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• W2040386323 abstract "Urease, a major virulence factor for Cryptococcus neoformans, promotes lethal meningitis/encephalitis in mice. The effect of urease within the lung, the primary site of most invasive fungal infections, is unknown. An established model of murine infection that utilizes either urease-producing (wt and ure1::URE1) or urease-deficient (ure1) strains (H99) of C. neoformans was used to characterize fungal clearance and the resultant immune response evoked by these strains within the lung. Results indicate that mice infected with urease-producing strains of C. neoformans demonstrate a 100-fold increase in fungal burden beginning 2 weeks post–infection (as compared with mice infected with urease-deficient organisms). Infection with urease-producing C. neoformans was associated with a highly polarized T2 immune response as evidenced by increases in the following: 1) pulmonary eosinophils, 2) serum IgE levels, 3) T2 cytokines (interleukin-4, -13, and -4 to interferon-gamma ratio), and 4) alternatively activated macrophages. Furthermore, the percentage and total numbers of immature dendritic cells within the lung-associated lymph nodes was markedly increased in mice infected with urease-producing C. neoformans. Collectively, these data define cryptococcal urease as a pulmonary virulence factor that promotes immature dendritic cell accumulation and a potent, yet non-protective, T2 immune response. These findings provide new insights into mechanisms by which microbial factors contribute to the immunopathology associated with invasive fungal disease. Urease, a major virulence factor for Cryptococcus neoformans, promotes lethal meningitis/encephalitis in mice. The effect of urease within the lung, the primary site of most invasive fungal infections, is unknown. An established model of murine infection that utilizes either urease-producing (wt and ure1::URE1) or urease-deficient (ure1) strains (H99) of C. neoformans was used to characterize fungal clearance and the resultant immune response evoked by these strains within the lung. Results indicate that mice infected with urease-producing strains of C. neoformans demonstrate a 100-fold increase in fungal burden beginning 2 weeks post–infection (as compared with mice infected with urease-deficient organisms). Infection with urease-producing C. neoformans was associated with a highly polarized T2 immune response as evidenced by increases in the following: 1) pulmonary eosinophils, 2) serum IgE levels, 3) T2 cytokines (interleukin-4, -13, and -4 to interferon-gamma ratio), and 4) alternatively activated macrophages. Furthermore, the percentage and total numbers of immature dendritic cells within the lung-associated lymph nodes was markedly increased in mice infected with urease-producing C. neoformans. Collectively, these data define cryptococcal urease as a pulmonary virulence factor that promotes immature dendritic cell accumulation and a potent, yet non-protective, T2 immune response. These findings provide new insights into mechanisms by which microbial factors contribute to the immunopathology associated with invasive fungal disease. Cryptococcus neoformans, an opportunistic fungal pathogen acquired through inhalation, causes severe pneumonia and subsequent central nervous system (CNS) infections, predominantly in immunocompromised individuals.1Berkefeld J Enzensberger W Lanfermann H Cryptococcus meningoencephalitis in AIDS: parenchymal and meningeal forms.Neuroradiology. 1999; 41: 129-133Crossref PubMed Scopus (37) Google Scholar, 2Hage CA Wood KL Winer-Muram HT Wilson SJ Sarosi G Knox KS Pulmonary cryptococcosis after initiation of anti-tumor necrosis factor-alpha therapy.Chest. 2003; 124: 2395-2397Crossref PubMed Scopus (94) Google Scholar, 3Kovacs JA Kovacs AA Polis M Wright WC Gill VJ Tuazon CU Gelmann EP Lane HC Longfield R Overturf G Masher AM Fauci AS Parrillo JE Bennett JE Masur H Cryptococcosis in the acquired immunodeficiency syndrome.Ann Intern Med. 1985; 103: 533-538Crossref PubMed Scopus (474) Google Scholar, 4Lee SC Dickson DW Casadevall A Pathology of cryptococcal meningoencephalitis: analysis of 27 patients with pathogenetic implications.Hum Pathol. 