The Metabolic Cytokine Adiponectin Inhibits Inflammatory Lung Pathology in Invasive Aspergillosis.

Systemic immunity and metabolism are coregulated by soluble factors, including the insulin-regulating adipose tissue cytokine adiponectin. How these factors impact detrimental inflammatory responses during fungal infection remains unknown. In this study, we observed that mortality, fungal burden, and tissue histopathology were increased in adiponectin-deficient mice in a neutropenic model of invasive aspergillosis. Lung RNA sequencing, quantitative RT-PCR, and subsequent pathway analysis demonstrated activation of inflammatory cytokine pathways with upstream regulation by IL-1 and TNF in adiponectin-deficient mice with decreased/inhibited anti-inflammatory genes/pathways, suggesting broad cytokine-mediated pathology along with ineffective fungal clearance. Quantitative RT-PCR analysis confirmed increased transcription of IL-1a, IL-6, IL-12b, IL-17A/F, and TNF in adiponectin-deficient mice at early time points postinfection, with a specific increase in intracellular TNF in alveolar macrophages. Although eosinophil recruitment and activation were increased in adiponectin-deficient mice, mortality was delayed, but not decreased, in mice deficient in both adiponectin and eosinophils. Interestingly, neutrophil depletion was required for increased inflammation in adiponectin-deficient mice in response to swollen/fixed conidia, suggesting that immune suppression enhances detrimental inflammation, whereas invasive fungal growth is dispensable. Our results suggest that adiponectin inhibits excessive lung inflammation in invasive aspergillosis. Our study has therefore identified the adiponectin pathway as a potential source for novel therapeutics in immune-compromised patients with detrimental immunity to invasive fungal infection.

Caspofungin Increases Fungal Chitin and Eosinophil and γδ T Cell-Dependent Pathology in Invasive Aspergillosis.

The polysaccharide-rich fungal cell wall provides pathogen-specific targets for antifungal therapy and distinct molecular patterns that stimulate protective or detrimental host immunity. The echinocandin antifungal caspofungin inhibits synthesis of cell wall ß-1,3-glucan and is used for prophylactic therapy in immune-suppressed individuals. However, breakthrough infections with fungal pathogen Aspergillus fumigatus are associated with caspofungin prophylaxis. In this study, we report in vitro and in vivo increases in fungal surface chitin in A. fumigatus induced by caspofungin that was associated with airway eosinophil recruitment in neutropenic mice with invasive pulmonary aspergillosis (IA). More importantly, caspofungin treatment of mice with IA resulted in a pattern of increased fungal burden and severity of disease that was reversed in eosinophil-deficient mice. Additionally, the eosinophil granule proteins major basic protein and eosinophil peroxidase were more frequently detected in the bronchoalveolar lavage fluid of lung transplant patients diagnosed with IA that received caspofungin therapy when compared with azole-treated patients. Eosinophil recruitment and inhibition of fungal clearance in caspofungin-treated mice with IA required RAG1 expression and γδ T cells. These results identify an eosinophil-mediated mechanism for paradoxical caspofungin activity and support the future investigation of the potential of eosinophil or fungal chitin-targeted inhibition in the treatment of IA.

Eosinophils are recruited in response to chitin exposure and enhance Th2-mediated immune pathology in Aspergillus fumigatus infection.

In patients infected with the fungus Aspergillus fumigatus, Th1 responses are considered protective, while Th2 responses are associated with increased morbidity and mortality. How host-pathogen interactions influence the development of these protective or detrimental immune responses is not clear. We compared lung immune responses to conidia from two fungal isolates that expressed different levels of the fungal cell wall component chitin. We observed that repeated aspirations of the high-chitin-expressing isolate Af5517 induced increased airway eosinophilia in the lungs of recipient mice compared to the level of eosinophilia induced by isolate Af293. CD4(+) T cells in the bronchoalveolar lavage fluid (BALF) of Af5517-aspirated mice displayed decreased gamma interferon secretion and increased interleukin-4 transcription. In addition, repeated aspirations of Af5517 induced lung transcription of the Th2-associated chemokines CCL11 (eotaxin-1) and CCL22 (macrophage-derived chemokine). Eosinophil recruitment in response to conidial aspiration was correlated with the level of chitin exposure during germination and was decreased by constitutive lung chitinase expression. Moreover, eosinophil-deficient mice subjected to multiple aspirations of Af5517 prior to neutrophil depletion and infection exhibited decreased morbidity and fungal burden compared to the levels of morbidity and fungal burden found in wild-type mice. These results suggest that exposure of chitin in germinating conidia promotes eosinophil recruitment and ultimately induces Th2-skewed immune responses after repeated aspiration. Furthermore, our results suggest that eosinophils should be examined as a potential therapeutic target in patients that mount poorly protective Th2 responses to A. fumigatus infection.

