Lyso-Gb3 (lyso-Ceramide trihexoside) was purchased from Matreya, LLC. Necrostatin (a RIP1 inhibitor) was purchased from Selleckchem. 3-methyladenine (3-MA, an autophagy inhibitor) and dimethyl sulfoxide were supplied by Sigma. GSK-872 (a RIP3 inhibitor) were obtained from MedChemExpress. Antibodies were purchased from the following vendors, as follows: VCAM-1, ICAM-1, and RIP3 (Santa Cruz Delaware); MLKL (Millipore); LC3 and beclin-1 (Cell Signaling Technology); RIP1 (Invitrogen); and α-tubulin (Sigma).

ARPE-19 cells (ATCC CRL-2302), a human retinal pigment epithelial cell line, were maintained in DMEM/F12 medium (Lonza) supplemented with 10% fetal bovine serum (FBS) (Gibco-Life Technologies), 50 U/ml penicillin, and 50 μg/ml streptomycin at 37°C in a 95% air-5% CO₂ atmosphere. Cells were cultured for 24 h, pretreated with 3-MA (10 mM), GSK'872 (30 μM), or necrostatin (30 μM), and stimulated with lyso-Gb3 (0.5 μM) for 24 h. Cells were harvested at the indicated time points. Human umbilical vein endothelial cells (HUVECs) were cultured in 0.2% gelatin-coated cell culture dishes in M200 medium (Gibco) containing 5% FBS and endothelial growth factor supplement (LSGS; Cascade biologics). Cells were used for experiments at passages 4, 5, or 6. Morphological changes were observed under a light microscope at ×100.

Cells were lysed in radioimmunoprecipitation assay lysis buffer supplemented with 1 mM phenylmethylsulfonyl fluoride and 0.01 mM protease inhibitor cocktail (Roche) and lysates were incubated on ice for 15 min and centrifuged at 15,000 × g for 10 min at 4°C. Protein concentrations were determined using a Bradford assay (Bio-Rad). Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes, followed by immunoblotting with primary antibodies (1:1,000 dilution) and corresponding secondary antibodies (1:4,000 dilution). Signals were visualized using electrochemiluminescence detection reagents (Millipore), according to the manufacturer’s instructions.

Non-reducing SDS/PAGE is carried out as regular SDS/PAGE, except that cell lysates were mixed with SDS sampling buffer without the reducing agent 2-Mercaptoethanol. MLKL oligomers were analyzed by non-reducing SDS/PAGE followed by Western blotting with anti-MLKL antibody.

The mRNA expressions of genes were determined by qRT-PCR as described previously [8]. Briefly, total amount of RNA was isolated with easy-BLUE (iNtRON), and reverse transcription was performed using TaqMan reverse transcription reagents according to the manufacturer’s instructions. qRT-PCR was performed on Power SYBR Green in an ABI PRISM 7500 unit using 1 μl of template cDNA. Quantification was performed using the efficiency-corrected ΔΔCq method.

The primers used to amplify DNA sequences were as follows:

RIP1 forward 5’ -GCCCAACCGCGCTGAGTACA-3’

and reverse 5’ -TGCCTTCTATGGCCTCCACGA-3’

RIP3 forward 5’ -TGTCAAGTTATGGCCTACTGGTGCG-3’

and reverse 5’ -AACCATAGCCTTCACCTCCCAGGAT-3’

MLKL forward 5’ -GGATTGCCCTGAGTTGTTGC-3’

and reverse 5’ -AACCGCAGACAGTCTCTCCA-3’

PCR conditions were as follows: preliminary denaturation at 50°C for 2 min, followed by 95°C for 10 min, 95°C for 15 sec, and 60°C for 1 min.

ARPE-19 cells were fixed with 4% buffered paraformaldehyde for 1 h, permeabilized with 0.2% Triton X-100 for 5 min at room temperature (RT), blocked with 5% normal goat serum in PBS-0.05% Tween 20, and incubated with anti-LC3B antibody (1:100) overnight at 4°C. Cells were then treated with Alexa Fluor 488 goat anti-rabbit secondary antibody at 1:200 (Invitrogen) for 1 h at RT. Nuclei were stained with DAPI for 30 min at RT, and cells were observed under a K1-Fluo confocal laser scanning microscope (Nanoscope Systems).

