Using two Aspergillus species (A. fumigatus and A. versicolor), we established a murine model of repeated pharyngeal aspiration (Figure 1A). Aspirated BALB/c mice were then rested for 2 weeks and sacrificed 3 days after a final challenge. BALF from each animal was analyzed by flow cytometry to measure airway leukocyte recruitment. The frequency of each population and total number of cells collected were used to calculate the total number of airway neutrophils, eosinophils, and CD4 and CD8 T cells (Figure 1B). Airway infiltration of CD45^hi leukocytes was increased in mice that aspirated conidia of A. fumigatus or A. versicolor (Figure 1C, left panel). Ly-6G^hi neutrophils were also increased in both exposure groups (Figure 1C, middle panel). Although aspiration of conidia increased airway eosinophils in both groups, mice that aspirated A. versicolor exhibited higher numbers in comparison to A. fumigatus aspirated mice (Figure 1C, right panel). Recruitment of CD4⁺ and CD8⁺ T lymphocytes were also analyzed by flow cytometry (Figure 1D, E). Although mice that aspirated conidia from either species of Aspergillus exhibited increased airway CD4⁺ T cells (Figure 1E, left panel), CD8⁺ T cells were more significantly increased in mice that aspirated A. fumigatus. This suggests that leukocyte populations recruited to the airways in response to aspiration of Aspergillus conidia varies between species.

To examine the effector functions of murine airway T cells in response to Aspergillus conidia, we performed ex vivo intracellular cytokine staining on BALF cells that had been stimulated with PMA/Ionomycin for 4 hours at 37°C in the presence of Brefeldin A. We examined intracellular production of IFN-γ and IL-4 in BALF CD4⁺ and CD8⁺ T cells (Figure 2A, bottom panels) in comparison to BALF cells stained with control rat-Ig (Figure 2A, top panels). Intracellular production of IFN-γ was significantly increased in both CD4⁺ and CD8⁺ T-cells in mice that aspirated A. fumigatus (Figure 2B, left and right panels, respectively). However, in response to A. versicolor, fewer CD4⁺ T cells produced IFN-γ. Low numbers of IL-4-producing CD4⁺ T cells were detected, although the number of IL-4-producing cells was increased in response to A. versicolor when compared to A. fumigatus (Figure 2B, middle panel). Therefore, in addition to differences in leukocyte recruitment, airway T-cell cytokine production in mice that aspirated Aspergillus conidia may vary between different species.

Next we compared the ability of Aspergillus conidia to germinate in the lungs. In comparison to control mice (Figure 3A, D), animals that aspirated A. versicolor (Figure 3B, E), or A. fumigatus (Figure 3C,F) exhibited airway inflammation and bronchiocentric infiltration of leukocytes. Fungal material was deposited in the terminal airways with a similar pattern in both groups of mice (Figure S1). However, with A. fumigatus, some conidia in the lungs appeared swollen or exhibited germ tube formation, while the morphology of A. versicolor conidia was unchanged (Figure 3G–I, and Table 1). Conidia from both species were equally able to be cultured from lung homogenates, indicating that viability was not significantly affected (data not shown). These results suggest that the ability of Aspergillus conidia to germinate in mouse lung tissue in our model may be species-dependent.

Since only A. fumigatus demonstrated evidence of lung germination following repeated conidia aspiration, we questioned whether airway immune responses to this species were influenced by metabolic activity of germinating conidia. We therefore examined airway recruitment of T cells in response to inactivated A. fumigatus. Paraformaldehyde-fixed or heat-inactivated A. fumigatus conidia were aspirated and flow cytometric analysis of BALF cells indicated a decreased total number of cells with fixed, but not heat-inactivated conidia in comparison to viable conidia (Figure 4A, left panels). This decrease was partially attributed to a significant decrease in neutrophils (Figure 4A, top middle panel), while eosinophil recruitment was not significantly affected (Figure 4A, top right panel). However, eosinophils were increased in response to heat-inactivated conidia (Figure 4A, bottom right panel), while neutrophils were unchanged (Figure 4A, bottom middle panel). Interestingly, CD4⁺ T cell recruitment was not decreased in response to conidia inactivated by either method, while CD8⁺ T cells were decreased regardless of method (Figure 4B, left panels, and 4C, left panels, respectively). IFN-γ-producing CD4⁺ T cells were significantly reduced only in response to heat-inactivated conidia (Figure 4B, right panels), while both methods of inactivation resulted in decreased IFN-γ-producing BALF CD8⁺ T cells (Figure 4C, right panels). Little or no BALF T-cell production of IL-4 was detected (data not shown). Histological examination of lung sections indicated that germ tubes or swollen conidia were only visible in mice that aspirated viable conidia, whereas in mice that aspirated non-viable conidia, no signs of lung germination were detected (Figure 4D, E, and Table 1). Together with the data comparing A. fumigatus and A. versicolor aspiration, these data indicate that airway CD8⁺ T-cell responses to A. fumigatus are correlated with germination of conidia in host lung tissue.

