IL-23 Dampens the Allergic Response to Cryptococcus neoformans through IL-17–Independent and –Dependent Mechanisms
2012; Elsevier BV; Volume: 180; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2011.12.038
ISSN1525-2191
AutoresWendy Szymczak, Rani S. Sellers, Liise‐anne Pirofski,
Tópico(s)Nail Diseases and Treatments
ResumoThe 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. 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. 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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. 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. 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. 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. 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). 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 cou
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