SA-β-gal staining was performed as previously described [9]. Briefly, HUVECs were seeded on 6-well plates, fixed with 4% formaldehyde at RT for 1 h, washed with PBS, and then stained using a SA-β-gal staining Kit (Sigma-Aldrich). The ratio of blue positive cells per 100 cells were measured by calculating under a light microscope at ×200.

ARPE-19 cells were cultured in 6 well plates until 80%–90% confluent. After removing the growth medium, the cells were incubated overnight in a serum-free ARPE-19 medium. Cells were pretreated with 3-MA for 1 h and then stimulated with Lyso-Gb3 for 24 h. The conditioned medium was collected and centrifuged at 3,000 × g for 10 min to eliminate cell debris. The levels of interleukin (IL)-1β, interferon gamma (IFN-γ), tumor necrosis factor α (TNF-α), and IL-6 secreted in the conditioned medium were determined using a human proinflammatory cytokine multiplex ELISA kit (arigo Biolaboratories) according to the manufacturer’s instructions.

ARPE-19 cells were plated in 6 wells and one day later when ~70% confluent cells were exposed to lyso-Gb3 (0.5 μM) in fresh medium without antibiotics for 24 h. After fixation, subsequent procedures were conducted as previously described [10]. For TEM studies, cell pellets were washed with PBS three times, fixed in 0.1 M sodium cacodylate (NaCac) buffer (pH 7.4) containing 2% glutaraldehyde, post-fixed in 0.1 M NaCac containing 2% osmium tetroxide, stained en bloc with 2% uranyl acetate, dehydrated using a graded ethanol series, embedded in Epon-Araldite resin, sectioned with a diamond knife, and stained with uranyl acetate and lead citrate. Cells were observed using a transmission electron microscope (JEM 1230-JEOL USA Inc.) at 110 kV and imaged with a CCD camera equipped with a first light digital camera controller (Gatan Inc.).

ARPE-19 cells were pretreated with vehicle, necrostatin (30 μM), GSK-872 (30 μM), or 3-MA (10 mM) for 1 h, and then incubated with lyso-Gb3 (0.5 μM) for 3 h. Media were then replaced with fresh medium, and CM were recovered after overnight culture. HUVECs were cultured in CMs for 24 h and then subjected to immunoblotting or cellular senescence assays.

Results in bar graphs are presented as means ± SDs. The significances of intergroup differences were assessed by Student’s t-test and multiple group comparisons using ANOVA followed by Bonferroni’s post-hoc test, and p-values of < 0.05 were considered statistically significant. Results are presented as the means of at least three independent experiments.

Lyso-Gb3 were found to induce autophagy in ARPE-19 cells (a human retinal pigment epithelial cell line). Electron microscopy showed typical vacuolar features of autophagy and fluorescence microscopy revealed puncta formation of LC3 in response to lyso-Gb3 (Fig. 1A, B). We found that lyso-Gb3 induced mixed types of autophay and ER-phagy was a major type of autophagy, suggesting that damaged ER membrane and constituents might be degraded or recycled through autophagy. In addition, lyso-Gb3-induced LC3 puncta formation and LC3B expression were suppressed by 3-MA (an autophagy inhibitor), suggesting lyso-Gb3 induced autophagy in retinal pigment epithelial cells (Fig. 1B, C). The effects of lyso-Gb3 on ARPE-19 cell viability were determined by MTT analysis. Lyso-Gb3 treatment slightly increased cell viability and similar phenotype was confirmed by morphological analysis under a light microscope (Fig. 1D, E). These results suggest that lyso-Gb3 induced ARPE-19 cell autophagy without affecting cell viability.

