Growth Factors Modulate Toll–Like Receptor Expression in Primary Cultured Fibroblasts Obtained From VKC Patients

Abstract

Purpose: : Vernal Keratoconjunctivitis (VKC) is a chronic inflammatory disorder of the ocular surface, characterized by inflammation and tissue remodeling. Nerve growth factor (NGF) and Transforming growth factor ß1 (TGFß1) are actively involved in VKC. We recently reported that Toll–like receptors (TLRs) are expressed in healthy human conjunctiva and undergo changes during VKC. The aim of this study was to investigate if growth factors might modulate primary fibroblast TLR expression.

Methods: : Primary cultures of healthy (n=5) and VKC (n=3) conjunctival fibroblasts (FBs) were obtained according to the explants technique. Serum–starved confluent FBs were incubated with NGF (1–100ng/mL) or TGFß1 (1–10 ng/mL) for 24 hrs. Total RNA and proteins were analysed for TLR4 and TLR9 expression by Western blotting/confocal analysis and real–time PCR.

Results: : Healthy conjunctival FBs express both TLR4 and TLR9. VKC FBs express less TLR4 and TLR9, as compared to healthy ones. The addition of NGF or TGFß1 induced a decrease of both TLR4 and TLR9mRNAs. TLR4mRNA down–regulation was more than those observed for TLR9mRNA, as observed for both growth factors. The molecular data were confirmed by the biochemical analysis.

Conclusions: : This is the first report of TLR4 and TLR9 expression in primary cultures of human healthy and VKC conjunctival FBs. The different expression of both TLRs in healthy and VKC FBs might reflect a different pattern of FBs differentiation, according to the activated FBs fenotype in VKC. This different expression might suggest that growth factors also modulate/influence local immune response via TLR expression.

Keywords: growth factors/growth factor receptors • conjunctiva • immunomodulation/immunoregulation