Aspergillus fumigatus Drives Tissue Damage via Iterative Assaults upon Mucosal Integrity and Immune Homeostasis.

The human lung is constantly exposed to Aspergillus fumigatus spores, the most prevalent worldwide cause of fungal respiratory disease. Pulmonary tissue damage is a unifying feature of Aspergillus-related diseases; however, the mechanistic basis of damage is not understood. In the lungs of susceptible hosts, A. fumigatus undergoes an obligatory morphological switch involving spore germination and hyphal growth. We modeled A. fumigatus infection in cultured A549 human pneumocytes, capturing the phosphoactivation status of five host signaling pathways, nuclear translocation and DNA binding of eight host transcription factors, and expression of nine host response proteins over six time points encompassing exposures to live fungus and the secretome thereof. The resulting data set, comprised of more than 1,000 data points, reveals that pneumocytes mount differential responses to A. fumigatus spores, hyphae, and soluble secreted products via the NF-κB, JNK, and JNK + p38 pathways, respectively. Importantly, via selective degradation of host proinflammatory (IL-6 and IL-8) cytokines and growth factors (FGF-2), fungal secreted products reorchestrate the host response to fungal challenge as well as driving multiparameter epithelial damage, culminating in cytolysis. Dysregulation of NF-κB signaling, involving sequential stimulation of canonical and noncanonical signaling, was identified as a significant feature of host damage both in vitro and in a mouse model of invasive aspergillosis. Our data demonstrate that composite tissue damage results from iterative (repeated) exposures to different fungal morphotypes and secreted products and suggest that modulation of host responses to fungal challenge might represent a unified strategy for therapeutic control of pathologically distinct types of Aspergillus-related disease.

Variation in Host Resistance to Blastomyces dermatitidis: Potential Use of Genetic Reference Panels and Advances in Immunophenotyping of Diverse Mouse Strains.

Host genetic determinants that underpin variation in susceptibility to systemic fungal infection are poorly understood. Genes responsible for complex traits can be identified by correlating variation in phenotype with allele in founder strains of wild mice with known genetic variation, assembled in genetic reference panels. In this work, we describe wide natural variation in both primary and acquired resistance to experimental pulmonary blastomycosis in eight founder strains, including 129, A/J, BL/6, CAST, NOD, NZO, PWK, and WSB of the Collaborative Cross collection, and the inbred DBA strain. These differences in susceptibility across strains were accompanied by sharp differences in the accumulation and function of immune cells in the lungs. Immune perturbations were mapped by identifying reagents that phenotypically mark immune cell populations in the distinct strains of mice. In particular, we uncovered marked differences between BL/6 and DBA/2 mouse strains in the development of acquired resistance. Our findings highlight the potential value in using genetic reference panels of mice, and particularly the BXD (recombinant inbred strains of mice from a cross of C57BL/6J and DBA/2J mice) collection harboring a cross between resistant BL/6 and susceptible DBA/2 mice, for unveiling genes linked with host resistance to fungal infection. IMPORTANCE Host genetic variation significantly impacts vulnerability to infectious diseases. While host variation in susceptibility to fungal infection with dimorphic fungi has long been recognized, genes that underpin this variation are poorly understood. We used a collection of seven mouse strains that represent nearly 90% of the genetic variation in mice to identify genetic variability among the strains in resistance to pulmonary infection with the dimorphic fungus Blastomyces dermatitidis. We analyzed differences between the strains in innate resistance by infecting naive mice and in acquired resistance by infecting vaccinated mice. We identified extreme variations in both innate and acquired resistance among the strains. In particular, we found sharp differences between C57BL/6 and DBA/2 strains in the ability to acquire vaccine-induced resistance. We also identified commercial reagents that allowed the phenotyping of immune cells from this strain collection of mice. Because there are additional mice harboring a genetic cross of the C57BL/6 and DBA/2 strains (BXD collection), such mice will permit future investigations to identify the genes that underlie differences in the ability to acquire resistance to infection.

Combination Adjuvants Enhance Recombinant Protein Vaccine Protection against Fungal Infection.

The development of effective vaccines against fungal infections requires the induction of protective, pathogen-specific cell-mediated immune responses. Here, we asked whether combination adjuvants based on delta inulin (Advax) formulated with Toll-like receptor (TLR) agonists could improve vaccine protection mediated by a fungal recombinant protein, Bl-Eng2 (i.e., Blastomyces endoglucanase 2), which itself harbors an immunodominant antigen and dectin-2 agonist/adjuvant. We found that Bl-Eng2 formulated with Advax3 containing TLR9 agonist or Advax8 containing TLR4 agonist provided the best protection against pulmonary infection with Blastomyces dermatitidis, being more effective than complete Freund's adjuvant or Adjuplex. Advax3 was most efficient in inducing gamma interferon (IFN-γ)- and interleukin-17 (IL-17)-producing antigen-specific T cells that migrated to the lung upon Blastomyces dermatitidis infection. Mechanistic studies revealed Bl-Eng2/Advax3 protection was tempered by neutralization of IL-17 and particularly IFN-γ. Likewise, greater numbers of lung-resident T cells producing IFN-γ, IL-17, or both IFN-γ and IL-17 correlated with fewer fungi recovered from lung. Protection was maintained after depletion of CD4+ T cells, partially reduced by depletion of CD8+ T cells, and completely eliminated after depletion of both CD4+ and CD8+ T cells. We conclude that Bl-Eng2 formulated with Advax3 is promising for eliciting vaccine-induced antifungal immunity, through a previously uncharacterized mechanism involving CD8+ and also CD4+ T cells producing IFN-γ and/or IL-17. Although no licensed vaccine exists as yet against any fungal disease, these findings indicate the importance of adjuvant selection for the development of effective fungal vaccines. IMPORTANCE Fungal disease remains a challenging clinical and public health problem. Despite medical advances, invasive fungal infections have skyrocketed over the last decade and pose a mounting health threat in immunocompetent and -deficient hosts, with worldwide mortality rates ranking 7th, even ahead of tuberculosis. The development of safe, effective vaccines remains a major hurdle for fungi. Critical barriers to progress include the lack of defined fungal antigens and suitable adjuvants. Our research is significant in identifying adjuvant combinations that elicit optimal vaccine-induced protection when formulated with a recombinant protective antigen and uncovering the mechanistic bases of the underlaying vaccine protection, which will foster the strategic development of antifungal vaccines.

LYSMD3: A mammalian pattern recognition receptor for chitin.

Chitin, a major component of fungal cell walls, has been associated with allergic disorders such as asthma. However, it is unclear how mammals recognize chitin and the principal receptor(s) on epithelial cells that sense chitin remain to be determined. In this study, we show that LYSMD3 is expressed on the surface of human airway epithelial cells and demonstrate that LYSMD3 is able to bind chitin, as well as ß-glucan, on the cell walls of fungi. Knockdown or knockout of LYSMD3 also sharply blunts the production of inflammatory cytokines by epithelial cells in response to chitin and fungal spores. Competitive inhibition of the LYSMD3 ectodomain by soluble LYSMD3 protein, multiple ligands, or antibody against LYSMD3 also blocks chitin signaling. Our study reveals LYSMD3 as a mammalian pattern recognition receptor (PRR) for chitin and establishes its role in epithelial cell inflammatory responses to chitin and fungi.