Necroptosis is induced by extracellular stimuli and mediated by RIP1, RIP3, and MLKL (mixed lineage kinase domain-like protein). Lyso-Gb3 dose-dependently increased RIP1 and RIP3 as well as phosphorylated levels of MLKL (Fig. 2A). To examine the role of autophagy in the regulation of necroptosis, ARPE-19 cells were exposed to lyso-Gb3 in the presence or absence of 3-MA (an autophagy inhibitor), and expressions of necroptosis-related molecules were then determined by Western blot and quantitative real time PCR. As shown in Fig. 2B, biochemical data showed that 3-MA significantly inhibited the up-regulations of autophagy-related proteins (LC3, beclin-1) and of necroptosis-associated markers (RIP1, RIP3, MLKL), and we also determined lyso-Gb3-induced MLKL oligomerization in ARPE-19 cells. Under non-reducing conditions, MLKL Western blotting detected the formation of around 250 kDa MLKL oligomers in ARPE-19 cells treated with lyso-Gb3 (Fig. 2C). On the other hand, in the cultures of ARPE-19 cells treated with the autophagy inhibitor 3-MA, low amounts of MLKL oligomers were detected, suggesting that MLKL phosphorylation leads to its oligomerization, creating pores for inflammatory cytokine release. In addition to protein expression, a similar pattern was observed for the mRNAs of RIP1, RIP3, and MLKL (Fig. 2D). Inflammatory cytokines are signaling molecules secreted by immune cells such as helper T cells, macrophages, and other cell types that promote inflammation, including IL-1β, IL-6, IL-12, IL-18, TNF-α, and IFN-γ. The innate immune response is mediated by inflammatory cytokines, which are primarily generated and implicated in the upregulation of the inflammatory response [11,12]. In recent years, it has been evident that inflammation and simultaneous activation of the innate immune system are common responses in FD and arise primarily due to accumulation of glycolipids (Gb3 and Lyso-Gb3), which function as risk signals. Lysosomal deposits have been shown to induce DAMP production in damaged cells through cell lysis, and the addition of lyso-Gb3 to normal control cells induces cytokine secretion [13-16]. Therefore, to investigate the role of autophagy in the inflammatory response initiation through necroptosis induced by lyso-Gb3, TNF-α, IL-1β and IFN-γ treatment, cells were exposed to autophagy inhibitor 3-MA prior to Lyso-Gb3, TNF-α, IL-1β, and IFN-γ, and the levels of proinflammatory molecules were measured using ELISA and Western blot (Fig. 2E, F). As shown in Fig. 2E, lyso-Gb3 increased the levels of IL-1β, IFN-γ, and TNF-α, whereas 3-MA pretreatment significantly inhibited these upregulations. In addition, 3-MA pretreatment significantly suppressed the protein expressions of necroptosis-related molecules by TNF-α, IL-1β and IFN-γ (Fig. 2F). Taken together, our findings suggest that lyso-Gb3 upregulates necroptosis and inflammation in an autophagy-dependent manner in ARPE-19 cells.

We next investigated whether lyso-Gb3 affects RIP1/RIP3/MLKL-induced epithelial necroptosis and inflammation in a cell-autonomous manner or indirectly by secreting molecules into conditioned media, which would indicate the triggering of neighboring endothelial cell necroptosis in a paracrine manner. As shown in Fig. 3A, ARPE-19 cells were pretreated with 30 μM of necrostatin (a RIP1 inhibitor), 30 μM GSK'872 (a specific inhibitor of RIP3), or 10 mM of 3-MA for 1 h, and then incubated with lyso-Gb3 (0.5 μM) for 3 h. Media were then replaced with fresh medium to remove remaining lyso-Gb3. After overnight culture, CM was transferred to HUVECs, and 24 h later, protein levels of necroptosis-associated markers (RIP1, RIP3, MLKL), autophagy-associated markers (LC3B, beclin-1), and inflammation-associated markers (VCAM-1 and ICAM-1) in HUVECs were analyzed by immunoblotting analysis. CM from lyso-Gb3 treated ARPE-19 cells was found to accelerate endothelial necroptosis and inflammation in HUVECs (Fig. 3B), and pretreatment of ARPE-19 cells with necrostatin, GSK'872, or 3-MA significantly suppressed CM-induced necroptosis and inflammation in HUVECs treated with CM of lyso-Gb3 treated ARPE-19 cells (Fig. 3B–D). These results showed lyso-Gb3 induced necroptosis via autophagy and that lyso-Gb3 treated retinal pigment epithelial cells could trigger endothelial necroptosis and inflammation via autophagy-dependent necroptosis.

Necroptosis and inflammation are closely associated with cellular senescence, and thus, we investigated whether lyso-Gb3 affects endothelial senescence via autophagy-dependent necroptosis in a paracrine manner. CM of lyso-Gb3 treated ARPE-19 cells induced positive staining by SA-β-gal in a time-dependent manner in HUVECs (Fig. 4A), and pretreatment with necrostatin, GSK'872, or 3-MA significantly suppressed this staining (Fig. 4B). These results suggest that lyso-Gb3 induces retinal pigment epithelial cell-derived endothelial dysfunction via an autophagy-dependent necroptosis pathway in retinal layers in FD.