Robust Th1 and Th17 Immunity Supports Pulmonary Clearance but Cannot Prevent Systemic Dissemination of Highly Virulent Cryptococcus neoformans H99
2009; Elsevier BV; Volume: 175; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2009.090530
ISSN1525-2191
AutoresYanmei Zhang, Fuyuan Wang, Kristin C. Tompkins, Andrew J. McNamara, Aditya V. Jain, Bethany B. Moore, Galen B. Toews, Gary B. Huffnagle, Michal A. Olszewski,
Tópico(s)Nail Diseases and Treatments
ResumoThe present study dissected the role of a Th2 bias in pathogenesis of Cryptococcus neoformans H99 infection by comparing inhalational H99 infections in wild-type BALB/c and IL-4/IL-13 double knockout mice. H99-infected wild-type mice showed all major hallmarks of Th2 but not Th1/Th17 immunity in the lungs and lung-associated lymph nodes. In contrast, the IL-4/13−/− mice developed robust hallmarks of Th1 and Th17 but not Th2 polarization. The IL-4/IL-13 deletion prevented pulmonary eosinophilia, goblet cell metaplasia in the airways and resulted in elevated serum IgE, and a switch from alternative to classical activation of macrophages. The development of a robust Th1/Th17 response and classical activation of macrophages resulted in significant containment of H99 in the lungs of IL-4/13−/− mice compared with unopposed growth of H99 in the lungs of wild-type mice. However, IL-4/13−/− mice showed only 1-week longer survival compared with wild-type mice. The comparison of brain and spleen cryptococcal loads at weeks 2, 3, and 4 postinfection revealed that the systemic dissemination in IL-4/13−/− mice occurred with an approximate 1-week delay but subsequently progressed with similar rate as in the wild-type mice. Furthermore, wild-type and IL-4/13−/− mice developed equivalently severe meningitis/encephalitis at the time of death. These data indicate that the Th2 immune bias is a crucial mechanism for pulmonary virulence of H99, whereas other mechanisms are largely responsible for its central nervous system tropism and systemic dissemination. The present study dissected the role of a Th2 bias in pathogenesis of Cryptococcus neoformans H99 infection by comparing inhalational H99 infections in wild-type BALB/c and IL-4/IL-13 double knockout mice. H99-infected wild-type mice showed all major hallmarks of Th2 but not Th1/Th17 immunity in the lungs and lung-associated lymph nodes. In contrast, the IL-4/13−/− mice developed robust hallmarks of Th1 and Th17 but not Th2 polarization. The IL-4/IL-13 deletion prevented pulmonary eosinophilia, goblet cell metaplasia in the airways and resulted in elevated serum IgE, and a switch from alternative to classical activation of macrophages. The development of a robust Th1/Th17 response and classical activation of macrophages resulted in significant containment of H99 in the lungs of IL-4/13−/− mice compared with unopposed growth of H99 in the lungs of wild-type mice. However, IL-4/13−/− mice showed only 1-week longer survival compared with wild-type mice. The comparison of brain and spleen cryptococcal loads at weeks 2, 3, and 4 postinfection revealed that the systemic dissemination in IL-4/13−/− mice occurred with an approximate 1-week delay but subsequently progressed with similar rate as in the wild-type mice. Furthermore, wild-type and IL-4/13−/− mice developed equivalently severe meningitis/encephalitis at the time of death. These data indicate that the Th2 immune bias is a crucial mechanism for pulmonary virulence of H99, whereas other mechanisms are largely responsible for its central nervous system tropism and systemic dissemination. Cryptococcus neoformans is a leading cause of fatal mycosis in HIV-positive individuals in numerous countries around the globe.1Kovacs JA Kovacs AA Polis M Wright WC Gill VJ Tuazon CU Gelmann EP Lane HC Longfield R Overturf G Macher AM Fauci AS Parrillo JE Bennett JE Masur H Cryptococcosis in the acquired immunodeficiency syndrome.Ann Intern Med. 1985; 103: 533-538Crossref PubMed Scopus (480) Google ScholarC. neoformans is also a problem in organ transplant recipients, patients with hematological malignancies, and those undergoing immunosuppressive therapies.2Pappas PG Perfect JR Cloud GA Larsen RA Pankey GA Lancaster DJ Henderson H Kauffman CA Haas DW Saccente M Hamill RJ Holloway MS Warren RM Dismukes WE Cryptococcosis in human immunodeficiency virus-negative patients in the era of effective azole therapy.Clin Infect Dis. 2001; 33: 690-699Crossref PubMed Scopus (501) Google Scholar Interestingly, over 50% of C. neoformans infections in the United States are reported in HIV negative patients, of which many express no apparent immune deficiencies.3Baddley JW Perfect JR Oster RA Larsen RA Pankey GA Henderson H Haas DW Kauffman CA Patel R Zaas AK Pappas PG Pulmonary cryptococcosis in patients without HIV infection: factors associated with disseminated disease.Eur J Clin Microbiol Infect Dis. 2008; 