title: Induction of beta defensin 2 by NTHi requires TLR2 mediated MyD88 and IRAK-TRAF6-p38MAPK signaling pathway in human middle ear epithelial cells.
abstract: 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.
authors.affiliation: The Gonda Department of Cell and Molecular Biology, House Ear Institute, Los Angeles, CA, USA. hlee@hei.org
authors.forename: Haa-Yung
authors.initials: HY
authors.lastname: Lee
authors.forename: Tamotsu
authors.initials: T
authors.lastname: Takeshita
authors.forename: Jun
authors.initials: J
authors.lastname: Shimada
authors.forename: Arsen
authors.initials: A
authors.lastname: Akopyan
authors.forename: Jeong-Im
authors.initials: JI
authors.lastname: Woo
authors.forename: Huiqi
authors.initials: H
authors.lastname: Pan
authors.forename: Sung K
authors.initials: SK
authors.lastname: Moon
authors.forename: Ali
authors.initials: A
authors.lastname: Andalibi
authors.forename: Rae-Kil
authors.initials: RK
authors.lastname: Park
authors.forename: Sung-Ho
authors.initials: SH
authors.lastname: Kang
authors.forename: Shin-Seok
authors.initials: SS
authors.lastname: Kang
authors.forename: Robert
authors.initials: R
authors.lastname: Gellibolian
authors.forename: David J
authors.initials: DJ
authors.lastname: Lim
mesh_terms: 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
chemical_list: 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