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IL-23 Dampens the Allergic Response to Cryptococcus neoformans through IL-17–Independent and –Dependent Mechanisms

  • Wendy A. Szymczak
    Affiliations
    Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York
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  • Rani S. Sellers
    Affiliations
    Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York

    Department of Pathology, Albert Einstein College of Medicine, Bronx, New York
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  • Liise-anne Pirofski
    Correspondence
    Address reprint requests to Liise-anne Pirofski, M.D., Albert Einstein College of Medicine, Forchheimer 709, 1300 Morris Park Ave, Bronx, NY 10461
    Affiliations
    Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York

    Division of Infectious Diseases, Department of Medicine, Montefiore Medical Center, Bronx, New York
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      The cytokines IL-23 and IL-17 have been implicated in resistance to cryptococcal disease, but it is not clear whether IL-23–mediated production of IL-17 promotes fungal containment following pulmonary challenge with Cryptococcus neoformans. We used mice lacking IL-23 (IL-23p19−/−) or IL-17RA (IL-17RA−/−), and wild type (WT) C57BL/6 mice to examine the IL-23/IL-17 axis after intranasal infection with the C. neoformans strain 52D. The absence of IL-23 or IL-17RA had no effect on pulmonary or brain fungal burden at 1 or 6 weeks after infection. However, survival of IL-23p19−/− mice was reduced compared to IL-17RA−/− mice. IL-I7 production by CD4 T cells and natural killer T (NKT) cells was impaired in IL-23p19−/− lungs, but was not completely abolished. Both IL-23p19−/− and IL-17RA−/− mice exhibited impaired neutrophil recruitment, increased serum levels of IgE and IgG2b, and increased deposition of YM1/YM2 crystals in the lung, but only IL-23p19−/− mice developed persistent lung eosinophilia. Although survival of IL-17RA−/− and WT mice was similar after 17 weeks of infection, only surviving IL-17RA−/− mice exhibited cryptococcal dissemination to the blood. These data demonstrate that IL-23 dampens the allergic response to cryptococcal infection through IL-17–independent suppression of eosinophil recruitment and IL-17–dependent regulation of antibody production and crystal deposition. Furthermore, IL-23, and to a lesser extent IL-17, contribute to disease resistance.
      Cryptococcus neoformans is a fungal pathogen that causes significant morbidity and mortality, primarily in immunocompromised, but also in immunocompetent individuals. In Africa, cryptococcal meningitis is the cause of death of 20% to 30% of HIV-infected patients.
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      Robust Th1 and Th17 immunity supports pulmonary clearance but cannot prevent systemic dissemination of highly virulent Cryptococcus neoformans H99.
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      In a chronic, systemic cryptococcal infection model, administration of recombinant IL-23 increased production of IL-17 by TH17 cells and non-T cells and prolonged survival,
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      • McClanahan T.
      • Kastelein R.A.
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      Administration of IL-23 engages innate and adaptive immune mechanisms during fungal infection.
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      IL-23 enhances the inflammatory cell response in Cryptococcus neoformans infection and induces a cytokine pattern distinct from IL-12.
      However, the role of IL-23 in the lungs in the setting of respiratory acquisition of C. neoformans, the natural route of infection, has not been examined.
      We sought to determine whether IL-23 promotes IL-17 production and subsequent control of chronic pulmonary infection caused by C. neoformans, strain 52D. Using IL-23p19−/− or IL-17RA−/− mice, we found that IL-23 and IL-17 did not significantly contribute to control of lung fungal burden or yeast dissemination to the brain during the first 6 weeks of infection, but the absence of IL-23 was associated with increased mortality. IL-23 and IL-17 both suppressed mediators of the allergic response to C. neoformans; specifically IgE production and the formation of YM1/YM2 crystals; however, IL-23 also inhibited eosinophil recruitment to the lung.

      Materials and Methods

      Mice

      Male 6- to 8-week-old C57BL/6 WT control mice were purchased from the National Cancer Institute (Charles River Laboratories, Wilmington, MA). IL-23p19−/− mice were obtained from Genentech (San Francisco, CA), and IL-17RA−/− mice were supplied by Amgen (Seattle, WA). IL-23p19−/− and IL-17RA−/− mice were previously backcrossed 10 generations onto the C57BL/6 background and were bred under pathogen-free conditions in the Institute for Animal Studies at the Albert Einstein College of Medicine (AECOM). All mice were given unrestricted access to food and water. All mouse experiments were conducted with prior approval from the Animal Care and Use Committee of AECOM following established guidelines.

      Cryptococcal Infection Model

      A serotype D strain (52D) of C. neoformans, ATCC 24067 (American Type Culture Collection, Manassas, VA), was used for intranasal (i.n.) infection of mice. Strain 52D has been used extensively to evaluate the immune response to experimental pulmonary cryptococcosis.
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      • McClanahan T.
      • Kastelein R.A.
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      IL-23 enhances the inflammatory cell response in Cryptococcus neoformans infection and induces a cytokine pattern distinct from IL-12.
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      IL-5 is required for eosinophil recruitment, crystal deposition, and mononuclear cell recruitment during a pulmonary Cryptococcus neoformans infection in genetically susceptible mice (C57BL/6).
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      Protective and nonprotective human immunoglobulin M monoclonal antibodies to Cryptococcus neoformans glucuronoxylomannan manifest different specificities and gene use profiles.
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      CCR2 expression determines T1 versus T2 polarization during pulmonary Cryptococcus neoformans infection.
      The C. neoformans strain was kept in 15% glycerol aliquots at −80°C until needed. Thawed aliquots of C. neoformans were grown in Difco Sabouraud Dextrose Broth (Becton Dickinson, Franklin Lakes, NJ) for 48 hours at 37°C with shaking, washed twice in PBS (Mediatech, Herndon, VA), and counted in a hemocytometer using Trypan Blue for viability. For the i.n. infection, mice were anesthetized with isoflurane (Halocarbon, River Edge, NJ) and placed in a vertical position. A volume of 20 μL containing 5 × 105 colony-forming units (CFU) of C. neoformans was administered via the nares. For survival studies, infected mice were observed at least once daily.

      Measurement of Tissue and Blood Fungal Burden

      Blood was collected from three to five animals per group by retro-orbital puncture under deep isoflurane anesthesia. After blood collection, mice were euthanized by cervical dislocation. Lungs and brains were removed and homogenized in 1 mL of HBSS (Lonza, Walkersville, MD). CFU were determined by making 10-fold serial dilutions of each tissue or twofold dilutions of blood and plating 20 μL of the sample and dilutions on Sabouraud Dextrose agar plates (BBL, Sparks, MD). Each sample was processed in duplicate. Plates were incubated at room temperature for 72 hours, after which colonies were visually counted.

      Measurement of Cytokine Levels

      Lung homogenates that were used to determine fungal burden were centrifuged at 3000 × g for 30 minutes at 4°C and the supernatants collected. The supernatants were centrifuged at 13,000 × g at 4°C for an additional 10 minutes to remove any remaining debris. Samples were stored at −80°C before use. All cytokine concentrations were determined using a DuoSet ELISA Development Kit (R&D Systems, Minneapolis, MN) according to the manufacturer's protocol.