1996; 27: 839-847Abstract Full Text PDF PubMed Scopus (149) Google Scholar Recent reports of invasive cryptococcosis in noncompromised individuals indicate that the organism can evade or neutralize intact host defense mechanisms.5Hoang LMN Maguire JA Doyle P Fyfe M Roscoe DL Cryptococcus neoformans infections at Vancouver Hospital and Health Sciences Centre (1997–2002): epidemiology, microbiology and histopathology.J Med Microbiol. 2004; 53: 935-940Crossref PubMed Scopus (162) Google Scholar, 6Zahra LV Azzopardi CM Scott G Cryptococcal meningitis in two apparently immunocompetent Maltese patients.Mycoses. 2004; 47: 168-173Crossref PubMed Scopus (19) Google Scholar, 7Hofman V Venissac N Mouroux C Butori C Mouroux J Hofman P [Disseminated pulmonary infection due to Cryptococcus neoformans in a non immunocompromised patient].Ann Pathol. 2004; 24: 187-191Crossref PubMed Scopus (12) Google Scholar Numerous cryptococcal virulence factors have been defined that influence the pathogenesis of C. neoformans infection.8Blackstock R Buchanan KL Cherniak R Mitchell TG Wong B Bartiss A Jackson L Murphy JW Pathogenesis of Cryptococcus neoformans is associated with quantitative differences in multiple virulence factors.Mycopathologia. 1999; 147: 1-11Crossref PubMed Scopus (17) Google Scholar, 9Buchanan KL Murphy JW What makes Cryptococcus neoformans a pathogen?.Emerg Infect Dis. 1998; 4: 71-83Crossref PubMed Scopus (232) Google Scholar, 10Panepinto JC Williamson PR Intersection of fungal fitness and virulence in Cryptococcus neoformans.FEMS Yeast Res. 2006; 6: 489-498Crossref PubMed Scopus (31) Google Scholar Quantitative differences in the expression of these factors have been shown to alter the host's ability to clear the primary infection or prevent systemic dissemination.11Blackstock R Buchanan KL Adesina AM Murphy JW Differential regulation of immune responses by highly and weakly virulent Cryptococcus neoformans isolates.Infect Immun. 1999; 67: 3601-3609PubMed Google Scholar, 12Huffnagle GB Chen GH Curtis JL McDonald RA Strieter RM Toews GB Down-regulation of the afferent phase of T cell-mediated pulmonary inflammation and immunity by a high melanin-producing strain of Cryptococcus neoformans.J Immunol. 1995; 155: 3507-3516PubMed Google Scholar, 13Olszewski MA Huffnagle GB Traynor TR McDonald RA Cook DN Toews GB Regulatory effects of macrophage inflammatory protein 1alpha/CCL3 on the development of immunity to Cryptococcus neoformans depend on expression of early inflammatory cytokines.Infect Immun. 2001; 69: 6256-6263Crossref PubMed Scopus (43) Google Scholar, 14Kawakami K Tohyama M Xie Q Saito A IL-12 protects mice against pulmonary and disseminated infection caused by Cryptococcus neoformans.Clin Exp Immunol. 1996; 104: 208-214Crossref PubMed Scopus (130) Google Scholar, 15Kawakami K Qifeng X Tohyama M Qureshi MH Saito A Contribution of tumour necrosis factor-α (TNF-α) in host defence mechanism against Cryptococcus neoformans.Clin Exp Immunol. 1996; 106: 468-474Crossref PubMed Scopus (70) Google Scholar However, the specific cellular and molecular mechanisms by which these factors enhance virulence remain largely unknown. Understanding these mechanisms may aid the development of new strategies designed to prevent and/or treat invasive fungal disease in humans.The cryptococcal urease gene (URE1) is a significant virulence factor associated with increased mortality in C. neoformans-infected mice.16Cox GM Mukherjee J Cole GT Casadevall A Perfect JR Urease as a virulence factor in experimental cryptococcosis.Infect Immun. 2000; 68: 443-448Crossref PubMed Scopus (385) Google Scholar, 17Olszewski MA Noverr MC Chen GH Toews GB Cox GM Perfect JR Huffnagle GB Urease expression by Cryptococcus neoformans promotes microvascular sequestration, thereby enhancing central nervous system invasion.Am J Pathol. 2004; 164: 1761-1771Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar The mechanisms responsible for this effect are not fully understood. We have previously shown that urease promotes microvascular sequestration within the brain, resulting in increased CNS invasion with resultant meningoencephalitis and death.17Olszewski MA Noverr MC Chen GH Toews GB Cox GM Perfect JR Huffnagle GB Urease expression by Cryptococcus neoformans promotes microvascular sequestration, thereby enhancing central nervous system invasion.Am J Pathol. 