Method: Primary human middle ear epithelial cells (HMEEC) were exposed to Der f crude body extract, LPS or both in different sequences, and the magnitude of each immunologic response produced by the HMEEC was compared. The mRNA expression level of mucin gene (MUC) 8, GM-CSF, TNF-a, TLR 4 and MD 2 were evaluated by using real-time polymerase chain reaction (qRT-PCR). The MUC proteins level before and after knocking out the TLR 4 and MD2 via siRNA transfection were assessed by Western blot analysis. Accordingly, the involved cell signaling pathway was evaluated by Western blot analysis and confocal microscopic image.
Results: The inflammatory response of cytokines (GM-CSF, TNF-a) and the expression of MUC 8 were augmented by the pretreatment of Der f followed by LPS, however, sequential treatment of HMEEC with LPS and Der f or adding together at the same time did not induce the same amount of response. The MUC expression was decreased by prior knockdown of TLR4 with siRNA but not by the MD2-siRNA. The MUC proteins level were increased by the pretreatment of Der f followed by LPS and decreased by the treatment of SB203580 (p38 inhibitor) and Bay (NF-kB inhibitor). The nuclear factor kB (NF-kB) translocation was demonstrated in the pretreatment of Der f followed by LPS condition.
Conclusion: Theses results suggest that Der f may act as a substitute for MD2 and make a strong augmentative response to the subsequent LPS infection. There was an increase in p38 and NF-kB activation within human middle epithelial cells, suggesting an important role for the development of OME in patients with concealed allergy airway sensitization.