Female BALB/c (H-2d) (National Cancer Institute/Charles River Laboratories, Boston, MA and The Jackson Laboratory, Bar Harbor, ME) and IL-17 receptor A knock out (IL-17RA−/−) mice (a kind gift of Amgen, Inc. Thousand Oaks, CA), all on the BALB/c background with an average weight of 20–25 grams, were used throughout these studies. 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), and mice were handled according to IACUC guidelines. All efforts were made to minimize animal suffering.
Strains and media
C. neoformans strain H99γ (serotype A, Mat α, an interferon-gamma producing strain derived from C. neoformans H99) was recovered from 15% glycerol stocks stored at −80°C prior to use in the experiments described herein. The strain was maintained on yeast-extract-peptone-dextrose (YPD) media (1% yeast extract, 2% peptone, 2% dextrose, and 2% Bacto agar) supplemented with nourseothricin. Yeast cells were grown for 18–20 h at 30°C 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. For tissue culture, complete medium consisted of RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U penicillin/ml, 100 μg of streptomycin/ml, and 50 mM 2-mercaptoethanol.
Pulmonary C. neoformans infections were initiated by nasal inhalation as previously described. 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 1 × 104 colony forming units (CFU) of C. neoformans strain H99γ in 50 μl 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 post-inoculation by CO2 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 30°C for 48 h.
Pulmonary leukocyte isolation
Lungs were excised at specific time points post-inoculation and digested enzymatically at 37°C 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 μm) (BD Biosciences) and washed with sterile HBSS to enrich for leukocytes. Erythrocytes were lysed by incubation in NH4Cl buffer (0.859% NH4Cl, 0.1% KHCO3, 0.0372% Na2EDTA [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 (800 × g) 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.
For neutrophil depletion experiments, mice received either 200 μg anti-Gr1 antibody (clone RB6-8C5) (BioXCell) or 200 μg anti-Ly6G antibody (clone 1A8) (BioXCell) in a volume of 100 μl injected intraperitoneally beginning 24 hours post-inoculation and continuing every other day throughout the study. For depletion of gamma-delta T cells, mice received 100 μg anti-gamma-delta T cell receptor antibody (eBioscience) via the intraperitoneal route beginning two days prior to inoculation and continued weekly throughout the experiment. These concentrations and schedules were chosen following studies testing different dosages and schedules in our laboratory to determine the optimum dosage and schedule for each antibody (data not shown). Controls for neutrophil and γδ+ T cell depletions included mice treated with IgG2a isotype control antibody (eBioscience Inc., San Diego, CA) via the intraperitoneal route. Mice were depleted of CD4+ and/or CD8+ T cell subsets via intraperitoneal administration of anti-CD4 (GK1.5, rat IgG2b) and anti-CD8α (2.43, rat IgG2b) antibodies (each from National Cell Culture Center, Minneapolis, MN). Each mouse received 200 μg of GK1.5 and/or 2.43 or control rat IgG2b (eBioscience Inc.) antibodies in a volume of 100 μl PBS 48 h prior to infection and weekly thereafter. Cellular depletions were confirmed for each experiment by flow cytometry using antibodies that adhere to epitopes distinct from those adhered to by the depletion antibodies. Antibodies depleted approximately 95% of neutrophils, 98% of γδ+ T cells, 98% of CD4+ T cells, and 98% of CD8+ T cells (data not shown).
For flow cytometry experiments, cells were incubated with CD16/CD32 (Fc Block™) (BD Biosciences, San Diego, CA) and the following antibodies conjugated to phycoerythrin (PE), allophycocyanin (APC), Alexa 647, or PECy7 were added: a cocktail of CD3, CD4, and CD8α; CD45, CD19, Siglec-F (BD Biosciences), 1A8, CD11c, CD11b, F4/80, NKp46, Fox3P, γδ, IL-17A, FcεR1α, CD117, CD34 (eBioscience Inc.), and F4/80 (Caltag Laoratories, Burlingame, CA).
Standard methodology was employed for the direct immunofluorescence of pulmonary leukocytes. Briefly, in 96-well U-bottom plates, 100 μl containing 1 × 106 cells in PBS + 2% FBS (FACS buffer) were incubated with 50 μl of Fc Block™ (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 μg/1 × 106 cells in 50 μl of FACS buffer) were added in various combinations to allow for dual or triple staining experiments, and plates were incubated for 30 minutes at 4°C. Following incubation, the cells were washed three times with FACS buffer and cells were fixed in 200 μl 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 4°C. 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™ on a BD FACSArray flow cytometer (BD Biosciences). Dead cells were excluded on the basis of forward angle and 90° 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+/CD11bint, CD19+, CD4+/CD3+, CD8+/CD3+, CD4+/Fox3p+, CD3-/NKp46+, CD3+/NKp46+, γδ+, Siglec-F+/CD11bint was determined by multiplying the percentage of each gated population by the total number of CD45+ cells.
γδ+ T cell ex vivo IL-17 production
Following leukocyte enrichment (see above), lung leukocytes from mice treated with either isotype control antibody or with anti-1A8 antibody were enriched for γδ+ T cells by positive selection using magnetic beads labeled with γδ antibody according to the manufacturer’s recommendations (Miltenyi Biotec, Auburn, CA). Cells were counted, and 1 × 105 cells/well were plated in triplicate in 96-well plates, with either complete media alone or with 100 μg/ml C. neoformans cell wall extract (CWE). CWE was tested for endotoxin contamination before use, and levels were confirmed to be <1 EU/μg protein (data not shown). Cells were incubated at 37°C, 5% CO2 for 24 hr. Following incubation, cells were centrifuged and supernatants were removed for quantification of IL-17A by ELISA. Remaining cells were harvested and used for intracellular IL-17A staining by flow cytometry.
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 μl) was taken to quantify the pulmonary fungal burden and an anti-protease 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 (800 × g) for 5 minutes. Supernatants from pulmonary homogenates were assayed using the Bio-Plex Protein Array System (Bio-Rad Laboratories) for the presence of IL-6, IL-10, IL-17A, and granulocyte-colony stimulating factor (G-CSF) expression, as well as the chemokine keratinocyte-derived chemokine (KC) (CXCL1). ELISA assays were performed according to manufacturer’s instructions to measure TGF-β, IL-23 (R&D Systems), and IL-21 (BD Biosciences) on pulmonary homogenates, and IL-17A ELISA (R&D Systems) was used to measure IL-17A in cell culture supernatants.
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). For multiple comparisons, a one-way ANOVA with the Tukey’s multiple comparison test was performed. Significant differences were defined as P < 0.05.