Assessment of Inhalation Exposures to Indoor and Occupational Aerosols

反复吸入真菌的小鼠模型--有害的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。Mold因毒理学特征而被NTP提名，基于方法学和专业知识，NIOSH被确定为对NTP提名的真菌物种进行亚慢性吸入暴露研究的合作者。NIOSH为这些研究开发的声学发生器系统(AGS)模拟了人们在真菌污染的环境中遇到的自然人类暴露。在这一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--第一阶段描述目标真菌物种的培养和气雾剂优化，第二阶段包括亚慢性研究。这一分项目还包括对在受污染的室内环境中发现的真菌多样性的评价。在2010财年期间，发表了两篇手稿，描述了产生三氯乙烯的水苏打菌的培养和气雾化，以及暴露后的肺部免疫反应。在这些研究中，使用了两株被命名为菌株A和菌株B的沙门氏菌，与菌株B相比，菌株A产生了更多的片段和真菌毒素。这些文献中的第一篇描述了活性和非活性沙门氏菌的培养，以及对AGS的优化，包括将沙门氏菌雾化并将其输送到关在仅鼻部暴露舱中的小鼠身上。然后，在进一步的研究中，利用暴露系统的特征和优化来测量重复真菌暴露后的肺免疫反应。每组小鼠每周吸入两次活的或热灭活的沙门氏菌分生孢子或经HEPA过滤的空气，持续4周和13周。暴露4周后，观察到辅助性T细胞2型(Th2)介导的反应。13周后，在接触菌株A后观察到混合T细胞反应，而在接触菌株B后观察到Th2介导的反应。两种菌株都在暴露后13周诱导了肺动脉重塑；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。总体而言，暴露于高片段产生菌株A的小鼠的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杂色曲霉后的肺部和全身毒性。反复暴露1周后，观察到天然免疫细胞的数量增加，随后在4周以上观察到额外的B细胞、T细胞和2型天然淋巴样细胞(ILC2s)的渗透增加。反复暴露于杂色曲霉导致局部和循环Th2细胞因子的产生增加，包括IL4和IL13，并在4周内增加ILC2。13周时，除ILC2s外，所有细胞类型的细胞浸润率均降低。对来自活的、不活的和仅有空气的对照样本的肺匀浆的miRNA、mRNA和蛋白质组数据集的分析仍在进行中。与烟曲霉和沙门氏菌相似，初步数据显示，在反复亚慢性接触杂色曲霉菌后，肺动脉组织重构，突显了心血管参与人类真菌暴露的可能性。 对杂色假单胞菌暴露后的大规模毒理学研究得出的miRNA、mRNA和蛋白质组数据集的分析正在进行中。正在使用独创性路径分析对数据集进行分析，以确定生物系统和疾病中的已知关联，以及确定与生物功能、路径和疾病的新的相互作用和关联。

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.