Clinical isolates of Aspergillus fumigatus (V45-07) and Aspergillus nidulans (V44-46) from CGD patients with invasive aspergillosis, used in all in vivo experiments, were a kind gift from Prof Paul E. Verweij (Nijmegen, NL). A. nidulans strains overexpressing Uge3 or UgeB, and the parent A26 strain, were kindly provided by Prof Don Sheppard and Dr Mark Lee, Montreal, Canada. Fluorescent Aspergillus Reporter (FLARE) conidia (Jhingran et al., 2012) were prepared from cultures of A. fumigatus Af293-dsRed conidia (a kind gift from Prof Tobias Hohl, Memorial Sloan-Kettering Cancer Centre, New York, USA).

All isolates were cultured in T-75 flasks (Grenier Bio-One) on glucose minimal media (GMM) agar slopes at 37°C and 5% CO₂. For the auxotrophic A. nidulans galactosaminogalactan (GAG) mutants (An-Uge3 and An-UgeB), and their parent strain (A26), GMM was supplemented with 0.5 mg/ml biotin. Conidia were harvested after 7 to 10 days by washing flasks with phosphate-buffered saline (PBS, Gibco) supplemented with 0.05% Tween 80 (Fisher Scientific). The resulting conidial suspension was filtered through a 40 μm sterile cell strainer (Fisher Scientific), washed twice in PBS, counted and then diluted to the appropriate concentration in the relevant media.

Aspergillus hyphal layers were obtained by seeding 96-well filter plates (Merck Millipore) with 1 x 10 (Henriet et al., 2012) conidia of A. fumigatus (V45-07) or A. nidulans (V44-46) in 100μl RPMI and incubating plates at 37°CC and 5% CO₂ for a minimum of 8 hours. The method for labelling Af293-dsRed conidia with the AF633 fluorophore was performed as described in Brunel et al. (2017).

All animals were bred and maintained at the Medical Research Facility, University of Aberdeen in accordance with the Home Office, UK, Animal Scientific Procedures Act, 1986. All experiments conformed to the terms and conditions of Home Office licences for research on animals and were approved by a local ethics review committee.

CYBB/C57BL/6 mice carrying a targeted disruption in the Cybb gene (Pollock et al., 1995) (referred to as gp91^-/-) were kindly provided by Prof Steve Holland (National Institute of Allergy and Infectious Diseases, NIH, Bethesda, USA). These mice were backcrossed with our in-house C57BL/6J mice to minimise genetic variation between gp91^-/- mice and our wild-type (WT) C57BL/6J controls. Maintenance of genetic integrity was regularly checked by PCR.

Following humane sacrifice, alveolar macrophages were harvested from gp91^-/- and WT mice by broncho-alveolar lavage (BAL). Lavage was performed four times using 1 ml ice cold PBS supplemented with 5 mM ultrapure ethylenediaminetetraacetic acid (EDTA, Invitrogen Life Technologies) via an 18 gauge (G) intravenous cannula (Introcan-W, Braun) inserted into the trachea. Samples were centrifuged at 500 x g for 5 min at 4°C, supernatants discarded, and the resulting cell pellet re-suspended in 300 μl RPMI supplemented with 10% HI FBS. Cells from a minimum of six mice per group were pooled, counted, and their viability checked using Trypan Blue.

Cell suspensions were adjusted to between 1 x 10⁵ and 5 x 10⁵ cells/ml and 100 μl of the resulting cell suspension transferred to a 96-well filter plate (Merck Millipore). Plates were then incubated for three hours at 37°C and 5% CO₂ to allow the macrophages to adhere. After three hours the media was refreshed before adding conidia to the wells at a ratio of 5 conidia to 1 AM. Plates were then incubated for 12 or 18 hours at 37°C and 5% CO₂ before fungal viability was assessed using a colorimetric XTT assay as described by Henriet et al. (Henriet et al., 2011)

