Allergy Asthma Immunol Res.  2016 Sep;8(5):445-456. 10.4168/aair.2016.8.5.445.

Synergistic Effect of Dermatophagoides farinae and Lipopolysaccharides in Human Middle ear Epithelial Cells

• Lee JE
• Kim YH
• Rhee CS
• Kim DY

• KMID: 2295154
• DOI: http://doi.org/10.4168/aair.2016.8.5.445

Abstract

PURPOSE
Although the concept of "one airway, one disease," which includes the middle ear space as part of the united airway is well recognized, the role of allergens in otitis media with effusion (OME) is not clearly understood. We aimed to investigate the effect of the interaction between Dermatophagoides farinae (Der f) and lipopolysaccharide (LPS) on the induction of epithelial inflammatory response in vitro.
METHODS
Primary human middle ear epithelial cells were exposed to Der f, LPS, or both in different sequences, and the magnitude of the immunologic responses was compared. The mRNA expressiona of mucin (MUC) 4, 5AC, 5B, 8, GM-CSF, TNF-α, TLR4, and MD-2 were evaluated using real-time PCR. MUC levels before and after siRNA-mediated knockout of TLR4 and MD-2 were assessed. Lastly, the involved cell signaling pathway was evaluated.
RESULTS
The expressiona of cytokines, and the MUC 4, 5AC, 5B, and 8 genes were augmented by pretreatment with Der f followed by LPS; however, reverse treatment or combined treatment did not induce the same magnitude of response. Increased MUC expression was decreased by TLR4 knockdown, but not by MD-2 knockdown. The signal intensity of MUC 8 was higher in MD-2 over-expressed cells than in those exposed to LPS only. The translocation of nuclear factor-κB was observed in cells pretreated with Der f followed by LPS.
CONCLUSIONS
When Der f treatment preceded LPS exposure, Der f and LPS acted synergistically in the induction of pro-inflammatory cytokines and the MUC gene, suggesting an important role in the development of OME in patients with concealed allergy airway sensitization.

Keyword

Dermatophagoides farinae; innate immunity; lipopolysaccharides; mucins; toll-like receptors

MeSH Terms

Allergens
Cytokines
Dermatophagoides farinae*
Ear, Middle*
Epithelial Cells*
Granulocyte-Macrophage Colony-Stimulating Factor
Humans*
Hypersensitivity
Immunity, Innate
In Vitro Techniques
Lipopolysaccharides*
Mucins
Otitis Media with Effusion
Pyroglyphidae*
Real-Time Polymerase Chain Reaction
RNA, Messenger
Toll-Like Receptors
Allergens
Cytokines
Granulocyte-Macrophage Colony-Stimulating Factor
Lipopolysaccharides
Mucins
RNA, Messenger
Toll-Like Receptors

Figure

• 

  Fig. 1 Study design. Cells were treated with medium only, a pre-determined, non-cytotoxic dose of Der f for 24/48 h or LPS for 24/48 h, or both in different sequences or simultaneously as indicated. UT, untreated; Df, Dermatophagoides farina; LPS, lipopolysaccharide; HMEEC, human middle ear epithelial cell.

• 

  Fig. 2 Real-time polymerase chain reaction to measure mucin mRNA level. (A) The expression of MUC 8 mRNA level was higher in cells exposed to Der f first and then to LPS than in cells exposed to Der f or LPS alone. The mRNA level did not increase when the cells were first exposed to LPS and then to Der f or were exposed to both agents simultaneously. (B) The levels of MUC 4, 5AC, and 5B mRNA showed similar patterns to MUC 8. (*P<0.05).

• 

  Fig. 3 Immunoblotting to determine MUC 8 and Toll-like receptor 4 signal intensity. (A) Treatment with Der f followed by LPS had a synergistic effect and increased MUC 8 production. (B) The signal intensity of MUC 8 and Toll-like receptor 4 induced by combined treatment with Der f and LPS was higher than that induced by treatment with LPS alone.

• 

  Fig. 4 Real-time PCR to measure the levels of various cytokine mRNAs. The mRNA levels of pro-inflammatory cytokines IL-1b, IL-33, GM-CSF, and TNF-α tended to increase when the cells were exposed to the Der f initially and then to LPS.

• 

  Fig. 5 mRNA and protein levels in cells transfected with small interfering RNA or in those overexpressing MD-2. (A, upper panel) The level of MD-2 or TLR4 mRNA was decreased in cells transfected with small interfering (si) RNA-MD-2 and siRNA-TLR4, respectively. (A, lower panel) The expressions of MUC 4, 5B, and 8 mRNA were also decreased in si-RNA-transfected cells. However, unlike in the cells transfected with si-RNA-TLR4, the expression of the target genes was not completely abolished in cells transfected with si-RNA-MD-2. (B) The decreased expression level of TLR4 and MUC 8 was confirmed at the protein level. The signal of MUC 8 protein was observed not only in the negative control, but also in the si-RNA-MD-2–transfected cells. (C) Overexpression of MD-2 enhanced the LPS-driven production of MUC 8. The signal intensity of MUC 8 was higher when LPS was added to the cell transfected with the Flag-pCMV-hMD-2 vector than with vector only. The pattern of increase when the cells were treated with LPS only for 24 hours was similar to that observed when those were treated first with Der f and then with LPS (Df48h/LPS24h).

• 

  Fig. 6 Confocal laser scanning image. (A) Red fluorescence indicates Der f expression; green, TLR4 expression; and blue, nuclear location. The lower right panel is a merged image of the other 3 panels. TLR4 was mainly expressed in the cell membrane, and it can be seen as yellow fluorescence after the overlap, suggesting that Der f and TLR4 co-localized in the cell membrane. Scale bar, 40 µm. (B) In high magnification, yellow fluorescence is seen not only in the negative control, but also in the membrane of cells transfected with si-RNA MD-2. Scale bar, 10 µm.

• 

  Fig. 7 Inhibition of MUC gene mRNA expression by SB203580 and Bay. (A) Expression of MUC 2, 5AC, and 8 mRNA was down-regulated by treatment with SB 203580 and Bay. (B) Down-regulation of MUC8 protein expression was confirmed by immunoblotting. Results are representative of those obtained from 3 independent experiments.

• 

  Fig. 8 Inhibition of phosphorylation of p38 MAPK and CREB, and translocation of nuclear factor κB (NF-κB) by SB203580 and Bay in HMEECs stimulated with Der f + LPS. (A) HMEECs were treated with Der f for 24 hours and then with SB203580 2 hours prior to LPS treatment. Subsequently, the cells were treated with LPS for 5, 15, and 30 minutes in the presence of Der f. The phosphorylation of p38 MAPK and CREB was analyzed using Western blotting. (B) Localization of NF-κB p65 was visualized under a fluorescence microscope after immunofluorescence staining with NF-κB p65 antibody (green). Cells were stained with DAPI to visualize nuclei (blue). NF-κB translocation in HMEECs stimulated with Der f + LPS was inhibited by treatment with SB203580 and Bay. Results are representative of those obtained from 2 independent experiments.

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