Characterization of the autophagic process in the inflammatory response of retinal pigment epithelium cells. Role of phospholipases D.

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

Killarney

Simposio; 18 th International Symposium on Retinal degeneration; 2018

Satellite Symposium of the XXIII MEETING OF THE INTERNATIONAL SOCIETY FOR EYE RESEARCH

Purpose: Autophagy is a catabolic process, highly active in retinal pigment epithelium cells (RPE) and photoreceptors (PR), responsible for the lysosomal degradation and recycling of proteins and organelles. Abnormal autophagy has been related to the pathogenesis of age-related macular degeneration (AMD) and other blinding retinal diseases due to defective lysosomal-autophagic degradation in the RPE. In addition, inflammation is a key factor in the pathogenesis of these diseases, ultimately leading to varying degrees of vision loss. Recent evidence demonstrated that autophagy participates in multicellular immunity and inflammatory processes. Our previous studies demonstrated for the first time that classical phospholipases D (PLDs) participates in the lipopolysaccharide (LPS)-induced inflammatory process in RPE cells. The aim of the present work is to study the autophagic process in RPE cells exposed to LPS, and its modulation by the PLD pathway.Approach: D407 and ARPE-19 human RPE cells were exposed to LPS (10 g/ml or 25 g/ml) for 24 and 48 h. Western Blot (WB) and immunofluorescence (IF) assays were performed in order to evaluate LC3II (a marker for autophagic structures) content and LC3-positive punctate structures, respectively. Furthermore, using bafilomycin A1 (BAF, 50 nM), a blocker of autophagosome fusion with lysosomes, we evaluated the autophagic flux by analyzing LC3 II and SQSTM1/p62 levels by WB. To study the role of the PLD pathway, cells were pre-incubated for 1 h with selective PLD1 (VU0359595, 0.5 or 5 μM) or PLD2 (VU0285655-1, 0.5 or 5 μM) inhibitors prior to LPS addition. Cell viability was measured using the MTT reduction assay. Early autophagy inhibitors, 3-methyladenin (3-MA, 5 mM) and LY294002 (10 M were tested to see if autophagy prevents cell damage.Results: In D407 cells, using IF and WB assays, we found that LPS increased LC3-positive punctate structures and LC3 II content respectively, after 24 and 48 h exposure. An increment in LC3-positive punctate structures was also observed in ARPE-19 cells exposed to LPS. In D407 cells, after blocking autophagic flux with BAF, we found an increase in both LC3 II and SQSTM1/p62 levels after LPS treatment compared with BAF control condition. In addition, PLD1 and PLD2 inhibition increases LC3II content in LPS-exposed D407 cells with respect to LPS condition. MTT assays in D407 cells showed that LPS reduces cell viability after 48 h treatment. Moreover, 3-MA and LY294002 worsened LPS-exposed cell viability.Conclusions: Our results show that LPS treatment reduces cell viability and enhances autophagy in RPE cells. Our findings also suggest that the activation of the autophagic process could be a protective mechanism triggered under inflammatory conditions. Moreover, both PLDs seem to modulate this process. Further experiments are certainly needed to fully characterize the autophagic process and the role of the PLD pathway in the inflammatory response of RPE cells.