      Isolation of Lung Cells

      In independent experiments, three to five mice per group were anesthetized with isoflurane, sacrificed by cervical dislocation, and lungs removed. Single-cell lung suspensions were obtained from using a gentleMACS Dissociator (Miltenyi Biotec, Auburn, CA) following the manufacturer's protocol for dissociation of mouse lung. Briefly, lungs were excised, washed in PBS, and added to gentleMACS Dissociator C tubes containing 5 mL of HEPES buffer, 2 mg/mL Collagenase D, and 40 U/mL DNase I (Roche, Indianapolis, IN). Lungs were briefly dissociated using the gentleMACS, incubated for 30 minutes at 37°C for tissue digestion, and then further dissociated with the gentleMACS to obtain a single-cell suspension. Red blood cells were lysed by addition of 0.17 mol/L NH4Cl (Sigma-Aldrich, St. Louis, MO), and the lung homogenate was passed through 70-μm filters (BD Biosciences, San Jose, CA) to remove debris.

      Flow Cytometry

      The phenotypes of isolated lung cells were determined by flow cytometry. Before staining for cell-surface markers, cells were incubated with CD16/32 in 1% bovine serum albumin–PBS for 10 minutes at 4°C to limit nonspecific binding. Cells were then stained for 15 minutes at 4°C with combinations of the following antibodies: CD45-Pacific Blue or Alexa 700, Ly6G-APC-Cy7, CD11b-Percp-Cy5.5, or APC-Cy7, CD11c-Pe-Cy7, MHCII-Pe, Ly6C-FITC, F4/80-Alexa 647, CD19-Pe-Cy7, B220-Percp-Cy5.5, IgD-Alexa 647, IgM-FITC, CD5-PE, CD49b-APC, CD4-APC-Cy7, CD8-Pacific Blue, CD3-Alexa 647. Antibodies were purchased from BD Biosciences (Franklin Lakes, NJ) with the exceptions of F4/80-Alexa 647 and CD11b-Percp-Cy5.5 (eBioscience, San Diego, CA), and CD45-Pacific Blue (Biolegend, San Diego, CA). Appropriate fluorescence minus one and isotype controls were also included. Data were collected on an LSRII (BD Biosciences) and analyzed with FlowJo software (Tree Star, Ashland, OR).

      Calculation of the Number of CD45+ Lung Leukocytes

      Isolated mammalian lung cells and extracellular yeast were counted using a Scepter handheld automated cell counter (Millipore, Billerica, MA). Cells ranging from 6 to 24 μm in size were included in the cell count, with >90% of the cells being between 6 and 14 μm. To arrive at the number of CD45+ lung leukocytes, the relative percentage of live lung cells as determined by forward and side scatter and CD45+ double gating was multiplied by the automated Scepter cell count, therefore excluding yeast and CD45 mammalian leukocytes.

      Intracellular Staining for IL-17a

      A Lympholyte M (Cedarlane, Ontario, Canada) gradient was used for enrichment of CD45+ leukocytes from lung cell suspensions. Isolated leukocytes were plated in 96-well plates at a concentration of 1 × 106 lung cells/200 μL of RPMI-10% bovine serum albumin. The lung cells were stimulated for 5 hours at 37°C with 50 ng/mL PMA, 1 μg/mL ionomycin (Sigma-Aldrich), and 1 μL of GolgiPlug (BD Biosciences). Leukocytes were washed and stained with LIVE/DEAD Fixable Violet Dead Cell stain (Invitrogen, Carlsbad, CA) for live-cell gating followed by staining with surface antibodies as described above. Leukocytes were then permeabilized in Cytofix/Cytoperm, and stained in Permwash buffer with anti–IL-17a-PE (BD Biosciences) at a 1:100 dilution for 30 minutes at 4°C.

      Determination of Serum Antibody Concentrations

      Concentrations of serum antibodies were determined by enzyme-linked immunosorbent assay as described previously.
      • Subramaniam K.S.
      • Datta K.
      • Marks M.S.
      • Pirofski L.A.
      Improved survival of mice deficient in secretory immunoglobulin M following systemic infection with Cryptococcus neoformans.
      EIA/RIA 96-well plates (Costar, Corning, NY) were coated with 10 μg/mL of goat anti-mouse IgM or IgG (SouthernBiotech, Birmingham, AL) or 2 μg/mL rat anti-mouse IgE (BD Biosciences) for 1 hour at 37°C. Plates were then blocked overnight with 1% bovine serum albumin–PBS (Sigma-Aldrich, St. Louis, MO) and washed with PBS-Tween 20 using an Aquamax 2000 plate washer (Molecular Devices, Bethesda, MD) before use. Serum was added at a dilution of 1:5 for IgE, 1:15 for total IgM, and 1:1000 for total IgG and IgG isotypes, and serially diluted 1:3 with 1% bovine serum albumin–PBS. Mouse IgM and IgG (SouthernBiotech) and IgE (BD Biosciences) standards were applied at a starting concentration of 10 μg/mL for IgM and IgG and 1 μg/mL for IgE. Samples and standards were incubated for 1 hour at 37°C, plates were washed, and then antibody detected with a 1:2500 dilution of goat anti-mouse IgM, IgG, IgG1, IgG2b, IgG2c, or IgG3 or 1:1000 dilution of goat anti-mouse IgE for 1 hour at 37°C. Plates were subsequently developed with 1 mg/mL p-nitrophenyl phosphate (Sigma-Aldrich) dissolved in bicarbonate buffer (pH 9.8). A titration curve was generated by curve-fit analysis using GraphPad Prism 5 software (San Diego, CA). The titer for IgG subtypes was defined as the point at which the titration curve crossed an optical density of 0.1 after subtraction of the background.

      Determination of GXM-Specific IgM and IgG

      Plates were coated with 10 μg/mL C. neoformans 24067 GXM for 3 hours at room temperature and processed as described.
      • Subramaniam K.S.
      • Datta K.
      • Marks M.S.
      • Pirofski L.A.
      Improved survival of mice deficient in secretory immunoglobulin M following systemic infection with Cryptococcus neoformans.
      Briefly, serum samples were added at starting dilution of 1:5 and serially diluted. GXM-specific antibody was detected by addition of goat anti-mouse IgM or IgG, plates were developed, and the titer determined as described above.

      Histology

      At 7, 42, and 123 days after infection, groups of three to four mice were anesthetized with isoflurane, sacrificed by cervical dislocation, and lungs and brains removed. Tissues were fixed in 10% neutral buffered formalin (Fisher Scientific, Pittsburgh, PA). Following 48 to 72 hours of fixation, samples were sent to the Histopathology Core of AECOM for routine processing into paraffin blocks. Five-micrometer lung and brain tissue sections were routinely stained with H&E and examined under a Zeiss AxioScope II microscope (Carl Zeiss, Thornwood, NY) by a board-certified veterinary pathologist (R.S.S.).

