Role of IL-17A on Resolution of Pulmonary C. neoformans Infection

The current studies evaluated the role of interleukin (IL)-17A in the induction of protective immunity against pulmonary cryptococcosis in mice. Protection against pulmonary infection with C. neoformans strain H99γ was associated with increased IL-17A production. Signaling through the IFN-γ receptor (R) was required for increased IL-17A production, however, a Th17-type cytokine profile was not observed. Neutrophils were found to be the predominant leukocytic source of IL-17A, rather than T cells, suggesting that the IL-17A produced was not part of a T cell-mediated Th17-type immune response. Depletion of IL-17A in mice during pulmonary infection with C. neoformans strain H99γ resulted in an initial increase in pulmonary fungal burden, but had no effect on cryptococcal burden at later time points. Also, depletion of IL-17A did not affect the local production of other cytokines. IL-17RA−/− mice infected with C. neoformans strain H99γ survived the primary infection as well as a secondary challenge with wild-type cryptococci. However, dissemination of the wild-type strain to the brain was noted in the surviving IL-17RA−/− mice. Altogether, our results suggested that IL-17A may be important for optimal protective immune responsiveness during pulmonary C. neoformans infection, but protective Th1-type immune responses are sufficient for protection against cryptococcal infection.

IL-17 has been shown in some infectious disease models, such as Staphylococcus aureus, Bordetella bronchiseptica, and Bacteroides fragilis, to contribute to exacerbated disease [33,34]. However, in other models of bacterial infection, IL-17 was associated with protective immune responses [33,[35][36][37][38][39]. Specifically, Th17 cells have been associated with protective immune responses in the lung against several bacterial pathogens, including Mycoplasma pneumoniae, Bordetella pertussis, and Mycobacterium tuberculosis (reviewed in [26]). Similarly, IL-17 has been shown to have roles in both resistance and susceptibility against a variety of fungal infections. Neutralization of IL-23 or IL-17 during disseminated and oral candidiasis as well as during pulmonary aspergillosis exacerbates pathology, demonstrated by decreased neutrophil infiltration, increased fungal burden, and reduced levels of chemokines [40][41][42]. IL-17 is also associated with protection against Pneumocystis carinii infection [43]. In contrast, studies have shown that Th17 cell activation promoted deleterious inflammation and defective fungal clearance in pulmonary aspergillosis and gastrointestinal candidiasis [44]. Further, in vitro C. neoformans studies showed that IL-17 treatment reduced yeast proliferation and yeast expulsion from macrophages compared to IL-4 and IL-13 treated macrophages [45]. Previous work in a mouse model of cryptococcal infection suggested that a Th17-type response and IL-17 production are important for modulating survival against cryptococcosis [46]. We have established a model in which infection with an interferongamma-producing C. neoformans strain, H99c, elicits protective host immunity against pulmonary cryptococcosis in mice [14,16]. This model system has been beneficial towards studying protective immunity against pulmonary cryptococcosis. These studies have shown that pulmonary infection with C. neoformans strain H99c, but not wild-type cryptococcci, results in increased pulmonary production of IL-17A [14]. Our observation of high levels of IL-17A in protected mice led to our hypothesis that IL-17A contributes to protective anti-cryptcoccal immune responses. The purpose of these studies was to determine the role of IL-17A in protection against Cryptococcus neoformans pulmonary infections.

