Toll-like receptor 2-mediated NF-kappa B pathway activation in ocular surface epithelial cells

• 作者: Aihua Hou Louis Tong Min Qi Tin
• 来源期刊: 眼视光学杂志(英文版)
• 学科: 医学
• 关键词: TLR2 NF-ΚB PATHWAY ACTIVATION OCULAR surface CELLS
• 年，卷（期）: 2018,（2）
• 页码范围: 34-40
• 页数: 7页
• 分类号: R73

摘要

Background:Gram-positive bacteria stimulate Toll-like receptor (TLR) 2 and then activate the pro-inflammatory nuclear factor-kappa B (NF-κB) pathway.As the human ocular surface is heavily colonised by gram-positive cocci bacteria,a balance of activation/repression of NF-κB target genes is essential to avoid uncontrolled infection or autoimmune-related inflammation.It is advantageous to test NF-κB targeting molecules in an ocular surface culture system that allows assessment of temporal NF-κB activation in a longitudinal fashion without destruction of cells.Such initial testing under standardised conditions should reduce the number of molecules that progress to further evaluation in animal models.This study aims to establish an in-vitro cell culture system to assess NF-κB activation in the context of ocular surface cells.Methods:NF-κB activity was evaluated through a secretory alkaline phosphatase reporter assay (SEAP).Immunoblots and immunofluorescence were used to examine lκBa phosphorylation and p65/p50 nuclear localization.Monocyte chemoattractant protein-1 (MCP-1) transcripts were evaluated by real time PCR and protein levels were measured by ELISA Results:NF-κB activity in HCE-T cells treated with TLR2 activator Pam3CSK4 was higher than control cells at both 6 and 24 h.Pam3CSK4-stimulated NF-κB activation was inhibited by lκK inhibitors,Wedelolactone and BMS-345541.In Pam3CSK4 treated cells,active NF-κB subunits p50 and p65 increased in cell nuclear fractions as early as 1.5 h.Although the level of total lκB-α remained constant,phospho-lκB-α increased with treatment over time.In the culture media of Pam3CSK4-stimulated cells,MCP-1 protein level was increased,which was suppressed in the presence of lκK inhibitors.Conclusion:NF-κB pathway can be activated by the TLR2 ligand and inhibited by lκK inhibitors in the ocular surface cell culture system.This cell culture system may be used to evaluate TLR-related innate defences in ocular surface diseases.