LIPOPOLYSACCHARIDE-INDUCED AUTOPHAGY IN RETINAL PIGMENT EPITHELIUM CELLS

VICENTE BERMÚDEZ; PAULA ESTEFANÍA TENCONI; NORMA MARÍA GIUSTO; MELINA VALERIA MATEOS

Buenos Aires

Congreso; REUNIÓN CONJUNTA DE SOCIEDADES DE BIOCIENCIAS; 2017

Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB)

Lipopolysaccharide (LPS) can reach the retinal pigment epithelium(RPE) in patients with bacterial endophthalmitis. This unusualpathology (consequence of intravitreal injections, trauma, eye surgery,or sepsis) has generally poor prognosis and often leads to visionloss. Our previous studies demonstrated for the first time thatclassical phospholipases D (PLDs) participates in the LPS-inducedinflammatory process in RPE cells. The aim of the present work is tostudy the autophagy process in RPE cells exposed to LPS.For this purpose D407 and ARPE-19 human RPE cells were exposedto LPS (25 μg/ml) for 24 and 48 h. Western Blot (WB) andimmunofluorescence (IF) assays were performed in order to evaluateLC3II (an autophagosome marker) content and LC3-positivepunctate structures, respectively. To study the role of the PLD pathway,cells were pre-incubated for 1 h with selective PLD1 (EVJ orVU0359595, 0.5 μM) or PLD2 (APV or VU0285655-1 0.5 μM) inhibitorsprior to LPS addition. Cell viability was measured using the MTTreduction assay. Statistical analysis was performed using ANOVAfollowed by Bonferroni´s test and p-values ≤ 0.05 were consideredstatistically significant.Our results demonstrate that in D407 cells LPS reduced cell viabilityafter 48 h treatment (p ≤ 0.001). LPS also increased LC3II/LC3Iratio and LC3-positive punctate structures after 24 and 48 h exposure(p ≤ 0.05). An increment in LC3-positive punctate structureswas also observed in ARPE-19 cells exposed to LPS. In addition,PLD1 and PLD2 inhibition highly increases LC3II content in LPS-exposedD407 cells with respect to LPS condition. Furthermore, bothPLD inhibitors restore cell viability to control condition levels in D407cells exposed to LPS.In conclusion, our results show that LPS treatment reduces cellviability and increases the autophagosome marker LC3II in RPEcells. Furthermore, the inhibition of both PLDs restores cell viability,possibly through the modulation of LPS-induced autophagy.Keywords: retinal pigment epithelium, lipopolysaccharide, autophagy,phospholipase D