Hypoxia-altered signaling pathways of toll-like receptor 4 (TLR4) in human corneal epithelial cells.
OBJECTIVE
Toll-like receptor 4 (TLR4), a member of the TLR family, is an important pattern recognition molecule that plays a role in the host's innate immune responses to lipopolysaccharide (LPS), a component of gram-negative bacteria. Contact lens wear is one of the risk factors for bacterial keratitis. The purpose of this study was to determine whether hypoxia or contact lens wear alters the TLR4 signaling pathways in human corneal epithelial cells (HCECs).


METHODS
A simian virus 40-immortalized human corneal epithelial cell (SV40-HCEC) line was cultured under 20% O2 or 2% O2 and exposed to LPS. The expression of TLR4, interleukin-6 (IL-6), and IL-8 was determined using a real-time reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and immunoblotting. Immunoblotting was also used to determine whether the nuclear factor kappa B (NFkappaB) was activated in the SV40-HCEs. HCECs were obtained from 17 healthy volunteers and 18 hydrogel soft contact lens (SCL) wearers using impression cytology (IC), and the expression of the mRNA of TLR4 was determined using real-time RT-PCR.


RESULTS
A reduction in the expression of the mRNA and protein of TLR4 was detected in SV40-HCECs cultured under hypoxic conditions. Hypoxia also attenuated both the LPS-induced expression of IL-6 and IL-8, and the activation of NFkappaB in SV40-HCECs. The expression of the mRNA of TLR4 was down-regulated in the HCECs of soft contact lens wearers.


CONCLUSIONS
These results indicate that hypoxia attenuates the TLR4 signaling pathway in HCECs, suggesting that the increase in the susceptibility to bacterial infections under hypoxic conditions may be related to the TLR4 signaling pathways.
Hara
Yuko
Y
Department of Ophthalmology, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime, Japan. yukoabc@m.ehime-u.ac.jp
Shiraishi
Atsushi
A
Ohashi
Yuichi
Y
D000328:Adult; D015687:Cell Hypoxia / Q000187:drug effects; D003261:Contact Lenses; D016207:Cytokines / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D019573:Epithelium, Corneal / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D015151:Immunoblotting; D008070:Lipopolysaccharides / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism; D010100:Oxygen / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*
D016207:Cytokines; D008070:Lipopolysaccharides; D016328:NF-kappa B; D012333:RNA, Messenger; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D010100:Oxygen