Adiponectin pathway activation dampens inflammation and enhances alveolar macrophage fungal killing via LC3-associated phagocytosis.

Although innate immunity is critical for antifungal host defense against the human opportunistic fungal pathogen Aspergillus fumigatus, potentially damaging inflammation must be controlled. Adiponectin (APN) is an adipokine produced mainly in adipose tissue that exerts anti-inflammatory effects in adipose-distal tissues such as the lung. We observed 100% mortality and increased fungal burden and inflammation in neutropenic mice with invasive aspergillosis (IA) that lack APN or the APN receptors AdipoR1 or AdipoR2. Alveolar macrophages (AMs), early immune sentinels that detect and respond to lung infection, express both receptors, and APN-/- AMs exhibited an inflammatory/M1 phenotype that was associated with decreased fungal killing. Pharmacological stimulation of AMs with AdipoR agonist AdipoRon partially rescued deficient killing in APN-/- AMs that was dependent on both receptors. Finally, APN-enhanced fungal killing was associated with increased activation of the non-canonical LC3 pathway of autophagy. Thus, our study identifies a novel role for APN in LC3-mediated killing of A. fumigatus.

Regulation of lung inflammation by adiponectin.

Adiponectin is an insulin sensitizing hormone that also plays a role in the regulation of inflammation. Although adiponectin can exert pro-inflammatory effects, more studies have reported anti-inflammatory effects, even in non-adipose tissues such as the lung. Obesity is considered an inflammatory disease, is a risk factor for lung diseases, and is associated with decreased levels of plasma adiponectin. The results of recent studies have suggested that adiponectin exerts anti-inflammatory activity in chronic obstructive pulmonary disease, asthma and invasive fungal infection. The signaling receptors of adiponectin, AdipoR1 and AdipoR2, are expressed by epithelial cells, endothelial cells, and immune cells in the lung. In this mini-review, we discuss the anti-inflammatory mechanisms of adiponectin in lung cells and tissues.

Survival Factor A (SvfA) Contributes to Aspergillus nidulans Pathogenicity.

Survival factor A (SvfA) in Aspergillus nidulans plays multiple roles in growth and developmental processes. It is a candidate for a novel VeA-dependent protein involved in sexual development. VeA is a key developmental regulator in Aspergillus species that can interact with other velvet-family proteins and enter into the nucleus to function as a transcription factor. In yeast and fungi, SvfA-homologous proteins are required for survival under oxidative and cold-stress conditions. To assess the role of SvfA in virulence in A. nidulans, cell wall components, biofilm formation, and protease activity were evaluated in a svfA-gene-deletion or an AfsvfA-overexpressing strain. The svfA-deletion strain showed decreased production of ß-1,3-glucan in conidia, a cell wall pathogen-associated molecular pattern, with a decrease in gene expression for chitin synthases and ß-1,3-glucan synthase. The ability to form biofilms and produce proteases was reduced in the svfA-deletion strain. We hypothesized that the svfA-deletion strain was less virulent than the wild-type strain; therefore, we performed in vitro phagocytosis assays using alveolar macrophages and analyzed in vivo survival using two vertebrate animal models. While phagocytosis was reduced in mouse alveolar macrophages challenged with conidia from the svfA-deletion strain, the killing rate showed a significant increase with increased extracellular signal-regulated kinase ERK activation. The svfA-deletion conidia infection reduced host mortality in both T-cell-deficient zebrafish and chronic granulomatous disease mouse models. Taken together, these results indicate that SvfA plays a significant role in the pathogenicity of A. nidulans.

The role of FIBCD1 in response to Aspergillus fumigatus in lung epithelial cells.

