Sulfatides Induce Human Astrocyte Cell Death

Sulfatide-mediated cell toxicity of astrocytes is less well studied in comparison to the effects of this lipid on oligodendrocytes. Therefore, the effects of sulfatides on human astrocyte survival were first investigated. Cultured human astrocytes were serum-starved for 4 h and then treated with sulfatides at the timepoints and concentrations indicated (Fig. 1A). Sulfatides reduced human astrocyte numbers in a concentration- and time-dependent manner: sulfatides treatment for 6 h modestly reduced astrocyte survival (20 µM, 92.6%; 50 µM, 88.3%; and 100 µM, 68.1%, compared with control; mean ± SEM). Treatment with sulfatides for 24 h and 48 h significantly reduced astrocyte cell survival (for 24 h; 20 µM, 74.2%; 50 µM, 65.8%; and 100 µM, 48.2%, compared with control; mean ± SEM) (for 48 h; 20 µM, 42.4%; 50 µM, 20.7%; and 100 µM, 4.7%, compared with control; mean ± SEM) (Fig. 1B and C). These data suggest that sulfatides induce astrocyte dysfunction and may contribute to demyelination in the associated disease.

Fig. 1

Sulfatides induce human astrocyte cell death. A Diagram of experimental timeline depicting astrocytes were incubated in serum-free media for 4 h and treated with sulfatides at concentrations indicated for 6 h or 24 h or 48 h. B Representative images of human astrocytes treated with 10 µM, 20 µM, 50 µM, and 100 µM sulfatides for either 6 h, 24 h, or 48 h. Cells were imaged under light microscopy. C Dose–response curves showing that sulfatides decreased human astrocyte viability in a concentration-dependent (5 µM, 10 µM, 20 µM, 50 µM, and 100 µM) and time-dependent (6 h (black diamond), 24 h (black square), 48 h (black circle)) manner (n = 5–10). Cell viability was quantified by measuring NADP(H) activity using an MTT assay (Vybrant® MTT Cell Proliferation Assay Kit, Life technologies). All values are given as a percentage normalized to the control group. Statistical analysis was performed using a log dose–response test with GraphPad Prism 5. Graphical data are presented as mean ± SD

Olaparib Reduces Sulfatide-Induced Toxicity in Human Astrocytes

Given the data above demonstrating that sulfatides induce cell toxicity in human astrocytes in a time-dependent manner, all further studies were carried out investigating the effects of sulfatide treatment at 24 h. Next, the protective effects of Olaparib on sulfatide-induced toxicity in human astrocytes were examined. Olaparib is a poly (ADP-ribose) polymerase (PARP) inhibitor, marketed as an anticancer therapeutic (Bochum et al. 2018). Human astrocytes were treated with sulfatides (1 µM, 5 µM, 10 µM, 20 µM, 50 µM, 100 µM, 500 µM, 1 mM, and 10 mM) and Olaparib (1 nM, 10 nM, 100 nM, and 1 µM) at the time points indicated (Fig. 2A). Olaparib demonstrated a protective effect at low doses (1 nM, 10 nM, and 100 nM) on sulfatide-induced astrocyte cell death (1 nM Olaparib: 10 µM, 83.8% vs 90.6%; 20 µM, 74.2% vs 89.6%; 50 µM, 65.8% vs 79.2%;100 µM, 48.2% vs 51.2%, mean ± SEM) (Fig. 2B), (10 nM Olaparib: 10 µM, 83.8% vs 95.4%; 20 µM, 74.2% vs 93.0%; 50 µM, 65.8% vs 88.3%;100 µM, 48.2% vs 51.6%, mean ± SEM) (Fig. 2C), (100 nM Olaparib: 10 µM, 83.8% vs 99.0%; 20 µM, 74.2% vs 97.1%; 50 µM, 65.8% vs 96.3%; 100 µM, 48.2% vs 59.1%, mean ± SEM) (Fig. 2D and E), (1 µM Olaparib: 10 µM, 83.8% vs 84.4%; 20 µM, 74.2% vs 75.3%; 50 µM, 65.8% vs 70.9%;100 µM, 48.2% vs 52.7%, mean ± SEM) (Fig. 2F). These results demonstrate Olaparib's efficacy in counteracting sulfatide-induced cytotoxicity in human astrocytes and further underline that the efficacy is most pronounced at the 100 nM dosage of Olaparib. Therefore, this concentration of Olaparib was selected for subsequent experiments.