Bone marrow derived macrophages (BMDMs) were obtained from gp91^-/- and WT mice using a protocol derived from that described by Zhang et al (Zhang et al., 2008) and Davies and Gordon (Davies and Gordon, 2005). After sacrifice by CO₂ asphyxiation and gentle cervical dislocation, the skin was sterilised using 70% ethanol. The lower limb skin and soft tissue was then removed, and the tibia and femur placed on ice in RPMI. Bones were immersed in 70% ethanol for one minute before washing twice in ice cold sterile PBS. The epiphyses were removed using sterile scissors and the bone marrow canals flushed with 5 ml ice cold RPMI supplemented with 2 mM ultrapure EDTA (Invitrogen Life Technologies). The resulting bone marrow plugs were washed through a sterile 70 µM cell strainer (Fisher Scientific) and collected in a 50 ml polypropylene tube (Grenier Bio-One).

Cell suspensions were then centrifuged at 500 x g for ten minutes at room temperature, the supernatant discarded, and the resulting cell pellet resuspended in 50 ml of macrophage complete medium (Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% HI FBS, 1% Penicillin-Streptomycin (Sigma) and 20% L929-cell conditioned media. The resulting cell suspensions from each mouse were then divided between two 15 cm petri dishes (Corning™ Falcon™, Fisher Scientific) and incubated at 37°C with 5% CO₂ for seven days to produce BMDMs. The media was refreshed every three days during this time. The method described by Mosser and Zhang (2008) was then used to generate classically activated macrophages from the differentiated BMDMs using 48-well plates.

For fungal killing assays, 1 x 10⁶ conidia in 250 μl DMEM plus 10% FBS were added to the appropriate wells of the 48-well plate (2 x 10⁵ BMDM/well; MOI 1:5) after a 24 or 46 hour LPS prime and the plates were then incubated for a further 18 hours at 37°C and 5% CO₂. Following incubation, fungal viability was assessed using an XTT assay (Henriet et al., 2011).

Bone marrow derived neutrophils (BMDNs) were obtained from gp91^-/- and WT mice using the protocol described by Swamydas and Lionakis (2013). A cell count was performed on the resulting cell suspensions using a haemocytometer with Trypan Blue exclusion to check neutrophil viability prior to each experiment. Neutrophils suspensions were adjusted to the appropriate concentration for each experiment.

For neutrophil anti-hyphal assays, BMDNs were added to hyphal layers at a neutrophil to conidium ratio of 50 to 1 and the plates incubated for 12 hours at 37°C and 5% CO₂. Following incubation, fungal viability was assessed using the XTT assay (Henriet et al., 2011).

FLARE conidia were added to 2 x 10⁶ BMDNs/ml in RPMI supplemented with 10% heat-inactivated FBS. Voriconazole was added at a final concentration of 2 μg/ml to inhibit conidial germination. After a 16-hour incubation at 37°C and 5% CO2, neutrophils and conidia were gently lifted from the wells using PBS with ultrapure EDTA, and the samples were prepared for flow cytometry analysis, following the procedures described in the flow cytometry section. The specific details of the antibodies and dyes used can be found in the supplementary data.

Age-matched wild-type C57BL/6J and gp91-/- mice, between seven and thirteen weeks of age, were infected under anaesthesia by intra-tracheal instillation of 5 x 10⁴ A. fumigatus (V45-07) or A. nidulans (V44-46) conidia in 40 μl PBS. Following infection, mice were kept in sterile-isolator cages with autoclaved food and bedding, and acidified (gp91-/- mice) or sterile (WT mice) drinking water was provided ad libitum. Mice were weighed daily and monitored at least twice daily. Humane end-points were weight loss ≥ 30% or a clinical score ≥ 5 using our in house clinical monitoring system. Upon reaching the pre-determined end-points, mice were culled with a lethal overdose of pentobarbital sodium (Euthatal, Merial Animal Health, Ltd.) followed by gentle cervical dislocation.

Fungal burden was determined using lung homogenised in 1 ml sterile PBS. Serial 1 in 2 dilutions of a 100 μl volume of the resulting homogenate were plated on glucose minimal media and cultured at 37°C and 5% CO₂. Colonies were counted daily for 5 days and colony forming units (cfu) per lung were calculated.