Results
Pulmonary C. neoformans strain H99c infection induces IL-17A production but not a Th17-type cytokine profile Previous studies in our laboratory have shown that infection with an IFN-c-producing C. neoformans strain, H99c, results in a significant increase in pulmonary IL-17A cytokine production on day 7 post-inoculation compared to mice infected with the parental C. neoformans strain H99 [14,16]. However, it remains unclear whether the increase in IL-17A production observed in the lungs of mice infected with C. neoformans strain H99c is coupled to an overall Th17-type cytokine response. We therefore determined the expression of cytokines associated with the induction of Th17cytokine responses (IL-6, IL-17A, IL-21, IL-23, and TGF-b) in total lung homogenates derived from mice infected with wild-type C. neoformans strain H99 or the transgenic C. neoformans strain H99c on day 7 post-inoculation. We observed a significant increase in IL-6 and IL-17 ( Figure 1) in lung homogenates derived from C. neoformans strain H99c infected mice compared to those obtained from mice infected with wild-type yeast (P,0.0001 for IL-6 and IL-17A) as previously described [14]. However, no significant differences in the production of IL-21, IL-23, or TGF-b were observed in the lungs of mice infected with wild-type C. neoformans strain H99 compared to mice infected with the transgenic C. neoformans strain H99c. Thus, the significant increase in IL-17A production observed in the lungs of mice during infection with the transgenic C. neoformans strain H99c appears not to be associated with a general induction of Th17-type cytokines.
Neutrophils are the predominant source of IL-17A in mice infected with C. neoformans strain H99c We sought to determine the leukocyte population that was the predominant source of IL-17A in the lungs of mice infected with C. neoformans strain H99c. Total leukocytes were isolated from lung tissues of naïve mice and C. neoformans strain H99c-infected mice on day 7 post-challenge, and the lymphocyte subpopulations characterized for intracellular IL-17A expression by flow cytometry. Leukocyte populations examined included CD4 + T cells, CD8 + T cells, cd T cells, regulatory T cells, natural killer T cells, macrophages, dendritic cells, neutrophils, B cells, eosinophils, and mast cells. Figure 2 demonstrates that the majority of intracellular IL-17A expression in both naïve mice and mice infected with C. neoformans strain H99c was observed in Ly6G + neutrophils (detected with the 1A8 monoclonal antibody). We also observed that mice infected with C. neoformans strain H99c had significantly higher intracellular IL-17A in total CD45 + cells, CD4 + T cells, and dendritic cells compared to naïve mice. Although neutrophils appear to be the predominant source of IL-17A, we cannot conclude that increased IL-17A production by other cell types in response to C. neoformans strain H99c infection does not contribute to the observed phenotype. Representative flow cytometry plots are shown in Figure S1.
Increased pulmonary IL-17A production in C. neoformans strain H99c infected mice involves IFN-c receptor (R) signaling Our studies suggest that signaling through the IFN-cR may be necessary for the increase in IL-17A production observed in the lungs of mice inoculated with C. neoformans strain H99c. To evaluate this hypothesis, IFN-cR 2/2 and wild-type (WT) BALB/c mice were inoculated with C. neoformans strain H99c or C. neoformans strain H99 and thereafter examined for pulmonary fungal burden, cytokine production, and leukocyte infiltration at day 7 post-infection. Results showed that IFN-cR 2/2 mice had significantly increased pulmonary fungal burden compared to WT mice when infected with C. neoformans strain H99c, but no change in fungal burden was detected between IFN-cR 2/2 mice and WT mice during infection with the wild-type strain, C. neoformans strain H99 ( Figure 3A). Furthermore, cytokine analysis showed that the IFN-cR 2/2 mice had significantly reduced levels of proinflammatory (IL-1a, IL-1b, and IL-6), IL-12p40, IL-17, and chemokine (G-CSF and CXCL1) production compared to WT mice during infection with C. neoformans strain H99c ( Figure 3B). No differences were observed in cytokine production in KO vs WT mice infected with C. neoformans strain H99.