      Statistical Analysis

      Mouse survival data were evaluated by comparing Kaplan-Meier survival curves with a log-rank (Mantel-Cox) test. For experiments in which only WT and IL-23p19−/− mice were compared, a Student's t-test was applied. For multiple comparisons between groups of WT, IL-23p19−/−, and IL-17RA−/− mice, data that exhibited equal variance and equal normality were analyzed for significance by one-way analysis of variance followed by Holm-Sidak post-test. For data that did not exhibit equal variance and normality, a Kruskal-Wallis rank-based one-way analysis of variance was performed followed by Dunn's post-test. A P value of <0.05 was considered significant. All statistical tests were performed using the advisory statistical software, SigmaStat 3.0 (SyStat, San Jose, CA).

      Results

      Tissue Fungal Burden in IL-23p19−/− and IL-17RA−/− Mice

      Previous studies have demonstrated impaired fungal clearance and reduced survival of IL-23p19−/− mice infected intraperitoneally or intravenously with C. neoformans, which was associated with reduced production of IL-17.
      • Kleinschek M.A.
      • Muller U.
      • Brodie S.J.
      • Stenzel W.
      • Kohler G.
      • Blumenschein W.M.
      • Straubinger R.K.
      • McClanahan T.
      • Kastelein R.A.
      • Alber G.
      IL-23 enhances the inflammatory cell response in Cryptococcus neoformans infection and induces a cytokine pattern distinct from IL-12.
      We sought to expand on these studies by determining whether IL-23 or IL-17 also contributes to control of pulmonary burden and/or yeast dissemination to the brain in an intranasal infection model of chronic cryptococcosis.
      • Arora S.
      • Hernandez Y.
      • Erb-Downward J.R.
      • McDonald R.A.
      • Toews G.B.
      • Huffnagle G.B.
      Role of IFN-gamma in regulating T2 immunity and the development of alternatively activated macrophages during allergic bronchopulmonary mycosis.
      IL-23p19−/−, IL-17RA−/−, and C57BL/6 WT mice were infected intranasally with 5 × 105 CFU of C. neoformans strain 52D, and the fungal burden in lungs, brain, and blood evaluated at days 7 and 42 after infection. Fungal burden in lungs of IL-23p19−/− or IL-17RA−/− mice were similar (P > 0.05) to WT at both time points examined (Figure 1A). No yeast was cultured (<50 CFU/mL) from the brain at day 7 or in blood at day 7 or day 42 from any WT, IL-23p19−/−, or IL-17RA−/− mice (n = 9 to 15). At day 42 after infection, the mean brain fungal burden was statistically comparable (P > 0.05) in all groups of mice (Figure 1A).
      Figure thumbnail gr1
      Figure 1IL-23 and IL-17RA do not significantly contribute to reduction in C. neoformans CFU, but survival of IL-23p19−/− mice is decreased. Fungal burden in lungs and brains (as labeled) was determined at the indicated time points after infection (A), (n = 9 to 20). Error bars represent the SEM from two to four independent experiments. Survival of IL-23p19−/− and C57BL/6 WT controls (B). Results are from two independent experiments, n = 6 (WT and IL-23p19−/−), n = 6 (WT, IL-23p19−/−, and IL-17RA−/−).
      We evaluated the survival of IL-23p19−/−, IL-17RA−/−, and WT mice. Although C57BL/6 mice can initially contain infection caused by strain 52D, the TH2-dominated allergic response to the fungus promotes yeast dissemination and death.
      • Arora S.
      • Olszewski M.A.
      • Tsang T.M.
      • McDonald R.A.
      • Toews G.B.
      • Huffnagle G.B.
      Effect of cytokine interplay on macrophage polarization during chronic pulmonary infection with Cryptococcus neoformans.
      • Huffnagle G.B.
      • Boyd M.B.
      • Street N.E.
      • Lipscomb M.F.
      IL-5 is required for eosinophil recruitment, crystal deposition, and mononuclear cell recruitment during a pulmonary Cryptococcus neoformans infection in genetically susceptible mice (C57BL/6).
      Survival 13 weeks post-infection was only 23% in IL-23p19−/− mice, whereas 83% of IL-17RA−/− and 75% of WT mice were alive at week 13 (Figure 1B). The decreased survival of IL-23p19−/− mice was significant (P < 0.05) compared to IL-17RA−/− mice, and there was a trend toward significance (P < 0.1) compared to WT (Figure 1B).

      Measurement of Lung Cytokines

      IL-17 is produced in response to IL-23,
      • Kleinschek M.A.
      • Muller U.
      • Schutze N.
      • Sabat R.
      • Straubinger R.K.
      • Blumenschein W.M.
      • McClanahan T.
      • Kastelein R.A.
      • Alber G.
      Administration of IL-23 engages innate and adaptive immune mechanisms during fungal infection.

      Kagami S, Rizzo HL, Kurtz SE, Miller LS, Blauvelt A: IL-23 and IL-17A, but not IL-12 and IL-22, are required for optimal skin host defense against Candida albicans. J Immunol 185:5453–5462