After sacrifice, the trachea was cannulated with an 18G (Introcan^®-W, Braun) and 1 ml of a 1 to 1 volume per volume (v/v) solution of 2 M sucrose and optimal cutting temperature (OCT) compound (Tissue-Tek) was used to inflate the left lung. The lung was then excised, placed in a cryomold (ProSci Tech, Australia) containing OCT and then covered with OCT. Samples were immediately frozen at -20°C before transfer to an -80°C freezer on dry ice. Six to eight μm cryostat sections were obtained in triplicate from at least six different levels in each lung using a Leica CM1900 cryostat. Following sectioning, samples were allowed to dry overnight before dehydrating in 100% ethanol for 5 minutes ready for staining. Samples were either stained with haematoxylin and eosin (H&E) or a modified Grocott Methanamine Silver (GMS) stain (Accustain^® Silver Stain, Sigma-Aldrich) and finished with a haematoxylin counter stain. Slides were examined using a light microscope and selected slides uploaded to the Zeiss Axioscan Z1 slide scanner.

For cytokine analyses, homogenised lung in 1 ml PBS containing protease inhibitors (cOmplete™ ULTRA, mini, EDTA-free, Roche) was placed in 1.8 ml round bottomed Nunc CryoTubes (Thermo Scientific). Samples were then centrifuged at 3000 x g for 5 minutes at 4°C before removing the supernatant and freezing at -80°CC in 200 μl aliquots. Samples were analysed using multiplex magnetic bead assays from BioRad (custom 9-plex Bio-Plex Pro™ Assay) and R&D Systems (ELISA; Mouse IL-1α and IL-1β DuoSet; custom Luminex^® assay) as per manufacturers instructions.

For flow cytometry analysis, the left lung was minced into small pieces in a 15 ml polypropylene tube. The lung pieces were placed on ice in 5 ml RPMI. The samples were then transferred to a gentleMACS C-tube and incubated with Liberase TL and DNase I. The gentleMACS dissociator (Miltenyi Biotec GmbH) was used to process the samples according to the manufacturer’s instructions. Following dissociation, the lung digest samples were filtered through a 40 μm cell filter into a 50 ml polypropylene tube with ice-cold PBS used to wash through any residual sample. The samples were centrifuged at 300 x g for 5 minutes at 4°C, and the supernatant was discarded. Red blood cell lysis was performed by resuspending the cell pellet with BD Pharm Lyse (BD Biosciences Inc) for three minutes and stopped by adding ice-cold PBS. The resulting cell suspensions in FACS-Buffer (PBS with 2%BSA, 2mMEDTA) were counted and transferred to FACS tubes. Control samples were prepared by combining cell suspensions from each mouse, including a live-dead control. The cell suspensions were incubated with Fc receptor block and a fixable viability dye, followed by staining with a mixture of fluorochrome-conjugated antibodies (see Supplementary Table 1 ). Corresponding compensation (capture beads) and FMO controls were also created. All samples were analysed using the BD LSRFortessa (BD Bioscience Inc) flow cytometer equipped with 355m 405, 488, 561, and 640 nm Laser. Data analysis was performed by using FlowJo v10 software (BD Bioscience Inc). Cell populations were identified using a gating strategy adapted from Misharin et al. (2013), including CD3+ lymphocytes and NK cells (see Supplementary Figure 1 ). T-distributed stochastic neighbourhood embedding (t-SNE) analysis was performed using FlowJo v10.3 and the t-SNE plug-in The live, single-cell, CD45+ populations were selected, concatinated for each sample group (WT or gp91-/-), and down-sampled to 100,000 events.

GraphPad Prism for Mac OS X version 7.0c (GraphPad Software, La Jolla, California, USA) was used for statistical analysis and to display graphical results. Kaplan-Meier curves, analysed using Mantel-Cox log-rank analysis, were used to demonstrate survival. Comparisons between 2 groups were performed using a Student t test (normal distributed data) or the non-parametric Mann Whitney U test (non-Gaussian data). One-way ANOVA with Bonferroni post-test was used to compare >2 groups (normal distributed) or Kruskal-Wallis with Dunn’s multiple comparison (not normally distributed). p<0.05 was considered statistically significant. Error bars in graphical results represent standard error of the mean (SEM) unless otherwise stated.

