Cell cultureCell line culture

N27 rat dopaminergic neuron cell line was cultured in RPMI 1640 medium. C6 rat astrocyte cell line and BV-2 mouse microglia cell line were cultured in DMEM high-glucose medium, all supplemented with 10% FBS, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 0.25 µg/mL amphotericin B, with 5% CO2 at 37 °C in a humidified atmosphere.

Primary mixed glial culture

For rat primary mixed glial culture, brain tissues were obtained from one-day postnatal Wistar rats. After dissection, the tissue was washed with PBS and digested with trypsin (0.15%). After adding DMEM high-glucose medium with 10% FBS, the digestion product was centrifuged at 800xg for 5 min at 4 °C. The pellet was resuspended in DMEM high-glucose medium containing 10% FBS, 100 U/ml penicillin, 0.1 µg/mL streptomycin, and 0.25 µg/mL amphotericin B, and cultured with 5% CO2 at 37 °C in a humidified incubator. Assays were performed on in vitro day 7. At this stage, cultures obtained contain 90% astrocytes, 5% microglia (1 microglial cell every 4 fields 600X), and 5% of other cell types [15]. This procedure was conducted following the approval of the Institutional Animal Care and Use Committee of the Universidad Nacional del Sur (IACUC, protocol number 225/2023-CDBByF Res: 567/23) and in conformity with the guidelines outlined in the Care and Use of Laboratory Animals (National Institutes of Health).

Experimental treatments

Cells at 80–90% confluence were treated with 20 µM MB (#45554, Sigma-Aldrich, USA) or vehicle (dimethyl sulfoxide 0.1%) for 24 h and/or pre-incubated with pharmacological inhibitors, agonist or antagonist of different components of the inflammation/resolution pathways: 20 µM luteolin (LUT, sc-203119, Santa Cruz Biotechnology, USA) for ALOX-15, 10 µM celecoxib (Cx, sc-217869, Santa Cruz Biotechnology, USA) and 100 µM acetylsalicylic acid (ASA #50-78-2, Sigma-Aldrich, USA) for cyclooxygenase-2 (COX-2), 10 µM PPOH (sc-205442, Santa Cruz Biotechnology, USA) for CYP450, 10 µM U0126 (sc-222395, Santa Cruz Biotechnology, USA) for ERK1/2, 10 µM YM-26734 (sc-204410, Santa Cruz Biotechnology, USA) for sPLA2, 0.1 µM MMK-1 (ALO-GPM-120, Alomone Labs, Ltd., Israel) as FPR2/ALX agonist and 10 µM Quin-C7 (ALO-Q-155, Alomone Labs, Ltd., Israel) as FPR2/ALX antagonist (viability assay testing Quin-C7 and MMK-1 concentrations are shown in Supplementary Fig. 5), lipoxin A4 0.2 nM and resolvin D1 0.2 nM (#490414 and #400384, respectively, Cayman Chemicals, USA), concentration was selected by performing a concentration screening.

Metabolomics

Metabolome analysis was assessed using the Mass Spectrometry service unit at Pontificia Universidad Católica de Chile. 106 cells/sample were pelleted and maintained at −80 °C. Pellets were suspended in 300 µL of cold methanol followed by 1 mL of methyl-ter-butyl ether and 250 µL of water. After separation, the upper phase and the lower phase were separately transferred to a vial. Samples (3 µl each) were analyzed by LC-MS (Elute UHPLC coupled with Bruker Compact QTOF mass spectrometer), in triplicates, in negative and positive modes and normalized by protein concentration. For the upper phase, a Luna C18 column was used, 1 mm x 100 mm, 1.6 μm particle, with solvent A containing 40% water + 60% acetonitrile + 0.1% formic acid + 10 mM ammonium formate and solvent B 90% isopropanol + 10% acetonitrile + 0.1% formic acid + 10 mM ammonium formate, at flow rate 300 µl/min. For the lower phase, a Luna C18 column was used with solvent A containing water + 0.1% formic acid and solvent B 90% acetonitrile + 0.1% formic acid, at flow rate 300 µl/min, and separately a HILIC column was used with solvent A containing water + 0.1% formic acid + 5 mM ammonium formate and solvent B 90% acetonitrile + 0.1% formic acid + 5 mM ammonium formate, at flow rate 250 µl/min. Metaboscape 4.0 software was used for tandem MS data analysis and metabolite identification. Using Qlucore Omics Explorer 3.0, missing value imputation was performed by first removing variables with more than 30% missing values, and for the remaining variables, the k-Nearest Neighbors (kNN) algorithm was used to impute missing values. A logarithm base 2 transformation was applied, followed by multiple t-test between the experimental conditions. For each comparison, fold change, p-value, and log2 ratio were reported. Finally, the data was uploaded to Ingenuity Pathway Analysis (IPA, QIAGEN), where the Connect, Path Explorer, and Molecule Activity Predictor (MAP) tools were used to build an interaction network. The fold change values were then overlaid onto this network and predictive activity for its nodes could be analyzed.