It is possible that the disparate airway T cell responses we observed were also influenced by differences in the ability of fungal conidia/antigen to persist in the lung tissue after aspiration. Therefore, we performed lung histological examination of tissue from mice that were sacrificed in the absence of a final challenge, two weeks after repeated conidia exposures (Figure 5A). Examination of H&E sections displayed lymphocytic granulomas in lungs of mice that had repeatedly aspirated both viable or non-viable A. fumigatus or A. versicolor conidia (Figure 5B–E and data not shown). These granulomas frequently contained what appeared to be intact conidia or in some instances fungal debris (Figure 5F–I). However, mice aspirated with viable A. fumigatus appeared to display a marked increase in inflammation, granuloma formation, and fungal particles in lung tissue in comparison to non-viable A. fumigatus or A. versicolor conidia treated mice (Figure 5C,F and data not shown). Therefore, persistence of fungal material is correlated with the ability to germinate in host lung tissue.

In order to examine the persistence of memory airway T cells, we harvested BALF and mediastinal lymph node (MLN) cells from mice at 40 days after the final conidia challenge (Figure 6A). Cell suspensions were then stained for surface markers associated with a lung memory phenotype [21] and analyzed by flow cytometry. Although BALF lung CD4⁺ T cell recruitment appeared different between viable and non-viable A. fumigatus or A. versicolor-treated mice, the differences were not statistically significant (Figure 6B). However, similar to results at d3 post-challenge, BALF CD8⁺ T cells persisted only in response to viable A. fumigatus conidia. Furthermore, BALF T cells expressed surface markers associated with lung T cell memory responses (Figure 6C). BALF T cells were CD11a^hi/loCD62L^loCD69^hiCD44^hi. Both CD4⁺ and CD8⁺ T cells expressed a memory phenotype at d40 after A. fumigatus challenge, with only CD4⁺ T cells present in the airways of mice that aspirated A. versicolor or non-viable A. fumigatus. However, all groups displayed populations of CD4⁺ and CD8⁺ memory-phenotype T cells in the draining lymph nodes (Figure 6D). These data suggest that the germination of A. fumigatus conidia at physiological temperature may play a role in the maintenance of airway anti-fungal memory CD8⁺, but not CD4⁺, T-cell responses.

Fungal isolates (A. fumigatus, strain Af293, A. versicolor, strain 44408) were purchased from the Fungal Genetics Stock Center (FGSC) and the American Type Culture Collection (ATCC), respectively. Fungi were grown on malt extract agar (MEA) plates at 25°C. Fungal spores were isolated from cultures kept in the dark at room temperature (RT) for 14 days, by applying 1 g of glass beads (0.5 mm, Braun-Melsungen, Melsungen, Germany) and gently shaken. The bead/conidia mixture was collected into a tube and suspended in 1 mL sterile phosphate buffered saline (PBS). The beads were vortexed and the supernatant containing the conidia was removed and counted with a hemacytometer. Conidia were subsequently resuspended to a concentration of 4×10⁷ conidia/ml. For inactivation, A. fumigatus conidia were fixed according to the method of Aimanianda et al. [38]. Briefly, 1 mL of a 4% paraformaldehyde solution was added to the bead/conidia mixture, vortexed, and incubated overnight at 4°C. Fixed conidia were centrifuged, then washed in 0.1 M ammonium chloride, followed by another wash and resuspension in 1 mL sterile PBS. As an alternative method of inactivation, conidia were autoclaved for 30 minutes [19]. Conidial inactivation by either method resulted in >99.9% reduction in viability by serial dilution. Vortexing glass beads with conidia in solution did not significantly alter viability.