2004; 164: 1761-1771Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar Although urease is not required for the acute growth of organism in the lungs,17Olszewski MA Noverr MC Chen GH Toews GB Cox GM Perfect JR Huffnagle GB Urease expression by Cryptococcus neoformans promotes microvascular sequestration, thereby enhancing central nervous system invasion.Am J Pathol. 2004; 164: 1761-1771Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar others have shown that chronic pulmonary infection, the primary portal of entry for C. neoformans,18Casadevall A Perfect JR Cryptococcus neoformans. ASM Press, Washington DC1998: 541Google Scholar is a prerequisite for cryptococcal dissemination into the CNS.19Manelis J Reichenthal E Merzbach D Haschman N Peyser E Cryptococcus neoformans meningitis. Report of a case and review of cryptococcosis in Israel.Confin Neurol. 1973; 35: 304-311Crossref PubMed Google Scholar, 20Poo QM Paul FM Cryptococcus neoformans meningitis in a school girl.J Singapore Paediatr Soc. 1977; 19: 17-23PubMed Google Scholar, 21Yalaburgi SB Mohapatra KC Cryptococcus neoformans meningitis: a case report.S Afr Med J. 1980; 57: 1011-1012PubMed Google Scholar Whether cryptococcal urease might also function as a virulence factor by contributing to the persistence of C. neoformans within the lung is unknown.The clearance of C. neoformans from the lung and the prevention of systemic dissemination is critically dependent on the development of an adaptive T1 immune response against the organism.22Huffnagle GB Lipscomb MF Lovchik JA Hoag KA Street NE The role of CD4+ and CD8+ T cells in the protective inflammatory response to a pulmonary cryptococcal infection.J Leukoc Biol. 1994; 55: 35-42PubMed Google Scholar, 23Kawakami K Koguchi Y Qureshi MH Yara S Kinjo Y Miyazato A Nishizawa A Nariuchi H Saito A Circulating soluble CD4 directly prevents host resistance and delayed-type hypersensitivity response to Cryptococcus neoformans in mice.Microbiol Immunol. 2000; 44: 1033-1041Crossref PubMed Google Scholar, 24Traynor TR Kuziel WA Toews GB Huffnagle GB CCR2 expression determines T1 versus T2 polarization during pulmonary Cryptococcus neoformans infection.J Immunol. 2000; 164: 2021-2027PubMed Google Scholar Both CD4+ and CD8+ T cells are required for this protective response.25Lindell DM Ballinger MN McDonald RA Toews GB Huffnagle GB Diversity of the T-cell response to pulmonary Cryptococcus neoformans infection.Infect Immun. 2006; 74: 4538-4548Crossref PubMed Scopus (22) Google Scholar In contrast, the development of a T2 response in the lungs is nonprotective and associated with persistence of C. neoformans.26Hernandez Y Arora S Erb-Downward JR McDonald RA Toews GB Huffnagle GB Distinct roles for IL-4 and IL-10 in regulating T2 immunity during allergic bronchopulmonary mycosis.J Immunol. 2005; 174: 1027-1036PubMed Google Scholar, 27Arora S Hernandez Y Erb-Downward JR McDonald RA Toews GB Huffnagle GB Role of IFN-gamma in regulating T2 immunity and the development of alternatively activated macrophages during allergic bronchopulmonary mycosis.J Immunol. 2005; 174: 6346-6356PubMed Google Scholar, 28Arora S McDonald RA Toews GB Huffnagle GB Effect of a CD4-depleting antibody on the development of Cryptococcus neoformans-induced allergic bronchopulmonary mycosis in mice.Infect Immun. 2006; 74: 4339-4348Crossref PubMed Scopus (23) Google Scholar, 29Olszewski MA Huffnagle GB McDonald RA Lindell DM Moore BB Cook DN Toews GB The role of macrophage inflammatory protein-1alpha/CCL3 in regulation of T cell-mediated immunity to Cryptococcus neoformans infection.J Immunol. 2000; 165: 6429-6436Crossref PubMed Scopus (77) Google Scholar Experimentally altering the T1 versus T2 balance profoundly affects pulmonary clearance of the organism. Mice lacking interferon-gamma (IFN-γ) signaling, the major cytokine component of the T1 response, lose their ability to successfully clear cryptococcal infection.30Chen GH McDonald RA Wells JC Huffnagle GB Lukacs NW Toews GB The gamma interferon receptor is required for the protective pulmonary inflammatory response to Cryptococcus neoformans.Infect Immun. 