Investigation of Bronchiolitis Obliterans Caused by Artificial Butter flavoring

人造黄油调味品引起闭塞性细支气管炎的调查

基本信息

批准号：
9143480
负责人：
DANIEL MORGAN
金额：
$ 72.93万
依托单位：
NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/9143480
关键词：
Affect Air Animal Model Anti-Inflammatory Agents Anti-inflammatory Apical Apoptosis Atrophic Biological Assay Biological Markers Breathing Bronchi Bronchiolitis Bronchiolitis Obliterans Butter CCL7 gene Characteristics Chemicals Chronic Collaborations Collagen Collagen Gene Culture Media Data Detection Development Diacetyl Disease Dose Down-Regulation Early Diagnosis Employee Epithelial Exposure to Extracellular Matrix Proteins Fibroblasts Fibrosis Flavoring Food Additives Gelatinase A Gene Expression Genes Goals Goblet Cells Graft Rejection Histopathology Hour IL8 gene Immune Inflammatory Inhalation Exposure Interleukin-1 Interleukin-10 Interleukin-6 Interstitial Collagenase Investigation Lactate Dehydrogenase Lesion Leukocytes Lung Lung Transplantation Lymphocyte Matrilysin Matrix Metalloproteinases Methods MicroRNAs Modeling Necrosis Occupational Organ Outcome Pathogenesis Pathway interactions Peptide Hydrolases Plants Play Prevention Protease Inhibitor Proteins Proteomics Rattus Running Sampling Stromelysin 1 Surface Survival Rate System TNF gene Testing Thick Time Tissue Fixation Tissue Inhibitor of Metalloproteinase-1 Tissue Inhibitor of Metalloproteinases Tissues Toxic effect Transplantation cell injury chemokine cytokine diketone effective therapy in vitro Model in vivo indium-bleomycin injured airway microwave electromagnetic radiation mimetics non-smoking prevent regenerative response therapeutic target vapor