Alveolar macrophages from gp91^-/- and WT mice demonstrated minimal antifungal activity against A. fumigatus following 18 hours co-incubation with resting conidia, with no significant difference between WT and gp91^-/- AMs ( Figure 1A ). In contrast, both gp91^-/- and WT AM significantly reduced the viability of A. nidulans conidia (p<0.01 for gp91^-/- AMs; p<0.001 for WT AMs) ( Figure 1A ). The anticonidial activity of activated BMDMs against A. fumigatus and A. nidulans conidia was comparable to our observations in AM, with both gp91^-/- and WT BMDM significantly reducing the viability of A. nidulans conidia (p<0.001; Figure 1B ).

We assessed if this enhanced susceptibility of A. nidulans conidia to macrophage killing could be explained by the decreased galactosaminogalactan in the A. nidulans cell wall by using A. nidulans strains overexpressing A. fumigatus Uge3 or A. nidulans UgeB. There was no significant difference observed ( Figure 1C ).

Co-incubation of WT or gp91^-/- BMDNs with Aspergillus hyphae resulted in a significant reduction in fungal metabolic activity of A. fumigatus (p<0.001 and p<0.0001 respectively) and A. nidulans hyphae (p<0.0001 for both WT and gp91^-/-). Surprisingly, co-incubation of gp91^-/- BMDN with A. fumigatus hyphae resulted in a greater reduction in metabolic activity than WT BMDN(p<0.05, Figure 2A ). However, BMDNs from gp91^-/- mice were less effective against A. nidulans hyphae than WT BMDNs (p<0.001). The overall reduction in fungal activity by gp91^-/- BMDNs was similar between the two organisms however A. nidulans hyphae were significantly more susceptible to the antifungal activity of WT BMDNs than A. fumigatus (p<0.0001).

To investigate the gp91^-/- BMDN anti-Aspergillus activity further, WT or gp91^-/- BMDNs were co-incubated with FLARE conidia. These A. fumigatus conidia emit a viability fluorophore (DsRed) that is extinguished upon conidial killing. The FLARE conidia are additionally coated in a second tracer fluorophore (AF633) to allow detection regardless of viability (Jhingran et al., 2012).

Surprisingly, gp91^-/- neutrophils were better able to kill A. fumigatus conidia than WT BMDNs, as demonstrated by the increased percentage of non-viable FLARE conidia after a 16-hour co-incubation with gp91^-/- neutrophils (p<0.01; Figure 2B ). However, this came at the expense of increased neutrophil death, with gp91^-/- neutrophils significantly more likely to die following co-incubation with FLARE conidia than WT neutrophils (p<0.0001). This increased risk of cell death was not apparent in unstimulated gp91^-/- BMDNs.

CD11b is a cell surface marker that is up regulated following neutrophil activation (Kolaczkowska and Kubes, 2013). Both WT and gp91^-/- neutrophils demonstrated a significant shift in CD11b MFI following co-incubation with FLARE conidia (p<0.0001 for both WT and gp91^-/- compared with unstimulated BMDNs). However this increase was more marked in gp91^-/- BMDNs (p<0.0001; Figure 2C ).

Significant early weight loss was seen following both A. fumigatus (p<0.0001) and A. nidulans (p<0.0001) infection in gp91^-/- mice compared with similarly infected WT mice, and was more pronounced and sustained in A. fumigatus infected gp91^-/- mice ( Figures 3C, D ). Mortality was 50% in A. fumigatus and 10% in A. nidulans infected gp91^-/- mice. None of the WT mice succumbed to infection (p=0.011 and p=0.317, resp., Figures 3A, B ).

Fungal burden assessed by quantitative culture demonstrated low numbers of CFUs with a high degree of variability (between 0 and 600 CFU/lung) in both WT and gp91-/- mice up to d7 post-infection. Cultures on d17 were negative except for gp91-/- mice infected with A. nidulans. Histology samples showed no significant inflammatory change in the lungs of WT mice infected with either A. fumigatus or A. nidulans at any time point. In contrast, and despite the reduced morbidity and mortality seen in gp91^-/- mice infected with A. nidulans, pulmonary pathology was of comparable severity in both infections, with extensive pulmonary infiltrates at day 7 and evidence of early granuloma formation at 17 days in both A. fumigatus and A. nidulans infection ( Figure 4 ).

