Abstract
Purpose: :
The innate immune Toll Like Receptor-3 (TLR3) recognizes double-stranded RNA throughout a viral replication process. The synthesis of cytokines such as IFN-beta and IL-8 is induced by the TLR3-ligand poly I:C. It has also been demonstrated that TLR3 activation drives apoptosis on viral infected cells. The amniotic membrane (AM) exerts anti-inflammatory and immunomodulatory effects and promotes the re-epithelialization in corneal diseases. Although several anti-inflammatory and immonomodulatory mechanisms of the AM have been described, its role in the inhibition of innate immunity receptors such as TLR3 is unknown. The aim of this study was to analyze the effect of AM on corneal limbal fibroblasts (CLF) TLR3 expression and to determine whether AM is capable to inhibit the synthesis of IL8 and IFNbeta in CLF.
Methods: :
Limbal corneal fibroblasts were obtained from cadaveric esclero-corneal rims. Frozen AM was enzimatically de-epithelized (de-AM). All the assays were carried out using 1x104 CLF in the presence or absence of de-AM incubated or not with poly (I:C) at a concentration of 1µg/ml during 24 h. After stimulation period, the total RNA was obtained retro-transcribed to cDNA. PCR assays for IL-8, IFN-beta and TLR3 were performed. Semi-quantitation of transcripts was performed using actin as a control gene.
Results: :
TLR3, IFNbeta and IL-8 were constitutively expressed by CLF; as expected, their expression was augmented when the cells were poly I:C stimulated. Interestingly, when CLF were culture on AM without poly I:C, TLR3, IFN-beta and IL-8 expression was diminished; meanwhile, poly I:C was able to restore the expression of these three transcripts on CLF cultured on AM.
Conclusions: :
Taken together these results suggest that AM is able to inhibit the expression of pro-inflammatory and anti-viral cytokines in CLF without any stimulation; this effect could be driven through the inhibition of the TLR3 signaling pathway. The capability of poly I:C to restore the expression of these three transcripts on CLF cultured on AM might enhance poly I:C-induced apoptosis in viral infected corneal stromal cells. These mechanisms could explain in part the immunomodulator effect of AM transplantation.
Keywords: cornea: stroma and keratocytes • immunomodulation/immunoregulation • cytokines/chemokines