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.
