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Hassan Alizadeh, Trivendra Tripathi, Mahshid Abdi, ; Characterization of A. castellanii Ligand that is Recognized by TLR4 on Corneal Epithelial Cells.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1888.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Previous results indicate that Acanthamoeba trophozoites activate TLR2 and TLR4 on human and Chinese hamster corneal epithelial cells and produce chemokines such as IL-8 or MIP-2 respectively. However, the components of the Acanthamoeba trophozoites that induce cytokine and chemokine production remain unknown. We sought to identify the trophozoite molecules that interact with TLR4 on the human corneal epithelial cells (HCE) and trigger the production proinflammatory chemokines.
Methods: HCE cells and TLR4 expressing HEK293 cells were incubated with or without Acanthamoeba castellanii, lipopolysaccharide for 12 and 24 h. Inhibition of TLR4 involved preincubating HEK293 and HCE cells for 1 h with neutralizing TLR4 antibody or with the control antibody followed by incubation with A. castellanii or LPS for 24 h. Acanthamoeba-membrane protein (AcMP) was isolated by homogenization of trophozoites using sterile glass beads in PBS and the resultant homogenates were centrifuged at 1000×g for 20 min at 40C. The supernatants were collected, solubilized, and membrane fractions separated by centrifugation using Mem-PERTM plus kit. AcMP chromatographed by fast protein liquid chromatography (FPLC) on Superdex 200 10/300 GL column. Fractions were pooled into four peaks and protein determined by polyacrylamide gels electrophoresis and western blotting. The ability of the membrane protein to stimulate the induction of IL-8 in HEK293 and HCE cells was examined by RT-PCR and ELISA.
Results: mRNA of several TLRs is expressed constitutively in HCE cells; however, only TLR2 and TLR4 are involved in Acanthamoeba recognition. A. castellanii induced significant upregulation of IL-8 production in HCE and TLR4 expressing HEK293 cells (P< 0.05). Anti-TLR4 antibody attenuated the production of IL-8 induced by A. castellanii treatment. Four fraction were identified by chromatography and were subjected to SDS-PAGE analysis. Only fraction #2 contain one band at approximately 20 KDa. Western blotting of AcMP fractions isolated by FPLC showed TLR4-antigen in fractions #2. Treatments of HEK293 and HCE cells with AcMP fractions 1-4 induce significant upregulation of IL-8 production by fraction# 2.
Conclusion: A.castellani recognize TLR4 on HCE and HEK293 cells by Acanthamoeba-cell membrane protein with a molecular mass of 20 KDa and induced up-regulation of IL-8.
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