Chitin, a polysaccharide, is ubiquitously found in nature and has been known to be an active immunogen in mammals, and interacts with Toll-like, mannose and glucan receptors, to induce cytokine and chemokine secretions. FIBCD1 is a tetrameric type II transmembrane endocytic vertebrate receptor that binds chitin, is found in human lung epithelium and modulates lung epithelial inflammatory responses to A. fumigatus cell wall polysaccharides. We previously reported the detrimental role of FIBCD1 in a murine model of pulmonary invasive aspergillosis. However, the effect that chitin and chitin-containing A. fumigatus conidia exerts on lung epithelium following exposure through FIBCD1 is not yet fully explored. Using both in vitro and in vivo strategies, we examined how lung and lung epithelial gene expression are modified after exposure to fungal conidia or chitin fragments in the presence or absence of FIBCD1. FIBCD1 expression was associated with a decrease in inflammatory cytokines with increasing size of chitin (dimer-oligomer). Thus, our results demonstrate that FIBCD1 expression modulates cytokine and chemokine expression in response to A. fumigatus conidia that is modified by the presence of chitin particles.

FIBCD1 Deficiency Decreases Disease Severity in a Murine Model of Invasive Pulmonary Aspergillosis.

Aspergillus fumigatus is a ubiquitous mold associated with the development of pulmonary diseases that include invasive pulmonary aspergillosis (IPA), an often fatal opportunistic infection. FIBCD1 is a transmembrane endocytic membrane receptor widely expressed on human epithelium. Although FIBCD1 was previously shown to bind chitin, modulate fungal colonization of the gut, and inhibit intestinal inflammation, the role of FIBCD1 in the context of lung fungal infection remains unknown. In this study, we observed that mortality, fungal burden, and tissue histopathology were decreased in the absence of FIBCD1 in murine IPA. Quantitative RT-PCR analyses demonstrated decreased inflammatory cytokines in the lungs of neutrophil-depleted FIBCD1-/- mice with IPA, when compared with wild-type controls. In contrast, inflammatory cytokines were increased in immune-competent FIBCD1-/- mice after fungal aspiration, suggesting that the presence of neutrophils is associated with cytokine modulation. In contrast to the clear IPA phenotype, FIBCD1-/- mice with systemic infection or bleomycin-induced lung injury exhibited similar morbidity and mortality when compared with their wild-type counterparts. Thus, our study identifies a detrimental role of FIBCD1 in IPA.

Murine models of airway fungal exposure and allergic sensitization.

Inhalation of common indoor filamentous fungi has been associated with the induction or exacerbation of allergic respiratory disease. The understanding of fungal inhalation and allergic sensitization has significantly advanced with the use of small animal models, especially mouse models. Numerous studies have employed different animal exposure and sensitization techniques, each with inherent advantages and disadvantages that are addressed in this review. In addition, most studies involve exposure of animals to fungal spores or spore extracts while neglecting the influence of hyphal or subcellular fragment exposures. Recent literature examining the potential for hyphae and fungal fragments to induce or exacerbate allergy is discussed. Innate immune recognition of fungal elements and their contribution to lung allergic inflammation in animal models are also reviewed. Though physical properties of fungi play an important role following exposure, host immune development is also critical in airway inflammation and allergy. We discuss the importance of environmental factors that influence early immune development and subsequent susceptibility to allergy. Murine studies that examine the role of intestinal microflora and prenatal or early life environmental factors that promote allergic sensitization are also evaluated. Future studies will require animal models that accurately reflect natural fungal exposures and identify environmental factors that influence immune development and thus promote respiratory fungal allergy and disease.

Reciprocal Inhibition of Adiponectin and Innate Lung Immune Responses to Chitin and Aspergillus fumigatus.

Chitin is a structural biopolymer found in numerous organisms, including pathogenic fungi, and recognized as an immune-stimulating pathogen associated molecular pattern by pattern recognition molecules of the host immune system. However, programming and regulation of lung innate immunity to chitin inhalation in the context of inhalation of fungal pathogens such as Aspergillus fumigatus is complex and our understanding incomplete. Here we report that the systemic metabolism-regulating cytokine adiponectin is decreased in the lungs and serum of mice after chitin inhalation, with a concomitant decrease in surface expression of the adiponectin receptor AdipoR1 on lung leukocytes. Constitutive lung expression of acidic mammalian chitinase resulted in decreased inflammatory cytokine gene expression and neutrophil recruitment, but did not significantly affect lung adiponectin transcription. Exogenous recombinant adiponectin specifically dampened airway chitin-mediated eosinophil recruitment, while adiponectin deficiency resulted in increased airway eosinophils. The presence of adiponectin also resulted in decreased CCL11-mediated migration of bone marrow-derived eosinophils. In contrast to purified chitin, aspiration of viable conidia from the high chitin-expressing A. fumigatus isolate Af5517 resulted in increased neutrophil recruitment and inflammatory cytokine gene expression in adiponectin-deficient mice, while no significant changes were observed in response to the isolate Af293. Our results identify a novel role for the adiponectin pathway in inhibition of lung inflammatory responses to chitin and A. fumigatus inhalation.