Fig. 2

Olaparib reduces sulfatide-induced human astrocyte cell death. A Diagram of experimental timeline and treatments. Human astrocytes were treated with 5 µM, 10 µM, 20 µM, 50 µM, and 100 µM sulfatides for 24 h with or without 100 nM Olaparib. B A dose–response curve showing changes in astrocyte viability after treatment with sulfatides (5 µM, 10 µM, 20 µM, 50 µM, and 100 µM, clear circle) with or without Olaparib treatment (1 nM, black square) for 24 h (n = 5). C A dose–response curve showing changes in astrocyte viability after treatment with sulfatides (5 µM, 10 µM, 20 µM, 50 µM, and 100 µM, clear circle) with or without Olaparib treatment (10 nM, black square) for 24 h (n = 5). D A dose–response curve showing changes in astrocyte viability after treatment with sulfatides (5 µM, 10 µM, 20 µM, 50 µM, and 100 µM, clear circle) with or without Olaparib treatment (100 nM, black square) for 24 h (n = 10). E Representative images of astrocytes treated with sulfatides (20 µM, 50 µM, and 100 µM) with or without Olaparib (100 nM) for 24 h. Cells were imaged under light microscopy. F A dose–response curve showing changes in astrocyte viability after treatment with sulfatides (5 µM, 10 µM, 20 µM, 50 µM, and 100 µM, clear circle) with or without Olaparib treatment (1 µM, black square) for 24 h (n = 5). Cell viability was quantified by measuring NADP(H) activity using an MTT assay (Vybrant® MTT Cell Proliferation Assay Kit, Life Technologies). Statistical analysis was performed using a log dose–response test with GraphPad Prism 5. Graphical data are presented as mean ± SD

Olaparib Decreases Sulfatide-Induced PARP-1 Activation in Human Astrocytes

Under normal physiological conditions, PARP-1 activity is involved in a myriad of cellular processes that includes detection and repair of DNA damage, stress response, and chromatin maintenance (Bai 2015). In neurodegenerative disorders including Parkinson’s disease and multiple sclerosis, hyperactivation of PARP-1 results in neuronal death via accumulation of PAR polymers as well as excessive glial cell activation (Alano et al. 2010). Hence, the effects of sulfatides and Olaparib on PARP-1 activation in human astrocytes were investigated by measuring the changes in PARP-1 expression in the nucleus. Treatment of human astrocytes with sulfatides (5 μM; 10 μM; 20 μM; 50 μM; 100 μM) for 24 h induced PARP-1 expression in astrocyte nuclei in a concentration-dependent manner (Fig. 3A and Supplementary Fig. 1C). Importantly, these changes in PARP-1 fluorescence were reduced by treatment with Olaparib (100 nM) (Ctrl: 8.0 vs 7.4; 5 μM 10.4 vs 8.6; 10 μM 23.4 vs 17.3, *p < 0.05; 20 μM 38.1 vs 16.8, **p < 0.01; 50 μM 42.9 vs 17.8, **p < 0.01; 100 μM 44.4 vs 36.2, *p < 0.05) (Fig. 2B). Results obtained through immunofluorescence were confirmed by Western blot where a detectable increase in PARP-1 protein (~132 kDa band) was observed in astrocytes after 24-h sulfatides exposure, with a consequent decrease following Olaparib treatment (Fig. 3C). The assessment of PAR synthesis serves as a representative marker of PARP-1 activation. Accordingly, PAR expression was quantified within the nuclei of astrocytes treated with sulfatides (5 μM; 10 μM; 20 μM; 50 μM; 100 μM) in the presence or absence of Olaparib (100 nM) (Fig. 3D and Supplementary Fig. 1D). Sulfatides induced a concentration-dependent elevation in PAR expression, which was significantly ameliorated by Olaparib treatment (Ctrl: 4.6 vs 2.2; 5 μM 7.7 vs 4.6; 10 μM 16.8 vs 7.2, *p < 0.05; 20 μM 25.2 vs 11.3, ***p < 0.001; 50 μM 27.8 vs 14.5, ***p < 0.001; 100 μM 29.7 vs 20.5, *p < 0.05) (Fig. 3E). Overall, these results support the idea that sulfatide-induced toxicity is at least in part mediated by PARP-1 overactivation and that Olaparib attenuates these effects.

