Distinct CD4+-T-Cell Responses to Live and Heat-Inactivated Aspergillus fumigatus Conidia

Aspergillus fumigatus is a ubiquitous mold that produces small, 2- to 3-μm spores (aka conidia) that are inhaled into lung alveoli. The hydrophobic nature of the spores and their small size allow them to remain airborne for long periods (30, 31). A. fumigatus spores are ubiquitous, and it is estimated that humans inhale several hundred conidia per day (31). In the lung airways, conidia are phagocytosed by alveolar macrophages, where they swell (the first stage of germination), thereby triggering the production of reactive oxygen intermediates (ROI) by the macrophage, a lethal event for the spore (30, 31, 34, 41, 44). Conidia that escape killing by alveolar macrophages germinate, eventually forming hyphae that invade tissues and blood vessels. Neutrophils are recruited to sites of fungal invasion, where they adhere to the hyphal surface and release ROI as well as hydrolytic enzymes that damage the fungal cell wall (22, 30, 33, 34, 44). Administration of immunosuppressive medications increases the incidence of invasive aspergillosis (IA) (31) and is responsible for the current status of Aspergillus fumigatus as one of the most prevalent airborne fungal pathogens (30, 31). The contributions of innate and adaptive immunity to protection of the immunocompetent host from invasive A. fumigatus infections are complex and incompletely defined. Clinical experience and studies in animal models implicate neutrophils and macrophages and their products, such as ROI and pentraxin 3, in innate immune defense against A. fumigatus (19, 22, 30, 31, 33, 34, 44). T cells are increasingly recognized as important mediators of protection from IA (53). Vaccination studies using dendritic cells pulsed with fungal antigens and adoptive transfer studies with T cells from A. fumigatus immune mice suggest that T cells can protect mice from invasive fungal infection (5, 6). Likewise, studies with cyclophosphamide-treated mice or with normal mice intravenously infected with conidia indicate that CD4+ T helper subsets influence the outcome of A. fumigatus infection (8-11). Inhibition of gamma interferon (IFN-γ) results in enhanced invasive disease after A. fumigatus challenge, suggesting that Th1 T cells mediate protection (8, 35). On the other hand, defense against IA is impaired by interleukin 4 (IL-4), and mice lacking this cytokine are more resistant to fungal invasion, suggesting that Th2 CD4+-T-cell responses are detrimental (7, 28, 37). A. fumigatus also causes allergic bronchopulmonary aspergillosis (ABPA), a disease that occurs in patients with asthma and exacerbates airway hyperactivity, peribronchial fibrosis, immunoglobulin E (IgE) production, and eosinophilia (15, 20, 31). A. fumigatus expresses a variety of allergens, several of which have been cloned by screening expression libraries with the sera of ABPA patients (17, 18). Most patients have been found to react to the ribotoxin Asp f I, the perixosome-like protein Asp f 3, the manganese superoxide dismutase Asp f 6, and the allergen Asp f 2 (17, 18). The presence of A. fumigatus allergen-specific antibodies in the sera of ABPA patients is an important diagnostic criterion for this disease and may play a pathogenic role (15, 20, 31). A mouse model that recapitulates the hallmarks of human ABPA has been used to dissect which components of the immune response contribute to pathogenesis (15, 20). A central role for CD4+ T cells in promoting the pathogenesis of ABPA has been demonstrated (12-14, 25, 29), with the Th2 cytokines IL-4, IL-5, and IL-13 contributing to pulmonary pathology (3, 4, 23, 27, 28, 38). The factors that determine when CD4+ T cells are activated in response to A. fumigatus exposure and whether the responding T cells will be biased to a Th1 or Th2 phenotype are unknown. In this study we assessed whether the metabolic state of the A. fumigatus spore influences CD4+-T-cell activation and differentiation by comparing responses to intratracheal challenge with live conidia or heat-inactivated conidia (HIC). We found disparate cytokine profiles in the two groups of mice, with Th1 type cytokines predominating upon exposure to live conidia while production of Th2 cytokines was more prominent following immunization with HIC. Although CD4+ T cells in draining mediastinal lymph nodes (MLN) proliferated in response to A. fumigatus antigens following immunization with live or heat-inactivated conidia, IFN-γ-producing CD4+ T cells specific for hyphae were present only in the airways of mice infected with live conidia. Humoral immune responses to A. fumigatus antigens were mounted in mice infected with live but not inactivated fungus. These results indicate that the immune system discriminates between inactivated and metabolically active spores, restricting optimal Th1 CD4+-T-cell responses and antibody generation for in vivo challenge with viable fungal spores.