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Barbara De Servi, Marisa Meloni, Stefano Barabino; IMMUNOCOMPETENT CELLS INFILTRATION INTO RECONTRUCTED HUMAN CORNEAL EPITHELIUM: APPLICATION TO DRY EYE SYNDROME. Invest. Ophthalmol. Vis. Sci. 2017;58(8):983.
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© ARVO (1962-2015); The Authors (2016-present)
The biological relevance of the recontructed human corneal epithelium (HCE) model is recognised to evaluate the eye irritation potential of chemicals and to predict irritation potential of eye drops. The aim of this research project was to use the HCE model to recapitulate in vitro the ocular surface immunity, its homeostatic mechanisms and their disruption in dry eye syndrome. HCE model was cultivated with activated immunocompetent cells (monocyte cell line-THP-1) to study infiltration of the cornea tissue in response to ocular surface damage and inflammation in modified culture conditions.
The HCE model was exposed to different stimuli: hyper-osmolarity (0,6M sorbitol for 16h) and dryness (40°C and Rh<40%). Undifferentiated THP-1 cells were cultured on the traswell filter (polycarbonate membrane) and once an adherent cell layer was formed, the immunocompetent cells were monitored to follow the infiltration and migration process after different time points of post incubation (4-24-48h) by applying a morphological (Immunohistochemistry of CD68, CD83 and CD14) and molecular approach (gene expression by qRT-PCR).
The preliminary results of the infiltration protocol showed that after the incubation step the THP-1 have migrated leading to resident immunocompetent cells in the suprabasal layers. Hyperosmolarity and dryness conditions have determined THP-1 cells migration and infiltration: CD68+ was relevant for macrophages, CD83+ for dendritic cells. CD14 was a marker of undifferentiated THP-1 cells. Quantification of the number of migrated cells have been performed by using the software Leica LASX applied to LEICA DM2500. The project was completed by transcriptomics analysis of the biomarkers related to dry eye symptoms: MMP9, TNFα, AQP3, MUC4, ZO1, OCLN, ITGB1, TGFB1, IL8. Cellular damage following corneal hyperosmolarity and dryness and co-cultered with THP-1 was evaluated by adenilate kinase release in the medium.
The developped ocular surface immunocompetent system on HCE demonstrate to be able to monitor macrophages and dendritic cells migration into the corneal tissue underling their differential positioning and immunological role within the corneal epithelium. The model is under development to investigate immunomodulatory therapies to effectively treat inflammatory based-ocular diseases as Sijodren syndrome.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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