Abstract
Purpose :
Vernal keratoconjunctivitis (VKC) is a severe ocular allergy with pathogenic mechanisms poorly understood and no efficacious treatment. Autophagy is involved in both induction and suppression of immune and inflammatory responses, including allergy. The aim of the study is to investigate the association between autophagy and VKC pathogenesis.
Methods :
Conjunctival biopsies obtained from active VKC patients and healthy subjects (CT) were analyzed by immunohistochemistry (IHC) with specific antibodies against LC3A, LC3B, Beclin-1, LAMP1, Cathepsin B and D. The positive IHC reaction was evaluated in the epithelium and in the sub-epithelial stroma.
Conjunctival cell cultures were treated with culture medium (CM) from PMA- and LPS-activated U937 cells, histamine, IL-1b, IL-4, or TNF-α and analyzed by qPCR, Western blotting (WB) and immunofluorescence for the expression of autophagic and lysosomal markers at 4, 10 and 24 hours after exposure. Since chloroquine (CQ) inhibits the autophagic flux, TNF-α treated fibroblasts were exposed to 100 µM CQ for 4, 10 and 24 hours and analyzed by WB for LC3B expression.
Results :
IHC analysis showed that LC3B, Cathepsin D, Beclin-1 and LAMP1 are over-expressed in VKC tissues compared to CT. Conjunctival fibroblasts treated with U937-activated CM showed increased expression of LC3B, Beclin-1, LAMP1 and p62. Conjunctival fibroblasts treated with TNF-α showed a significantly increased expression of LC3A, LC3B, Cathepsin D and p62 after 4 and 24 hours of exposure. WB analysis revealed cleavage and lipidation of LC3B quantified as an increased LC3BII/LC3BI ratio after TNF-α treatment. TNF-α + CQ significantly decreased the expression of LC3BI and LC3BII after 24 hr of exposure. Double immunofluorescence showed that Cathepsin D and LAMP1 were co-localized within the fibroblasts lysosomes at 4, 10, 24 hours after TNF-α exposure. Histamine, IL-1b and IL-4 did not show relevant changes in the expression of autophagy markers.
Conclusions :
The increased tissue expression of LC3B, Beclin 1 and Cathepsin D demonstrated that autophagy is activated in VKC. Among the pro-inflammatory mediators tested in the in vitro model, TNF-α significantly stimulated autophagy by increasing the autophagosome adaptor LC3B and the cargo adaptor p62 in conjunctival fibroblasts.
This is a 2020 ARVO Annual Meeting abstract.