Oxidized Lipoprotein Uptake Through the CD36 Receptor Activates the NLRP3 Inflammasome in Human Retinal Pigment Epithelial Cells.
Accumulation of oxidized phospholipids/lipoproteins with age is suggested to contribute to the pathogenesis of AMD. We investigated the effect of oxidized LDL (ox-LDL) on human RPE cells.



Primary human fetal RPE (hf-RPE) and ARPE-19 cells were treated with different doses of LDL or ox-LDL. Assessment of cell death was measured by lactate dehydrogenase release into the conditioned media. Barrier function of RPE was assayed by measuring transepithelial resistance. Lysosomal accumulation of ox-LDL was determined by immunostaining. Expression of CD36 was determined by RT-PCR; protein blot and function was examined by receptor blocking. NLRP3 inflammasome activation was assessed by RT-PCR, protein blot, caspase-1 fluorescent probe assay, and inhibitor assays.



Treatment with ox-LDL, but not LDL, for 48 hours caused significant increase in hf-RPE and ARPE-19 (P < 0.001) cell death. Oxidized LDL treatment of hf-RPE cells resulted in a significant decrease in transepithelial resistance (P < 0.001 at 24 hours and P < 0.01 at 48 hours) relative to LDL-treated and control cells. Internalized ox-LDL was targeted to RPE lysosomes. Uptake of ox-LDL but not LDL significantly increased CD36 protein and mRNA levels by more than 2-fold. Reverse transcription PCR, protein blot, and caspase-1 fluorescent probe assay revealed that ox-LDL treatment induced NLRP3 inflammasome when compared with LDL treatment and control. Inhibition of NLRP3 activation using 10 μM isoliquiritigenin significantly (P < 0.001) inhibited ox-LDL induced cytotoxicity.



These data are consistent with the concept that ox-LDL play a role in the pathogenesis of AMD by NLRP3 inflammasome activation. Suppression of NLRP3 inflammasome activation could attenuate RPE degeneration and AMD progression.
Schepens Eye Research Institute/Massachusetts Eye and Ear, Boston, Massachusetts, United States 2Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States.
Gopalan
G
Gnanaguru
Program in Liberal Medical Education, Warren Alpert Medical School of Brown University, Providence, Rhode Island, United States.
Ariel R
AR
Choi
Schepens Eye Research Institute/Massachusetts Eye and Ear, Boston, Massachusetts, United States 2Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States.
Dhanesh
D
Amarnani
Schepens Eye Research Institute/Massachusetts Eye and Ear, Boston, Massachusetts, United States 2Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States 4Department of Pathology, Harvard Medical School, Boston, Massachusetts, United States.
Patricia A
PA
D'Amore
D018955:CD36 Antigens / Q000378:metabolism*; D016923:Cell Death; D002460:Cell Line; D005786:Gene Expression Regulation; D006801:Humans; D015151:Immunoblotting; D058847:Inflammasomes / Q000378:metabolism*; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008268:Macular Degeneration / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein / Q000096:biosynthesis / Q000235:genetics*; D010084:Oxidation-Reduction; D012313:RNA / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D055213:Retinal Pigment Epithelium / Q000196:embryology / Q000378:metabolism*; D015398:Signal Transduction
D018955:CD36 Antigens; D058847:Inflammasomes; D008077:Lipoproteins, LDL; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; C453881:NLRP3 protein, human; C099252:oxidized low density lipoprotein; D012313:RNA