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Zheng Wang, Hua Yang, J. Mario Wolosin, Peter S. Reinach; In Human Corneal Epithelial Cells TLR2/4 Innate Immune Responses are Fully Dependent on TRPV1 Activity Mediated Through both MyD88-dependent and Independent Signaling Pathways. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1832.
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© ARVO (1962-2015); The Authors (2016-present)
To test the hypothesis that in human corneal epithelial cells (HCEC) transient receptor potential vanilloid1 (TRPV1) activation induces innate immune responses through the same signaling pathways linked to Toll-like receptor (TLR) TLR2 and 4.
SV40-immortalized HCEC were transduced with a MyD88 shRNAmir lentiviral vector to establish a stable MyD88 deficient subline and TRPV1 expression was silenced with siRNAs. Respective non coding sequences were used to control for non specific effects. Phosphorylation levels were determined by immunoblotting and immunoprecipitation was used to determine MyD88-TRPV1 interaction. Enzyme-linked immunosorbent assay (ELISA) evaluated interleukin and CCL5/RANTES release rates.
Capsaicin (10 µM; selective TRPV1 agonist) or TLR2 and 4 selective agonists, lipoteichoic acid (LTA, 10 µg/ml), and lipopolysaccharide (LPS, 10 µg/ml) caused indistinguishable increases in the phosphorylation of transforming growth factor kinase1 (TAK1) and mitogen-activated protein kinases (MAPKs: ERK1/2, p-38 and JNK1/2) and release rates of interleukin 6 and 8. Preincubation with the TRPV1 antagonist capsazepine (CPZ, 10 µM) or TRPV1 gene silencing with siRNA abolished or markedly attenuated the TLR agonist induced responses. In the MyD88 shRNA subline, in which MyD88 protein expression was knocked down by 85%, increases in NF-ΚB activation and interleukin 6 and interleukin 8 releases were blocked by these agonists. In MyD88 immuno-precipitates, isolated from parental HCEC cells following CAP exposure, Western blots identified TRPV1 protein. TRPV1 siRNA knockdown eliminated LTA and CAP-induced increases in p-IRAK4 formation. LPS-induced increases in p-IRF3 formation and CCL5/RANTES release were blocked by dynasore, a specific TRIF inhibitor. CPZ also inhibited LPS-induced p-IRF3 formation and CCL5/RANTES release.
Both TLRs elicit through TRPV1 activation of MyD88 dependent signaling increases in interleukin 6 and interleukin 8. TLR4-induced increases in CCL5/RANTES release are mediated through TRPV1 activation of the independent pathway. TRPV1 is a potential drug target to modulate innate immune responses induced by TLR2 and 4 activation.
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