Maturation and localization of macrophages and microglia during infection with a neurotropic murine coronavirus.

Macrophages and microglia are critical in the acute inflammatory response and act as final effector cells of demyelination during chronic infection with the neutrotropic MHV-JHM strain of mouse hepatitis virus (MHV-JHM). Herein, we show that "immature" F4/80(+)Ly-6C(hi) monocytes are the first cells, along with neutrophils, to enter the MHV-JHM-infected central nervous system (CNS). As the infection progresses, macrophages in the CNS down-regulate expression of Ly-6C and CD62L, consistent with maturation, and a higher frequency express CD11c, a marker for dendritic cells (DCs). Microglia also express CD11c during this phase of the infection. CD11c(+) macrophages in the infected CNS exhibit variable properties of immature antigen-presenting cells (APCs), with modestly increased CD40 and MHC expression, and equivalent potent antigen uptake when compared with CD11c(-) macrophages. Furthermore, CDllc(+) and F4/80(+) macrophages and microglia are localized to areas of demyelination, in some instances directly associated with damaged axons. These results suggest that chronic CNS infection results in the appearance of CD11c-expressing macrophages from the blood that exhibit properties of immature APCs, are closely associated with areas of demyelination, and may act as final effectors of myelin destruction.

Role of IFN-gamma responsiveness in CD8 T-cell-mediated viral clearance and demyelination in coronavirus-infected mice.

Immunocompetent, but not RAG1(-/-) mice infected with MHV-JHM develop demyelination. Transferred CD8 T cell-enriched splenocytes reconstitute demyelination, and this ability is dependent on donor IFN-gamma. We used IFN-gammaR1(-/-) mice to examine the target of IFN-gamma in CD8 T cell-mediated demyelination. In IFN-gammaR1(-/-)RAG1(-/-) recipients, demyelination is decreased, but not eliminated, while viral titers are significantly increased when compared to IFN-gammaR1(+/+)RAG1(-/-) recipients. IFN-gammaR1(-/-) CD8 T cells retain virus-specific effector function regardless of IFN-gammaR1 expression. Although IFN-gammaR1 responsiveness is critical for maximal demyelination, increased levels of infectious virus coupled with adoptive transfer of CD8 T cells may result in myelin destruction independent of IFN-gammaR1 expression.

Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis.

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse models of pulmonary fungal infection. Although multiple methods are used to assess fungal burden, quantitative polymerase chain reaction (qPCR) of fungal DNA has emerged as a technique with several advantages over previous culture-based methods. Currently, a comprehensive assessment of lung pathology, leukocyte recruitment, fungal burden, and gene expression in mice with invasive aspergillosis (IA) necessitates the use of a significant number of experimental and control animals. Here the quantification of lung histological staining to determine fungal burden using a reduced number of animals was examined in detail. Lung sections were stained to identify fungal structures with Gomori's modified methanamine silver (GMS) staining. Images were taken from the GMS-stained sections from 4 discrete fields of each formalin-fixed paraffin-embedded lung. The GMS stained areas within each image were quantified using an image analysis program, and from this quantification, the mean percentage of stained area was determined for each sample. Using this strategy, eosinophil-deficient mice exhibited decreased fungal burden and disease with caspofungin therapy, while wild-type mice with IA did not improve with caspofungin. Similarly, fungal burden in mice lacking γδ T cells were also improved by caspofungin, as measured by qPCR and GMS quantification. GMS quantification is therefore introduced as a method for the determination of relative lung fungal burden that may ultimately reduce the quantity of experimental animals required for comprehensive studies of invasive aspergillosis.

FIBCD1 Binds Aspergillus fumigatus and Regulates Lung Epithelial Response to Cell Wall Components.