      • Meeks K.D.
      • Sieve A.N.
      • Kolls J.K.
      • Ghilardi N.
      • Berg R.E.
      IL-23 is required for protection against systemic infection with Listeria monocytogenes.
      as well as other stimuli.
      • Doisne J.M.
      • Soulard V.
      • Becourt C.
      • Amniai L.
      • Henrot P.
      • Havenar-Daughton C.
      • Blanchet C.
      • Zitvogel L.
      • Ryffel B.
      • Cavaillon J.M.
      • Marie J.C.
      • Couillin I.
      • Benlagha K.
      Cutting edge: crucial role of IL-1 and IL-23 in the innate IL-17 response of peripheral lymph node NK1.1- invariant NKT cells to bacteria.
      • Kimura A.
      • Naka T.
      • Kishimoto T.
      IL-6-dependent and -independent pathways in the development of interleukin 17-producing T helper cells.
      • Korn T.
      • Bettelli E.
      • Gao W.
      • Awasthi A.
      • Jager A.
      • Strom T.B.
      • Oukka M.
      • Kuchroo V.K.
      IL-21 initiates an alternative pathway to induce proinflammatory T(H)17 cells.
      • Yabu M.
      • Shime H.
      • Hara H.
      • Saito T.
      • Matsumoto M.
      • Seya T.
      • Akazawa T.
      • Inoue N.
      IL-23-dependent and -independent enhancement pathways of IL-17A production by lactic acid.
      • Torchinsky M.B.
      • Garaude J.
      • Martin A.P.
      • Blander J.M.
      Innate immune recognition of infected apoptotic cells directs T(H)17 cell differentiation.
      We asked whether IL-23 was required for IL-17 production during C. neoformans infection. IL-17 levels were increased in both WT (P < 0.001) and IL-23p19−/− (P < 0.01) lungs at day 7 after infection compared to naive WT and IL-23p19−/− mice, respectively, but IL-17 was lower (P < 0.01) in IL-23p19−/− and IL-17RA−/− lungs at day 7 compared to WT (Figure 2). At day 42, there was no difference (P > 0.05) in IL-17 levels between WT and IL-23p19−/− lungs (Figure 2). We also examined other cytokines that can be regulated by IL-23/IL-17 and/or influence cryptococcal containment,
      • Chen G.H.
      • McNamara D.A.
      • Hernandez Y.
      • Huffnagle G.B.
      • Toews G.B.
      • Olszewski M.A.
      Inheritance of immune polarization patterns is linked to resistance versus susceptibility to Cryptococcus neoformans in a mouse model.
      • Kawakami K.
      • Tohyama M.
      • Xie Q.
      • Saito A.
      IL-12 protects mice against pulmonary and disseminated infection caused by Cryptococcus neoformans.
      • Song C.
      • Luo L.
      • Lei Z.
      • Li B.
      • Liang Z.
      • Liu G.
      • Li D.
      • Zhang G.
      • Huang B.
      • Feng Z.H.
      IL-17-producing alveolar macrophages mediate allergic lung inflammation related to asthma.
      • Fossiez F.
      • Djossou O.
      • Chomarat P.
      • Flores-Romo L.
      • Ait-Yahia S.
      • Maat C.
      • Pin J.J.
      • Garrone P.
      • Garcia E.
      • Saeland S.
      • Blanchard D.
      • Gaillard C.
      • Das Mahapatra B.
      • Rouvier E.
      • Golstein P.
      • Banchereau J.
      • Lebecque S.
      T cell interleukin-17 induces stromal cells to produce proinflammatory and hematopoietic cytokines.
      • Huffnagle G.B.
      • Boyd M.B.
      • Street N.E.
      • Lipscomb M.F.
      IL-5 is required for eosinophil recruitment, crystal deposition, and mononuclear cell recruitment during a pulmonary Cryptococcus neoformans infection in genetically susceptible mice (C57BL/6).
      • Arora S.
      • Hernandez Y.
      • Erb-Downward J.R.
      • McDonald R.A.
      • Toews G.B.
      • Huffnagle G.B.
      Role of IFN-gamma in regulating T2 immunity and the development of alternatively activated macrophages during allergic bronchopulmonary mycosis.
      • Scurlock A.M.
      • Frazer L.C.
      • Andrews Jr., C.W.
      • O'Connell C.M.
      • Foote I.P.
      • Bailey S.L.
      • Chandra-Kuntal K.
      • Kolls J.K.
      • Darville T.
      Interleukin-17 contributes to generation of Th1 immunity and neutrophil recruitment during Chlamydia muridarum genital tract infection but is not required for macrophage influx or normal resolution of infection.
      but we did not find differences (P > 0.05) in levels of IL-4, IL-5, IL-13, IL-12, IFN-γ, IL-10, TGF-β, or IL-6 (Figure 2).
      Figure thumbnail gr2
      Figure 2The absence of IL-23 or IL-17RA results in decreased IL-17 levels in lungs early after infection but has no influence on production of other cytokines. Lung homogenates were used to quantitate cytokine production in naive mice or 7 days (D7) and 42 days (D42) after infection (n = 3 to 8). **P < 0.001, ***P < 0.001 versus WT D7; ††P < 0.01 versus IL-23p19−/− naive; †††P < 0.001 versus WT naive mice. Error bars represent the SEM of one to two experiments.
      To identify leukocytes that were producing IL-17 in WT and IL-23p19−/− mice, we performed intracellular staining for IL-17a followed by flow cytometry. We found that >95% of the IL-17a–producing leukocytes in the lungs of WT and IL-23p19−/− mice were CD3+ (Figure 3, A and B), whereas no CD11b+ cells were IL-17a+ (Figure 3A). Further analysis revealed that the IL-17a+ cells were primarily composed of CD3+CD4+ T cells and CD3+CD49b+ NKT cells (Figure 3, A and B). The percentage of CD45+ leukocytes producing IL-17a was similar (P > 0.05) between WT and IL-23p19−/− mice (Figure 3C), as well as the absolute number (mean × 104 ± SEM, n = 5) of IL-17a+ leukocytes (WT: 3.00 ± 0.22, IL-23p19−/−: 3.28 ± 0.22), but the median fluorescent intensity of the IL-17a+ cells was decreased (P < 0.01) in IL-23p19−/− compared to WT (Figure 3D).
      Figure thumbnail gr3
      Figure 3CD4T and NKT cells in lungs of IL-23p19−/− mice produce decreased amounts of IL-17 compared to WT mice. At day 7 post-infection, lung leukocytes were isolated from WT (top panels) and IL-23p19−/− (bottom panels) mice, and intracellular production of IL-17a was assessed by flow cytometry (A). Graphs represent the percentage of CD45+IL-17a+ cells that were CD3+, CD3+CD4+ T cells, or CD3+CD49b+ NKT cells (B); percentage of CD45+ lung leukocytes producing IL-17a (C); and median fluorescent intensity (MFI) of CD3+IL-17a+ cells (D), (n = 5). *P < 0.05 versus WT.

      Cellular Inflammation in Lungs

      IL-23–mediated IL-17 production enhances neutrophil and monocyte recruitment to sites of inflammation,
      • Aggarwal S.
      • Ghilardi N.
      • Xie M.H.
      • de Sauvage F.J.
      • Gurney A.L.
      Interleukin-23 promotes a distinct CD4 T cell activation state characterized by the production of interleukin-17.
      • Ye P.
      • Rodriguez F.H.
      • Kanaly S.
      • Stocking K.L.
      • Schurr J.
      • Schwarzenberger P.
      • Oliver P.
      • Huang W.
      • Zhang P.
      • Zhang J.
      • Shellito J.E.
      • Bagby G.J.
      • Nelson S.
      • Charrier K.
      • Peschon J.J.
      • Kolls J.K.
      Requirement of interleukin 17 receptor signaling for lung CXC chemokine and granulocyte colony-stimulating factor expression, neutrophil recruitment, and host defense.

      Roussel L, Houle F, Chan C, Yao Y, Berube J, Olivenstein R, Martin JG, Huot J, Hamid Q, Ferri L, Rousseau S: IL-17 promotes p38 MAPK-dependent endothelial activation enhancing neutrophil recruitment to sites of inflammation. J Immunol 184:4531–4537

