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David De La O-Altamirano, Karina de la Luz Miranda Sánchez, Brenda Magaly Cesár Moreno, Atzin Robles-Contreras; Functional Analysis of TLR9 in human Iris Pigmented Epithelium (hIPE) cells.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5752.
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© ARVO (1962-2015); The Authors (2016-present)
Toll like receptors (TLR) are the most important family of PRR receptors. As part of the innate immune response, its main function is the rapid recognition of pathogens. Expression and function of TLR has been mainly studied in corneal tissue. TLR9 recognizes unmethylated CpG sequences of viral DNA. In iris expression of TLR9 has already been described, but whether present activity in the hIPE before antigen challenge. So the purpose of this study was analyze the functionality of TLR-9 by cytokines secretion.
We isolated and cultivated hIPE cells from tissue obtained from iridectomies in patients with primary open-angle glaucoma. We made a phenotypic characterization with Vimentin and CK12; Once characterized hIPE cells, we stimulated with ODN2006 (TLR9 agonist) (TLR9a+ group) we used phorbol-12-Myristate-13-acetate (PMA)-Ionomicina as a positive stimulation control (C+ group), we have a negative control(C- group) using only medium (RPMI-1640). We collected supernatants at 24 hours of stimulation and stored at -20°C. Then we processed the supernatants by CBA (Human Th1/Th2/Th17 kit, BD Biosciences). A Mann-Withney test was performed to compare each cytokine between groups
K12 expression was 14.2%, and vimentin was 15.3%. Cells are able to secrete all the cytokines. We did not found any statistically significant differences between the three groups: IL2: C- 2.3±1.8pg/mL, C+ 3.6±1.4pg/mL, TLR9a+ 2.4±1.3pg/mL; IL4 C- 0.7±0.6pg/mL, C+ 0.6±0.6pg/mL, TLR9a+ 0.9±0.5pg/mL; IL6 C- 0pg/mL, C+ 0.03±0.1pg/mL, TLR9a+ 0pg/mL; IL10 C- 0pg/mL, C+ 0.1±0.2pg/mL, TLR9a+ 0.1±0.2pg/mL; TNFa C- 0.7±0.6pg/mL, C+ 0.8±1.0pg/mL, TLR9a+ 0.6±0.9pg/mL; IFNg C- 0.1±0.3pg/mL, C+ 0.2±0.4pg/mL, TLR9a+ 0.2±0.6pg/mL; IL17A C- 5.8±7.3pg/mL, C+ 18.0±42.7pg/mL, TLR9a+ 7.2±5.9pg/mL.
With this study we conclude that TLR9 in hIPE cells is not functional, because there was not a difference in the secretion of cytokines. Conduct more studies are needed, as the analysis of inflammatory cytokines like IFN type I and II as well as studies of different concentrations of ODN2006 at different stimulation times.
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