Additional file 6: Figure S5. of Remodeling of lipid bodies by docosahexaenoic acid in activated microglial cells

Reduction of oxidative stress in microglial cells treated with DHA. Oxidative stress in N9 microglial cells following treatment with DHA (10, 25, 50 μM), in the presence or absence of LPS (100 ng/mL) for 1 h. Hydrogen peroxide (20 μM) was included as a positive control. N9 microglial cells were loaded with CM-H2DCFDA (10 μM, 30 min), then exposed to treatment. Following treatment, intracellular fluorescence was imaged in live cells using a fluorescence microscope. Four fields were imaged per treatment, and 30 cells were analyzed per field. Shown are average relative intracellular fluorescence ± SEM (fold increase of control set to 1) from three independent experiments. **p < 0.001 compared to control; ##p < 0.001 compared to LPS. (TIF 1344 kb

Additional file 1: Figure S1. of Remodeling of lipid bodies by docosahexaenoic acid in activated microglial cells

Viability of microglial cells exposed to DHA treatment. A–B. Viability of N9 microglial cells in response to DHA (5–100 μM) after 24 h, in the presence or absence of LPS (100 ng/mL). Following treatment, cells were labeled with Hoechst 33258 and incubated with Alexa 488-labeled Annexin V (A) or propidium iodide (B) for 15 min, after which they were imaged under a high-throughput fluorescence microscope. Five fields were imaged per well, and cells were analyzed using the Columbus software. Shown are mean percentages of total cells positive for Annexin V or propidium iodide ± SEM from three independent experiments. *p < 0.01. C. Viability of N9 microglial cells treated with DHA (5–100 μM) as determined by cell counting after 24 h, in the presence or absence of LPS (100 ng/mL). Following treatment, cells were labeled with Hoechst 33258 and imaged under a high-throughput fluorescence microscope. Five fields were imaged per well, and three wells were included per treatment. The number of nuclei was counted using the Columbus software. Shown are mean percentage values ± SEM as compared to the untreated controls from three independent experiments. *p < 0.01. (TIF 2392 kb

Additional file 5: Figure S4. of Remodeling of lipid bodies by docosahexaenoic acid in activated microglial cells

Mitochondrial membrane potential of microglial cells exposed to DHA. A. Change in mitochondrial membrane potential in N9 microglial cells exposed to DHA and LPS for 6 h. Mitochondrial membrane potential of N9 microglial cells treated with DHA (50 μM) for 6 h. Following treatment, the cells were incubated with TMRE (50 nM) for 20 min, after which the media was refreshed and cells were imaged under a fluorescence microscope. Three fields were imaged per treatment, and ten cells were imaged per field. Shown are average fold increase in intracellular relative fluorescence intensities ± SEM as compared to untreated control (set to 1) from two independent experiments. *p < 0.01; **p < 0.001 B. Change in mitochondrial membrane potential in N9 microglial cells exposed to DHA (5–50 μM) for 24 h. Following treatment, cells were incubated with TMRE (50 nM) for 20 min, after which the media was refreshed and cells were imaged under a fluorescence microscope. Four fields were imaged per treatment, and two cells were imaged per field. Shown are average fold increase in intracellular relative fluorescence intensities ± SEM as compared to untreated control (set to 1) from three independent experiments. Cells imaged in the absence of TMRE and cells treated with FCCP were negative controls. *p < 0.01. (TIF 1214 kb

Additional file 8: Figure S7. of Remodeling of lipid bodies by docosahexaenoic acid in activated microglial cells

Nuclear fragmentation (between arrowheads) following treatment with LPS. n = nucleus. (TIF 8550 kb

Additional file 4: Figure S3. of Remodeling of lipid bodies by docosahexaenoic acid in activated microglial cells

Microglial cell morphology, phagocytosis, and mitochondrial alterations following stimulation with LPS. A–B: Microglial cell showing nuclear budding (arrowhead), a phenomenon suggestive of ongoing mitosis, and an accumulation of mitochondria with various alterations (m; inset). In particular, mitochondria partially to completely devoid of cristae and partially to completely devoid of a double external membrane can be seen. The adjacent microglial cell is representative of the most frequently observed morphological phenotype following treatment with LPS, showing several lipid vacuoles (v), similarly to what has been described previously. er = endoplasmic reticulum. C–D: microglial cell showing an accumulation of small mitochondria often devoid of cristae (inset). Budding from larger mitochondria can sometimes be seen (arrowhead). g = golgi apparatus. E–F: Microglial cell containing mitochondria of various sizes, from small to large, and often showing alteration (inset), as well as a large phagocytic inclusion (in). To be noted, only mitochondria showing alteration are annotated in the three insets. (TIF 18662 kb

Modulation of Abundance and Location of High-Mobility Group Box 1 in Human Microglia and Macrophages under Oxygen–Glucose Deprivation

