Assessment of Inhalation Exposures to Indoor and Occupational Aerosols

重复真菌吸入暴露的小鼠模型-不良健康影响与微生物生长和潮湿的室内环境有关；然而，与真菌暴露相关的毒性机制仍未得到充分研究。霉菌被提名到NTP进行毒理学表征，并根据方法和专业知识，NIOSH被确定为合作者，对NTP提名的真菌物种进行亚慢性吸入暴露研究。NIOSH为这些研究开发的声学发生器系统（AGS）模拟了人类在真菌污染环境中自然暴露的情况。在这个子项目下进行的研究描述了与反复接触真菌有关的毒理学和肺部免疫反应。这些研究分为两个阶段-第一阶段表征目标真菌物种的培养和气溶胶优化，第二阶段包括亚慢性研究。该子项目还包括对污染室内环境中发现的真菌多样性的评估。在2020财年，发表了两篇论文，描述了产生毛霉烯的Stachybotrys chartarum的培养和雾化，以及暴露后的肺部免疫反应。在这些研究中，使用了两株被称为菌株A和菌株B的chartarum菌株，菌株A比菌株B产生更多的片段和霉菌毒素。这些出版物首先描述了活的和非活的chartarum菌株的培养，以及优化AGS，包括将S. chartarum雾化和给药给住在只有鼻子的暴露舱中的小鼠。暴露系统的表征和优化随后被用于进一步的研究，以测量反复真菌暴露后的肺部免疫反应。各组小鼠吸入活的或热灭活的沙氏葡萄球菌分生孢子，或hepa过滤的空气，每周两次，持续4周和13周。暴露后4周，观察到t辅助细胞2型（Th2）介导的应答。13周后，暴露于菌株a后观察到混合t细胞反应，而暴露于菌株B后观察到th2介导的反应。暴露后13周，两种菌株均诱导肺动脉重构；然而，暴露于菌株a的小鼠比暴露于菌株b的小鼠进展得更快。灌洗液由炎性细胞群组成，包括嗜酸性粒细胞、中性粒细胞和巨噬细胞。总的来说，在暴露于高片段产生菌株A的小鼠中，免疫病理反应发生得更早，这表明真菌片段的存在增加了暴露并促成了观察到的反应。NIOSH目前正在评估一项大型研究的结果，该研究评估了反复暴露于花斑草后的肺部和全身毒性。反复暴露1周后，观察到先天免疫细胞数量增加，随后在4周内，b细胞、t细胞和2型先天淋巴样细胞（ILC2s）的浸润增加。反复暴露于花斑草导致局部和循环Th2细胞因子的产生增加，包括IL4和IL13，以及ILC2s增加4周。到13周时，除ILC2s外，所有细胞类型的细胞浸润均减少。从活菌、非活菌和纯空气对照样本的肺匀质中提取的miRNA、mRNA和蛋白质组学数据集的分析仍在进行中。与烟曲霉和chartarum类似，初步数据显示反复亚慢性暴露于a.s vericolor后肺动脉组织重塑，突出了人类真菌暴露可能涉及心血管的可能性。

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.