Aspergillus fumigatus (A. fumigatus) is a ubiquitous fungus of clinical importance associated with development of various pulmonary diseases and allergic hypersensitivity reactions. It is protected against environmental stress by a cell wall that contains polysaccharides such as chitin. We previously demonstrated that fibrinogen C domain-containing protein 1 (FIBCD1) is a membrane-bound protein that binds chitin through a conserved S1 binding site and is expressed in intestinal epithelium and salivary glands. Here, we further localized FIBCD1 protein expression at the surface of bronchial and alveolar human lung epithelium, observed recognition of A. fumigatus cell wall with S1 site-independent recognition. We observed FIBCD1-mediated suppression of IL-8 secretion, mucin production, and transcription of genes associated with airway inflammation and homeostasis in FIBCD1-transfected lung epithelial cells. These modulations were generally enforced by stimulation with A. fumigatus cell wall polysaccharides. In parallel, we demonstrated a FIBCD1-mediated modulation of IL-8 secretion induced by TLR2,-4, and -5. Collectively, our findings support FIBCD1 as a human lung epithelial pattern recognition receptor that recognizes the complex A. fumigatus cell wall polysaccharides and modulates the lung epithelial inflammatory response by suppressing inflammatory mediators and mucins.

Pathogenesis of acute and chronic central nervous system infection with variants of mouse hepatitis virus, strain JHM.

Infection of mice with variants of mouse hepatitis virus, strain JHM (MHV-JHM), provide models of acute and chronic viral infection of the central nervous system (CNS). Through targeted recombination and reverse genetic manipulation, studies of infection with MHV-JHM variants have identified phenotypic differences and examined the effects of these differences on viral pathogenesis and anti-viral host immune responses. Studies employing recombinant viruses with a modified spike (S) glycoprotein of MHV-JHM have identified the S gene as a major determinant of neurovirulence. However, the association of S gene variation and neurovirulence with host ability to generate anti-viral CD8 T cell responses is not completely clear. Partially protective anti-viral immune responses may result in persistent infection and chronic demyelinating disease characterized by myelin removal from axons of the CNS and associated with dense macrophage/microglial infiltration. Demyelinating disease during MHV-JHM infection is immune-mediated, as mice that lack T lymphocytes fail to develop disease despite succumbing to encephalitis with high levels of infectious virus in the CNS. However, the presence of T lymphocytes or anti-viral antibody can induce disease in infected immunodeficient mice. The mechanisms by which these immune effectors induce demyelination share an ability to activate and recruit macrophages and microglia, thus increasing the putative role of these cells in myelin destruction.

Lung eosinophil recruitment in response to Aspergillus fumigatus is correlated with fungal cell wall composition and requires γδ T cells.

The differential recognition of fungal cell wall polysaccharides that program innate and adaptive immunity to the human opportunistic fungal pathogen Aspergillus fumigatus has been a focus of considerable interest. In a mouse model of fungal conidia aspiration, decreased relative levels of cell wall core carbohydrates ß-1,3-glucan to chitin in A. fumigatus isolates and mutant strains were correlated with increased airway eosinophil recruitment. In addition, an increase in fungal surface chitin exposure induced by the ß-1,3-glucan synthesis-targeting drug caspofungin was associated with increased murine airway eosinophil recruitment after a single challenge of conidia. The response to increased A. fumigatus chitin was associated with increased transcription of IL-17A after a single aspiration, although this cytokine was not required for eosinophil recruitment. Rather, both RAG1 and γδ T cells were required, suggesting that this subset of innate-like lymphocytes may be an important regulator of potentially detrimental type 2 immune responses to fungal inhalation and infection.

Co-recognition of ß-glucan and chitin and programming of adaptive immunity to Aspergillus fumigatus.

The prevalence of fungal infections has increased concurrently with increases in immune suppressive therapies and susceptible individuals. Opportunistic fungal pathogens such as Aspergillus fumigatus may exhibit invasive growth and dissemination resulting in a high mortality rate. Herein, we discuss how immune sensing of germination directs innate immune responses and programs adaptive responses that could promote or impair immune protection during periods of heightened susceptibility. In infected individuals, Th1 responses are the most protective, while Th2 responses lead to poor disease outcomes. In particular, the roles of ß-glucan and chitin co-recognition in shaping Th1- and Th2-type immunity to fungal infection are explored. We discuss how fungal responses to environmental stresses could result in decreased immune protection from infection, particularly in response to anti-fungal drugs that target ß-glucan synthesis. Furthermore, we consider how experimental modulation of host-pathogen interactions might elucidate the mechanisms of protective and detrimental immunity and the potential of current and future studies to promote the development of improved treatments for patients that respond poorly to existing therapies.

