Koichi Kobayashi1, Kazuya Koyama1, Maho Suzukawa2, Sayaka Igarashi1, Akira Hebisawa1, Takahide Nagase3, Ken Ohta1. 1. National Hospital Organization Tokyo National Hospital, Tokyo, Japan. 2. National Hospital Organization Tokyo National Hospital, Tokyo, Japan. Electronic address: suzukawam@tokyo-hosp.jp. 3. Department of Respiratory Medicine, University of Tokyo, Tokyo, Japan.
Abstract
BACKGROUND: Epithelial-mesenchymal transition (EMT) is reported to promote airway remodeling in asthmatics, which is the main histological change that causes complex and severe symptoms in asthmatics. However, little is known about whether EMT also plays a role in acute exacerbations of asthma evoked by respiratory tract infections. METHODS: A human lung adenocarcinoma line, A549, was incubated with TGF-β1 at 10 ng/ml to induce EMT. Then the cells were stimulated with CpG ODN. Expression of surface and intracellular molecules was analyzed by flow cytometry. IL-6, IL-8 and MCP-1 in the culture supernatant were measured by Cytometric Bead Assay, and the expression of mRNA was quantitated by real-time PCR. CpG ODN uptake was analyzed by flow cytometry. RESULTS: The culture supernatant levels of IL-6, IL-8 and MCP-1 and the expression of mRNA for these cytokines in CpG ODN-stimulated A549 cells that had undergone EMT was significantly higher compared to those that had not. Addition of ODN H154, a TLR9-inhibiting DNA, significantly suppressed the CpG ODN-induced production of those cytokines. However, flow cytometry found the level of TLR9 expression to be slightly lower in A549 cells that had undergone EMT compared to those that had not. On the other hand, CpG ODN uptake was increased in cells that had undergone EMT. CONCLUSIONS: EMT induction of A549 cells enhanced CpG ODN uptake and CpG ODN-induced production of IL-6, IL-8 and MCP-1. These results suggest that EMT plays an important role in exacerbation in asthmatics with airway remodeling by enhancing sensitivity to extrinsic pathogens.
BACKGROUND: Epithelial-mesenchymal transition (EMT) is reported to promote airway remodeling in asthmatics, which is the main histological change that causes complex and severe symptoms in asthmatics. However, little is known about whether EMT also plays a role in acute exacerbations of asthma evoked by respiratory tract infections. METHODS: A humanlung adenocarcinoma line, A549, was incubated with TGF-β1 at 10 ng/ml to induce EMT. Then the cells were stimulated with CpG ODN. Expression of surface and intracellular molecules was analyzed by flow cytometry. IL-6, IL-8 and MCP-1 in the culture supernatant were measured by Cytometric Bead Assay, and the expression of mRNA was quantitated by real-time PCR. CpG ODN uptake was analyzed by flow cytometry. RESULTS: The culture supernatant levels of IL-6, IL-8 and MCP-1 and the expression of mRNA for these cytokines in CpG ODN-stimulated A549 cells that had undergone EMT was significantly higher compared to those that had not. Addition of ODN H154, a TLR9-inhibiting DNA, significantly suppressed the CpG ODN-induced production of those cytokines. However, flow cytometry found the level of TLR9 expression to be slightly lower in A549 cells that had undergone EMT compared to those that had not. On the other hand, CpG ODN uptake was increased in cells that had undergone EMT. CONCLUSIONS: EMT induction of A549 cells enhanced CpG ODN uptake and CpG ODN-induced production of IL-6, IL-8 and MCP-1. These results suggest that EMT plays an important role in exacerbation in asthmatics with airway remodeling by enhancing sensitivity to extrinsic pathogens.