There was some heterogeneity in pulmonary lesions within individual mice. However, in gp91^-/- mice infected with A. fumigatus lesions were typically well circumscribed, with a densely packed central neutrophil core, a surrounding layer of epitheliod cells, and an outer layer of more loosely organised macrophages, polymorphonuclear cells and lymphocytes ( Figures 4A–F ). The lesions present in mice infected with A. nidulans similarly had dense areas of neutrophil accumulation but these were less well delineated and a surrounding epitheliod cell layer was not evident until later in infection.

The utilisation of t-distributed stochastic neighbourhood embedding (tSNE) to objectively analyse all CD45+ cell populations present in lung digest samples allows simultaneous visualisation of all analysed pulmonary cell populations in a two-dimensional map ( Figure 5 ). Neutrophils clearly represent the predominant CD45+ cells present in the gp91^-/- lung at each time point following both A. fumigatus and A. nidulans infection. This is in contrast to WT mice where, similar to uninfected lung samples from both WT and gp91^-/- mice, T-cells remain the most prevalent cell type ( Figure 5 ).

Neutrophil numbers were significantly increased in gp91-/- lung digests within 24 hrs of infection, and at all subsequent time points in both infections compared with similarly infected WT mice and uninfected gp91-/- mice ( Figures 6A, B ). Neutrophil recruitment persisted over the 7 days following infection with no evidence of resolution in neutrophilic infiltration in gp91-/- mice. In contrast, there was no significant increase in neutrophil proportion or cell count in WT mice at any time following infection with either organism ( Figures 6A, B ; Supplementary Figure 2 ).

Neutrophil populations were phenotypically different in gp91-/- mice compared with WT mice. Similar to our findings during the in vitro experiments, neutrophil cell surface expression of the β2-integrin CD11b, was increased in uninfected gp91-/- mice compared with WT mice (16715 ± 436.6 versus 12271 ± 232.1 respectively, p<0.0001) and at all observed time points following both A. fumigatus and A. nidulans infection compared to similarly infected WT mice (p<0.0001 at 24 hours and 3 days following both A. fumigatus and A. nidulans infection; p<0.0001 at 7 days following A. nidulans infection and; p<0.05 at 7 days following A. fumigatus infection).

The proportion of AM present in gp91^-/- lungs was significantly lower than similarly infected WT mice at each time point following both A. fumigatus and A. nidulans infection ( Figure 6C ; Supplementary Figure 3 ). Prior to infection, gp91^-/- mice had reduced AM populations compared with WT mice (p<0.05). However both WT and gp91^-/- mice had significant reductions in AM populations following Aspergillus infection when compared with corresponding healthy lung samples. This reduction persisted over the seven days following infection (p<0.0001 in gp91-/- mice at d7 following A. fumigatus and A. nidulans infection compared with uninfected samples) and is also clearly evident on tSNE analysis ( Figure 5 ).

A clear phenotypic shift in CD11b expression in gp91-/- AM was observed following both infections, with these classically CD11b negative cells expressing increasing levels of the CD11b integrin ( Figure 6E ). This could not be accounted for by baseline differences in CD11b expression between uninfected WT and gp91-/- mice (CD11b MFI 236 ± 6 versus 269 ± 18 respectively, p = ns).

Alongside the reduction in resident AM populations following A. fumigatus and A. nidulans infection, pro-inflammatory macrophage populations were recruited to the gp91-/- lung. In A. fumigatus infected mice these infiltrating macrophages were predominantly interstitial macrophages (shown in purple on tSNE analysis, Figure 5 ), with subtle shifts in Ly6C^hi and Ly6C^lo monocyte/macrophage populations (shown in yellow and black respectively on tSNE plots, Figure 5 ). Within 24 hours of A. fumigatus infection in gp91-/- mice interstitial macrophage populations had increased significantly compared with uninfected gp91-/- mice and similarly infected WT mice (p<0.0001, Figure 6D ). In contrast, following A. nidulans infection there was no significant change in the gp91-/- pulmonary interstitial macrophage population compared with uninfected gp91-/- mice and similarly infected WT mice ( Figure 6D ).