27: 937-943Crossref PubMed Scopus (119) Google Scholar This may be attributed to the emergence of new high-virulence strains of C. neoformans and a high potential of the organism to adapt to extreme environmental conditions and a variety of hosts.3Baddley JW Perfect JR Oster RA Larsen RA Pankey GA Henderson H Haas DW Kauffman CA Patel R Zaas AK Pappas PG Pulmonary cryptococcosis in patients without HIV infection: factors associated with disseminated disease.Eur J Clin Microbiol Infect Dis. 2008; 27: 937-943Crossref PubMed Scopus (119) Google Scholar, 4Hoang LM 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 (167) Google Scholar, 5Zahra LV Azzopardi CM Scott G Cryptococcal meningitis in two apparently immunocompetent Maltese patients.Mycoses. 2004; 47: 168-173Crossref PubMed Scopus (20) Google Scholar, 6Hofman 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 The invasion of an immunocompetent host is possible when the microbes develop mechanisms that allow them to evade and/or modulate the immune responses to cause “immune deviation.” Reports from C. neoformans infection models suggest that C. neoformans may exploit both of these mechanisms to achieve its virulence.7Huffnagle 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, 8Osterholzer JJ Surana R Milam JE Montano GT Chen GH Sonstein J Curtis JL Huffnagle GB Toews GB Olszewski MA Cryptococcal urease promotes the accumulation of immature dendritic cells and a non-protective T2 immune response within the lung.Am J Pathol. 2009; 174: 932-943Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar Dissemination into the central nervous system (CNS) and the subsequent development of meningitis/encephalitis is the major cause of mortality in uncontrolled cryptococcosis.5Zahra LV Azzopardi CM Scott G Cryptococcal meningitis in two apparently immunocompetent Maltese patients.Mycoses. 2004; 47: 168-173Crossref PubMed Scopus (20) Google Scholar, 9Chretien F Lortholary O Kansau I Neuville S Gray F Dromer F Pathogenesis of cerebral Cryptococcus neoformans infection after fungemia.J Infect Dis. 2002; 186: 522-530Crossref PubMed Scopus (171) Google Scholar, 10Lortholary O Improvisi L Nicolas M Provost F Dupont B Dromer F Fungemia during murine cryptococcosis sheds some light on pathophysiology.Med Mycol. 1999; 37: 169-174PubMed Google Scholar The CNS infection is caused by secondary dissemination of C. neoformans from primary sites of infection (lung) and it occurs readily when pulmonary growth of C. neoformans is not controlled by the infected host. 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Recently, Th17 immune response was also shown to play a protective role in C. neoformans infection.18Kleinschek MA Muller U Brodie SJ Stenzel W Kohler G Blumenschein WM Straubinger RK McClanahan T Kastelein RA Alber G IL-23 enhances the inflammatory cell response in Cryptococcus neoformans infection and induces a cytokine pattern distinct from IL-12.J Immunol. 2006; 176: 1098-1106PubMed Google Scholar, 19Muller 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 In contrast, defects in the immune responses [T cell, tumor necrosis factor (TNF)-α, or interferon (IFN)-γ deficiencies]7Huffnagle 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; 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73: 1788-1796Crossref PubMed Scopus (71) Google Scholar and/or deviation to a Th2 immunity promote alternative activation of macrophages (AAM).17Arora 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, 23Hernandez 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, 24Mowen KA Glimcher LH Signaling pathways in Th2 development.Immunol Rev. 2004; 202: 203-222Crossref PubMed Scopus (200) Google Scholar The alternatively activated macrophages have been demonstrated to harbor C. neoformans and their presence is associated with uncontrolled growth of C. neoformans and severe lung pathology.8Osterholzer JJ Surana R Milam JE Montano GT Chen GH Sonstein J Curtis JL Huffnagle GB Toews GB Olszewski MA Cryptococcal urease promotes the accumulation of immature dendritic cells and a non-protective T2 immune response within the lung.Am J Pathol. 2009; 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165: 6429-6436Crossref PubMed Scopus (78) Google Scholar, 30Stenzel W Muller U Kohler G Heppner FL Blessing M McKenzie AN Brombacher F Alber G IL-4/IL-13-dependent alternative activation of macrophages but not microglial cells is associated with uncontrolled cerebral cryptococcosis.Am J Pathol. 2009; 174: 486-496Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar C. neoformans H99 is a human isolate that expresses one of the highest virulence levels among C. neoformans strains used for experimental infections. This strain grows in the lungs in an uncontrolled fashion, readily disseminates into CNS, and causes 100% mortality in a variety of immunocompetent mouse strains such as C57BL/6, BALB/c, CB6129F2, and CBA/J mice.8Osterholzer JJ Surana R Milam JE Montano GT Chen GH Sonstein J Curtis JL Huffnagle GB Toews GB Olszewski MA Cryptococcal urease promotes the accumulation of immature dendritic cells and a non-protective T2 immune response within the lung.Am J Pathol. 