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A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva
Published on: July 5, 2017
Regulation of Toll-like receptor expression in human conjunctival epithelial cells
Jing Li1, Melina Setiawan1, Hong Wu2
1Department of Ophthalmology, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, 1665 Kong Jiang Road, Shanghai 200092, China.
Abstract:
Previous studies showed marked decrease of multiple Toll-like receptor (TLR) expression in corneal and conjunctival epithelial cells upon culture in vitro. The aim of this study was to identify factor(s) which regulate TLR expression. Primary human conjunctival epithelial cells and immortal conjunctival (IOBA-NHC) and corneal epithelial cell lines (HCET) were used. The effect of various cytokines, hypoxia, mechanical wounding, and airlifting culture on TLR expression was examined by quantitative PCR and western blot analysis. Ligand stimulated TLR activation was analyzed. TLR mRNA expression increased modestly when cultured monolayered cells were stimulated by TNF-α, IL-1α, IL-1β, IL-6, IL-8, IFN-γ (about 2-fold), hypoxia (2.1- to 4.8-fold selectively), and wounding (3.1- to 9.3-fold). In airlifted multilayered cells, TLR expression increased 7.8- to 25.9-fold compared to monolayered cells. Airlifted cells showed increased response to low concentrations of lipopolysaccharide (LPS) and peptidoglycan (PGN) stimulation. NF κ B inhibition prevented the formation of cell sheets and led to the collapse of already-formed multilayered structure and the simultaneous reduction of TLR mRNA level. In conclusion, our study showed that the conjunctival epithelial cell expressed TLR was sensitive to various stimulants, and a multilayered epithelium-like structure was needed to maintain TLR expression.
Insights
Toll-like receptor (TLR) expression decreases in cultured eye cells. A multilayered structure, like that found in vivo, is crucial for maintaining TLR expression and function in conjunctival epithelial cells.
Area of Science:
- Ophthalmology
- Immunology
- Cell Biology
Background:
- Toll-like receptor (TLR) expression is significantly reduced in cultured corneal and conjunctival epithelial cells.
- Understanding the regulation of TLRs in ocular surface epithelia is critical for immune function.
Purpose of the Study:
- To identify factors regulating Toll-like receptor (TLR) expression in human conjunctival and corneal epithelial cells.
- To investigate the role of epithelial structure in maintaining TLR expression and responsiveness.
Main Methods:
- Quantitative PCR and western blot analysis were used to assess TLR expression.
- Primary human conjunctival epithelial cells and immortalized cell lines (IOBA-NHC, HCET) were cultured under various conditions (cytokines, hypoxia, wounding, airlifting).
- Ligand-stimulated TLR activation and the effect of NF-κB inhibition were analyzed.
Main Results:
- TLR mRNA expression increased modestly with cytokine stimulation, hypoxia, and mechanical wounding in monolayer cultures.
- Airlifted, multilayered epithelial structures showed significantly higher TLR expression (7.8- to 25.9-fold) compared to monolayers.
- Multilayered cells exhibited enhanced responses to lipopolysaccharide (LPS) and peptidoglycan (PGN) stimulation.
- NF-κB inhibition disrupted multilayer formation and reduced TLR mRNA levels.
Conclusions:
- Conjunctival epithelial cell TLR expression is sensitive to various stimuli.
- A multilayered, in vivo-like epithelial structure is essential for maintaining robust TLR expression and responsiveness in ocular surface cells.
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