2005; 73: 1788-1796Crossref PubMed Scopus (68) Google Scholar In contrast, depletion of the crucial T2 cytokines interleukin (IL)-4 or IL-13 reduces cryptococcal burden and increases host survival.31Blackstock R Murphy JW Role of interleukin-4 in resistance to Cryptococcus neoformans infection.Am J Respir Cell Mol Biol. 2004; 30: 109-117Crossref PubMed Scopus (39) Google Scholar, 32Muller U Stenzel W Kohler G Werner C Polte T Hansen G Schutze N Straubinger RK Blessing M McKenzie AN Brombacher F Alber G IL-13 induces disease-promoting type 2 cytokines, alternatively activated macrophages and allergic inflammation during pulmonary infection of mice with Cryptococcus neoformans.J Immunol. 2007; 179: 5367-5377PubMed Google Scholar Thus, the balance between a T1 versus T2 adaptive immune responses is a strong determinant defining the outcome of infections with C. neoformans.Pulmonary dendritic cells (DCs) play an important role in defining the T1/T2 balance within the lung.33Vermaelen K Pauwels R Pulmonary dendritic cells.Am J Respir Crit Care Med. 2005; 172: 530-551Crossref PubMed Scopus (220) Google Scholar Studies of DC interactions with Candida albicans have shown that differential expression of fungal factors may significantly alter DC phenotype, subsequent T cell polarization, and the outcome of the infection with this opportunistic pathogen.34Romani L Bistoni F Puccetti P Fungi, dendritic cells and receptors: a host perspective of fungal virulence.Trends Microbiol. 2002; 10: 508-514Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 35Bellocchio S Montagnoli C Bozza S Gaziano R Rossi G Mambula SS Vecchi A Mantovani A Levitz SM Romani L The contribution of the toll-like/IL-1 receptor superfamily to innate and adaptive immunity to fungal pathogens in vivo.J Immunol. 2004; 172: 3059-3069PubMed Google Scholar We have recently shown that large numbers of conventional DCs are recruited to the lung in response to cryptococcal infection.36Osterholzer JJ Curtis JL Polak T Ames T Chen GH McDonald R Huffnagle GB Toews GB CCR2 mediates conventional dendritic cell recruitment and the formation of bronchovascular mononuclear cell infiltrates in the lungs of mice infected with cryptococcus neoformans.J Immunol. 2008; 181: 610-620PubMed Google Scholar We have also shown that the adoptive transfer of immature DCs to mice infected with C. neoformans can bias the resultant immune response toward a T2 phenotype.37Herring AC Falkowski NR Chen GH McDonald RA Toews GB Huffnagle GB Transient neutralization of tumor necrosis factor alpha can produce a chronic fungal infection in an immunocompetent host: potential role of immature dendritic cells.Infect Immun. 2005; 73: 39-49Crossref PubMed Scopus (53) Google Scholar To date, specific cryptococcal-derived factors that influence polarization of the adaptive immune response against C. neoformans have not been identified. We hypothesized that in addition to its effects on microvascular sequestration during fungemia, cryptococcal urease might impair local host defense within the lungs. We investigated this hypothesis using an established murine model comparing the effect of infection with urease-producing or urease-deficient strains of C. neoformans in mice.17Olszewski MA Noverr MC Chen GH Toews GB Cox GM Perfect JR Huffnagle GB Urease expression by Cryptococcus neoformans promotes microvascular sequestration, thereby enhancing central nervous system invasion.Am J Pathol. 2004; 164: 1761-1771Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar Specifically, we sought to determine whether cryptococcal urease expression influences the clearance of the organism and whether (and how) it might alter the development of T1 or T2 adaptive immune responses within the lung.Materials and MethodsMiceMice (C57BL/6, Jackson Laboratories, Bar Harbor, ME) were housed under specific pathogen-free conditions in enclosed filter top cages at the University of Michigan Laboratory Animal Facility. Clean food and water were given ad libitum. The mice were handled and maintained using microisolator techniques with daily veterinarian monitoring. This study complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Dept. of Health, Education, & Welfare Publication No. 80-32) and followed a protocol approved by the Animal Care Subcommittee of the local Institutional Review Board.C. neoformansThree strains of C. neoformans were used in this study were kindly provided by Drs. Gary Cox