项目摘要

2,3-Butanedione (diacetyl), a reactive diketone used in artificial butter flavoring has been associated with obliterative bronchiolitis (OB) in employees at microwave popcorn packaging, and flavoring manufacturing plants. We have shown that diacetyl and a related flavoring 2,3-pentanedione (PD), cause OB-like lesions in rats after inhalation exposure. We have used this rat model to study the pathogenesis of OB. Recently we found that in PD-exposed fibrotic bronchi, over 2500 genes were differentially altered with a majority of genes being up-regulated in affected pathways. Tgf-beta2 and downstream genes implicated in fibrosis were significantly up-regulated in fibrotic lesions. Genes for collagens and extracellular matrix proteins were highly up-regulated. In addition, expression of genes for peptidases and for peptidase inhibitors were significantly altered suggesting tissue remodeling that may contribute to fibrosis. Recent studies have shown that downregulation of microRNA-29 (miR-29) is key to the development of fibrosis in the bleomycin lung model. We hypothesized that a similar mechanism is involved in development of airway fibrosis in OB, and that treatment with a miR-29 mimetic will prevent the development of PD-induced OB. Prior to testing this hypothesis, we have validated methods for extraction and detection of miRNAs in the rat lung. Lungs have been collected from rats exposed to a fibrogenic dose of PD. The miRNA-29 levels will be compared with levels in lungs of air-exposed control rats to establish whether miRNA-29 is downregulated in fibrotic airways. Subsequent studies will be conducted to determine if a miRNA-29 mimetic can prevent airway fibrosis in PD-exposed rats. EpiAirway-full thickness (FT) tissues, which have donor-matched fibroblasts embedded in a sub-epithelial stromal/collagen-rich matrix, were used as an in vitro model of diacetyl (DA) vapor-induced airway injury/toxicity. The presence of fibroblasts in this model is critical since airway fibrosis is a characteristic feature of obliterative bronchiolitis (OB). EpiAirway-FT tissues were treated with 0 (vehicle only) or 25 mM DA for 1 hour using vapor cups on days 0, 2 and 4 as previously described for standard (non-FT) EpiAirway tissues. Some tissues were similarly treated with 10 mM 2,3-hexanedione (Hex) as a control compound since Hex is structurally/chemically similar to DA but is less toxic than DA in vivo. The vapor exposure concentration for both DA and Hex was estimated 1000 ppm. The apical surface of the tissue was rinsed with PBS prior to exposure on days 0, 2, 4 and also on day 6 prior to tissue fixation (for histopathology). Lactate dehydrogenase (LDH) activity in the apical wash (an indicator of cellular injury) increased over time following exposure to DA, but not Hex, vapors. DA, but not Hex, vapors induced degenerative/regenerative histopathologic changes on day 6 which included decreased ciliated and goblet cells, increased apoptosis/necrosis, atrophy/erosion, basal spongiosis and karyomegaly. Sub-epithelial changes observed following DA, but not Hex, vapor exposure on day 6 included fibroblast nucleomegaly and hyperchromasia (suggestive of increased fibroblast activity). Day 3 and 5 culture media samples (24 hours after the 2nd and 3rd vapor exposures, respectively) were collected for multiplex protein assays. A 41-plex assay was used to look at the cytokine/chemokine profile of EpiAirway-FT tissues in response to DA vapor exposure. TGFα, IL-1α, sIL-1Rα, IL-6, IL-8, TNFα and MCP-3 were significantly increased (compared to vehicle controls) following exposure to DA, but not Hex, vapors. IL-10, which is a potent immune-regulatory/anti-inflammatory cytokine, was significantly increased following exposure to Hex, but not DA, vapors. A multiplex assay was also used to look at the secretion profile of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), which play a key role in tissue re-modeling and fibrosis, following exposure to DA vapors. MMP-1, MMP-3 and TIMP-1 were significantly increased (compared to vehicle controls) following exposure to DA, but not Hex, vapors. Conversely, MMP-2, MMP-7 and TIMP-2 were significantly decreased (compared to vehicle controls) following exposure to DA, but not Hex, vapors. Interestingly, all of these changes (LDH, histopathology and multiplex) in response to DA vapor exposure occurred in the absence of inflammatory leukocytes/lymphocytes. The multiplex assays were also run in parallel on day 3 and 5 culture media samples from donor-matched TBE-20 (non-FT) tissues following exposure to DA vapors to determine if the responses were dependent upon the presence of fibroblasts/matrix. IL-6, MCP-3, TNFα, all MMPs and all TIMPs were significantly increased following DA vapor exposure (compared to vehicle controls) only when fibroblasts/matrix were present (i.e. in the FT tissues) which also suggests increased fibroblast activity induced by DA vapor exposure. The EpiAirway-FT system can serve as a useful in vitro model for mechanistic studies of DA vapor-induced pulmonary toxicity and OB. OB is also a common outcome of lung transplantation. In addition, we performed a study in collaboration with the lung transplantation group at DUMC in which EpiAirway (non-FT) tissues from 4 healthy, non-smoking donors were exposed to DA vapor or vehicle control (as described above) for proteomics/secretomics and phospho-proteomics analyses in order to identify common biomarkers of exposure and potential therapeutic targets for OB.