Fig. 3

Olaparib decreases sulfatide-induced PARP-1 activation and expression in human astrocytes. A Representative confocal images displaying PARP-1 (red) and Hoechst (blue) immunostaining under treatment conditions indicated. Confocal images were captured at ×40 magnification. B Treatment with Olaparib (100 nM) reduced sulfatide-induced PARP-1 translocation to nuclei (5 µM, 10 µM, 20 µM, 50 µM, and 100 µM). A number of 15 images were analyzed per condition. Semi-quantitative analysis of PARP-1-associated fluorescence in the nuclei of astrocytes. C To confirm the effects of sulfatides and Olaparib on PARP-1, a western blot was conducted and showed that PARP-1 protein levels were increased after sulfatide treatment in a concentration-dependent manner. Olaparib reduced sulfatide-induced PARP-1 expression. D Representative confocal images displaying PAR (purple) and Hoechst (blue) immunostaining under treatment conditions indicated. Confocal images were captured at ×63 magnification. E Treatment with Olaparib (100 nM) reduced sulfatide-induced PAR expression in nuclei (5 µM, 10 µM, 20 µM, 50 µM, and 100 µM). A number of 15 images were analyzed per condition. Data are presented as mean ± SEM (n = 5), one-way ANOVA followed by Turkey’s post hoc test, *p < 0.05, **p < 0.01, ***p < 0.001

Sulfatide-Induced Impairment in Human Astrocyte Morphology is Partially Reversed by Olaparib

To further examine the cytotoxic effects of sulfatides and the potential rescue by Olaparib, the effects of these treatments were investigated on changes in astrocyte morphology. Specifically, the protective effect of Olaparib on sulfatide-induced toxicity was examined by immunocytochemistry of type III intermediate filament astrocyte marker vimentin, which is involved in cytoskeleton formation in astrocytes. Treatment of human astrocytes with sulfatides (5 μM; 10 μM; 20 μM; 50 μM; 100 μM) for 24 h induced a reduction in vimentin expression in astrocytes, particularly in the cell processes, suggesting deregulation of the cellular cytoskeleton (Fig. 4A and Supplementary Fig. 1A and B). This change likely reflected the rounding of astrocytes before detachment induced by sulfatides. Importantly, these sulfatide-induced changes in vimentin localization were reduced by treatment with Olaparib (100 nM) (Ctrl: 3.6 vs 3.4; 10 µM, 2.7 vs 3.2, *p < 0.05; 20 µM, 1.6 vs 2.3, ***p < 0.001; 50 µM, 1.4 vs 2.5, ***p < 0.001, 100 µM, 0.7 vs 1.3, *p < 0.05) (Fig. 4B). The analysis of vimentin intensity fluorescence confirmed that Olaparib reverses sulfatide-induced astrocytes morphology impairment at 10 µM (7.46 vs 11.43, *p < 0.05), 20 µM (5.24 vs 13.36, *p < 0.05), and 100 µM (1.04 vs 4.20, *p < 0.05) (Fig. 4C). Overall, these results support the idea that altered cytoskeleton structure preceding sulfatide-induced cell death in human astrocytes is also attenuated by Olaparib.

