Assessment of Inhalation Exposures to Indoor and Occupational Aerosols - Murine Models of Repeated Fungal Inhalation Exposure

National Institute Of Environmental Health Sciences

Investigators

Abstract

Adverse health effects have been associated with microbial growth and damp indoor environments; however, knowledge gaps exist as to the mechanisms underlying the responses to fungal exposure. To address these knowledge gaps, NIOSH, in collaboration with the NTP and NIEHS, conducted subchronic inhalation studies to examine the pulmonary immunological and toxicological effects following repeated exposure to Aspergillus fumigatus, Stachybotrys chartarum, and Aspergillus versicolor. A computer-controlled acoustical generator system (AGS), based on a modified acoustically powered particle (Pitt-3) generator, was utilized for these studies and models a natural human exposure one would encounter in a fungal contaminated environment. The overall goal of these studies is to characterize the toxicological and pulmonary responses associated with repeated exposure to fungi commonly found in damp, indoor environments. Previously, a large study assessing the pulmonary and systemic toxicity following a 1-, 2-, 3-, 4-, 8-, and 13-week repeated exposure to Aspergillus versicolor was completed. Data showed increased innate immunity cells 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. Analysis of miRNA, mRNA, and proteomic datasets derived from this study is ongoing. Similar to the reported responses following A. fumigatus and S. chartarum exposure, pulmonary arterial tissue remodeling was observed following repeated subchronic exposure to A. versicolor. Recovery of pulmonary arterial remodeling was also assessed in FY22, in which mice were repeatedly exposed to A. versicolor for 4 and 13 weeks and then allowed to recover for varying intervals of 1 week to 4 weeks. Results did not show any recovery of the pulmonary arterial tissues. Additional recovery studies are planned for FY23. In FY22, additional exposure studies utilizing genetically modified mouse strains were initiated to further characterize specific mechanisms influencing the pulmonary arterial remodeling observed in previous IAA studies following exposure to A. fumigatus, S. chartarum, and to A. versicolor. Specifically, expression of critical Th2 cytokines known to be involved in pulmonary arterial remodeling, IL4 and IL13, have individually been eliminated in mouse models that are then being used in S. chartarum and A. versicolor exposure studies. Cardiac and respiratory functional measurements are being collected, as well as additional study endpoints including pathology, immune cell profiling via flow cytometry, cytokine quantification, and proteomic and RNA expression profiling. Preliminary results from these studies showed that fungal exposure impaired cardiac function as measured by heart rate and ejection fraction, both of which were restored in the IL13 knockout mouse. Previous sequencing-based studies conducted at NIOSH have also identified fungal yeasts as sources of personal exposure. Basidiomycota yeasts are prominent in indoor contaminated environments, but their role in adverse health effects has remained relatively uncharacterized. To address this knowledge gap, studies in which mice were repeatedly exposed to Cryptococcus victoriae were completed in FY22. The resultant pulmonary immune response was compared to the response elicited from exposure to Cryptococcus neoformans, a yeast known to exacerbate allergic airway disease. Results showed that while both C. neoformans and V. victoriae cells were detectable in the lungs 21 days post final exposure, repeated C. neoformans exposure initiated myeloid and lymphoid cellular infiltration into the lung that worsened over time, whereas repeated V. victoriae exposure induced a strong CD4+ T cell-driven lymphoid response that started to resolve by 21 days post final exposure. Results from these studies provide a better understanding of the role that fungal yeasts play in respiratory health disease. A 13-week study examining the toxicological responses to repeated A. versicolor exposure concluded in FY22. Future studies will investigate endpoints such as cardiopulmonary and neurological responses to fungal exposure.