Abstract
BackgroundToll-like receptors (TLRs) are recognized as important contributors to the initiation and modulation of the inflammatory response in the eye. This study investigated the precise expression patterns and functionality of TLRs in human corneal epithelial cells (HCE) and in conjunctival fibroblasts (HCF).MethodsThe cell surface expression of TLRs 2-4, TLR7 and TLR9 in HCE and HCF was examined by flow cytometry with or without stimulation with lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (poly I:C). The mRNA expression of the TLRs was determined by real-time PCR. The protein content levels of interleukin (IL)-6, IL-8, IL-1β and tumor necrosis factor-α (TNF-α) were measured in HCE and HCF using multiplex fluorescent bead immunoassay (FBI).ResultsThe surface expression of TLR3 and TLR4 was detected on both HCE and HCF. Following incubation with LPS, the percentage of HCE cells staining for TLR4 decreased from 10.18% to 0.62% (P < 0.001). Incubation with poly I:C lowered the percentage of HCE cells positive for TLR3 from 10.44% to 2.84% (P < 0.001). The mRNA expression of TLRs2, 4, 7 and 9 was detected in HCE only. Activation of HCE with LPS complex elicited protein secretion up to 4.51 ± 0.85-fold higher levels of IL-6 (P < 0.05), 2.5 ± 0.36-fold IL-8 (P > 0.05), 4.35 ± 1.12-fold IL-1β (P > 0.05) and 29.35 ± 2.3-fold TNFα (P < 0.05) compared to cells incubated in medium.ConclusionsHCF and HCE both express TLRs that respond to specific ligands by increasing cytokine expression. Following activation, the surface expression of TLR3 and TLR4 on HCE is decreased, thus creating a negative feedback loop, mitigating the effect of TLR activation.
Highlights
Toll-like receptors (TLRs) are recognized as important contributors to the initiation and modulation of the inflammatory response in the eye
human corneal epithelial cells (HCE) cells express TLR3 and TLR4 on the cell surface Our aim was to determine whether HCE cells express TLR3 and TLR4 on their cell surface
Human conjunctival fibroblasts (HCF) cells express TLR3 and TLR4 on the cell surface We found that both TLR3 and TLR4 were present on the cell surface of HCF cells (Figure 1) using flow cytometry analysis
Summary
Toll-like receptors (TLRs) are recognized as important contributors to the initiation and modulation of the inflammatory response in the eye. This study investigated the precise expression patterns and functionality of TLRs in human corneal epithelial cells (HCE) and in conjunctival fibroblasts (HCF). The corneal epithelium, in particular, serves a critical function of the mucosal innate immune system, as it is constantly exposed to microorganisms and their virulent products [2]. An important characteristic of the Microorganisms possess highly conserved motifs and pathogen associated molecular patterns (PAMP) that are recognized by pattern recognition receptors (PRR) found on cells of the innate immune system. Toll-like receptors (TLR) are a family of PRR, capable of recognizing and responding to various PAMP. Ten different functional TLRs were demonstrated in humans, each presents a tendency towards a specific PAMP. TLR4 recognizes lipopolysaccharide (LPS), the endotoxin of Gram-negative bacteria, while TLR3 is a sensor of viral dsRNA [4]
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