and John Perfect from Duke University: 1) wt, the wild-type H99 strain that expresses urease; 2) ure1, a urease-negative transformant (knockout) of H99 with disruption of the native urease gene; and 3) ure1::URE1, a urease-expressing (revertant) strain in which strain ure1 has a wild-type copy of URE1 reintroduced. For the infection, yeast that were recovered from 10% glycerol stocks were grown to stationary phase (at least 72 hours) at 36°C in Sabouraud dextrose broth (1% neopeptone, 2% dextrose; Difco, Detroit, MI) on a shaker. The cultures were then washed in non-pyrogenic saline (Travenol, Deerfield, IL), counted on a hemocytometer, and diluted to 3.3 × 105 yeast cells/ml or to 3.3 × 107 in sterile non-pyrogenic saline.Experimental DesignIn all experiments, primary cryptococcal infection was induced in the lungs by intratracheal inoculation of C57BL/6 mice with either a urease-producing (wt or ure1::URE1) or urease-deficient (ure1) strain of C. neoformans on day 0. Thereafter, the following experiments were performed: 1) Analysis of pulmonary cryptococcal burden, by lung CFU assay, at weeks 1, 2, and 3 postinfection; 2) Enumeration of lung leukocytes, by visual identification, at weeks 1, 2, and 3; 3) Quantification of serum IgE levels, by enzyme-linked immunosorbent assay (ELISA), at week 3; 4) Histological assessment of the lungs, by H&E staining and immunohistochemistry performed on lung sections, at week 3; 5) Determination of the expression of IL-4, IL-13, and IFN-γ by lung leukocyte cultures, using ELISA, at week 2, or intracellular cytokine staining using flow cytometric analysis; IL-4 and IFN-γ; at week 2; and 6) Enumeration and immunophenotyping of pulmonary DC in lung and lung associated lymph nodes by flow cytometric analysis, at weeks 1 and 2. Comparative analysis was performed between experimental groups infected with either urease-producing (wt or ure1::URE1) or urease-deficient (ure1) strains of C. neoformans.Surgical Intratracheal InoculationMice were anesthetized via intraperitoneal injection of ketamine/xylazine mix (ketamine/xylazine 100/6.8 mg/kg/BW) and were restrained on a foam plate. A small incision was made through the skin over the trachea and the underlying tissue was separated. A bent 30-gauge needle (Becton Dickinson, Rutherford, NJ) was attached to a tuberculin syringe (BD & Co, Franklin Lakes, NJ) filled with the diluted C. neoformans culture. The needle was inserted into the trachea and 30 μl of inoculum dispensed into the lungs (104 yeast cells except where indicated). The skin was closed with cyanoacrylate adhesive. The mice recovered with minimal visible trauma.Lung CFU AssayAliquots of the lung digest solutions were collected for lung CFU assays. Lung suspensions were serially diluted in sterile water. Dilution samples (10 μl each) were plated on Sabouraud dextrose agar and incubated at room temperature for 48 hours. Colony counts were performed and adjusted to reflect the total lung colony-forming units.Detection of Urease Activity within Lung TissueC57BL/6 mice were inoculated by the intratracheal route with 106 wt or ure1 strains of C. neoformans. One week postinfection, lungs were dissected, homogenized, and incubated in 2 ml Christensen urea broth (Becton Dickinson, Cockeysville, MD.) at 37°C for 2 hours. Media color change (from light orange to red) indicates urease activity in the homogenized lung tissue.Lung Leukocyte IsolationThe lungs from each mouse were excised, washed in PBS, minced with scissors, and digested enzymatically at 37°C for 30 to 35 minutes in 15 ml/lung of digestion buffer [RPMI, 5% fetal calf serum, antibiotics, and 1 mg/ml collagenase (Boehringer Mannheim Biochemical, Chicago, IL), and 30 μg/ml DNase (Sigma-Aldrich, St Louis, MO)]. The cell suspension and tissue fragments were further dispersed by repeated aspiration through the bore of a 10-ml syringe and were centrifuged. Erythrocytes in the cell pellets were lysed by addition of 3 ml of NH4Cl buffer (0.829% NH4Cl, 0.1% KHCO3, 0.0372% Na2EDTA, and pH 7.4) for 3 minutes, followed by a tenfold excess of RPMI. Cells were resuspended and a second cycle of syringe dispersion and filtration through a sterile 100 μm nylon screen (Nitex Kansas City, MO) was performed. The filtrate was centrifuged for 25 minutes at 1500 × g in