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，知识差距存在的机制，潜在的反应真菌暴露。为了解决这些知识空白，NIOSH与国家毒理学规划和NIEHS合作，进行了亚慢性吸入研究，以检查反复暴露于烟曲霉、葡萄曲霉和花斑曲霉后的肺部免疫和毒理学效应。基于改进的声动力粒子（Pitt-3）发生器的计算机控制声发生器系统（AGS）被用于这些研究，并模拟了人类在真菌污染环境中自然暴露的情况。这些研究的总体目标是描述与反复暴露于潮湿室内环境中常见的真菌相关的毒理学和肺部反应。此前，一项大型研究评估了1、2、3、4、8和13周反复暴露于花斑曲霉后的肺部和全身毒性。数据显示，在重复暴露1周后，先天免疫细胞增加，随后在4周内，额外的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。来自本研究的miRNA、mRNA和蛋白质组学数据集的分析正在进行中。与烟曲霉和沙氏葡萄球菌暴露后的反应类似，反复亚慢性暴露于花斑葡萄曲霉后观察到肺动脉组织重塑。FY22还评估了肺动脉重塑的恢复情况，在该研究中，小鼠反复暴露于花青草4周和13周，然后允许恢复1周至4周的不同间隔。结果显示肺动脉组织没有任何恢复。计划在23财年进行更多的回收研究。在FY22，利用转基因小鼠品系的额外暴露研究被启动，以进一步表征在先前的IAA研究中观察到的暴露于烟曲霉、沙曲霉和花斑曲霉后影响肺动脉重塑的具体机制。具体来说，已知参与肺动脉重塑的关键Th2细胞因子IL4和IL13的表达在小鼠模型中被单独消除，然后用于沙棘和花斑草暴露研究。正在收集心脏和呼吸功能测量数据，以及其他研究终点，包括病理学、流式细胞术免疫细胞谱、细胞因子定量、蛋白质组学和RNA表达谱。这些研究的初步结果表明，真菌暴露会损害心脏功能（通过心率和射血分数测量），而在il - 13敲除小鼠中，这两项功能都恢复了。先前在NIOSH进行的基于测序的研究也确定了真菌酵母菌是个人暴露的来源。担子菌酵母在室内污染环境中表现突出，但其在不良健康影响中的作用仍相对不明确。为了解决这一知识差距，在2022财年完成了小鼠反复暴露于维多利亚隐球菌的研究。由此产生的肺部免疫反应与暴露于隐球菌引起的反应进行了比较，隐球菌是一种已知会加剧过敏性气道疾病的酵母菌。结果表明，虽然最终暴露21天后肺部均可检测到新生梭状芽孢杆菌和维多利亚梭状芽孢杆菌细胞，但反复暴露于新生梭状芽孢杆菌会引发骨髓和淋巴细胞浸润，并随着时间的推移而恶化，而反复暴露于维多利亚梭状芽孢杆菌会引起强烈的CD4+ T细胞驱动的淋巴细胞反应，这种反应在最终暴露21天后开始消退。这些研究的结果更好地理解了真菌酵母在呼吸系统疾病中的作用。一项为期13周的研究检查了反复接触花斑田鼠的毒理学反应，该研究于22财年结束。未来的研究将调查终点，如对真菌暴露的心肺和神经反应。

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.