Clearance of pulmonary cryptococcosis in IL-17A deficient mice
Previous studies have suggested that IL-17A may play a role in protection against experimental pulmonary crytococcosis [14]. We therefore examined the effects of IL-17A depletion on the development of protection in mice given an experimental pulmonary infection with C. neoformans strain H99c. Mice were treated intranasally with anti-IL-17A monoclonal antibody or isotype-control antibody beginning at 4 hours post-inoculation and continued every 4 days throughout the experiment. We observed a significant reduction in IL-17A production in lung homogenates of mice treated with the anti-IL-17A antibody at days 7 and 14 postinfection compared to mice treated with the isotype control antibody ( Figure 4A). Examination of other cytokines and chemokines in lung homogenates revealed no significant differences between isotype-control and anti-IL-17A antibody treated mice (Table 1). Mice treated with anti-IL-17A antibody had significantly increased pulmonary fungal burden compared to mice treated with the isotype control antibody (P,0.002) at day 7 post-infection ( Figure 4B). No differences in pulmonary fungal burden were observed at day 14 post-infection ( Figure 4B). Although all leukocytes are significantly increased in infected mice compared to naïve controls, we observed no significant differences in leukocyte populations in H99c-infected isotype control treated mice compared to IL-17A depleted mice at each time point tested ( Figure 5).
Although antibody depletion significantly reduced the amount of IL-17A in lung homogenates, the depletion was not absolute. Therefore, we evaluated survival of IL-17 receptor A knock-out (IL-17RA 2/2 ) mice given a pulmonary infection with C. neoformans strain H99c. IL-17RA 2/2 mice still have the ability to produce IL-17A, but signaling through its receptor, IL-17RA, is abrogated [47]. Figure 6A demonstrates 100% survival of WT and IL-17RA 2/2 mice given a primary pulmonary infection with C. neoformans strain H99c through day 35 post-inoculation. All surviving WT and IL-17RA 2/2 mice were subsequently rechallenged with the non-IFN-c producing wild-type C. neoformans strain H99 on day 35 post-primary inoculation and also demonstrated 100% survival through day 28 post-secondary challenge ( Figure 5B). Culture of tissue homogenates derived from re-challenged WT and IL-17RA 2/2 mice at day 28 postsecondary inoculation indicated no significant difference in fungal burden in lung or brain tissues ( Figure 6C). Evidence of dissemination to the spleen was not observed in any surviving wild-type mice, but was observed in one surviving knock-out mouse. Interestingly, following differential plating on YPD media with and without nourseothricin (a selectable marker for the H99c strain), colonization of spleen and brain was due to the challenge organism, strain H99, while the colonization of the lungs was due to the immunizing organism, strain H99c. Our results suggest that IL-17A contributes to, but is not required for, the resolution of acute infection with C. neoformans strain H99c. However, IL-17RA signaling may be needed to prevent dissemination of cryptococci from the lung to the CNS of immunized mice following rechallenge with WT cryptococci.