Fig. 4

Sulfatide-induced changes in vimentin in human astrocytes are partially reversed by Olaparib. Human astrocytes were treated with sulfatides (5 µM, 10 µM, 20 µM, 50 µM, and 100 µM for 24 h) with or without the PARP inhibitor Olaparib (100 nM for 24 h). A Representative confocal images displaying Hoechst (blue) and vimentin (green) staining under treatment conditions indicated. Confocal images were captured at ×20 magnification showing that treatment with 100 nM Olaparib (bottom) attenuates astrocyte morphology impairment induced by sulfatides (5 µM, 10 µM, 20 µM, 50 µM, and 100 µM, top). A number of 15 images were analyzed per condition. B Bar graph illustrating changes in astrocytes area after the treatment with sulfatides (5 µM, 10 µM, 20 µM, 50 µM, and 100 µM) and with or without Olaparib (100 nM) for 24 h. C Bar graph illustrating changes in the intensity of vimentin fluorescence after the treatment with sulfatides (5 µM, 10 µM, 20 µM, 50 µM, and 100 µM) and with or without Olaparib (100 nM) for 24 h. Data are presented as mean ± SEM (n = 5), one-way ANOVA followed by Dunnett post hoc test, *p < 0.05, ***p < 0.001

Olaparib Attenuates Sulfatide-Induced Oxidative Stress Damage and Mitochondrial Stress in Human Astrocytes

Next, oxidative stress in human astrocytes was assessed using DCFH-DA. Following exposure to sulfatides (5 μM, 10 μM, 20 μM, 50 μM, 100 μM), the fluorescence intensity of DCFH-DA was elevated compared to the control group (Fig. 5A and Supplementary Fig. 1D). Treatment with 100 nM Olaparib significantly attenuated sulfatide-induced reactive oxygen species (ROS) levels (Ctrl: 1.0 vs 1.08; 5 μM 1.10 vs 0.87; 10 μM 1.32 vs 0.90, *p < 0.05; 20 μM 2.31 vs 1.29, **p < 0.01; 50 μM 3.11 vs 1.50, ***p < 0.001; 100 μM 3.16 vs 2.27) (Fig. 5A). Increasing evidence now suggests the involvement of PARP in alterations of electron transport and loss of mitochondrial membrane potential (ΔΨm) (Wang et al. 2018). Here, the ΔΨm was measured using the membrane-permeant dye tetraethylbenzimidazolylcarbocyanine iodide (JC-1), which exhibits potential-dependent accumulation in mitochondria. An increase in JC‐1 monomers indicates a loss of mitochondrial membrane potential. Cultured human astrocytes were serum-starved and treated with sulfatides (5 μM, 10 μM, 20 μM, 50 μM, 100 μM) with or without Olaparib (100 nM). Cells were then loaded with 1 μM JC-1 and after 30 min the emission spectra were measured. Sulfatides treatment of astrocytes caused loss of ΔΨm compared with control, which is indicative of increased mitochondrial stress (Fig. 5B and Supplementary Fig. 1E). Notably, treatment with Olaparib attenuated the sulfatide-induced increase of mitochondrial stress (Ctrl: 0.0 vs 1.29; 5 μM 11.16 vs 6.76; 10 μM 35.87 vs 21.01, **p < 0.01; 20 μM 82.27 vs 58.23, **p < 0.01; 50 μM 83.44 vs 73.84; 100 μM 97.64 vs 76.01) (Fig. 5B). These data further demonstrate astrocyte cell damage in the presence of sulfatides and support the involvement of PARP-1 in modulating oxidative stress and mitochondrial dysfunction.

