Role of Germination in Murine Airway CD8 + T-Cell Responses to Aspergillus Conidia Steven P. Templeton 1 *, Amanda D. Buskirk 1,2 , Brandon Law 1 , Brett J. Green 1 , Donald H. Beezhold 1 1 Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia, United States of America, 2 Department of Microbiology, Immunology, and Cell Biology, West Virginia University School of Medicine, Morgantown, West Virginia, United States of America Abstract Pulmonary exposure to Aspergillus fumigatus has been associated with morbidity and mortality, particularly in immunocompromised individuals. A. fumigatus conidia produce b-glucan, proteases, and other immunostimulatory factors upon germination. Murine models have shown that the ability of A. fumigatus to germinate at physiological temperature may be an important factor that facilitates invasive disease. We observed a significant increase in IFN-c-producing CD8 + T cells in bronchoalveolar lavage fluid (BALF) of immunocompetent mice that repeatedly aspirated A. fumigatus conidia in contrast to mice challenged with A. versicolor, a species that is not typically associated with invasive, disseminated disease. Analysis of tissue sections indicated the presence of germinating spores in the lungs of mice challenged with A. fumigatus, but not A. versicolor. Airway IFN-c + CD8 + T-cells were decreased and lung germination was eliminated in mice that aspirated A. fumigatus conidia that were formaldehyde-fixed or heat-inactivated. Furthermore, A. fumigatus particles exhibited greater persistence in the lungs of recipient mice when compared to non-viable A. fumigatus or A. versicolor, and this correlated with increased maintenance of airway memory-phenotype CD8 + T cells. Therefore, murine airway CD8 + T cell-responses to aspiration of Aspergillus conidia may be mediated in part by the ability of conidia to germinate in the host lung tissue. These results provide further evidence of induction of immune responses to fungi based on their ability to invade host tissue. Citation: Templeton SP, Buskirk AD, Law B, Green BJ, Beezhold DH (2011) Role of Germination in Murine Airway CD8 + T-Cell Responses to Aspergillus Conidia. PLoS ONE 6(4): e18777. doi:10.1371/journal.pone.0018777 Editor: Kirsten Nielsen, University of Minnesota, United States of America Received January 4, 2011; Accepted March 9, 2011; Published April 13, 2011 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This work was supported by an interagency agreement with the National Institute of Occupational Safety and Health (Y1-ES-0001). No additional external funding was received for this study. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: STempleton@cdc.gov Introduction Filamentous fungi are ubiquitous microorganisms in indoor and outdoor environments and acquire nutrients from a wide variety of substrates such as decaying plant matter or water-damaged building materials [1]. The small size of conidia (i.e. asexual spores) of many fungal species allows particles to easily become airborne and inhaled, with a potential for deposition in the terminal airways of the lungs. Small amounts of inhaled conidia are quickly phagocytosed and degraded by alveolar macrophages [2,3]. However, repeated exposure to large numbers may result in persistence of conidia and induction of airway inflammation. Conidia from the genus Aspergillus have been associated with allergic sensitization as well as exacerbation of allergy and asthma in otherwise healthy individuals [2,3]. However, the pathology of Aspergillus-associated invasive disease varies between fungal species. Aspergillus fumigatus is the etiologic agent of allergic bronchopul- monary aspergillosis (ABPA), has been associated with hypersen- sitivity pneumonitis, and is a primary cause of invasive aspergillosis in immunocompromised individuals [3]. In contrast, A. versicolor is not typically associated with invasive pulmonary infection. The ability of A. fumigatus to colonize the respiratory tract of susceptible individuals has been attributed to several biological properties. In contrast to A. versicolor, A. fumigatus can maintain growth within a wide range of temperatures, from below 20uC up to 70uC [3,4]. Furthermore, A. fumigatus conidia exhibit an ability to persist inside macrophages after phagocytosis or produce factors that inhibit phagocytosis [5,6,7]. Conidia that persist in the lungs of immunocompromised individuals may germinate and form hyphal structures that invade surrounding tissue. Furthermore, release of immunostimulatory molecules such as b-glucan and allergens have been shown in germinating, but not resting, conidia [8,9]. The ability of A. fumigatus to exhibit invasive growth in the respiratory tract is believed to be mediated in part by the ability to germinate at physiological temperatures and by the secretion of fungal proteases [7]. In support of these hypotheses, recent studies of gene-targeted mutants have demonstrated that decreased thermo- tolerance or protease secretion resulted in significantly decreased virulence of A. fumigatus in murine models of invasive infection [10,11]. Based on the results of these studies, lung persistence and tissue invasion are characteristics of Aspergillus conidia that may be species-dependent. Both innate and adaptive immunity are critical in the development of immune protection from invasive aspergillosis. In addition to phagocytosis by resident alveolar macrophages, inhaled A. fumigatus conidia are prevented from germination and the establishment of early invasive infection by infiltrating neutrophils [12,13]. However, adaptive immune responses also provide protection from invasive infection. Adoptive transfer of fungal-specific CD4 + T h 1 lymphocytes confers protection from PLoS ONE | www.plosone.org 1 April 2011 | Volume 6 | Issue 4 | e18777