Discussion
IL-17A and the Th17 response have been shown to be important in protection against multiple infectious pathogens. Previous studies suggesting that IL-17A is associated with protective immune responses against C. neoformans [16] led us to investigate its contribution in our model of complete protection against experimental pulmonary cryptococcosis. Contrary to reports suggesting that IFN-c suppresses IL-17A production [39,48], we found that in the absence of IFN-cR signaling, IL-17A production is significantly reduced during pulmonary infection with C. neoformans strain H99c. Previous results in our laboratory have shown that IL-17A production in IFN-c KO mice is similar to that observed in WT mice following infection with C. neoformans strain H99c [49]. Therefore, signaling through the IFNc receptor may directly or indirectly influence IL-17A production in our model system, but is not entirely responsible for IL-17A production. Further, the presence or absence of the IFN-c cytokine does not affect IL-17A production [49]. These results suggest that in our model, IL-17A works in collaboration with the protective Th1-type immune response as reported in studies of protection against other infectious pathogens [18,45].
Elevated IL-17A production in the lungs correlates with protective immune responses against cryptococcal infection and resolution of infection [14,16]. Indeed, our results herein suggest that IL-17A may be involved in the protective response at early time points, since increased fungal burden is evident in mice treated with anti-IL-17A at day 7 post-inoculation with C. neoformans strain H99c. One important function of IL-17A, that of a neutrophil chemoattractant [21,31], was not observed in our studies ( Figure 5). Also, depletion of IL-17A during infection with C. neoformans strain H99c resulted in no changes in any other pulmonary cytokine tested. Despite early differences in pulmonary fungal burden, mice depleted of IL-17A and IL-17RA 2/2 mice eventually resolve the pulmonary infection, suggesting that alternative protective mechanisms may be present in mice lacking IL-17A or in mice lacking signalling through the IL-17 receptor. Interestingly, the surviving IL-17R 2/2 animals all had detectable brain colonization with C. neoformans strain H99, suggesting that these animals had difficulty preventing dissemination of the wildtype cryptococci, even though they showed no signs or symptoms of disease.
Our studies examining IL-17A expression within lung leukocytes during C. neoformans strain H99c infection suggest that neutrophils are the predominant source of IL-17A, rather than CD4 + T cells, which has been shown in other systems [22,50,51]. Results from other fungal infection model systems also suggested that multiple cell types can produce IL-17A (reviewed in [28]). Our data suggest that the IL-17A produced during protective anti-C. neoformans immune responses is not associated with an overall Th17-type cytokine response. Th17 responses are associated with the production of IL-21, IL-23, and TGF-b; none of which are significantly increased in the lungs of protected mice in our model ( Figure 1). IL-17RA deficiency as well as treatment with anti-IL-17A neutralizing antibodies has been shown to decrease neutrophil infiltration and production of G-CSF [23]. Further, IL-17A regulates neutrophils by inducing G-CSF production and controls expansion of IL-17A-producing neturophil regulating T cells through the IL-17RA [23]. In contrast, we did not observe any change in neutrophil recruitment in mice treated with anti-IL-17A antibodies ( Figure 5), further suggesting that IL-17A alone was not required to induce neutrophil recruitment to the lungs in response to C. neoformans H99c infection.
Since complete protection is only observed during acute infection in mice infected with C. neoformans strain H99c and not in mice infected with wild-type cryptococci, this model has been beneficial in evaluating the impact of various components of host immunity on the resolution of experimental pulmonary cryptococcosis. In our model, infection of mice with an IFN-c-producing C. neoformans strain, H99c, leads to increased production of IL-17A, resolution of the acute infection, and protection against challenge with wild-type C. neoformans [14,16,52]. Our studies show that the significant increase in IL-17A production requires IFN-c receptor signaling and is likely not associated with a Th17-type immune response. Furthermore, neutrophils appeared to be the predominant leukocyte population expressing IL-17A. Taken together, our studies show that although IL-17A may contribute to immune defenses against pulmonary infection with C. neoformans  . Induction of IL-17A production during C. neoformans strain H99c infection requires IFN-cR expression. BALB/c and IFN-cR 2/2 mice were given an intranasal inoculation with either C. neoformans strain H99 or H99c. Naïve mice were used as controls for pulmonary cytokines. Lungs were excised at day 7 post-inoculation, and pulmonary cryptococcal burden (A) and cytokine production (B) quantified. Fungal burden results are expressed as mean log CFU per milliliter 6 standard errors of the means. A. Asterisks (*) indicate where significant decreases in CFU were observed in IFN-cR 2/2 mice compared to wild-type BALB/c mice (P,0.01). B. Asterisks (*) indicate where significant differences in cytokines were observed (P,0.02). Data are cumulative of two experiments using 5 mice per group. doi:10.1371/journal.pone.0017204.g003 strain H99c, alternative mechanisms, namely Th1-type host responses, are predominantly responsible for the resolution of pulmonary C. neoformans infection.

Ethics
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Mice were housed at The University of Texas at San Antonio Small Animal Laboratory Vivarium. These animal experiments were approved by The University of Texas at San Antonio Institutional Animal Care and Use Committee (IACUC), approved protocol number MU021-11/ 11A2, and mice were handled according to IACUC guidelines. All efforts were made to minimize animal suffering.

Mice
Female BALB/c (H-2 d ) (National Cancer Institute/Charles River Laboratories, Boston, MA and The Jackson Laboratory, Bar Harbor, ME), IFN-cR 2/2 (The Jackson Laboratory), and IL-17 receptor A knock out (IL-17RA 2/2 ) mice (a kind gift of Jay K. Kolls, Louisiana State University Health Sciences Center, New Orleans, LA), all on the BALB/c background with an average weight of 20-25 grams, were used throughout these studies.
Strains and media C. neoformans strains H99 (serotype A, Mat a) and H99c (serotype A, Mat a, an interferon-gamma producing strain derived from C. neoformans H99 [14]) were recovered from 15% glycerol stocks stored at -80uC prior to use in the experiments described herein. The strains were maintained on yeast-extract-peptonedextrose (YPD) media (1% yeast extract, 2% peptone, 2% dextrose, and 2% Bacto agar). Yeast cells were grown for 18-20 h at 30uC with shaking in YPD broth (Becton Dickinson and Company, Sparks, MD), collected by centrifugation, washed three times with sterile phosphate-buffered saline (PBS), and viable yeast quantified using trypan blue dye exclusion in a hemacytometer.

