Murine Models of Repeated Fungal Inhalation Exposure - Adverse health effects have been associated with microbial growth and damp indoor environments; however, the mechanisms of toxicity associated with fungal exposure remain understudied. Mold was nominated to the NTP for toxicological characterization and based on the methodologies and expertise, NIOSH was identified as a collaborator to conduct sub-chronic inhalation exposures studies with NTP-nominated fungal species. The Acoustical Generator System (AGS) developed by NIOSH for these studies models a natural human exposure one would encounter in a fungal contaminated environment. The studies conducted under this subproject characterize the toxicological and pulmonary immune responses associated with repeated fungal exposure. These studies are separated into two phases ? the first phase characterizing the cultivation and aerosol optimization of the targeted fungal species and the second phase consists of subchronic studies. Also included in this subproject is the evaluation of fungal diversity found within contaminated indoor environments. During FY20, two manuscripts characterizing the cultivation and aerosolization of trichothecene-producing Stachybotrys chartarum, as well as the pulmonary immune response following exposure were published. For these studies, two strains of S. chartarum designated as strain A and strain B were utilized with strain A producing a higher amount of fragments and mycotoxin compared to strain B. The first of these publications characterized the cultivation of viable and non-viable S. chartarum, as well as the optimization of the AGS including the aerosolization and delivery of S. chartarum to mice housed in nose-only exposure pods. The characterization and optimization of the exposure system was then utilized in further studies to measure the pulmonary immune responses following repeated fungal exposure. Groups of mice inhaled viable or heat-inactivated S. chartarum conidia, or HEPA?filtered air twice per week for 4 and 13 weeks. At 4 weeks after exposure, a T-helper cell type 2 (Th2)?mediated response was observed. After 13 weeks, a mixed T-cell response was observed following exposure to strain A compared with a Th2?mediated response after strain B exposure. Both strains induced pulmonary arterial remodeling at 13 weeks following exposure; however, strain A?exposed mice progressed more quickly than strain B?exposed mice. Lavage fluid was composed of inflammatory cell populations including eosinophils, neutrophils, and macrophages. Overall, the immunopathological responses occurred earlier in mice exposed to high fragment-producing strain A, suggesting that the presence of fungal fragments increased exposure and contributed to the observed responses. NIOSH is currently evaluating the results from a large study that assessed pulmonary and systemic toxicity following repeated exposure to A. versicolor. An increased number of innate immune cells were observed after 1 week of repeated exposure followed by the increasing infiltration of additional B-cells, T-cells, and type 2 innate lymphoid cells (ILC2s) over 4 weeks. Repeated exposure to A. versicolor led to the increased production of local and circulating Th2 cytokines, including IL4 and IL13, as well increased ILC2s by 4 weeks. By 13 weeks, cellular infiltration was decreased for all cell types except ILC2s. The analysis of miRNA, mRNA, and proteomic datasets derived from the lung homogenates of viable, nonviable, and air-only control samples are still ongoing. Similar to A. fumigatus and S. chartarum, preliminary data revealed pulmonary arterial tissue remodeling following repeated subchronic exposure to A. versicolor, highlighting the potential for cardiovascular involvement in human fungal exposures. An analysis of miRNA, mRNA, and proteomic datasets derived from the large toxicology study following A. versicolor exposure is ongoing. The datasets are being analyzed using Ingenuity Pathway Analysis to identify known associations within biological systems and diseases, as well as to identify novel interactions and associations with biological functions, pathways and diseases.