2,3-丁二酮（二乙酰）是一种用于人造黄油调味剂的反应性二酮，它与微波爆米花包装和调味品制造厂员工的闭塞性细支气管炎 (OB) 有关。我们已经证明，二乙酰和相关调味剂 2,3-戊二酮 (PD) 在吸入暴露后会导致大鼠发生 OB 样病变。我们使用该大鼠模型来研究 OB 的发病机制。最近我们发现，在PD暴露的纤维化支气管中，超过2500个基因发生了差异性改变，其中大多数基因在受影响的通路中上调。 Tgf-beta2 和与纤维化有关的下游基因在纤维化病变中显着上调。胶原蛋白和细胞外基质蛋白的基因高度上调。此外，肽酶和肽酶抑制剂的基因表达显着改变，表明组织重塑可能导致纤维化。最近的研究表明，microRNA-29 (miR-29) 的下调是博莱霉素肺模型中纤维化发展的关键。我们假设 OB 气道纤维化的发生涉及类似的机制，并且用 miR-29 模拟物治疗将阻止 PD 诱导的 OB 的发生。在检验这一假设之前，我们已经验证了大鼠肺中 miRNA 的提取和检测方法。从暴露于纤维化剂量的 PD 的大鼠中采集肺部。将 miRNA-29 水平与暴露于空气的对照大鼠肺部水平进行比较，以确定 miRNA-29 在纤维化气道中是否下调。后续研究将确定 miRNA-29 模拟物是否可以预防 PD 暴露大鼠的气道纤维化。 EpiAirway 全层 (FT) 组织具有嵌入上皮下基质/富含胶原蛋白的基质中的供体匹配的成纤维细胞，被用作二乙酰 (DA) 蒸气诱导的气道损伤/毒性的体外模型。该模型中成纤维细胞的存在至关重要，因为气道纤维化是闭塞性细支气管炎 (OB) 的一个特征。如之前针对标准（非 FT）EpiAirway 组织所述，在第 0、2 和 4 天使用蒸气杯将 EpiAirway-FT 组织用 0（仅载体）或 25 mM DA 处理 1 小时。一些组织同样用 10 mM 2,3-己二酮 (Hex) 作为对照化合物进行处理，因为 Hex 在结构/化学上与 DA 相似，但体内毒性低于 DA。 DA 和 Hex 的蒸气暴露浓度估计为 1000 ppm。在第 0、2、4 天暴露之前以及在组织固定（用于组织病理学）之前的第 6 天，用 PBS 冲洗组织的顶端表面。暴露于 DA 蒸汽（而非 Hex）蒸汽后，根尖洗液中的乳酸脱氢酶 (LDH) 活性（细胞损伤的指标）随着时间的推移而增加。 DA（而非 Hex）蒸气在第 6 天诱导退行性/再生性组织病理学变化，包括纤毛细胞和杯状细胞减少、细胞凋亡/坏死增加、萎缩/糜烂、基底海绵组织增生和核肿大。第 6 天，在 DA（而非 Hex）蒸气暴露后观察到的上皮下变化包括成纤维细胞核肿大和深染（提示成纤维细胞活性增加）。收集第 3 天和第 5 天的培养基样品（分别在第二次和第三次蒸气暴露后 24 小时）用于多重蛋白质测定。使用 41 重检测来观察 EpiAirway-FT 组织响应 DA 蒸气暴露的细胞因子/趋化因子谱。暴露于 DA 蒸气后，TGFα、IL-1α、sIL-1Rα、IL-6、IL-8、TNFα 和 MCP-3 显着增加（与载体对照相比），但不暴露于 Hex 蒸气。 IL-10 是一种有效的免疫调节/抗炎细胞因子，在接触 Hex 蒸汽后显着增加，但不增加 DA 蒸汽。还使用多重测定来观察基质金属蛋白酶 (MMP) 和金属蛋白酶组织抑制剂 (TIMP) 的分泌情况，它们在暴露于 DA 蒸气后在组织重塑和纤维化中发挥着关键作用。暴露于 DA 蒸气后，MMP-1、MMP-3 和 TIMP-1 显着增加（与媒介物对照相比），但不暴露于 Hex 蒸气。相反，暴露于 DA 蒸气后，MMP-2、MMP-7 和 TIMP-2 显着降低（与媒介物对照相比），但未接触 Hex 蒸气。有趣的是，所有这些因 DA 蒸气暴露而发生的变化（LDH、组织病理学和多重变化）都是在不存在炎症白细胞/淋巴细胞的情况下发生的。在暴露于 DA 蒸气后的第 3 天和第 5 天，来自供体匹配的 TBE-20（非 FT）组织的培养基样品也并行进行多重测定，以确定反应是否依赖于成纤维细胞/基质的存在。仅当存在成纤维细胞/基质（即在 FT 组织中）时，IL-6、MCP-3、TNFα、所有 MMP 和所有 TIMP 在 DA 蒸气暴露后（与媒介物对照相比）显着增加，这也表明 DA 蒸气暴露诱导的成纤维细胞活性增加DA 蒸气暴露。 EpiAirway-FT 系统可作为有用的体外模型，用于 DA 蒸气引起的肺毒性和 OB 的机制研究。 OB 也是肺移植的常见结果。此外，我们与 DUMC 的肺移植小组合作进行了一项研究，其中将来自 4 名健康、不吸烟捐献者的 EpiAirway（非 FT）组织暴露于 DA 蒸气或载体对照（如上所述）以进行蛋白质组学/分泌组学研究和磷酸蛋白质组学分析，以确定常见的暴露生物标志物和 OB 的潜在治疗靶点。