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Hua Yang, Zheng Wang, Peter S. Reinach; Dependence of Toll Like Receptor induced Responses on TRPV1 Activation in Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5317.
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© ARVO (1962-2015); The Authors (2016-present)
Toll-like receptors (TLRs) activation in the corneal epithelium induces innate immune responses, which are essential mediators of host defense during pathogen infection. Transient receptor potential vanilloid 1 (TRPV1) channel activation by severe corneal injury also induces many of the responses elicited by TLR activation. This commonality between their effects prompted us to determine in human corneal epithelial cells (HCEC) if TLR-induced responses occur as a consequence of TRPV1 activation.
Western blot analysis and immunostaining probed for TLRs expression. TRPV1 and TLR interaction was assessed using coimmunoprecipitation. Calcium imaging evaluated Ca2+ transients in fura2-loaded HCEC. TLR2,3 and 4 agonists used were lipopolysaccharide (LPS), lipoteichoic acid (LTA) and polyinosinic polycytidylic acid [poly(I:C)], respectively. TRPV1 agonist/antagonist pair was capsaicin (CAP) and capsazepine (CPZ). Gene silencing studies used TRIF siRNA and the stably transfected shRNA MyD88 subline. ELISA determined proinflammatory cytokine and chemoattractant release.
TLR2,3 and 4 expression was identified in scrambled shRNA cells. CAP (10 µM) and LTA (5 µg/ml)-induced more than 3-fold increases in IL-6 release that were fully suppressed in TRPV1 siRNA transfected cells. Following exposure of the scrambled shRNA subline to either LPS (10 ng/ml), CAP(10 µM) or LTA (5 µg/ml), p-IRAK4 formation was detected in the MyD88 immunoprecipitates. However, in the MyD88 shRNA subline, none of these agonists induced p-IRAK4 formation were measurable. CAP and poly(I:C) (10 µg/ml)-induced three and 4 fold increases in CCL5/ Rantes release, respectively, whereas in TRIF-siRNA transfected cells these rises failed to occur. LPS-induced a 2.6-fold Ca2+ transient in scrambled shRNA cells, which was fully blocked during exposure to CPZ. LPS-induced 3-fold increases in IL-6/IL-8 release, which was similar to rises elicited by CAP. On the other hand, during exposure to CPZ, neither LPS nor CAP had any effect on IL-6/IL-8 levels.
TLR2, and 4 mediate through TRPV1 activation stimulation of MyD88 dependent signaling leading to increases in IL-6/IL-8 release. TLR3 is also dependent on TRPV1 activation to induce increases in CCL5/Rantes release through MyD88 independent signaling. These results suggest that TRPV1-linked signaling provides a potential drug target for modulating responses induced by TLR2,3 and 4 activation.
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