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Expression and function of toll-like receptor-3 and -9 in human corneal myofibroblasts
Nobuyuki Ebihara1, Satoru Yamagami, Lizhong Chen
1Department of Ophthalmology, Juntendo University School of Medicine, Hongo, Tokyo, Japan. ebihara@med.juntendo.ac.jp
Purpose:
To investigate the expression and function of toll-like receptor (TLR)-3 and -9 in corneal myofibroblasts.
Methods:
Two types of human keratocytes were used, which were freshly isolated keratocytes from donor corneas and cultured keratocytes. Expression of the mRNAs for various molecular markers was analyzed in these cells by RT-PCR, and TLR-2, -3, -4, and -9 mRNAs were also analyzed by RT-PCR. Expression of TLR-3 and -9 at the protein level was assessed by flow cytometry. In addition, an antibody array and ELISA were used to detect chemokines and cytokines in the supernatant of cultured keratocytes, with or without stimulation by poly inosine-polycytidylic acid (poly (I:C)) or CpG-DNA. Furthermore, a phagocytosis assay was performed to evaluate whether signaling via TLR-3 and -9 enhances phagocytosis.
Results:
Keratocytes cultured for three passages underwent differentiation into corneal myofibroblasts. TLR-3 and -9 were detected in corneal myofibroblasts at the mRNA and protein levels, but not in freshly isolated keratocytes. Stimulation of corneal myofibroblasts with poly (I:C) or CpG-DNA enhanced the production of IL-6, IL-8, GRO, ENA-78, and RANTES compared with that by untreated cells. Phagocytic activity of myofibroblasts was upregulated by signaling via TLR-3 and -9.
Conclusions:
This is the first report on the in vitro expression and function of TLR-3 and -9 in corneal myofibroblasts. The findings suggest that the keratocyte phenotype determines the expression of TLR-3 and -9 and that corneal myofibroblasts may have an important role in bacterial and viral clearance.
Insights
Toll-like receptor (TLR)-3 and -9 are expressed in corneal myofibroblasts, enhancing their function in pathogen clearance. This suggests a role for these receptors in the innate immune response of the cornea.
Area of Science:
- Immunology
- Ophthalmology
- Cell Biology
Background:
- Corneal myofibroblasts are crucial for wound healing.
- Toll-like receptors (TLRs) are key components of the innate immune system.
- The role of TLR-3 and TLR-9 in corneal myofibroblasts is not well understood.
Purpose of the Study:
- To investigate the expression of TLR-3 and TLR-9 in corneal myofibroblasts.
- To determine the functional significance of TLR-3 and TLR-9 signaling in these cells.
Main Methods:
- Human keratocytes were isolated and cultured.
- RT-PCR was used to analyze mRNA expression of TLRs and other markers.
- Flow cytometry assessed TLR protein levels.
- Chemokine and cytokine production was measured via ELISA and antibody arrays.
- Phagocytosis assays evaluated cellular function.
Main Results:
- Cultured keratocytes differentiated into myofibroblasts, expressing TLR-3 and TLR-9 at mRNA and protein levels.
- TLR-3 and TLR-9 stimulation upregulated inflammatory cytokine production (IL-6, IL-8, GRO, ENA-78, RANTES).
- Signaling via TLR-3 and TLR-9 enhanced myofibroblast phagocytic activity.
Conclusions:
- This study is the first to report on TLR-3 and TLR-9 expression and function in corneal myofibroblasts in vitro.
- Keratocyte phenotype dictates TLR-3 and TLR-9 expression.
- Corneal myofibroblasts expressing TLR-3 and TLR-9 may contribute to clearing bacterial and viral infections.
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