While stroke represents one of the main causes of death worldwide, available effective drug treatment options remain limited to classic thrombolysis with recombinant tissue plasminogen activator (rtPA) for arterial-clot occlusion. Following stroke, multiple pathways become engaged in producing a vicious proinflammatory cycle through the release of damage-associated molecular patterns (DAMPs) such as high-mobility group box 1 (HMGB1) and heat shock protein 70 kDa (HSP72). HMGB1, in particular, can activate proinflammatory cytokine production when acetylated (AcHMGB1), a form that prefers cytosolic localization and extracellular release. This study aimed at determining how HMGB1 and HSP72 are modulated and affected following treatment with the anti-inflammatory compound resveratrol and novel platelet membrane-derived nanocarriers loaded with rtPA (CSM@rtPA) recently developed by our group for ischemic artery recanalization. Under ischemic conditions of oxygen–glucose deprivation (OGD), nuclear abundance of HMGB1 and AcHMGB1 in microglia and macrophages decreased, whereas treatment with CSM@rtPA did not alter nuclear or cytosolic abundance. Resveratrol treatment markedly increased the cytosolic abundance of HSP72 in microglia. Using proximity ligation assays, we determined that HSP72 interacted with HMGB1 and with acetylated HMGB1. The interaction was differentially affected under the OGD conditions. Resveratrol treatment under the OGD further decreased HSP72-HMGB1 interactions, whereas, in contrast, treatment increased HSP72-AcHMGB1 interactions in microglia. This study points out a salient molecular interaction suited for a two-pronged nanotherapeutic intervention in stroke: enhancement of rtPA’s thrombolytic activity and modulation of cytosolic interactions between HMGB1 and HSP72 by resveratrol

Sulfated Hyperbranched and Linear Polyglycerols Modulate HMGB1 and Morphological Plasticity in Neural Cells

The objective of this study was to establish if polyglycerols with sulfate or sialic acid functional groups interact with high mobility group box 1 (HMGB1), and if so, which polyglycerol could prevent loss of morphological plasticity in excitatory neurons in the hippocampus. Considering that HMGB1 binds to heparan sulfate and that heparan sulfate has structural similarities with dendritic polyglycerol sulfates (dPGS), we performed the experiments to show if polyglycerols can mimic heparin functions by addressing the following questions: (1) do dendritic and linear polyglycerols interact with the alarmin molecule HMGB1? (2) Does dPGS interaction with HMGB1 influence the redox status of HMGB1? (3) Can dPGS prevent the loss of dendritic spines in organotypic cultures challenged with lipopolysaccharide (LPS)? LPS plays a critical role in infections with Gram-negative bacteria and is commonly used to test candidate therapeutic agents for inflammation and endotoxemia. Pathologically high LPS concentrations and other stressful stimuli cause HMGB1 release and post-translational modifications. We hypothesized that (i) electrostatic interactions of hyperbranched and linear polysulfated polyglycerols with HMGB1 will likely involve sites similar to those of heparan sulfate. (ii) dPGS can normalize HMGB1 compartmentalization in microglia exposed to LPS and prevent dendritic spine loss in the excitatory hippocampal neurons. We performed immunocytochemistry and biochemical analyses combined with confocal microscopy to determine cellular and extracellular locations of HMGB1 and morphological plasticity. Our results suggest that dPGS interacts with HMGB1 similarly to heparan sulfate. Hyperbranched dPGS and linear sulfated polymers prevent dendritic spine loss in hippocampal excitatory neurons. MS/MS analyses reveal that dPGS-HMGB1 interactions result in fully oxidized HMGB1 at critical cysteine residues (Cys23, Cys45, and Cys106). Triply oxidized HMGB1 leads to the loss of its pro-inflammatory action and could participate in dPGS-mediated spine loss prevention. LPG-Sia exposure to HMGB1 results in the oxidation of Cys23 and Cys106 but does not normalize spine density

Dendritic Polyglycerol Sulfates in the Prevention of Synaptic Loss and Mechanism of Action on Glia

Dendritic polyglycerols (dPG), particularly dendritic polyglycerol sulfates (dPGS), have been intensively studied due to their intrinsic anti-inflammatory activity. As related to brain pathologies involving neuroinflammation, the current study examined if dPG and dPGS can (i) regulate neuroglial activation, and (ii) normalize the morphology and function of excitatory postsynaptic dendritic spines adversely affected by the neurotoxic 42 amino acid amyloid-β (Aβ₄₂) peptide of Alzheimer disease (AD). The exact role of neuroglia, such as microglia and astrocytes, remains controversial especially their positive and negative impact on inflammatory processes in AD. To test dPGS effectiveness in AD models we used primary neuroglia and organotypic hippocampal slice cultures exposed to Aβ₄₂ peptide. Overall, our data indicate that dPGS is taken up by both microglia and astrocytes in a concentration- and time-dependent manner. The mechanism of action of dPGS involves binding to Aβ₄₂, i.e., a direct interaction between dPGS and Aβ₄₂ species interfered with Aβ fibril formation and reduced the production of the neuroinflammagen lipocalin-2 (LCN2) mainly in astrocytes. Moreover, dPGS normalized the impairment of neuroglia and prevented the loss of dendritic spines at excitatory synapses in the hippocampus. In summary, dPGS has desirable therapeutic properties that may help reduce amyloid-induced neuroinflammation and neurotoxicity in AD