the presence of 20% Percoll (Sigma) to separate leukocytes from cell debris and epithelial cells. Leukocyte pellets were resuspended in 5 ml of media, and enumerated on a hemocytometer following dilution in Trypan blue (Sigma).Lung-Associated Lymph Node Leukocyte IsolationIndividual lung-associated lymph nodes (LALNs) were excised. To collect LALN leukocytes, nodes were dispersed using a 3-ml sterile syringe plunger and a flushed through a 70-μm cell strainer (BD Falcon) with complete media into a sterile tube. Following erythrocyte lysis (as above), cells were resuspended in complete medium and enumerated in the presence of trypan blue using a hemocytometer.Visual Identification of Leukocyte PopulationsTo obtain differential cell counts of lung cell suspensions isolated from the lung digest, samples were cytospun (Shandon Cytospin, Pittsburgh, PA) onto glass slides and stained by a modification of the Diff-Quik whole blood stain (Diff-Quik, VWR Scientific Products). Samples were fixed/prestained for 2 minutes in a one-step, methanol-based, Wright-Giemsa stain (Harleco, EM Diagnostics, Gibbstown, NJ), rinsed in water, and stained using steps two and three of the Diff-Quik stain. This modification of the Diff-Quik stain procedure improves the resolution of eosinophils from neutrophils in the mouse. A total of 200 cells were counted for each sample from randomly chosen, high-power microscope fields.HistologyLungs were fixed by inflation with 1 ml of 10% neutral buffered formalin (Fisher Scientific, Fair Lawn, NJ), excised en bloc and immersed in neutral buffered formalin. After paraffin embedding, 5 μm sections were cut and stained with H&E ± counterstained with mucicarmine. Quantification of YM1 crystal deposition was assessed by identifying the number of crystals observed per high power (at ×400 magnification) field within inflamed regions of lungs from infected mice. Data represent the mean (±SEM) of at least 15 fields per experimental condition (n = 2 to 3 mice each). For immunohistochemistry, unstained paraffin sections were dewaxed and pretreated with hydrogen peroxide (Sigma) to eliminate residual peroxidase activity. Anti-Arginase1 antibody (BD Pharmingen) staining coupled with immunodetection using VECTOR M.O.M. Peroxidase Immunodetection kit (Vector Laboratories, Burlingame, CA) was performed according to manufacturer's instructions. Sections were analyzed with light microscopy and microphotographs taken using Nikon Digital Microphotography system DFX1200 and ACT-1 software (Nikon Instrum. Inc., Melville, NY).Cytokine ProductionIsolated lung leukocytes were adjusted to 5 × 106 cells/ml in media, plated, and cultured at 37°C, 95% O2, and 5% CO2. After 24 hours, supernatants were separated from cells by centrifugation, collected, and frozen until tested. Reactions were performed in 96-well ELISA plates (Costar Corning Inc., Corniny, NY) following the manufacturer's instructions. The optical densities were read on a microplate reader (Versa Max, MolecularDevices, Sunnyvale, CA) interpolation of sample optical density values with an appropriate standard by a four-parameter curve-fitting program.Total Serum IgE AnalysisBlood was obtained by tail vein bleed of the mice. Following centrifugation to separate cells, serum was removed and total IgE concentrations were assessed using an IgE specific sandwich ELISA (BD Pharmingen, San Diego, CA), read and analyzed as cytokine ELISAs described above.Flow Cytometric AnalysisLeukocytes for the lung and LALN were harvested and single cell suspensions were generated as described above. Antibody staining, including blockade of Fc receptors, was performed as previously described.38Traynor TR Herring AC Dorf ME Kuziel WA Toews GB Huffnagle GB Differential roles of CC chemokine ligand 2/monocyte chemotactic protein-1 and CCR2 in the development of T1 immunity.J Immunol. 