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-丁烷二酮（二乙酰基）是一种用于人造黄油调味的反应性二酮，与Microwave Popcorn包装的员工中的闭塞性支气管炎（OB）有关，并与调味厂的制造厂有关。我们已经表明，二乙酰和相关的调味剂2,3-戊二酮（PD）会在吸入后导致大鼠的ob样病变。我们已经使用此大鼠模型研究了OB的发病机理。最近，我们发现在PD暴露的纤维化支气管中，超过2500个基因被差异改变，大多数基因在受影响的途径中被上调。在纤维化病变中，与纤维化有关的TGF-BETA2和与纤维化有关的下游基因显着上调。胶原蛋白和细胞外基质蛋白的基因高度上调。另外，肽酶和肽酶抑制剂的基因表达显着改变，表明组织重塑可能有助于纤维化。最近的研究表明，microRNA-29（miR-29）的下调是博来霉素肺模型中纤维化发展的关键。我们假设OB中气道纤维化的发展涉及类似的机制，而用miR-29模拟物进行治疗将阻止PD诱导的OB的发展。在检验该假设之前，我们已经验证了大鼠肺中miRNA的提取和检测方法。从暴露于PD纤维剂量的大鼠中收集了肺。将将miRNA-29水平与暴露于空气对照大鼠的肺的水平进行比较，以确定miRNA-29是否在纤维化气道中下调。将进行随后的研究，以确定miRNA-29模拟物是否可以防止暴露于PD的大鼠气道纤维化。 Epiairway-full厚度（FT）组织具有嵌入在上皮下基质/胶原蛋白富基质中的供体匹配的成纤维细胞，用作二乙酰基（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支气管炎（OB）的特征。如前所述，使用0、2和4的蒸气杯对Epiairway-ft组织用0（仅车）或25 mm DA处理1小时，如先前所述的标准（非FT）epiairway组织。由于HEX在结构上/化学上与DA相似，但与DA相似，但与10 mm 2,3-己二二酮（HEX）类似地处理了一些组织作为对照化合物。 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，将组织的顶部表面用PBS冲洗，以及在组织固定之前的第6天（用于组织病理学）。在暴露于DA的DA，但不是十六进制后，乳酸脱氢酶（LDH）活性随着时间的流逝而增加。 DA，但不是十六进制，蒸气会在第6天引起退化/再生组织病理学变化，其中包括减少纤毛和杯状细胞，凋亡/坏死，萎缩/侵蚀，基础海绵病和核心肿大增加。在DA之后观察到的下皮下皮变化，但没有HEX，第6天的蒸气暴露包括成纤维细胞核瘤和高染色症（暗示着成纤维细胞活性增加）。收集了第3和5天的培养基样品（分别在第二蒸气和第三蒸气暴露后24小时）进行多重蛋白质测定。使用41个PLEX分析来研究Epiairway-FT组织的细胞因子/趋化因子谱，以响应DA蒸气暴露。暴露于DA但不是HEX，蒸气后，TGFα，IL-1α，SIL-1Rα，IL-6，IL-8，TNFα和MCP-3显着增加（与媒介物对照相比）。 IL-10是一种有效的免疫调节/抗炎细胞因子，在暴露于十六进制（而不是DA）蒸气后显着增加。多重测定也用于查看基质金属蛋白酶（MMP）和金属蛋白酶的组织抑制剂（TIMP）的分泌曲线，这些酶（TIMP）在暴露于DA蒸发后在组织重新建模和纤维化中起关键作用。暴露于DA（而不是六角形）蒸气后，MMP-1，MMP-3和TIMP-1显着增加（与媒介物对照相比）。相反，在暴露于DA（但不是十六进制）蒸气后，MMP-2，MMP-7和TIMP-2显着降低（与车辆对照相比）。有趣的是，在没有炎性白细胞/淋巴细胞的情况下，所有这些变化（LDH，组织病理学和多重）响应于DA蒸气。在暴露于DA蒸气后，从第3天和5次培养基的TBE-20（非FT）组织中并联运行多重测定，以确定响应是否取决于成纤维细胞/矩阵的存在。 DA蒸汽暴露后（与媒介物对照相比）IL-6，MCP-3，TNFα，所有MMP和所有TIMP显着增加（在FT组织中存在成纤维细胞/基质时），这也表明，这也表明DA Vapor暴露诱导的成纤维细胞活性增加。 Epiairway-FT系统可以作为DA蒸汽诱导的肺毒性和OB的机械研究的有用体外模型。 OB也是肺移植的共同结果。此外，我们与DUMC的肺移植组合作进行了一项研究，在该研究中，来自4个健康，非吸烟供体的Epiairway（非FT）组织暴露于DA Vapor或DA Vapor或车辆对照（如上所述），以确定常见的曝光和潜在的生物标志的蛋白质组学和磷酸化蛋白质组学分析，以确定常见的生物标志和潜在的目标。