      • Shahrara S.
      • Pickens S.R.
      • Mandelin 2nd, A.M.
      • Karpus W.J.
      • Huang Q.
      • Kolls J.K.
      • Pope R.M.
      IL-17-mediated monocyte migration occurs partially through CC chemokine ligand 2/monocyte chemoattractant protein-1 induction.
      therefore, we assessed lung leukocyte phenotype in IL-23p19−/−, IL-17RA−/−, and WT mice before and after 7 and 42 days of infection by flow cytometry. The number of CD45+ lung leukocytes was similar (P > 0.05) between all strains at all time points examined (Table 1). However, there were differences in the proportions of leukocyte populations. Naive IL-17RA−/− and IL-23p19−/− mice exhibited an increase (P < 0.001, P < 0.01, respectively) in CD3+CD8+ T cells in lungs compared to WT (Figure 4). There were also more (P < 0.001) CD3+CD49b+ NKT cells in naive IL-17RA−/− lungs compared to WT (Figure 4). At day 7 after infection, SiglecF+CD11cSSChi eosinophils were increased (P < 0.001) in lungs of IL-23p19−/− mice compared to WT, whereas IL-17RA−/− lungs contained more (P < 0.001) CD3+CD4+ T cells and NKT cells (Figure 4). At day 42, eosinophils remained increased (P < 0.001) in IL-23p19−/− lungs, and CD11b+CD11cF4/80+ macrophages were decreased (P < 0.001) compared to WT. Ly6GhiCD11b+ neutrophils were diminished in both IL-23p19−/− (P < 0.05) and IL-17RA−/− lungs (P < 0.01) compared to WT at day 42 (Figure 4). There was no difference (P > 0.05) in numbers of CD11chiCD11blo alveolar macrophages, CD11c+CD11b+ dendritic cells, CD11cCD11b+Ly6Chi monocytes, CD49b+CD3 NK cells, or CD19+B220hi B cells between the strains at any time point (data not shown).
      Table 1Lung CD45+ Leukocytes
      Day 0Day 7Day 42
      WT1.53 ± 0.05
      Mean number of CD45+ leukocytes ± SEM × 105, (n = 4 to 9).
      3.09 ± 0.065.46 ± 0.12
      IL-23p19−/−1.61 ± 0.403.04 ± 0.105.62 ± 0.20
      IL-17RA−/−1.41 ± 0.353.93 ± 0.255.85 ± 0.51
      low asterisk Mean number of CD45+ leukocytes ± SEM × 105, (n = 4 to 9).
      Figure thumbnail gr4
      Figure 4Eosinophils are increased in lungs of infected IL-23p19−/− mice, whereas CD4 T and NKT cells are increased in lungs of infected IL-17RA−/− mice. Lung cells were analyzed by flow cytometry to determine the relative percentages of the indicated leukocyte populations after gating on CD45+ cells in WT, IL-23p19−/−, and IL-17RA−/− lungs before and during infection (n = 4 to 9). *P < 0.05, **P < 0.01, and ***P < 0.001versus WT. Data represent the mean ± SEM of one to two experiments.

      Antibody Response

      Lung eosinophil influx is enhanced by IgE,
      • Coyle A.J.
      • Wagner K.
      • Bertrand C.
      • Tsuyuki S.
      • Bews J.
      • Heusser C.
      Central role of immunoglobulin (Ig) E in the induction of lung eosinophil infiltration and T helper 2 cell cytokine production: inhibition by a non-anaphylactogenic anti-IgE antibody.
      and both eosinophil recruitment and IgE production are associated with cryptococcal disease susceptibility.
      • Chen G.H.
      • McNamara D.A.
      • Hernandez Y.
      • Huffnagle G.B.
      • Toews G.B.
      • Olszewski M.A.
      Inheritance of immune polarization patterns is linked to resistance versus susceptibility to Cryptococcus neoformans in a mouse model.
      • Jain A.V.
      • Zhang Y.
      • Fields W.B.
      • McNamara D.A.
      • Choe M.Y.
      • Chen G.H.
      • Erb-Downward J.
      • Osterholzer J.J.
      • Toews G.B.
      • Huffnagle G.B.
      • Olszewski M.A.
      Th2 but not Th1 immune bias results in altered lung functions in a murine model of pulmonary Cryptococcus neoformans infection.
      We found that serum IgE was significantly increased (P < 0.05) in naive IL-17RA−/− mice (Figure 5A) and at day 7 in IL-23p19−/− and IL-17RA−/− mice compared to WT, but at day 42, there was no difference (P > 0.05) between the strains (P > 0.05) (Figure 5).
      Figure thumbnail gr5
      Figure 5IgG and IgE is elevated in serum of IL-23p19−/− and IL-17RA−/− mice. Concentration of serum IgE, IgM, and IgG at the indicated time points (A), titers of serum IgG isotypes 7 days after infection (B), and titers of serum cryptococcal GXM-specific IgM and IgG 7 days after infection (C), (n = 5 to 10). *P < 0.05, **P < 0.01, and ***P < 0.001 versus WT. Data represent the mean ± SEM from one to two experiments.
      In contrast to IgE, secretion of IgM and antigen-specific IgG can contribute to resistance to cryptococcosis.
      • Feldmesser M.
      • Mednick A.
      • Casadevall A.
      Antibody-mediated protection in murine Cryptococcus neoformans infection is associated with pleotrophic effects on cytokine and leukocyte responses.

      Subramaniam KS, Datta K, Quintero E, Manix C, Marks MS, Pirofski LA: The absence of serum IgM enhances the susceptibility of mice to pulmonary challenge with Cryptococcus neoformans. J Immunol 184:5755–5767

      • Yuan R.R.
      • Casadevall A.
      • Oh J.
      • Scharff M.D.
      T cells cooperate with passive antibody to modify Cryptococcus neoformans infection in mice.
      • Shapiro S.
      • Beenhouwer D.O.
      • Feldmesser M.
      • Taborda C.
      • Carroll M.C.
      • Casadevall A.
      • Scharff M.D.
      Immunoglobulin G monoclonal antibodies to Cryptococcus neoformans protect mice deficient in complement component C3.
      Total serum IgM concentration did not differ (P > 0.05) between IL-23p19−/−, IL-17RA−/−, and WT mice, but total serum IgG was significantly elevated in naive IL-17RA−/− and in both IL-23p19−/− and IL-17RA−/− mice compared to WT at day 7 and day 42 after infection (Figure 5A). We determined the titer of specific IgG isotypes and found that serum titers of IgG2b were increased (P < 0.001) in IL-23p19−/− and IL-17RA−/− mice at day 7 compared to WT, whereas differences in IgG1, IgG2c, and IgG3 were not significant (P > 0.05), (Figure 5B). Despite the increase in total serum IgG in both IL-23p19−/− and IL-17RA−/− mice at day 7, titers of anti-cryptococcal GXM were only significantly elevated (P < 0.01) in IL-17RA−/− mice (Figure 5C).