Isolate-dependent growth, virulence, and cell wall composition in the human pathogen Aspergillus fumigatus.

The ubiquitous fungal pathogen Aspergillus fumigatus is a mediator of allergic sensitization and invasive disease in susceptible individuals. The significant genetic and phenotypic variability between and among clinical and environmental isolates are important considerations in host-pathogen studies of A. fumigatus-mediated disease. We observed decreased radial growth, rate of germination, and ability to establish colony growth in a single environmental isolate of A. fumigatus, Af5517, when compared to other clinical and environmental isolates. Af5517 also exhibited increased hyphal diameter and cell wall ß-glucan and chitin content, with chitin most significantly increased. Morbidity, mortality, lung fungal burden, and tissue pathology were decreased in neutropenic Af5517-infected mice when compared to the clinical isolate Af293. Our results support previous findings that suggest a correlation between in vitro growth rates and in vivo virulence, and we propose that changes in cell wall composition may contribute to this phenotype.

Pulmonary immune responses to Aspergillus fumigatus in an immunocompetent mouse model of repeated exposures.

Aspergillus fumigatus is a filamentous fungus that produces abundant pigmented conidia. Several fungal components have been identified as virulence factors, including melanin; however, the impact of these factors in a repeated exposure model resembling natural environmental exposures remains unknown. This study examined the role of fungal melanin in the stimulation of pulmonary immune responses using immunocompetent BALB/c mice in a multiple exposure model. It compared conidia from wild-type A. fumigatus to two melanin mutants of the same strain, Δarp2 (tan) or Δalb1 (white). Mass spectrometry-based analysis of conidial extracts demonstrated that there was little difference in the protein fingerprint profiles between the three strains. Field emission scanning electron microscopy demonstrated that the immunologically inert Rodlet A layer remained intact in melanin-deficient conidia. Thus, the primary difference between the strains was the extent of melanization. Histopathology indicated that each A. fumigatus strain induced lung inflammation, regardless of the extent of melanization. In mice exposed to Δalb1 conidia, an increase in airway eosinophils and a decrease in neutrophils and CD8(+) IL-17(+) (Tc17) cells were observed. Additionally, it was shown that melanin mutant conidia were more rapidly cleared from the lungs than wild-type conidia. These data suggest that the presence of fungal melanin may modulate the pulmonary immune response in a mouse model of repeated exposures to A. fumigatus conidia.

A murine inhalation model to characterize pulmonary exposure to dry Aspergillus fumigatus conidia.

Most murine models of fungal exposure are based on the delivery of uncharacterized extracts or liquid conidia suspensions using aspiration or intranasal approaches. Studies that model exposure to dry fungal aerosols using whole body inhalation have only recently been described. In this study, we aimed to characterize pulmonary immune responses following repeated inhalation of conidia utilizing an acoustical generator to deliver dry fungal aerosols to mice housed in a nose only exposure chamber. Immunocompetent female BALB/cJ mice were exposed to conidia derived from Aspergillus fumigatus wild-type (WT) or a melanin-deficient (Δalb1) strain. Conidia were aerosolized and delivered to mice at an estimated deposition dose of 1×105 twice a week for 4 weeks (8 total). Histopathological and immunological endpoints were assessed 4, 24, 48, and 72 hours after the final exposure. Histopathological analysis showed that conidia derived from both strains induced lung inflammation, especially at 24 and 48 hour time points. Immunological endpoints evaluated in bronchoalveolar lavage fluid (BALF) and the mediastinal lymph nodes showed that exposure to WT conidia led to elevated numbers of macrophages, granulocytes, and lymphocytes. Importantly, CD8+ IL17+ (Tc17) cells were significantly higher in BALF and positively correlated with germination of A. fumigatus WT spores. Germination was associated with specific IgG to intracellular proteins while Δalb1 spores elicited antibodies to cell wall hydrophobin. These data suggest that inhalation exposures may provide a more representative analysis of immune responses following exposures to environmentally and occupationally prevalent fungal contaminants.