Following A. fumigatus infection the increase in interstitial macrophages was associated with almost complete loss of CD11b+ dendritic cells (DCs) in the gp91-/- lung ( Figures 5 , 6C–E ; p<0.0001 at all time points following infection compared with uninfected gp91-/- mice). In A. nidulans infection a significant reduction in CD11b+ DCs was observed in the gp91-/- lung (p<0.0001 at all time points post-infection compared with uninfected samples), however, a small population did remain evident on FACS analysis ( Figure 7 ). Pulmonary CD103+ DCs were also reduced in gp91-/- mice at d3 and d7 following both A. fumigatus and A. nidulans infection ( Figures 7A, B ). DC populations did not change significantly in WT mice following either infection.

T-cells represented a significantly smaller proportion of CD45+ cells in the uninfected gp91-/- lung compared with uninfected WT mice (p<0.05, Figure 8A ). Following infection with either A. fumigatus or A. nidulans the expansion in inflammatory macrophage and neutrophil populations resulted in significant proportionate reductions in T-cell populations in the gp91-/- lung compared with uninfected gp91-/- mice and similarly infected WT mice (p<0.0001 for all time points) whereas the proportion of CD3+ cells did not change in WT mice infected with A. nidulans. In contrast to the significant reduction in the CD3+ cell populations in gp91-/- mice following A. fumigatus infection, there was a significant expansion in CD3+ cells on d3 following A. fumigatus infection in WT mice compared with uninfected WT mice (p<0.001). Comparing A. nidulans and A. fumigatus infected gp91-/- mice, a comparatively greater proportion of CD3+ cells remained in the lungs of mice infected with A. fumigatus ( Figure 8A ). Natural killer (NK) cell populations were proportionately reduced in the gp91-/- lung following both A. fumigatus and A. nidulans infection. This was more evident following A. nidulans infection, however NK cell populations were significantly reduced compared to similarly infected WT and uninfected gp91-/- mice following both infections. There was no significant change in NK cell populations in WT mice following either infection ( Figure 8B ).

Wild-type mice developed eosinophilia, with significant increases in pulmonary eosinophils at 24 hours (p<0.05 and p<0.0001 for A. fumigatus and A. nidulans respectively), 3 days (p<0.0001 and p<0.0001), and 7 days (p<0.0001 and p<0.05) compared to uninfected WT mice. In both infections, maximum eosinophil response occurred on d3 and was resolving by d7 post-infection ( Figure 8C ). In contrast, there was no evidence of eosinophil recruitment in gp91-/- mice infected with A. fumigatus and the eosinophil population was almost completely absent on flow cytometry ( Figure 5 ). Following A. nidulans infection, gp91-/- mice did demonstrate evidence of eosinophil recruitment at d3 post-infection compared with uninfected gp91-/- mice (p<0.001), however the response was significantly lower than that mounted by WT mice (p<0.0001, Figure 8C ).

A rapidly induced, and steadily increasing, IL-1 response was observed in gp91^-/- mice over the 17 days following infection with both A. fumigatus and A. nidulans. In gp91^-/- mice, pulmonary IL-1β in particular increased over 100-fold by day 17 following both A. fumigatus (25024 ± 3169 pg/ml, p<0.0001) and A. nidulans infection (24051 ± 6305 pg/ml, p<0.0001) compared with uninfected samples (150 ± 78 pg/ml) and showed no evidence of resolution ( Figure 9B ).

Pulmonary IL-1α increased steadily over the 7 days post-infections, increasing over 100-fold compared with uninfected mice (p<0.0001 for both infections), and peaked at d7 ( Figure 9A ). There was no significant difference in IL-1α or IL-1β response in gp91^-/- mice between A. nidulans and A. fumigatus infection, apart from on d3 following A. nidulans infection when IL-1β, but not IL-1α, was significantly lower (p<0.05) in mice infected with A. nidulans compared with A. fumigatus. However, by day 7 IL-1β concentrations resumed their parity with A. fumigatus infected gp91^-/- mice ( Figure 9B ).

