Induction of beta defensin 2 by NTHi requires TLR2 mediated MyD88 and IRAK-TRAF6-p38MAPK signaling pathway in human middle ear epithelial cells.
BACKGROUND
All mucosal epithelia, including those of the tubotympanium, are secreting a variety of antimicrobial innate immune molecules (AIIMs). In our previous study, we showed the bactericidal/bacteriostatic functions of AIIMs against various otitis media pathogens. Among the AIIMs, human beta-defensin 2 is the most potent molecule and is inducible by exposure to inflammatory stimuli such as bacterial components or proinflammatory cytokines. Even though the beta-defensin 2 is an important AIIM, the induction mechanism of this molecule has not been clearly established. We believe that this report is the first attempt to elucidate NTHi induced beta-defensin expression in airway mucosa, which includes the middle ear.


METHODS
Monoclonal antibody blocking method was employed in monitoring the TLR-dependent NTHi response. Two gene knock down methods - dominant negative (DN) plasmid and small interfering RNA (siRNA) - were employed to detect and confirm the involvement of several key genes in the signaling cascade resulting from the NTHi stimulated beta-defensin 2 expression in human middle ear epithelial cell (HMEEC-1). The student's t-test was used for the statistical analysis of the data.


RESULTS
The experimental results showed that the major NTHi-specific receptor in HMEEC-1 is the Toll-like receptor 2 (TLR2). Furthermore, recognition of NTHi component(s)/ligand(s) by TLR2, activated the Toll/IL-1 receptor (TIR)-MyD88-IRAK1-TRAF6-MKK3/6-p38 MAPK signal transduction pathway, ultimately leading to the induction of beta-defensin 2.


CONCLUSIONS
This study found that the induction of beta-defensin 2 is highest in whole cell lysate (WCL) preparations of NTHi, suggesting that the ligand(s) responsible for this up-regulation may be soluble macromolecule(s). We also found that this induction takes place through the TLR2 dependent MyD88-IRAK1-TRAF6-p38 MAPK pathway, with the primary response occurring within the first hour of stimulation. In combination with our previous studies showing that IL-1alpha-induced beta-defensin 2 expression takes place through a MyD88-independent Raf-MEK1/2-ERK MAPK pathway, we found that both signaling cascades act synergistically to up-regulate beta-defensin 2 levels. We propose that this confers an essential evolutionary advantage to the cells in coping with infections and may serve to amplify the innate immune response through paracrine signaling.
Lee
Haa-Yung
HY
The Gonda Department of Cell and Molecular Biology, House Ear Institute, Los Angeles, CA, USA. hlee@hei.org
Takeshita
Tamotsu
T
Shimada
Jun
J
Akopyan
Arsen
A
Woo
Jeong-Im
JI
Pan
Huiqi
H
Moon
Sung K
SK
Andalibi
Ali
A
Park
Rae-Kil
RK
Kang
Sung-Ho
SH
Kang
Shin-Seok
SS
Gellibolian
Robert
R
Lim
David J
DJ
D000818:Animals; D002460:Cell Line; D004432:Ear, Middle / Q000166:cytology* / Q000276:immunology / Q000382:microbiology; D004847:Epithelial Cells / Q000276:immunology* / Q000382:microbiology; D006193:Haemophilus influenzae / Q000276:immunology* / Q000472:pathogenicity; D006801:Humans; D053592:Interleukin-1 Receptor-Associated Kinases / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D053594:Myeloid Differentiation Factor 88 / Q000378:metabolism*; D015398:Signal Transduction; D048029:TNF Receptor-Associated Factor 6 / Q000378:metabolism; D051195:Toll-Like Receptor 2 / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation; D023083:beta-Defensins / Q000235:genetics / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
C106641:DEFB4A protein, human; D053594:Myeloid Differentiation Factor 88; D048029:TNF Receptor-Associated Factor 6; D051195:Toll-Like Receptor 2; D023083:beta-Defensins; C507768:IRAK1 protein, human; D053592:Interleukin-1 Receptor-Associated Kinases; D048051:p38 Mitogen-Activated Protein Kinases