Der p2 Internalization by Epithelium Synergistically Augments Toll-like Receptor-Mediated Proinflammatory Signaling.
OBJECTIVE
House-dust-mite (HDM) major allergen Der p2 shares homology and function with Toll-like receptor (TLR) signaling protein myeloid differentiation-2 (MD2) and may lead to airway inflammation. Should Der p2 be internalized by human airway epithelium, it has the theoretical propensity to potentiate epithelium activation. This study aimed to demonstrate the internalization of Der p2 by airway epithelium and to investigate the effects of Der p2 on MD2 expression and epithelium activation.


METHODS
Internalization of recombinant, enhanced green fluorescent protein-labelled Der p2 (rDer p2-EGFP) into human airway epithelium (BEAS-2B) was tracked by laser confocal microscopy and confirmed by immunoblotting. Reverse-transcription polymerase chain reaction (RT-PCR), immunoblotting, and immunohistochemical staining were used to determine the effect of Der p2 on MD2 expression in vitro and ex vivo. Expression of messenger RNA (mRNA) encoding receptors/cytokines was measured by RT-PCR. Secretion of interleukin-6/interleukin-8 (IL-6/IL-8) was measured by enzyme-linked immunosorbent assay (ELISA).


RESULTS
Internalization of Der p2 by BEAS-2B was confirmed by confocal microscopy and immunoblotting using rDer p2-EGFP and rDer p2, respectively. Expression of MD2 protein was increased in BEAS-2B and human nasal polyp airway epithelium cultured with rDer p2. Recombinant Der p2-cultured BEAS-2B caused little spontaneous IL-6/IL-8 secretion but significantly augmented by TLR ligand LPS. IL-6 secretion was up-regulated after MD2 transfection. Internalization of Der p2 was reduced by TLR2 RNA knockdown. Dexamethasone, calcitriol, anti-MD2/anti-TLR2 antibodies, and signalling inhibitors significantly reduced LPS+Der p2-induced IL-6/IL-8 secretion.


CONCLUSIONS
Human airway epithelium may internalize Der p2, which potentiates the response to environmental proinflammatory stimuli through MD2 and TLRs. This study highlights a novel mechanism and alleviates IL-6/IL-8 secretion in mite-induced airway inflammation.
Center for Translational Medicine, Department of Medical Research, Taichung Veterans General Hospital, Taichung, Taiwan.
Sui Chu
SC
Yin
Center for Translational Medicine, Department of Medical Research, Taichung Veterans General Hospital, Taichung, Taiwan.; Department of Bioindustry Technology, Da-Yeh University, Changhua, Taiwan.; Department of Medical Technology, Jen-Teh Junior College of Medicine, Nursing and Management, Miaoli, Taiwan.
En Chih
EC
Liao
Instrumentation Center, Department of Medical Research, Taichung Veterans General Hospital, Taichung, Taiwan.
Chih Liang
CL
Chiu
Division of Allergy, Immunology and Rheumatology, Department of Internal Medicine, Taichung Veterans General Hospital, Taichung, Taiwan.
Ching Yun
CY
Chang
Division of Allergy, Immunology and Rheumatology, Department of Internal Medicine, Taichung Veterans General Hospital, Taichung, Taiwan.; Institute of Biomedical Sciences, National Chung-Hsing University, Taichung, Taiwan.; Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan. jawji@vghtc.gov.tw.
Jaw Ji
JJ
Tsai
Der p2; Internalization; Toll-like receptor 2; calcitriol; human airway epithelium; inflammation