN-γ to the protective immune response in fungal keratitis, C57BL/6 mice were immunized s.c., and neutralizing Abs to IL-17A or IFN-γ were injected into the conjunctiva 2 h prior to infection with live Aspergillus or Fusarium conidia. We found that infected corneas from mice given neutralizing Abs had no detectable IL-17A (Fig. 4A). Further, anti–IL-17A–treated C57BL/6 mice exhibited significantly elevated corneal opacity, similar to unimmunized mice (Fig. 4B, Supplemental Fig. 4A). CFUs were also significantly higher in mice receiving anti–IL-17A compared with mice given control IgG (Fig. 4C).
As a second approach to determine the role of IL-17 in fungal keratitis, Il17a−/− mice were immunized and infected with Aspergillus or Fusarium conidia. Il17a−/− mice exhibited significantly elevated corneal opacity (Fig. 4D, Supplemental Fig. 4B), similar to the effect of IL-17A–neutralizing Ab. CFU were also significantly elevated in infected Il17a−/− mice on either a C57BL/6 or BALB/c background (Fig. 4E, 4F).
In contrast, although IFN-γ was not detected in infected corneas following a subconjunctival injection of anti–IFN-γ (Fig. 4G), there was no significant difference in corneal opacity between mice given neutralizing IFN-γ and those given control IgG (Fig. 4H, Supplemental Fig. 4C, 4D). There was also no significant difference in CFU at 48 or 72 h after Aspergillus or Fusarium infection (Fig. 4I). Taken together, these data show that IL-17A but not IFN-γ is required for the protective immune response in fungal keratitis.
Immunized Rag2−/− mice exhibit intermediate protective immunity in fungal keratitis
To examine the role of T cells in IL-17–dependent protective immunity, Rag2−/− mice, which have no functional T or B cells, were immunized s.c. and infected intrastromally with A. fumigatus. At 48 h postinfection, Th17 cells (CD4+IL-17+) were present in C57BL/6, but not in Rag2−/−, corneas; however, there was no significant difference in the number of IL-17A+ neutrophils in infected corneas of Rag2−/− compared with C57BL/6 mice (Fig. 5A).
There were significantly less CFUs in immunized C57BL/6 and Rag2−/− mice compared with unimmunized counterparts, indicating enhanced killing activity in the absence of T cells (Fig. 5B); however, protection was only partial, as CFUs recovered from immunized, infected Rag2−/− corneas were significantly higher than immunized C57BL/6. Total and percent corneal opacity were also significantly higher in immunized, infected Rag2−/− compared with C57BL/6 mice, which is consistent with the impaired fungal clearance (Fig. 5C, 5D).
Collectively, these findings demonstrate that immunized, infected Rag2−/− mice have an intermediate phenotype between unimmunized and immunized C57BL/6 mice and also indicate that Th17 cells have a significant role in fungal keratitis.
Depletion of neutrophils ablates protective immunity in fungal keratitis
To determine the role of neutrophils in fungal keratitis in immunized mice, neutrophils were depleted by i.p. injection of 200 μg NIMP-R14 after immunization and 24 h prior to corneal infection. We found that NIMP-R14–treated mice had no detectable neutrophils in the bone marrow (Fig. 6A); further, IL-17A production in Fusarium- or Aspergillus-infected corneas was undetectable in neutrophil-depleted compared with mice given control IgG (Fig. 6B), consistent with neutrophils being the predominant source of IL-17A at this time point. Further, CFU were significantly higher in neutrophil-depleted mice compared with mice given control IgG (Fig. 6C), and the percent and total corneal opacity was significantly higher (Fig. 6D, Supplemental Fig. 4E).
These data clearly demonstrate that in immunized mice, neutrophils are the major source of IL-17A 24 h after corneal infection with Fusarium or Aspergillus, and that neutrophils have an essential role in limiting fungal growth and corneal disease.
Filamentous molds, which are the major worldwide cause of fungal keratitis, are ubiquitous in the environment, and conidia are present in the air we breathe. The report that Aspergillus-specific T cell IFN-γ responses can be detected in healthy individuals demonstrates that we can respond to airborne spores in the absence of disease manifestations (24). Based on this finding and on reports that the concentration of spores in the air can reach very high levels in some environments (25, 26), we examined the possibility that T cell responses contribute to the outcome of fungal keratitis in regions with high airborne spore counts. As the prevalence of fungal keratitis is very high in rural India and China, and the incidence increases further during harvest seasons (6, 7, 27), the study by Karthikeyan and colleagues (17) showed evidence of T cell involvement in infected corneas from patients in southern India who had undergone corneal transplantation. CD3+ and CD4+ cells were detected in Aspergillus- and Fusarium-infected corneas, which also express elevated IFN-γ and IL-17A compared with uninfected corneas, whereas IL-4 expression was low (17). Although non-CD4 cells can also produce IL-17 (28), these observations are consistent with the presence of Th1 and Th17 cells.