      Histopathology of Lungs and Brains

      Eosinophil recruitment to the lungs of C57BL/6 mice infected with C. neoformans results in deposition of Charcot-Leyden–like crystals, resulting in tissue damage.
      • Huffnagle G.B.
      • Boyd M.B.
      • Street N.E.
      • Lipscomb M.F.
      IL-5 is required for eosinophil recruitment, crystal deposition, and mononuclear cell recruitment during a pulmonary Cryptococcus neoformans infection in genetically susceptible mice (C57BL/6).
      These crystals are composed of the chitinases YM1 and YM2 that are produced by alternatively activated macrophages; therefore, it is hypothesized that eosinophils induce crystal formation by influencing macrophage activation.
      • Arora S.
      • Hernandez Y.
      • Erb-Downward J.R.
      • McDonald R.A.
      • Toews G.B.
      • Huffnagle G.B.
      Role of IFN-gamma in regulating T2 immunity and the development of alternatively activated macrophages during allergic bronchopulmonary mycosis.
      • Guo L.
      • Johnson R.S.
      • Schuh J.C.
      Biochemical characterization of endogenously formed eosinophilic crystals in the lungs of mice.
      • Liu Q.
      • Cheng L.I.
      • Yi L.
      • Zhu N.
      • Wood A.
      • Changpriroa C.M.
      • Ward J.M.
      • Jackson S.H.
      p47phox deficiency induces macrophage dysfunction resulting in progressive crystalline macrophage pneumonia.
      Lung sections from C. neoformans–infected mice were examined by H&E staining to determine whether the increased eosinophilia in IL-23p19−/− mice was associated with amplified lung pathology. On day 7 after infection, there was no significant difference in lung pathology between IL-23p19−/−, IL-17RA−/−, and WT mice (data not shown). However, at day 42, there were increased amounts of acidophilic YM1/YM2 crystals in lungs of both IL-23p19−/− and IL-17RA−/− mice (Figure 6, Table 2). The inflammation was primarily eosinophilic and neutrophilic except in the WT mice, which had notably more neutrophilic inflammation. In the WT mice, the neutrophils were generally associated with focal tissue destruction (Table 2). We also examined brain sections at day 42 after infection, but there were no notable differences in cryptococcal burden or inflammatory infiltrate (Table 2).
      Figure thumbnail gr6
      Figure 6IL-23p19−/− and IL-17RA−/− mice exhibit increased YM1/YM2 crystal formation in lungs during chronic infection compared to WT. Representative H&E-stained lung sections from WT (A and B), IL-23p19−/− (C and D), and IL-17RA−/− (E and F) 42 days after infection (n = 3 to 4). Large arrowheads designate YM1/YM2 crystals, and small arrows indicate eosinophils. Original magnification: ×10 (A, C, and E); ×40 (B, D, and F).
      Table 2Histopathology of Lungs and Brains 42 Days after Infection
      WTIL-23p19−/−IL-17RA−/−
      Brain
       Mouse number12341231234
       Cryptococcal nodules
      Severity modifiers: 0 = no lesion, 1 = minimal lesion, 2 = mild lesion, 3 = moderate lesion, 4 = marked lesion, 5 = severe lesion.
      301–21–21121112
        With pyogranulomatous inflammation
      Severity modifiers: 0 = no lesion, 1 = minimal lesion, 2 = mild lesion, 3 = moderate lesion, 4 = marked lesion, 5 = severe lesion.
      20000000002
       Infiltrate, perivascular, chronic
      Severity modifiers: 0 = no lesion, 1 = minimal lesion, 2 = mild lesion, 3 = moderate lesion, 4 = marked lesion, 5 = severe lesion.
      10010011100
      Lung
       Mouse number12341231234
       Granulomatous pneumonia
      Granulomatous pneumonia refers to dense pyogranulomatous and eosinophilic inflammation.
      (# lobes/5)
        Minimal00000000011
        Mild00010002244
        Moderate13133112200
        Marked12412231100
        Severe30000210000
       Acidophilic crystalline material
      Pneumonia severity modifiers: 1 = 1% to 10%, 2 = 10% to 25%, 3 = 25% to 45%, 4 = 45% to 75%, 5 = >75%.
      12123444442
       Neutrophils in inflammation
      Pneumonia severity modifiers: 1 = 1% to 10%, 2 = 10% to 25%, 3 = 25% to 45%, 4 = 45% to 75%, 5 = >75%.
      33321111111
      low asterisk Severity modifiers: 0 = no lesion, 1 = minimal lesion, 2 = mild lesion, 3 = moderate lesion, 4 = marked lesion, 5 = severe lesion.
      Granulomatous pneumonia refers to dense pyogranulomatous and eosinophilic inflammation.
      Pneumonia severity modifiers: 1 = 1% to 10%, 2 = 10% to 25%, 3 = 25% to 45%, 4 = 45% to 75%, 5 = >75%.
      After 17 weeks of infection (day 123), four of five surviving WT mice displayed little granulomatous inflammation in lungs (Figure 7, A–D, Table 3), and one WT mouse exhibited minimal to mild acidophilic macrophage pneumonia with YM1/YM2 crystals (Table 3). Cryptococci were not apparent in lungs of any WT mice (Table 3). By contrast, all IL-17RA−/− mice (n = 4), exhibited acidophilic macrophage pneumonia in lungs, ranging from mild to severe, with cryptococci. Cryptococcal foci contained mild numbers of eosinophils and aggregates of fragmented neutrophils that were associated with necrosis, presumably resulting from tissue damage from pointed YM1/YM2 crystals (Figure 7, A–D, Table 3).
      Figure thumbnail gr7
      Figure 7Surviving IL-17RA−/− mice display acidophilic macrophage pneumonia and fungal dissemination to the blood after 17 weeks of infection. Representative H&E-stained lung sections from WT (A and B) and IL-17RA−/− mice (C and D), (n = 4 to 5). Fungal burden (Log10 CFU/mL) in blood of WT and IL-17RA−/− mice (E), (n = 4 to 5). Original magnification: ×10 (A and C); ×40 (B and D). KO, knockout.
      Table 3Histopathology of Lungs and Brains of Surviving WT and IL-17RA−/− Mice 123 Days after Infection
      WTIL-17RA−/−
      Brain
       Mouse number123451234
       Cryptococcal nodules
      Severity modifiers: 0 = no lesion, 1 = minimal lesion, 2 = mild lesion, 3 = moderate lesion, 4 = marked lesion, 5 = severe lesion.
      000000003
       Granulomatous inflammation
      Severity modifiers: 0 = no lesion, 1 = minimal lesion, 2 = mild lesion, 3 = moderate lesion, 4 = marked lesion, 5 = severe lesion.
      000200202
       Infiltrate, perivascular, chronic
      Severity modifiers: 0 = no lesion, 1 = minimal lesion, 2 = mild lesion, 3 = moderate lesion, 4 = marked lesion, 5 = severe lesion.
      000000102
      Lung
       Mouse number123451234
       Granulomatous pneumonia
      Granulomatous pneumonia refers to dense pyogranulomatous and eosinophilic inflammation.
      (# lobes/5)
        No lesions405034543
        Minimal150321010
        Mild000200002
       Acidophilic
       Macrophage pneumonia
      Acidophilic macrophage pneumonia refers to dense inflammation composed of macrophages with acidophilic YM1/YM2 crystals.
        No lesions010002242
        Minimal020000011
        Mild020000000
        Moderate000000302
        Marked000001000
        Severe000002000
      low asterisk Severity modifiers: 0 = no lesion, 1 = minimal lesion, 2 = mild lesion, 3 = moderate lesion, 4 = marked lesion, 5 = severe lesion.
      Granulomatous pneumonia refers to dense pyogranulomatous and eosinophilic inflammation.
      Acidophilic macrophage pneumonia refers to dense inflammation composed of macrophages with acidophilic YM1/YM2 crystals.
      The brains of WT mice at week 17 had focal areas of chronic inflammation with no typical cryptococcal organisms (Table 3). Two of four IL-17RA−/− mice had similar inflammatory infiltrates, with one containing aggregates of viable-appearing organisms. Cryptococci were cultured from the blood of IL-17RA−/− mice at week 17, but were below the detection limit (<50 CFU/mL) in WT (Figure 7E).

