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Alfredo Domínguez, Rodrigo Bolanos, Jeanet Serafín, Jessica Nieves-Hernández, Yonathan Garfias; TLR3, RIG-1 and MDA5 are constitutively expressed on human corneal limbal fibroblasts and induce proinflammatory response. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2070.
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The ocular surface is exposed to a wide array of microorganisms some of which could corneal opacification and loss of vision. The principle cause of corneal damage is an exacerbated immune response. Innate immunity is the first line of defense against corneal infection. In response to virus infections, viral components are recognized by pattern recognition receptors (PRRs). Upon ligand binding, PRRs activate cytokine genes that set into motion a complex inflammatory cascade. We previously reported that human corneal limbal fibroblast (HCLF) stimulated with polyI:C elicited the elevated production and mRNA expression of TLR3 and great variety of pro-inflammatory cytokines. It was recently reported that RIG-I and MDA-5 also recognize polyI:C. In this study, we investigated whether RIG-I and/or MDA-5 contribute to polyI:C inducible responses in HCLF.
HCLF were obtained from cadaveric sclero-corneal rims. All the assays were carried out using 1x104 HCLF incubated or not with poly I:C at a concentration of 10µg/ml during 12 h. After stimulation period, the cell culture supernatant was used to detect 36 different cytokines. The total RNA was obtained retro-transcribed to cDNA, were performed PCR assays for pro-inflammatory cytokines, TLR3, RIG-1 and MDA-5. On the other hand, the total protein were isolated and separated by SDS-PAGE and transferred to a nitrocellulose membrane for immunoblot analysis with rabbit anti-RIG-I or rabbit anti-MDA5 or rabbit anti-TLR3 antibodies.
The mRNA levels of both the pro-inflammatory cytokines (IL-6, IL-8, CCL5, CCL2, CXCL1, CXCL10 and IFN-β), and receptors TLR3, and RIG were augmented when the cells were poly I:C stimulated; similarly, the levels the pro-inflammatory cytokines in the culture supernatants of HCLF were also augmented. Interestingly, TLR3 protein was constitutively presented and increased its expression when HLCF were poly I:C stimulated.
Our results showed that HLCF express transcripts and proteins for PRR such as TLR3, MID-5 and RIG-1. After poly I:C stimulation, these cells are able to respond increasing the synthesis, production and secretion of proinflammatory cytokines.
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