Research article

Induction of beta defensin 2 by NTHi requires TLR2 mediated MyD88 and IRAK-TRAF6-p38MAPK signaling pathway in human middle ear epithelial cells

Haa-Yung Lee¹ , Tamotsu Takeshita^1,2 , Jun Shimada^1,3 , Arsen Akopyan¹ , Jeong-Im Woo¹ , Huiqi Pan¹ , Sung K Moon¹ , Ali Andalibi¹ , Rae-Kil Park^1,4 , Sung-Ho Kang⁵ , Shin-Seok Kang¹ , Robert Gellibolian¹ and David J Lim^1,6,7

¹The Gonda Department of Cell and Molecular Biology, House Ear Institute, Los Angeles, CA, USA

²Department of Otorhinolaryngology, Hamamatsu University School of Medicine, Hamamatsu, Japan

³Department of Otolaryngology and Head & Neck Surgery, Wakayama Medical University, Wakayama, Japan

⁴Department of Microbiology, Wonkwang University School of Medicine, Iksan, South Korea

⁵Department of Otolaryngology, Konkuk University School of Medicine, Chungju, South Korea

⁶Department of Otolaryngology, University of Southern California, Los Angeles, CA, USA

⁷Department of Cell and Neurobiology, University of Southern California, Los Angeles, CA, USA

author email corresponding author email

BMC Infectious Diseases 2008, 8:87doi:10.1186/1471-2334-8-87

Published:	25 June 2008

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 β-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 β-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 β-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 β-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 β-defensin 2.

Conclusion

This study found that the induction of β-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-1α-induced β-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 β-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.