Fig. 5

Olaparib suppresses reactive oxygen species (ROS) generation caused by sulfatides in human astrocytes and reduces mitochondrial stress. A Quantification of ROS levels was determined by 2’-7’ dichlorofluorescin diacetate (DCFH-DA) staining in human astrocytes treated with 5 µM, 10 µM, 20 µM, 50 µM, and 100 µM of sulfatides (clear circle) with or without 100 nM of Olaparib (black square). Data are presented as the mean absorbance levels (at 490 nm). B To confirm that PARP-1 activation had negligible effects on cellular stress, the JC-1 assay was performed to measure changes in the mitochondrial membrane potential of sulfatides (5 µM, 10 µM, 20 µM, 50 µM, and 100 µM) and Olaparib (100 nM). Data are presented as the mean absorbance levels (at 570 nm). The measures of fluorescence signal are reported in the graphs as fold change (FC) or percentage of each condition versus the control and presented as mean ± SD (n = 5). Statistical significance was determined by paired t-test, *p < 0.05, **p < 0.01, ***p < 0.001

Olaparib Reduces Calcium Influx in Sulfatide-Stimulated Astrocytes

Activation of PARP-1 has been observed to facilitate the expression of Ca2+ permeable channels and to alter mitochondrial Ca2+ homeostasis in ischemic or traumatic brain injury, and in a NMDA toxicity model in rat primary cortical neurons (Vosler et al. 2009; Gerace et al. 2015). Our results above show that sulfatides increase PARP-1 expression in human astrocytes (Fig. 2), hence the effects of sulfatides in the presence or absence of Olaparib on activation, influx, and oscillations of Ca2+ in human astrocytes were investigated. Astrocyte cultures were pre-treated with sulfatides (5 μM, 10 μM, 20 μM, 50 μM, 100 μM) with or without Olaparib (100 nM) for 24 h and then loaded with Cal-520AM calcium dye. Time-lapse Ca2+ imaging was performed. After a 20 s baseline recording, cells were treated with 300 μM ATP and imaged for a further 3 min (to determine the proportions of astrocytes that display ATP-induced Ca2+ oscillations). Sulfatide-treated astrocytes displayed ATP-induced Ca2+ peaks that were larger in amplitude than those elicited by astrocytes cotreated with sulfatides and Olaparib (Fig. 6A-G). Measurement of the area under the curve (AUC) of 20 µM sulfatides group showed the largest AUC Ca2+ response to ATP perfusion compared to the control group (1321.6 vs 3042.8, *p < 0.05). Interestingly, Olaparib significantly reduced this sulfatide-induced AUC Ca2+ response to ATP perfusion (10 µM, 2851.6 vs 1293.2, *p < 0.05; 20 µM, 3042.8 vs 1630.8, *p < 0.05) (Fig. 6H). Sulfatides treatment did not affect the halftime of Ca2+ release (Fig. 6I). Taken together with the data above showing sulfatide-induced PARP-1 overexpression and that Olaparib reduces Ca2+ oscillations in response to ATP in sulfatide-stimulated astrocytes, we speculate a cross modulation between the PARP-1 expression and Ca2+ signaling, that is regulated in an opposing manner by sulfatides and Olaparib.

Fig. 6

Olaparib decreases Ca2+changes caused by sulfatides in human astrocytes. Human astrocytes were treated with 5 µM, 10 µM, 20 µM, 50 µM, and 100 µM sulfatides for 24 h and with or without 100 nM Olaparib and loaded with 3 µM Cal-520AM calcium indicator for 2 h in Hank's balanced salt solution (HBSS) without calcium (Ca2+) and magnesium (Mg2+). Immediately prior to Ca2+ imaging experiments, astrocytes were transferred to a fresh DMEM medium. Time-lapse Ca2+ imaging experiments were performed at a rate of 0.99 frames per second over a 3 min 20 s period, which included 20 s baseline, and 3 min of exposure to 300 μM ATP. A Representative confocal images (×20 magnification) of Ca2+ influx in response to 300 µM ATP in control and reactive astrocytes pre-treated with sulfatides (5 µM, 10 µM, 20 µM, 50 µM, and 100 µM) and with or without 100 nM Olaparib for 24 h. B-G Representative traces showing changes in cytosolic Ca2+ levels in controls and sulfatide-stimulated astrocytes (continuous line) with or without 100 nM Olaparib (broken line). There was a 30 s baseline, at which point astrocytes were stimulated with ATP for a further 3 min. H-I Olaparib (black square) caused a larger decrease of both the peak amplitude and AUC in 10 µM and 20 µM sulfatide-treated astrocytes (clear circle) but didn’t change the time half. Data for the total 3.20 min’ treatment period are presented as mean ± SEM (n = 5) and were analyzed using a two-way ANOVA followed by Bonferroni post-hoc test, *p < 0.05