Female BALB/c mice, aged 5–7 weeks, were obtained from Jackson Laboratory (Jackson Laboratory, Bar Harbor, ME) and allowed to rest approximately one week before initial exposures. The NIOSH animal facility is an environmentally controlled barrier facility fully accredited by the Association for the Assessment and Accreditations of Laboratory Animal Care International. Fungal suspensions were delivered by involuntary aspiration as previously described [39]. Briefly, mice were anesthetized with isoflurane and suspended on a slant board. The tongue of the animal was held in full extension as a 50 µL suspension of 2×10⁶ spores in PBS was placed at the base of the tongue. The tongue was restrained briefly for approximately two breaths while the mice inhaled the conidial suspension, after which anesthetized mice were returned to the cage and allowed to recover. To examine lung deposition of aspirated conidia, mice were sacrificed one hour after aspiration, and lungs were inflated with air, and tissue was fixed for histological analysis. To assess responses to repeated exposures, mice aspirated conidia twice a week for two weeks (Figure 1A, Figure 5A, and Figure 6a). Some mice were challenged after a two week rest and others were harvested with no final challenge. After the final challenge (d3 or d40; Figure 1A, Figure 5A, and Figure 6a) mice were sacrificed with an intraperitoneal injection of sodium pentobarbital and the lungs were perfused with 10 mL PBS prior to collection of bronchoalveolar lavage fluid (BALF). For histological preparation, lungs were perfused with 5 mL of PBS followed by further perfusion and inflation of the lungs with 10% buffered formalin phosphate (5 mL and 1 mL, respectively) (Fisher Scientific, Fair Lawn, NJ). Tissue processing, embedding, and hematoxylin and eosin (H&E) staining was performed by the West Virginia University Tissue Bank (Morgantown, WV). To detect fungal germination in the lungs, Grocott's Methanamine Silver (GMS) stain was performed on lung sections by the tissue pathology laboratory of the Pathology and Physiology Research Branch (CDC-NIOSH, Morgantown, WV). To obtain the frequency of germinated conidia in lung tissues, 100 conidia from one section per sample were randomly counted for signs of germination. Germinating conidia were defined by conidial swelling (2–3× normal size) or by the formation of germ tubes. BALF was collected by exposing and nicking the trachea followed by insertion of a catheter tied off with suture to prevent leakage. A syringe containing 1 mL of PBS was attached to the tracheal catheter, with the liquid injected into the lungs and subsequently removed. This process was repeated until 3 mL of BALF was collected. All animal procedures were approved by the National Institute of Occupational Safety and Health Animal Care and Use Committee (protocol# 08-ST-M-015).

All reagents were obtained by BD biosciences (BD biosciences San Jose, CA) unless otherwise specified. BALF cell composition was determined by flow cytometric analysis of recovered lavage cells in suspension. BALF was centrifuged for 5 min at 1500 rpm, the supernatant removed, and the cell pellet resuspended and washed in 1 mL of FACS buffer (PBS, 5% fetal bovine serum, 0.05% sodium azide). The washed pellet was resuspended and stained in FACS buffer, 10% rat serum, Fc-receptor blocking antibody (clone 24G2) and the following antibodies: rat anti-mouse Ly-6G FITC, rat anti-mouse Siglec-F PE, pan-leukocyte rat anti-mouse CD45 PerCP, and rat anti-mouse CD11c APC. After staining for 30 minutes in the dark on ice, cells were washed and fixed with BD Cytofix, and resuspended in FACS buffer. Populations of cells were evaluated by flow cytometric analysis on a BD FACSCalibur, or in the case of memory T cells, on a BD LSRII (BD Biosciences, San Jose, CA). Neutrophils were defined as CD45^hiLy-6G^hiCD11c^lo, eosinophils were defined as Ly-6G^loSiglec-F^hiCD11c^lo and alveolar macrophages were Ly-6G^loSiglec-F^hiCD11c^hi, as previously reported [40]. In a separate tube, BALF T cells were quantified using rat anti-mouse CD4 FITC and CD4 PerCp antibodies. Total numbers of each cell population were obtained by multiplying the frequency of the specific population by the total number of BALF cells recovered for each animal. For examination of airway memory cells, BALF cells were harvested from mice 40 days after the final aspiration and stained for airway memory T cell markers [21]; these included rat anti-mouse CD4 APC-Cy7, CD8 FITC, CD11a PE, CD62L PerCp Cy5.5, CD69 PE-Cy7, and CD44 APC.

All reagents were obtained by BD biosciences unless otherwise specified. T-cell cytokine production was determined by fluorescent intracellular cytokine staining (ICS) as previously described [41]. Briefly, the BALF suspension was centrifuged for 5 min at 1500 rpm and washed in 1 mL of complete medium. The supernatant was discarded and a solution of Leukocyte Activation Cocktail with GolgiPlug in 0.2 mL complete medium was added to each sample for stimulation of cytokine production and simultaneous inhibition of cytokine secretion. Cells were incubated at 37°C for 4 hrs. After incubation, the cells were washed in FACS buffer and stained for flow cytometry using rat-anti-mouse CD4 PerCP and rat-anti-mouse CD8 FITC on ice. After a 30 min incubation, cells were washed in FACS buffer and centrifuged, and cell pellets were resuspended in BD Cytofix/Cytoperm for 15 minutes to allow for fixation and permeabilization required for subsequent intracellular cytokine staining. Cells were washed with 1 mL BD Permwash, and resuspended in Permwash. Each sample was equally divided into two tubes and stained with rat-anti-mouse IFN-γ APC and rat-anti-mouse IL-4 PE, or with control isotype antibodies (eBioscience, San Diego, CA). Cell populations were analyzed on a BD FACSCalibur, with lymphocytes gated on the basis of low forward and side scatter, then subsequently gated on CD4⁺ or CD8⁺ populations to determine intracellular expression of cytokines.

Analysis of flow cytometric samples was performed with FlowJo software (TreeStar, Ashland, OR). GraphPad Prism was used for generation of graphs and figures and for statistical analyses (GraphPad Software, La Jolla, CA). Unpaired t-tests were performed to measure statistical significance, and differences between experimental groups that resulted in a p value of less than 0.05 were considered significant.