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. NFκB 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.
Highlights
Toll-like receptors (TLRs) are a family of pattern-recognition receptors [1]
No changes in TLR mRNA expression were measured by Quantitative PCR (qPCR) in monolayered cells cultured in the basic medium with additional insulin, recombinant human epidermal growth factor (hEGF), hydrocortisone, fetal bovine serum (FBS), or cholera toxin
Increased TLR mRNA expression was first detected by qPCR in cells 3 days after airlifting and continued to increase as cells form stratified sheets
Summary
Toll-like receptors (TLRs) are a family of pattern-recognition receptors [1]. Ten TLR proteins have been identified in human cells. Conjunctival and corneal epithelial cells, were known to express multiple TLRs [4,5,6]. Cultured monolayer cells were most often used to study the expression, function, and regulation of TLR in epithelial cells. While the most TLR expressed in epithelial cells showed responses to its ligands in cultured cells, contradictory results were reported on the biological activity of TLR2 and TLR4 in corneal and conjunctival epithelial cells [7,8,9]. We explored the effect of multiple factors on TLR expression and report that the multilayered structure was a key factor to maintain a normal level of TLR expression in conjunctival and corneal epithelial cells
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