Olaparib Inhibits Pro-Inflammatory Cytokine and Chemokine Release from Sulfatide-Treated Astrocytes

MLD patients exhibit augmented levels of pro-inflammatory cytokines and chemokines including CCL2, IL-1Ra, IL-8, and CCL4 in the cerebral spinal fluid. PARP-1 has a key role in chronic inflammation in the context of many inflammatory-driven pathologies. Therefore, the effects of sulfatides treatment on the expression of pro-inflammatory cytokines and chemokines from human astrocytes were examined in the presence or absence of PARP-1 inhibition. The levels of pro-inflammatory cytokines IL-6, IL-17, IL-8, and CX3CL1 secreted from human astrocytes treated with sulfatides (5 μM, 10 μM, 20 μM, 50 μM, 100 μM) for 24 h, showed a concentration-dependent increase compared to control group (Fig. 7A-D and Supplementary Fig. 1F-I). Treating astrocytes with 100 nM Olaparib for 24 h, in the presence of sulfatides, significantly decreased the release of IL-6 (Ctrl 32.8 vs. 25.3 pg/ml; 20 μM 125.4 vs. 50.6 pg/ml, *p < 0.05; 50 μM 145.3 vs. 63.0 pg/ml, **p < 0.01; Fig. 7A), IL-17 (Ctrl 7.4 vs. 10.1 pg/ml; 20 μM 78.1 vs. 35.1 pg/ml, *p < 0.05; 50 μM 105.4 vs. 54.4 pg/ml, *p < 0.05; Fig. 7B), IL-8 (Ctrl 14.9 vs. 24.5 pg/ml; 20 μM 88.0 vs. 29.3 pg/ml, *p < 0.05; 50 μM 112.1 vs. 17.9 pg/ml, *p < 0.05; Fig. 7C), and CX3CL1 (Ctrl 0.68 vs. 0.55 ng/ml; 20 μM 9.4 vs. 5.0 ng/ml, *p < 0.05; 50 μM 10.5 vs. 6.0 ng/ml, *p < 0.05; Fig. 7D). Interestingly, the overall expression of these cytokines is negatively correlated with astrocyte survival measured by MTT (Fig. 7E). Taken together, these data suggest that sulfatides exposure promotes inflammation and that Olaparib can attenuate this by inhibiting the secretion of pro-inflammatory cytokines and chemokines. Astrocytic PARP-1 overexpression may, therefore, promote CNS neuroinflammation.

Fig. 7

PARP-1 modulates cytokine and chemokine release from human astrocytes. Cytokine and chemokine release from control and sulfatide-stimulated astrocytes (clear circle) were measured after 24 h of Olaparib exposure (black square). Olaparib (100 nM) showed a significant decrease in A IL-6, B IL-17, C IL-8, and D CX3CL1 release into the cell culture medium, as measured by ELISA. A Olaparib causes a significant reduction in IL-6 secretion from astrocytes when compared to the sulfatides group, B Olaparib also caused a significant decrease in IL-17 secretion from sulfatide-stimulated astrocytes. C Olaparib reduces IL-8 secretion and D CX3CL1 from sulfatide-stimulated astrocytes. E Correlation between total cytokines level and astrocyte survival. The levels of the 4 cytokines were significantly positively correlated with cell death. Data from the ELISA assays are presented as mean ± SEM. IL-6, IL-17, IL-8, and CX3CL1 ELISA's were repeated n = 5 (using supernatants generated from MTT assay experiments). CX3CL1 values were multiplied by 1000 and divided by 100 to adjust Fig. E. Statistical analysis was performed using a paired t-test; *p < 0.05, **p < 0.01

Olaparib Reduces the Chemoattraction of Human Immune Cells Towards the Soluble Microenvironment of Sulfatide-Treated Astrocytes