2002; 168: 4659-4666PubMed Google Scholar Antibodies were purchased from BD Biosciences PharMingen (San Diego, CA). Data were collected on a FACScan flow cytometer using Cell Quest software (both from Becton Dickinson Immunocytometry Systems, Mountain View, CA) and analyzed using FlowJo software (Tree Star Inc., San Carlos, CA). A minimum of 50,000 cells was analyzed per sample. Initial gates were set based on light scatter characteristics to exclude debris, red cells, and cell clusters. Identification and enumeration of DCs as cells expressing CD11c (FL-1 channel; fluorescein isothiocyanate) and MHC Class II (FL-2 channel; phycoerythrin) within single-cell suspensions generated from total lung leukocytes was performed using a gating strategy previously described in detail.39Osterholzer JJ Ames T Polak T Sonstein J Moore BB Chensue SW Toews GB Curtis JL CCR2 and CCR6, but not endothelial selectins, mediate the accumulation of immature dendritic cells within the lungs of mice in response to particulate antigen.J Immunol. 2005; 175: 874-883PubMed Google Scholar These cells also express CD11b but not B220 and can therefore be defined as “conventional” DCs, (cDCs).36Osterholzer JJ Curtis JL Polak T Ames T Chen GH McDonald R Huffnagle GB Toews GB CCR2 mediates conventional dendritic cell recruitment and the formation of bronchovascular mononuclear cell infiltrates in the lungs of mice infected with cryptococcus neoformans.J Immunol. 2008; 181: 610-620PubMed Google Scholar, 40Shortman K Naik SH Steady-state and inflammatory dendritic-cell development.Nat Rev Immunol. 2007; 7: 19-30Crossref PubMed Scopus (955) Google Scholar Similarly, cDCs were defined within the LALN as cells expressing CD11c (fluorescein isothiocyanate) and MHC class II (I-Ab, phycoerythrin) as previously described.38Traynor TR Herring AC Dorf ME Kuziel WA Toews GB Huffnagle GB Differential roles of CC chemokine ligand 2/monocyte chemotactic protein-1 and CCR2 in the development of T1 immunity.J Immunol. 2002; 168: 4659-4666PubMed Google Scholar, 41Chen GH McNamara DA Hernandez Y Huffnagle GB Toews GB Olszewski MA Inheritance of immune polarization patterns is linked to resistance versus susceptibility to Cryptococcus neoformans in a mouse model.Infect Immun. 2008; 76: 2379-2391Crossref PubMed Scopus (70) Google Scholar Cytometer parameters and gate position were held constant during analysis of all samples. The percent cDCs within each lung and LALN population was obtained after subtracting background events occurring within this gate in isotype control populations. The percentage of cDCs obtained from flow cytometry was used to calculate the total number of DC from each tissue by multiplying the frequency of cDCs by the total cell count for that sample.Three-color analysis was then performed to assess the expression of CD80 on cDCs identified within LALN populations.41Chen GH McNamara DA Hernandez Y Huffnagle GB Toews GB Olszewski MA Inheritance of immune polarization patterns is linked to resistance versus susceptibility to Cryptococcus neoformans in a mouse model.Infect Immun. 2008; 76: 2379-2391Crossref PubMed Scopus (70) Google Scholar Specifically, the expression of CD80 was determined using biotinylated anti-CD80 antibody, followed by streptavidin-conjugated peridinin chlorophyll. CD80 expression was defined (in the FL3 channel) as either high or low based on values of mean fluorescence intensity relative to an isotype control. The total number of CD80hi and CD80lo DCs was determined by multiplying their frequency by the total number of LALN leukocytes.Calculations and StatisticsData (mean ± SE) for each experimental group were derived from at least 2 independent infections (of compared groups of animals) and analyzed using Primer of Biostatistics Software, Version 4.0 (McGraw Hill, Columbus, OH) via t-test, one way or two-way analysis of variance, depending on the experimental design. For individual comparisons of multiple groups, Student-Newman-Keuls posthoc test was used to calculate P values. To calculate IL-4:IFN-γ ratios, the ratio of paired cytokine samples for each mouse was determined and then averaged. A Kruskal-Wallis analysis was performed followed by a Dunn's posthoc test to compare ratios between individual groups. Means with P < 0.05 were considered statistically significant.ResultsCryptococcal Urease Is Associated with Impaired Clearance of C. neoformans from the LungOur previous study demonstrated that urea" @default.
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• W2040386323 title "Cryptococcal Urease Promotes the Accumulation of Immature Dendritic Cells and a Non-Protective T2 Immune Response within the Lung" @default.
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