IL-6 and KC levels peaked at 24 hrs and then gradually subsided, but remained elevated 17 days post-infection ( Figures 9C, D ). The initial magnitude of the KC response was similar between the two infections but subsequently dropped at d3 following A. nidulans infection, resulting in significantly lower KC (p<0.0001) compared with A. fumigatus infected mice at this time point. A drop in IL-6 was also evident at d3 post-infection in gp91-/- mice infected with A. nidulans, although the initial peak in IL-6 following A. nidulans infection was significantly lower than that seen in A. fumigatus infected mice (p<0.001). The gp91^-/- G-CSF and GM-CSF responses paralleled IL-6 and KC peaking at 24 hrs post-infection and subsequently resolving ( Figures 9K, L ). Whereas the CXCL2 response mirrored that of IL-1, increasing steadily over the first 7d post-infection ( Figure 9M ). No significant difference in G-CSF, GM-CSF or CXCL2 response was seen between A. nidulans and A. fumigatus infected gp91^-/- mice ( Figures 9K–M ). Significant increases were also seen in CCL2 (also known as monocyte chemoattractant protein (MCP)-1; p<0.0001 and p<0.01 for A. nidulans and A. fumigatus respectively), CCL3 (also known as macrophage inflammatory protein-1-alpha (MIP-1α); p<0.0001 for both infections) and CCL4 (also known as macrophage inflammatory protein-1-beta (MIP-1β); p<0.0001 for A. nidulans and p<0.01 for A. fumigatus) within 24 hrs of infection. The pattern of chemokine secretion was not significantly different between A. fumigatus and A. nidulans infection ( Figures 9N-P ). The IFN-γ response was minimal following both A. fumigatus and A. nidulans infection in gp91^-/- and WT mice. However, the increase in IFN-γ did reach significance at day 7 following A. nidulans infection in gp91^-/- mice compared with uninfected gp91^-/- mice (p<0.001) and was also significantly greater than similarly infected WT mice at this time point (p<0.001, Figure 9H ).

Pulmonary IL-17 concentrations were significantly increased in gp91^-/- mice at d7 following both A. fumigatus (p<0.0001) and A. nidulans (p<0.0001) infection compared with uninfected gp91^-/- mice and similarly infected WT mice (p<0.0001 for both infections, Figure 9I ). However, the IL-17 peak following A. nidulans infection was significantly lower than that following A. fumigatus infection (p<0.0001). A. fumigatus also induced significantly more of the Th-17 sustaining cytokine, IL-23, in gp91^-/- lung homogenates than A. nidulans (p<0.001, Figure 9J ) suggesting A. fumigatus may preferentially induce a Th-17 promoting environment in the gp91^-/- lung.

Wild-type mice showed a significant pulmonary IL-12 response at 3 days (p<0.01, Figure 9E ) following A. nidulans infection. This response was significantly greater than that seen in similarly infected gp91^-/- lung homogenates (p<0.05). In addition, A. nidulans infection induced significantly more TNF-α than A. fumigatus at d3 and d7 post-infection (p<0.0001 and p<0.05 respectively) in WT mice. Similarly, in gp91-/- mice, A. nidulans induced significantly more TNF-α than A. fumigatus (p<0.01 at d3 and p<0.0001 at d7 post-infection). However, the peak in TNF-α in gp91-/- mice following A. nidulans infection appeared relatively delayed compared with WT mice, with maximum TNF-α seen at 7 days (784 ± 237 pg/ml) compared with a 3 days in WT mice (963 ± 293 pg/ml, Figure 9F ). A gradual increase in IL-10 in gp91^-/- lung homogenates following A. fumigatus infection was observed reaching significance at d17 post-infection compared to WT mice (p<0.05). Both WT and gp91^-/- mice showed a small, gradual increase in pulmonary IL-10 following A. nidulans infection but this did not reach statistical significance ( Figure 9G ).