Pulmonary infections
Pulmonary C. neoformans infections were initiated by nasal inhalation as previously described [16,53,54]. BALB/c mice were anesthetized with 2% isoflurane using a rodent anesthesia device (Eagle Eye Anesthesia, Jacksonville, FL) and then given a yeast inoculum of 1610 4 colony forming units (CFU) of C. neoformans strains H99 or H99c in 50 ml of sterile PBS pipetted directly into the nares. The inocula used were verified by quantitative culture on YPD agar. The mice were fed ad libitum and were monitored by inspection twice daily. Mice were euthanized at specific time points postinoculation by CO 2 inhalation followed by cervical dislocation, and lung tissues were excised using aseptic technique. Tissues were homogenized in 1 ml of sterile PBS, followed by culture of 10-fold dilutions of each tissue on YPD agar supplemented with chloramphenicol (Mediatech, Inc., Herndon, VA). CFU were enumerated following incubation at 30uC for 48 h. Alternatively, mice intended for survival analysis were monitored by inspection twice daily and euthanized if they appeared to be in pain or moribund.

Pulmonary leukocyte isolation
Lungs were excised at specific time points post-inoculation and digested enzymatically at 37uC for 30 minutes in 10 ml of digestion buffer (RPMI 1640 and 1 mg/ml of collagenase type IV [Sigma-Aldrich, St. Louis, MO.]) with intermittent (every 10 min) stomacher homogenizations. The enzymatically-digested tissues were then successively filtered through sterile nylon filters of various pore sizes (70 and 40 mm) (BD Biosciences) and washed with sterile HBSS to enrich for leukocytes. Erythrocytes were lysed by incubation in NH 4 Cl buffer (0.859% NH 4 Cl, 0.1% KHCO 3 , 0.0372% Na 2 EDTA [pH 7.4]; Sigma-Aldrich) for 3 minutes on ice followed by the addition of a 10-fold excess of PBS. The resulting leukocyte population was then collected by centrifugation (8006g) for 5 minutes, washed twice with sterile PBS, resuspended in sterile PBS+ 2% heat-inactivated fetal bovine serum (FACS buffer) and enumerated in a hemacytometer using trypan blue dye exclusion. Flow cytometric analysis was used to determine the percentage of each leukocyte population as well as the absolute number of total leukocytes (CD45 + ) within the lung cell suspension for standardization of hemacytometer counts.

Cytokine depletions
For anti-IL-17A experiments, mice received 100 mg anti-IL-17A in a volume of 25 ml pipetted directly into the nares, beginning 4 hours post-inoculation and continuing every 4 days throughout the study. Controls for these experiments included mice treated with isotype control antibody via the intranasal route.

Antibodies
For flow cytometry experiments, cells were incubated with CD16/CD32 (Fc Block TM ) (BD Biosciences, San Diego, CA) and

Flow cytometry
Standard methodology was employed for the direct immunofluorescence of pulmonary leukocytes. Briefly, in 96-well U-bottom plates, 100 ml containing 1610 6 cells in PBS + 2% FBS (FACS buffer) were incubated with 50 ml of Fc Block TM (BD Biosciences) diluted in FACS buffer for 5 minutes to block non-specific binding of antibodies to cellular Fc receptors. Subsequently, an optimal concentration of fluorochrome-conjugated antibodies (between 0.06-0.5 mg/1610 6 cells in 50 ml of FACS buffer) were added in various combinations to allow for dual or triple staining experiments, and plates were incubated for 30 minutes at 4uC. Following incubation, the cells were washed three times with FACS buffer and cells were fixed in 200 ml of 2% ultrapure formaldehyde (Polysciences, Inc., Warrington, PA) diluted in FACS buffer (fixation buffer). For intracellular staining, cells remained in fixation buffer for 10 min at room temperature. After fixation, the cells were washed and permeabilized with 0.1% saponin for 10 min at room temperature. While permeabilized, the cells were intracellularly stained with anti-IL-17A (eBioscience Inc.) and/or anti-Fox3P (regulatory T cell) (eBioscience Inc.) for 30 min at 4uC. Cells were then washed 3 times with 0.1% saponin and then resuspended in fixation buffer before flow cytometry was performed. Cells incubated with either FACS buffer alone or single fluorochrome-conjugated antibodies were used to determine positive staining and spillover/compensation calculations, and the flow cytometer determined background fluorescence. The samples were analyzed using BD FACSArray software TM on a BD FACSArray flow cytometer (BD Biosciences). Dead cells were excluded on the basis of forward angle and 90u light scatter. For data analyses, 30,000 events (cells) were evaluated from a predominantly leukocytic population identified by backgating from CD45 + -stained cells. The absolute number of total leukocytes was quantified by multiplying the total number of cells observed by hemacytometer counting by the percentage of CD45 + cells determined by flow cytometry. The absolute number of each leukocyte subset (1A8, F4/80 + , CD11c + /CD11b + , CD11c + / CD11b + /MHC class II + , CD11c + /CD11b + /CD86 + , B220 + , MHC class II + , CD4 + /CD3 + CD8 + /CD3 + , CD4 + /Fox3p + , CD4 + /DX5 + , Siglec-F + /CD11b + , FceR1a + /CD117 + /CD34 + was determined by multiplying the percentage of each gated population by the total number of CD45 + cells.

