Oxidized Lipoprotein Uptake Through the CD36 Receptor Activates the NLRP3 Inflammasome in Human Retinal Pigment Epithelial Cells

Abstract

Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Figure 1

Ox-LDL induces RPE cytotoxicity in a dose-dependent manner. (A) We treated ARPE-19 cells with 50, 100, and 300 μg/mL LDL or ox-LDL or serum-free media; conditioned media were collected after 48 hours and LDH release was measured. Growth of ARPE-19 cells in 100 μg/mL or 300 μg/mL ox-LDL led to a significant increase in LDH release. (B) We treated hf-RPE cells with 100, 300, and 500 μg/mL LDL or ox-LDL or serum-free media; conditioned media were collected after 48 hours and LDH was measured. Growth of hf-RPE cells in 500 μg/mL ox-LDL led to a significant increase in LDH release. *** P < 0.001.

Figure 2

Treatment of Ox-LDL disrupts RPE barrier properties. Human fetal RPE cells grown on 0.4-μm transwell membranes for 2 to 4 weeks were treated with LDL or ox-LDL for 48 hours and then examined for actin cytoskeletal organization using AlexaFluor 488 phalloidin. (A) Control hf-RPE cells treated with PBS and (B) Human fetal cells treated with LDL (500 μg/mL) exhibited intact hexagonal RPE morphology. (C) In comparison with (A) and (B), treatment of hf-RPE with ox-LDL (500 μg/mL) led to a disrupted cytoskeletal organization. Scale bar: 50 μm (A–C). (D) Measurements of TER in hf-RPE cells treated with 500 μg/mL LDL or ox-LDL were used to assess RPE barrier properties. There was no change in TER following 24 hours of LDL treatment, whereas ox-LDL–treated RPE cells exhibited a dramatic decrease in TER, which continued to decline over the next 24 hours. *** P < 0.001, ** P < 0.01.

Figure 3

Oxidized LDL taken up by RPE is targeted to the lysosomes through the CD36 receptor. hf-RPE and ARPE-19 cells grown on laminin-coated coverslips for a week were treated with 10 μg/mL DiI-ox-LDL and then visualized at 15 hours by immunofluorescent staining for LAMP-1 to localize the lysosomes. For function blocking assay, ARPE-19 cells were treated with 10 μg/mL DiI-ox-LDL in the presence of 20 or 40 μg/mL of control IgA or anti-CD36 IgA. At 15 hours posttreatment, there was clear localization of DiI-ox-LDL to LAMP-1-staining lysosomes in (A–C) hf-RPE and (D–F) ARPE-19. Anti-CD36 IgA treatment (40 μg/mL) significantly decreased DiI-ox-LDL uptake in ARPE-19 cells (H). (I) Quantification of the number of DiI-ox-LDL particles present per field of view. ** P < 0.01. Arrows in (C) and (F) show the location of DiI-ox-LDL within LAMP-1–positive lysosomes. Scale bars: 10 μm (A–F) and 50 μm (G, H).

Figure 4

Treatment with Ox-LDL increases CD36 expression level. We treated (A) hf-RPE and (C) ARPE-19 cells with LDL or ox-LDL for 24 hours and CD36 mRNA levels were measured by RT-PCR. Treatment with LDL did not alter the expression of CD36 mRNA, whereas ox-LDL treatment increased CD36 mRNA levels in both cell types by approximately over 2-fold. We treated (B) hf-RPE and (D) ARPE-19 cells with LDL or ox-LDL for 72 hours and 48 hours were analyzed for CD36 protein expression levels. Treatment with LDL did not alter CD36 protein level relative to the untreated control. In contrast, ox-LDL treatment significantly increased CD36 protein levels in hf-RPE cells and ARPE-19 cells as compared with LDL and control cells. ** P < 0.01.

Figure 5

Oxidized LDL increases NLRP3 mRNA expression in RPE cells. We incubated (A) ARPE-19 cells grown for 2 to 3 weeks were incubated with 40 μg/mL of control IgA or anti-CD36 IgA in the presence or absence of 100 μg/mL of ox-LDL. After 48 hours of treatment, conditioned media were collected to measure LDH levels. We found that ARPE-19 cells that were grown in the presence ox-LDL showed a significant increase (P < 0.01) in LDH level. Treatment of ARPE-19 cells with anti-CD36 IgA significantly (P < 0.05) decreased ox-LDL induced cytotoxicity. We treated (B) hf-RPE and (C) ARPE-19 cells with LDL or ox-LDL for 24 hours and then analyzed for NLRP3 mRNA levels by RT-PCR. Cells treated with Ox-LDL showed nearly a 6-fold increase (P < 0.01) in NLRP3 mRNA expression in hf-RPE cells and more than a 5-fold (P < 0.001) increase in ARPE-19 cells relative to LDL or untreated control cells. *** P < 0.001, ** P < 0.01, * P < 0.01.

Figure 6

Oxidized LDL leads to caspase-1 activation in RPE cells. (A) We treated hf-RPE cells with 500 μg/mL LDL or ox-LDL for 72 hours and then analyzed for pro- and cleaved caspase-1 by protein immunoblot. Human fetal RPE cells treated with ox-LDL exhibited a significant increase in pro-caspase-1 (P < 0.01) and cleaved caspase-1 (P < 0.01) compared with untreated control and LDL-treated cells. Each sample represents hf-RPE from three different fetal eyes. (B) We treated ARPE-19 cells for 48 hours with 100 μg/mL LDL or ox-LDL in the presence of fluorescent-labeled inhibitor of caspase-1 (FLICA, FAM-YVAD-FMK). Untreated and LDL-treated cells displayed little to no caspase-1-positive cells (top left and middle), whereas cells treated with ox-LDL showed increased number of caspase-1 positive cells (top right). Scale bar: 100 μm. (C) We treated ARPE-19 cells with LDL or ox-LDL for 48 hours, conditioned media were collected and IL-1β release was measured by protein blot. Pro- and mature IL-1β were observed in ox-LDL–treated cells but not in untreated and LDL-treated cells.

Figure 7

Inhibition of NLRP3 inflammasome activation prevents ox-LDL–induced RPE cell death. We treated ARPE-19 cells for 48 hours with 100 μg/mL ox-LDL in the presence or absence of 10 μM isoliquiritigenin, and conditioned media were collected to measure LDH release. Oxidized LDL induced significant RPE cell death, and inclusion of isoliquiritigenin nearly completely inhibited ox-LDL–induced cell death. Arrow on the top-left image denotes cells with abnormal morphology, which are presumably undergoing cell death. Scale bar: 100 μm. *** P < 0.001.