2009; 174: 932-943Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 13Olszewski 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 (204) Google Scholar, 31Cox 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 (400) Google Scholar, 32Noverr MC Cox GM Perfect JR Huffnagle GB Role of PLB1 in pulmonary inflammation and cryptococcal eicosanoid production.Infect Immun. 2003; 71: 1538-1547Crossref PubMed Scopus (100) Google Scholar, 33Wormley Jr, FL Perfect JR Steele C Cox GM Protection against cryptococcosis by using a murine gamma interferon-producing Cryptococcus neoformans strain.Infect Immun. 2007; 75: 1453-1462Crossref PubMed Scopus (139) Google Scholar The infections with H99 were previously shown to result in up-regulation of several hallmarks of Th2 immunity in the lungs. This was associated with the expression of virulence factors by H99 including urease and PLB1.8Osterholzer JJ Surana R Milam JE Montano GT Chen GH Sonstein J Curtis JL Huffnagle GB Toews GB Olszewski MA Cryptococcal urease promotes the accumulation of immature dendritic cells and a non-protective T2 immune response within the lung.Am J Pathol. 2009; 174: 932-943Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 32Noverr MC Cox GM Perfect JR Huffnagle GB Role of PLB1 in pulmonary inflammation and cryptococcal eicosanoid production.Infect Immun. 2003; 71: 1538-1547Crossref PubMed Scopus (100) Google Scholar These previous studies have suggested that promoting a shift to a non-protective Th2 immune response could be a mechanism of H99-induced virulence. The goal of the present study was to test the hypothesis that inducing a Th2 immune bias is an important mechanism for H99 virulence. Using the double deletion of Th2-driving cytokines interleukin (IL)-4 and IL-13 in mice, we demonstrate that in the absence of IL-4 and IL-13, H99-infected mice switch from a robust immune response with the Th2 phenotype and AAM to that of a mixed Th1/Th17 phenotype and CAM. This response significantly improves control of pulmonary cryptococcal growth in the lungs. However, it does not protect against brain dissemination of C. neoformans and the high mortality associated with an H99 infection. This is the first report that a strong Th1/Th17 immune response and CAM is insufficient for significant protection against CNS invasion and high lethality associated with inhalation of a highly virulent C. neoformans H99. IL-4/13−/− mice were generated and originally provided by Dr. Andrew McKenzie, Medical Research Council, London, UK34McKenzie GJ Fallon PG Emson CL Grencis RK McKenzie AN Simultaneous disruption of interleukin (IL)-4 and IL-13 defines individual roles in T helper cell type 2-mediated responses.J Exp Med. 1999; 189: 1565-1572Crossref PubMed Scopus (293) Google Scholar and were bred at the University of Michigan Animal Research Facility in specific pathogen-free conditions. Age matched wild-type control BALB/c mice were purchased from Charles River Laboratories, (Wilmington, MA). Following at least 1-week acclimatization period at the Arbor VA Medical Center Mice Veterinary Medical Unit, wild-type and IL-4/13−/− mice were infected and subsequently housed in pressurized BSL2 cubicles in microisolator cages covered with filter tops. Food/water was provided ad libitum and mice were monitored daily for the period of infection. At the time of data collection mice were humanely euthanized by CO2 inhalation. All experimental procedures were approved by the University Committee on the Use and Care of Animals and VA Institutional Animal Care and Use and Committee. C. neoformans strain H99 (ATCC 208821) was recovered from 10% glycerol frozen stocks stored at −80°C and 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 in sterile non-pyrogenic saline.8Osterholzer JJ Surana R Milam JE Montano GT Chen GH Sonstein J Curtis JL Huffnagle GB Toews GB Olszewski MA Cryptococcal urease promotes the accumulation of immature dendritic cells and a non-protective T2 immune response within the lung.Am J Pathol. 2009; 174: 932-943Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar Mice were anesthetized via intraperitoneal injection of ketamine/xylazine (ketamine/xylazine 100/6.8 mg/kg/bw) and were restrained on a foam plate. A small incision was made through the skin covering the trachea. The underlying salivary glands and muscles were separated. Infection was performed by intratracheal injection of 30 μl (104 CFU) via 30-gauge needle actuated from a 1-ml tuberculin syringe with C. neoformans suspension (3.3 × 105/ml). After inoculation, the skin was closed with cyanoacrylate adhesive.8Osterholzer JJ Surana R Milam JE Montano GT Chen GH Sonstein J Curtis JL Huffnagle GB Toews GB Olszewski MA Cryptococcal urease promotes the accumulation of immature dendritic cells and a non-protective T2 immune response within the lung.Am J Pathol. 