      Discussion

      Our findings reveal IL-17–independent and -dependent roles of IL-23 in limiting the allergic response during chronic pulmonary cryptococcal disease. Although IL-23 was not required for induction of IL-17, it enhanced IL-17 production by NKT and CD4T cells during early cryptococcal infection. Both IL-23 and IL-17 suppressed serum IgE, IgG2b, and formation of pulmonary YM1/YM2 crystals, suggesting that IL-23–dependent production of IL-17 contributes to control of cryptococcal-induced allergy. However, an IL-17–independent function of IL-23 was also evident, since IL-23p19−/− mice had increased numbers of eosinophils in the lung at all time points after cryptococcal infection. This was in sharp contrast to IL-17RA−/− and WT mice, which did not exhibit these findings. Based on these data, pulmonary eosinophilia might promote mortality, because IL-23p19 null mice had higher mortality than IL-17 mutant mice, despite having a similar cryptococcal burden in lung and brains.
      Cryptococcal infection in C57BL/6 mice results in a TH2-dominated response that is accompanied by allergic markers that are similar to those observed in humans with allergic bronchopulmonary mycosis.
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      Although our data indicates that IL-23 dampens the allergic bronchopulmonary mycosis phenotype of C. neoformans–infected mice, IL-23 enhanced allergic lung inflammation and eosinophilia in experimental models of ovalbumin-induced asthma in C57BL/6 × 129 and BALB/c mice.
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      The contrast between the roles of IL-23 in cryptococcal versus ovalbumin-induced allergy suggests that cryptococcal antigens drive an allergic response that is unique from that of ovalbumin-induced allergy and in which IL-23 is immunoregulatory. In support of this hypothesis, recent work has demonstrated that the mechanisms of eosinophil recruitment induced by fungal chitin and ovalbumin are distinct.
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      Fungal chitin from asthma-associated home environments induces eosinophilic lung infiltration.
      Furthermore, an immunoregulatory role for IL-23 and IL-17a has been indicated in chronic asthma induced by Aspergillus fumigatus or house dust mite antigen. Lung inflammation is exacerbated in asthmatic TLR6 null mice as a result of decreased IL-23 production, and IL-17a inhibits goblet cell metaplasia in asthmatic C57BL/6 mice.
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      The protective role of TLR6 in a mouse model of asthma is mediated by IL-23 and IL-17A.
      Taken together, these studies indicate that the role of IL-23 and IL-17 signaling in the lung is complex, and likely dependent on many immune factors that are modulated by exogenous antigens.
      Eosinophils are held to be deleterious in the mouse model of cryptococcosis. Early studies demonstrated that IL-5–dependent eosinophil recruitment results in crystal formation in macrophages and lung damage.
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      • Lipscomb M.F.
      IL-5 is required for eosinophil recruitment, crystal deposition, and mononuclear cell recruitment during a pulmonary Cryptococcus neoformans infection in genetically susceptible mice (C57BL/6).
      More recent work, using mice that lack eosinophils, confirm these findings and demonstrate decreased fungal burden in the absence of eosinophils.
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      Eosinophils contribute to IL-4 production and shape the T-helper cytokine profile and inflammatory response in pulmonary cryptococcosis.
      These findings contrast with those in rat models of cryptococcosis, in which primary infection can resolve and eosinophils contribute to a protective TH1 response.
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      Rat eosinophils stimulate the expansion of Cryptococcus neoformans-specific CD4(+) and CD8(+) T cells with a T-helper 1 profile.
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      Eosinophils elicit proliferation of naive and fungal-specific cells in vivo so enhancing a T helper type 1 cytokine profile in favour of a protective immune response against Cryptococcus neoformans infection.
      Our data support a destructive role for eosinophils, possibly by promoting tissue damage and subsequent mortality of IL-23p19−/− mice. Eosinophilic granule proteins are toxic to bystander cells, resulting in lung damage,
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      and contribute to airway constriction and impaired respiration.
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      Human eosinophil major basic protein induces airway constriction and airway hyperresponsiveness in primates.
      Additional deleterious functions of eosinophils were demonstrated in an Aspergillus-induced allergic asthma model, in which mice lacking eosinophils or CCR3, an eosinophil chemokine receptor, displayed reduced mucous production and decreased transcription of genes involved in coagulation.
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      Similar to our data, survival of IL-23p19−/− mice infected intraperitoneally or intravenously with C. neoformans strain 1841D is decreased.
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      IL-23 enhances the inflammatory cell response in Cryptococcus neoformans infection and induces a cytokine pattern distinct from IL-12.
      In the foregoing systemic infection model, the absence of IL-23 resulted in increased hepatic fungal burden and decreased infiltration of inflammatory cells into the brain, but not increased brain fungal burden.
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      We could not attribute the increased mortality of IL-23p19−/− mice infected intranasally with C. neoformans-52D to increased brain or lung fungal burden or differences in brain inflammation. However, we did find that their earlier time to death was associated with a higher number of lung eosinophils. One explanation for the increased mortality of IL-23p19−/− mice in our model is that in combination, lung eosinophils and YM1/YM2 crystals could impair respiration, whereas crystals without increased eosinophils do not impact pulmonary physiology in IL-17RA−/− mice. Although alveolar accumulation of needle-shaped YM1/YM2 crystals can induce mechanical damage, lung consolidation, insufficient respiration, and fatality without eosinophilia,
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      Pulmonary pathology of the motheaten mouse.
      comparable crystal formation in the lungs of IL-23p19−/− and IL-17RA−/− mice suggests that additional factors, such as lung-damaging eosinophils, could have contributed to respiratory demise and mortality in infected-IL-23p19−/− mice.
      Eosinophil recruitment to lungs can be mediated by CD4 T-cell production of TH2 cytokines.
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      IL-13 induces disease-promoting type 2 cytokines, alternatively activated macrophages and allergic inflammation during pulmonary infection of mice with Cryptococcus neoformans.
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      IL-5 is required for eosinophil recruitment, crystal deposition, and mononuclear cell recruitment during a pulmonary Cryptococcus neoformans infection in genetically susceptible mice (C57BL/6).
      • Hernandez Y.
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      Distinct roles for IL-4 and IL-10 in regulating T2 immunity during allergic bronchopulmonary mycosis.
      Although the increase in lung eosinophils in IL-23p19−/− mice was not accompanied by an increase in lung levels of IL-5, IL-4, or IL-13, it is possible that non–T-cell sources masked a decrease in CD4 T-cell production of one or more of these cytokines in whole-lung homogenates. At present, it is not known whether the cellular source and/or the location of TH2 cytokine production are important factors in eosinophil emigration into the lung during cryptococcal infection. Given that ligation of Ox40, a T-cell–activating receptor, on CD4 T cells limits eosinophil recruitment in an IFN-γ–dependent manner during cryptococcal infection, signaling via OX40 could be decreased in IL-23p19−/− mice.
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      OX40 ligation on activated T cells enhances the control of Cryptococcus neoformans and reduces pulmonary eosinophilia.
      Alternatively, eosinophil generation and release from bone marrow or migration from the circulation into the lung could be mediated by other factors. However, we found that transcription of eotaxin, an eosinophil chemoattractant that can promote eosinophil recruitment,
      • Ganzalo J.A.
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      Mouse eotaxin expression parallels eosinophil accumulation during lung allergic inflammation but it is not restricted to a Th2-type response.
      was not increased in lungs of IL-23p19−/− mice at day 7 or 21 after infection (data not shown). Furthermore, we note that the increase in eosinophils in lungs of IL-23p19−/− mice could reflect a defect in eosinophil clearance, rather than an increase in eosinophil recruitment. Though not specific to eosinophils, IL-23 was recently shown to inhibit cellular infiltration into the lungs in an A. fumigatus allergy model,
      • Moreira A.P.
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      • Uematsu S.
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      • Hogaboam C.M.
      The protective role of TLR6 in a mouse model of asthma is mediated by IL-23 and IL-17A.
      but the mechanism was not examined. Therefore, more work is needed to determine how IL-23 directly or indirectly inhibits eosinophil recruitment in fungal infections.
      IgE secretion during cryptococcal infection is associated with reduced lung function.
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      Th2 but not Th1 immune bias results in altered lung functions in a murine model of pulmonary Cryptococcus neoformans infection.
      It appears that IL-23 and IL-17RA restrain IgE production, since IgE was elevated in IL-23p19−/− and IL-17RA−/− mice during infection. Increased IgE levels also occurred in naive IL-17RA−/− mice. To our knowledge, elevated IgE has not been reported in naive IL-17RA−/− mice. To confirm that increased IgE was not a response to parasites,
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      Infection with Syphacia obvelata (pinworm) induces protective Th2 immune responses and influences ovalbumin-induced allergic reactions.
      the cecum and colon were evaluated histologically to confirm the lack of intestinal parasites, and sentinel mice were routinely verified to be parasite free. Interestingly, a link between IL-23/IL-17 and IgE regulation is also seen in humans. Individuals with hyper-IgE syndrome exhibit a genetic mutation in STAT3 that results in impaired IL-23 and IL-17 production; however, the cause of the elevated IgE is not known. These individuals also have increased susceptibility to infections by the fungi Candida and Aspergillus, as well as to various bacteria.
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      In addition to elevated IgE, the absence of IL-23 and IL-17RA resulted in increased serum IgG, primarily IgG2b. Elevated total serum IgG in naive IL-17RA−/− mice and IgG2b after Porphyromonas gingivalis has also been reported.
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      An essential role for IL-17 in preventing pathogen-initiated bone destruction: recruitment of neutrophils to inflamed bone requires IL-17 receptor-dependent signals.
      It is not clear from our studies whether the increase in IgG in IL-23p19−/− mice or IL-17RA−/− has any effect on immunity. Previous studies have demonstrated that GXM-specific IgG2b can have protective functions in mice infected with a lethal dose of C. neoformans,
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      but IgG2b has also been shown to be a weaker opsonin than IgG2a and IgG1,
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      Opsonization of Cryptococcus neoformans by a family of isotype-switch variant antibodies specific for the capsular polysaccharide.
      and is associated with decreased survival time when administered to mice before cryptococcal infection in comparison to other antibody isotypes.
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      Another possibility is that the elevated IgG to IgM ratio in IL-23p19−/− and IL-17RA−/− mice impacts binding of IgM, which is diluted by the excess IgG. Secreted IgM promotes phagocytosis of C. neoformans, inflammation, and granuloma formation that is associated with disease resistance.