To elucidate the effects of sulfatide accumulation and Olaparib treatment on immune cell infiltration to the inflamed CNS, peripheral blood lymphocyte, NK cell, and T cell migration towards human astrocyte conditioned media was examined. Human astrocytes were treated, for 24 h, with sulfatides (5 μM, 10 μM, 20 μM, 50 μM, 100 μM) with or without 100 nM Olaparib. Migration of the isolated peripheral blood immune cells towards the astrocyte-conditioned medium (ACM) was measured using a transwell chemotaxis assay (Fig. 8A). Data are presented as fold-change (FC) of lymphocyte, NK cell, and T cell migration towards the lower chamber of the transwell. Data showed migration of immune cells towards the positive control (DMEM + 20% FBS) was significantly higher compared with the negative control (DMEM) demonstrating functionality of the chemotaxis system (Lymphocytes, Neg Ctrl vs Pos Ctrl: 1 vs 6.16, *p < 0.05), (T cells, Neg Ctrl vs Pos Ctrl: 1 vs 4.80, *p < 0.05), (NK cells, Neg Ctrl vs Pos Ctrl: 1 vs 3.64, *p < 0.05). The conditioned media from astrocytes treated with sulfatides increased migration of lymphocytes, T cells, and NK cells towards the lower chamber, compared to the negative control (Fig. 8B-D and Supplementary Fig. 1J-L). Importantly, conditioned medium from astrocytes treated with sulfatides and Olaparib significantly attenuated immune cell migration compared to conditioned medium from astrocytes treated with sulfatides: (Lymphocytes, 10 μM: 11.34 vs 5.01, *p < 0.05; 20 μM: 13.29 vs 5.36, *p < 0.05; Fig. 8B); (T cells, 20 μM: 24.85 vs 11.35, *p < 0.05; 50 μM: 24.94 vs 11.84, *p < 0.05; Fig. 8C); (NK cells, 20 μM: 7.92 vs 0.73, *p < 0.05; Fig. 8D). Of note, at higher concentrations of sulfatides (100 μM) there was a decrease in some cytokine release from astrocytes (IL-17 and IL-8) and immune cell migration. Together with the data from cell viability, calcium and ROS assays, it is likely that this is due to excessive sulfatide-induced astrocyte toxicity. Overall, these data suggest that the sulfatide-induced changes in pro-inflammatory response of human astrocytes are paralleled by an increased potential to recruit lymphocytes. Importantly, these changes can be attenuated by treatment with Olaparib.

Fig. 8

Olaparib reduced the chemoattraction of human immune cells towards the soluble microenvironment of sulfatide-treated astrocytes. Lymphocyte, T cell, and NK cell migration towards astrocyte-conditioned medium (ACM) from control and sulfatide-stimulated astrocytes were measured after 24 h of 100 nM Olaparib exposure. A Diagram of experimental timeline and treatments. B Bar charts showing the fold change migration of lymphocytes towards the negative control (serum-free DMEM (-)), positive control (DMEM + 20% FBS (+)), vehicle control (Control), or ACM from, astrocytes treated with sulfatides (clear circle) (5 µM, 10 µM, 20 µM, 50 µM, and 100 µM) with or without 100 nM Olaparib (black square). C Bar charts showing fold change migration of T cells towards the negative control (-), positive control (+), vehicle control (Control), or ACM from astrocytes treated with sulfatides (clear circle (5 µM, 10 µM, 20 µM, 50 µM, and 100 µM)) with or without 100 nM Olaparib (black square). D Bar charts showing FC migration of NK cells to negative control (-), positive control (+), vehicle control or ACM from astrocytes treated with sulfatides (clear circle (5 µM, 10 µM, 20 µM, 50 µM, and 100 µM)) with or without 100 nM Olaparib (black square). Data from the chemotaxis assays are presented as mean ± SEM (n = 6, using supernatants generated from MTT assay experiments). Statistical analysis was performed using a paired t-test; *p < 0.05, **p < 0.01