2009; 174: 932-943Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 26Chen 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 (71) Google Scholar For determination of microbial burden in the lungs, small aliquots of dispersed lungs were collected following the digest procedure. For determination of brain and spleen CFU, the brains and spleens were dissected using sterile instruments, placed in 2 ml of sterile water, and homogenized. Series of 10-fold dilutions of the lung, spleen, and brain samples were plated on Sabouraud dextrose agar plates in duplicates in 10-μl aliquots and incubated at room temperature. C. neoformans colonies were counted 2 days later and the number of CFU were calculated on a per-organ basis.8Osterholzer JJ Surana R Milam JE Montano GT Chen GH Sonstein J Curtis JL Huffnagle GB Toews GB Olszewski MA Cryptococcal urease promotes the accumulation of immature dendritic cells and a non-protective T2 immune response within the lung.Am J Pathol. 2009; 174: 932-943Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 26Chen 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 (71) Google Scholar The lungs from each mouse were excised, washed in RPMI, minced with scissors, and digested enzymatically at 37°C for 30 minutes in 15 ml of digestion buffer per mouse [RPMI, 5% fetal calf serum, antibiotics, 1 mg/ml collagenaseA (Roche Diagnostics, Indianapolis, IN), and 30 μg/ml DNase (Sigma)] and processed as previously described.11Olszewski 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, 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 (78) Google Scholar 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, and 0.0372% Na2-EDTA, pH 7.4) for 3 minutes followed by a 10-fold 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 complete RPMI media and enumerated on a hemocytometer following dilution in Trypan Blue (Sigma). Individual lung-associated lymph nodes (LALNs) were excised. To collect LALN leukocytes, nodes were dispersed using a 3-ml sterile syringe plunger and flushed through a 70-μm cell strainer (BD Falcon) with complete media into a sterile tube, as described previously.8Osterholzer JJ Surana R Milam JE Montano GT Chen GH Sonstein J Curtis JL Huffnagle GB Toews GB Olszewski MA Cryptococcal urease promotes the accumulation of immature dendritic cells and a non-protective T2 immune response within the lung.Am J Pathol. 2009; 174: 932-943Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar After being centrifuged at 12,000 rpm/min for 10 minutes, the supernatant was removed and the cell pellets were saved at −70°C for gene expression analysis by real-time reverse transcription polymerase chain reaction (PCR). Isolated pulmonary leukocytes (10 × 106 cells/ml) were seeded in six-well plates and cultured at 37°C for 1.5 hours. Plates were washed twice using phosphate-buffered saline to remove non-macrophage cells. Total RNA was collected from adherent cells and used for real-time reverse transcription PCR analysis. Macrophages, neutrophils, eosinophils, monocytes, and lymphocytes were visually counted in Wright-Giemsa-stained samples of lung cell suspensions cytospun onto glass slides. Samples were fixed/pre-stained for 2 minutes in a one-step methanol based Wright-Giemsa stain (Harleco, EM Diagnostics, Gibbstown, NJ) 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 300 cells were counted for each sample from randomly chosen high-power microscope fields. The percentages of leukocyte subsets were multiplied by the total number of leukocytes to give the absolute number of specific leukocyte subsets in the sample. Isolated lung leukocytes were diluted to 5 × 106 cells/ml and were cultured in 24-well plates with 2 ml of complete RPMI medium at 37°C and 5% CO2 for 24 hours. Supernatants were separated from cells by centrifugation, collected, and frozen until tested. The cytokines TNF-α, IFN-γ, IL-12p35, IL-4, and IL-10 were quantified by enzyme-linked immunosorbent assay (ELISA) using DuoSet kits (R&D Systems, Minneapolis, MN) and OPT-EIA kits (BD Biosciences, San Jose, CA) following the manufacturer’s specifications. All plates were read on a Versamax plate reader (Molecular Devices, Sunnyvale, CA).8Osterholzer JJ Surana R Milam JE Montano GT Chen GH Sonstein J Curtis JL Huffnagle GB Toews GB Olszewski MA Cryptococcal urease promotes the accumulation of immature dendritic cells and a non-protective T2 immune response within the lung.Am J Pathol. 2009; 174: 932-943Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 26Chen 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. 20
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