      Subramaniam KS, Datta K, Quintero E, Manix C, Marks MS, Pirofski LA: The absence of serum IgM enhances the susceptibility of mice to pulmonary challenge with Cryptococcus neoformans. J Immunol 184:5755–5767

      Although the absence of IL-17RA did not impact mortality up to 17 weeks, our data suggest that IL-17 contributes to disease resistance by limiting dissemination of yeast to the blood late in infection. The presence of cryptococci in blood of IL-17RA−/− mice correlated with increased acidophilic YM1/YM2 crystal deposition in lungs, which progressed between weeks 6 and 17 after infection. A role for IL-17 late in infection is also demonstrated in a vaccination model in which C. neoformans-H99γ, a γ-interferon–producing H99 strain, elicits protection from subsequent infection of BALB/c mice with H99.
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      IL-17 is not required for survival, but vaccinated IL-17RA−/− mice exhibited dissemination to the brain after rechallenge, indicating protective functions of IL-17.
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      • Wormley Jr., F.L.
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      However, our findings contrast with this vaccination model, in which IL-17RA also contributed transiently to control of pulmonary burden.
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      • Wormley Jr., F.L.
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      We found that lung cryptococcal burden was similar between WT and IL-17RA−/− mice at day 7 and day 42 after infection, suggesting that there are compensatory pathways that can sufficiently control fungal burden during early infection of C57BL/6 mice with cryptococcal strain 52D. IL-17RA−/− lungs did contain elevated numbers of CD8T and NKT cells before infection and CD4T and NKT cells at day 7 after infection, and these cellular effectors are known to contribute to cryptococcal disease resistance.
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      Generation of antifungal effector CD8+ T cells in the absence of CD4+ T cells during Cryptococcus neoformans infection.
      Although our studies did not address a potential deleterious role of the IL-23/IL-17 axis, it is important to note that IL-17 is also associated with cryptococcal disease susceptibility. Increased production of IL-17 by alveolar macrophages is associated with accelerated mortality in mice challenged with a highly virulent mucoid strain of C. neoformans
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      Cryptococcus neoformans variants generated by phenotypic switching differ in virulence through effects on macrophage activation.
      or in mice lacking secretory IgM.

      Subramaniam KS, Datta K, Quintero E, Manix C, Marks MS, Pirofski LA: The absence of serum IgM enhances the susceptibility of mice to pulmonary challenge with Cryptococcus neoformans. J Immunol 184:5755–5767

      Also, increasing IL-17 levels are associated with mortality in HIV patients with immune reconstitution syndrome following cryptococcal meningitis.
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      Future studies are needed to understand whether yeast virulence factors, the timing of IL-17 production, the cellular sources of IL-17, or additional host factors influence the outcome of IL-17 signaling.
      The findings presented herein provide evidence that IL-23 and IL-17 may contribute to disease resistance by limiting the allergic response to cryptococcal infection. Since IL-23 has opposing effects in non-fungal allergy models,
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      • Yang X.O.
      • Chang S.H.
      • Yang J.
      • Dong C.
      IL-23 signaling enhances Th2 polarization and regulates allergic airway inflammation.
      • Wakashin H.
      • Hirose K.
      • Maezawa Y.
      • Kagami S.
      • Suto A.
      • Watanabe N.
      • Saito Y.
      • Hatano M.
      • Tokuhisa T.
      • Iwakura Y.
      • Puccetti P.
      • Iwamoto I.
      • Nakajima H.
      IL-23 and Th17 cells enhance Th2-cell-mediated eosinophilic airway inflammation in mice.
      it will be important to further examine IL-23 signaling in specific IL-23–responsive leukocyte populations and determine how cryptococcal antigen and host factors influence IL-23 activity.

      Acknowledgments

      We thank the AECOM Flow Cytometry Core Facility under the support of the AECOM National Cancer Institute ( P30CA013330 ) for assistance with data acquisition.

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