Cytokine analysis
Cytokine levels in lung tissues were analyzed using the Bio-Plex Protein Array System (Luminex-based technology) (Bio-Rad Laboratories, Hercules, CA). Briefly, lung tissue was excised and homogenized in ice-cold sterile PBS (1 ml). An aliquot (50 ml) was taken to quantify the pulmonary fungal burden and an antiprotease buffer solution (1 ml) containing PBS, protease inhibitors (inhibiting cysteine, serine, and other metalloproteinases) and 0.05% Triton X-100 was added to the homogenate. Samples were then clarified by centrifugation (8006g) for 5 minutes. Supernatants from pulmonary homogenates were assayed using the Bio-

Statistical analysis
The unpaired Student's t test (two-tailed) was used to analyze fungal burden, pulmonary cell populations, and cytokine/ chemokine data using GraphPad Prism version 5.00 for Windows (GraphPad Prism Software, San Diego California USA). Survival data was analyzed using the log-rank test (GraphPad Prism Software). Significant differences were defined as P,0.05. Figure S1 Lung neutrophils are the predominant leukocyte population expression IL-17A during pulmonary infection with C. neoformans strain H99c. BALB/c mice received an intranasal inoculum of 1610 4 CFU of C. neoformans strain H99c in 50 ml of sterile PBS. Naïve Balb/c mice are shown as controls. The lungs were excised at day 7 post-inoculation and a single cell suspension generated using enzymatic digestion. The Figure 6. Lack of IL-17R signaling does not significantly affect resolution of infection or survival to cryptococcal infection. BALB/c mice or IL-17RA 2/2 mice received an intranasal inoculum of C. neoformans strain H99c. Mice were assayed for survival through 35 days of primary infection with C. neoformans strain H99c (A). Surviving mice were then challenged with C. neoformans strain H99 and assayed for survival through 28 days of secondary challenge (B). Culture of lung, spleen, and brain tissues at day 28 post-secondary challenge were determined from mice with positive cultures (C) and numbers of mice with positive cultures are shown above each bar. Survival data show one experiment containing nine WT and eight IL-17RA 2/2 mice. Asterisks (*) indicate where significant differences in fungal burden were observed in IL-17RA 2/2 mice compared to WT mice following infection with C. neoformans strain H99 (P,0.0001). doi:10.1371/journal.pone.0017204.g006 leukocytes were stained with anti-mouse antibodies (CD45, 1A8 (Neut) CD4, CD8, F4/80 (Mac), CD11b/CD11c (DC), CD4/ Fox3p (Treg), CD4/DX5 (NKT),cd, B220 (B cell), SiglecF/ CD11b (Eosinophil), FceR1a/CD117/CD34 (Mast cell)), fixed, permeabilized, and incubated with anti-mouse antibodies specific for IL-17A and quantified by flow cytometry. Flow cytometry dot plots are representative data of five independent experiments using pooled leukocytes from 5 mice per group per experiment. Results shown in the upper right quadrant of each plot are the percentage of leukocytes expressing the indicated surface markers and IL-17A. (TIF)