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

对健康的不利影响与微生物生长和潮湿的室内环境有关；然而，对于真菌暴露反应的机制存在知识差距。为了解决这些知识差距，NIOSH 与 NTP 和 NIEHS 合作，进行了亚慢性吸入研究，以检查反复接触烟曲霉、黑穗霉和杂色曲霉后对肺部免疫学和毒理学的影响。这些研究使用了基于改进的声动力粒子 (Pitt-3) 发生器的计算机控制声学发生器系统 (AGS)，并模拟了人们在真菌污染环境中会遇到的自然暴露情况。这些研究的总体目标是表征与反复接触潮湿室内环境中常见真菌相关的毒理学和肺部反应。此前，一项大型研究已完成，评估重复暴露于杂色曲霉 1、2、3、4、8 和 13 周后的肺部和全身毒性。数据显示，重复暴露 1 周后先天免疫细胞增加，随后 4 周内额外 B 细胞、T 细胞和 2 型先天淋巴细胞 (ILC2) 的浸润增加。反复接触云芝会导致局部和循环 Th2 细胞因子（包括 IL4 和 IL13）的产生增加，并使 ILC2 增加 4 周。到第 13 周时，除 ILC2 之外的所有细胞类型的细胞浸润均减少。对本研究得出的 miRNA、mRNA 和蛋白质组数据集的分析正在进行中。与报道的暴露于烟曲霉和黄曲霉后的反应类似，反复亚慢性暴露于花斑曲霉后观察到肺动脉组织重塑。肺动脉重塑的恢复也在 2022 财年进行了评估，其中小鼠反复暴露于杂色曲霉 4 周和 13 周，然后允许以 1 周至 4 周的不同间隔恢复。结果没有显示肺动脉组织有任何恢复。计划在 2023 财年进行更多恢复研究。 2022 财年，启动了利用转基因小鼠品系的额外暴露研究，以进一步表征影响先前 IAA 研究中观察到的暴露于烟曲霉、黄曲霉和云芝后肺动脉重塑的具体机制。具体而言，已知参与肺动脉重塑的关键 Th2 细胞因子 IL4 和 IL13 的表达已在小鼠模型中单独消除，然后用于 S. Chartarum 和 A. versicolor 暴露研究。正在收集心脏和呼吸功能测量结果，以及其他研究终点，包括病理学、通过流式细胞术进行的免疫细胞分析、细胞因子定量以及蛋白质组和 RNA 表达分析。这些研究的初步结果表明，通过心率和射血分数测量，真菌暴露损害了心脏功能，而 IL13 敲除小鼠的这两项功能都得到了恢复。 NIOSH 之前进行的基于测序的研究也发现真菌酵母是个人接触的来源。担子菌门酵母菌在室内污染环境中很常见，但其在不良健康影响中的作用仍然相对不为人所知。为了解决这一知识差距，2022 财年完成了将小鼠反复暴露于胜利隐球菌的研究。将由此产生的肺部免疫反应与暴露于新型隐球菌（一种已知会加剧过敏性气道疾病的酵母）引起的反应进行比较。结果显示，虽然最终暴露后 21 天，肺部可检测到新型隐球菌和胜利弧菌细胞，但重复的新型隐球菌暴露引发了骨髓和淋巴细胞向肺部的浸润，这种浸润随着时间的推移而恶化，而重复的胜利弧菌暴露则诱导了强烈的 CD4 T 细胞驱动的淋巴反应，该反应在最终暴露后 21 天开始消退。这些研究的结果使人们更好地了解真菌酵母在呼吸系统疾病中所起的作用。一项为期 13 周的研究检查了重复接触杂色曲霉的毒理学反应，于 2022 财年得出结论。未来的研究将调查诸如心肺和神经系统对真菌暴露的反应等终点。

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.