To examine the role of IL-17 and IFN-γ in fungal keratitis, we developed a murine model in which Th17 and Th1 cells are induced systemically by either intratracheal or s.c. immunization with heat-killed, swollen conidia. As with human corneas, CD4 cells, IL-17A and IFN-γ were detected in Aspergillus- and Fusarium-infected mouse corneas. However, although Th1 and Th17 cells are generated systemically following immunization, Th17 cells are recruited to infected corneas earlier than Th1 cells. These finding are consistent with production of the Th17 chemokines CCL20 and CCL22, which were detected prior to the Th1 chemokines CXCL9 and CXCL10 and produced by corneal fibroblasts in response to IL-17. We also found that CD4 cells are not the only source of IL-17 in fungal keratitis, as ∼50% total neutrophils recruited to the cornea 24 h postinfection expressed intracellular IL-17A and were the predominant source of IL-17A at this time point. The number of neutrophils in infected corneas of immunized mice was the same as unimmunized mice, indicating that immunization does not affect neutrophil recruitment. This is likely due to elevated production of neutrophil chemokines CXCL1 and CXCL2 in both groups of animals, which were produced by corneal fibroblasts and macrophages. Neutrophils also produce CXC chemokines (29).
Although IFN-γ was produced in infected corneas, there was no effect of IFN-γ neutralization on either CFU or corneal disease. In contrast, neutralization or deletion of IL-17A ablated the protective immune response, indicating an essential role for this cytokine in regulating fungal growth. Depletion of neutrophils or eliminating lymphoid cells (T cells and γδ T cells) as a source of IL-17 by using Rag2−/− mice each impaired fungal killing, indicating an essential role for both cell types.
Individuals who have impaired IL-17 responses due to production of autoantibodies to IL-17 are more susceptible to fungal infections, especially mucosal candidiasis (30, 31). Similarly, mutations in STAT3, which is required for IL-17 production, result in increased susceptibility to Candida infections (32, 33). Murine models of oral candidiasis also show a requirement for IL-17, with the cell sources being Th17 and innate lymphoid cells (34–36). Th17 cells and neutrophils were also found to be a source of IL-17 in a model of pulmonary aspergillosis and systemic histoplasmosis (23, 37, 38), and IL-17–producing neutrophils were reported in models of Bacillus anthracis and Yersinia pestis infection and LPS-induced lung inflammation (39–41). In addition to microbial infections, IL-17–producing neutrophils have been reported in murine models of kidney ischemia reperfusion, early-stage collagen-induced arthritis, and human psoriasis tissue and T cell lymphomas (42–45). Taken together, these reports not only support our current findings, but also indicate that IL-17–producing neutrophils are involved in multiple infectious and inflammatory diseases. Although T cells reportedly contribute to IL-17 production by neutrophils in pulmonary aspergillosis (38), the current study clearly shows IL-17–producing neutrophils in Rag2−/− mice, indicating that T cells are not required. IL-23 has been implicated in IL-17 production by neutrophils (38, 46); however, our recent studies show that in addition to IL-23, IL-6 is required for IL-17A production and that these cytokines also induce expression of a functional IL-17R (47).
IL-17 also mediates corneal infections with HSV, Pseudomonas aeruginosa, and Candida albicans (48–50). The latter study shows neutrophils and CD4 cells as a source of IL-17 during Candida infection (50), which is consistent with our findings; however, these investigators reported increased fungal killing and less corneal disease in the absence of CD4 cells or IL-17. The differences between their study and ours may relate to infection with Candida compared with Aspergillus or Fusarium or to differences between the models, as they examined unimmunized IL-17A−/− mice on a BALB/c background. However, although these investigators had reported differences between BALB/c and C57BL/6 mice (51), we show in the current study that C57BL/6/IL-17A−/− and BALB/c/IL-17A−/− both exhibited impaired fungal clearance compared with the parent strains. The difference between our results and theirs has therefore yet to be determined.
A broadly accepted role for IL-17 in infection and autoimmunity is that IL-17 produced by Th17 and innate lymphoid cells such as NK T cells and γδ T cells activate fibroblasts and epithelial cells, which constitutively express the IL-17RA and IL-17RC subunits (52). IL-17A then induces production of proinflammatory and chemotactic cytokines, which mediate neutrophil recruitment, resulting in microbial killing and tissue damage. In fungal keratitis, IL-17 produced by neutrophils and Th17 cells likely activates both these mechanisms; however, we reported that IL-17–producing neutrophils also express functional IL-17 receptors and showed that IL-17–producing neutrophils generate more ROS and have increased fungal killing activity than their IL-17–negative counterparts (47). Therefore, IL-17 produced by neutrophils and Th17 cells likely contribute to the increased ROS production and fungal killing by these cells.
IL-17 has multiple functions in inflammation, including inducing lymphatic endothelial cells to produce vascular endothelial growth factor-D, and IL-17 can stimulate lymphangiogenesis in the normally avascular cornea (53). This mechanism could also contribute to the pathogenesis of fungal keratitis by promoting cellular infiltration to the corneal stroma. Future studies will examine the role of IL-17 in promoting inflammation that leads both to microbial clearance and to IL-17–associated tissue damage in the cornea and other tissues in which IL-17 regulates the severity of disease.
The authors have no financial conflicts of interest.
We thank the core modules of the Visual Sciences Research Center core facilities, including Scott Howell, Denice Major, and Dawn Smith for outstanding technical assistance and Dave Nethery for technical support on the intratracheal immunization model.
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.), and P30 EY011373 (to E.P.), the Research to Prevent Blindness Foundation, and the Ohio Lions Eye Research Foundation (to E.P.).
The online version of this article contains supplemental material.
Abbreviations used in this article:
- Aspergillus hyphal extract
- Fusarium hyphal extract
- quantitative PCR
- reactive oxygen species.
- Received August 21, 2013.
- Accepted January 22, 2014.
- Copyright © 2014 by The American Association of Immunologists, Inc.