Fatty acid (FA) profiling by GC-MS

Lipid extraction was performed in cells by the Bligh & Dyer method [16] followed by derivatization to obtain total FA methyl esters (FAME). Briefly, methanolysis was carried out in 1% sulfuric acid in dry methanol, at 45 °C for 24 h. After the reaction, partition with distilled water and chloroform was performed to recover the organic phase [17]. After dryness, FAMEs were dissolved in ethyl ether for separation by thin layer chromatography (TLC) in silica gel G plates with hexane: ethyl ether 95:5 (v/v) as mobile phase. After elution, samples were dried and resuspended in hexane until analysis. GC-MS analysis was performed on an HP 6890 Chromatograph with a 5972 A Mass Selective Detector (MSD) (single hyperbolic quadrupole analyzer) in the Shared Use Instrumental Laboratory– LIUC– Chemistry Department– Universidad Nacional del Sur. Chromatographic separation was performed on an HP5-MS 30 m x 0.25 mm x 0.25 μm column. The mobile phase was composed of Helium at 0.6 mL/min constant flow. The gradient program was the following: injection temperature of 250 °C, initial temperature of 195 °C for 20 min, and oven ramp of 2 °C/min to 220 °C for 8 min. The injection volume was 1uL on split mode. The ionization source was 70 eV electron ionization. Source temperature 250 °C. Quadrupole temperature 150 °C. Data acquisition delay 20 min. Scan range 33 to 320 AMU. Confirmation of structures was made by comparison with GC/MS NIST Library MS 2.0 data.

Neuron-glia crosstalk experiments

For secretome assays, donor cells were exposed to MB or vehicle in their corresponding serum-free medium at 80% confluence for 24 h with or without pharmacological modulators (see Experimental treatments). The secretome was collected directly from the culture dish, either for lipid extraction (Bligh & Dyer method) or for additional experiments with receptor cells. Receptor cells were incubated for 24 h with the secretome collected immediately prior to use, their lipid extracts (resuspended in a serum-free medium containing 0.01% DMSO), and MB or vehicle, with or without inhibitors, agonists, or antagonists, for further analysis. Depending on the cell type, the secretomes were named as follows: N-SEC (neuronal secretome), A-SEC (astrocytic secretome), G-SEC (primary glial cultures secretome).

Cell viability

Cell viability was assessed by the MTT reduction assay. MTT (3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide; #475989-IGM, Calbiochem, USA), a water-soluble tetrazolium salt, is metabolically reduced by viable cells to a colored, water-insoluble formazan salt. After treatments, cells seeded in 96-well plates (1.5 × 104 cells/well) or 24-well plates (7.5 × 104 cells/well) were incubated for 2 h at 37 °C in a serum-free medium containing 500 µg/mL MTT. The medium was removed, and the formazan salt was dissolved with 20% (w/v) sodium dodecyl sulfate (SDS pH 4.6). The reaction was quantified spectrophotometrically at 570 nm and corrected by 650 nm measurement. Results were expressed as a percentage of control [4].

Proliferation assay

To identify proliferating cells, a bromodeoxyuridine (BrdU) incorporation assay was performed as follows: cells were plated in 24-well plates with round glass coverslips [18]. Cells were treated and incubated along with 10 μm BrdU for 24 h. The medium was removed and washed with D-PBS. Cells were fixed with cold ethanol 70% (−20 °C) for 20 min and incubated with HCl 2 N for strictly 10 min to denaturalize DNA and then incubated with borate solution for 10 min (A:B 1:1, A: Borax 1.9%, B: Boric Acid 1.25%) at room temperature to neutralize any residual acid. After washing with PBS, coverslips were incubated with sheep polyclonal anti-BrdU (1:200, BIODESIGN International, USA) for 1 h at room temperature, washed with PBS, and incubated with the corresponding fluorescent secondary antibody (donkey anti-sheep IgG (1:200; Jackson ImmunoResearch Laboratories Inc., USA) for 1 h at room temperature and DAPI (1:10000, D9542, Sigma-Aldrich, USA) as nuclear staining for 4 min. Coverslips were mounted and analyzed with a Nikon Eclipse E600 microscope. At least 100 cells per condition were counted in triplicate, and results were expressed as percentage of BrdU-positive nuclei over total nuclei and compared to controls. Quantification was performed using the FIJI platform [19].

Immunofluorescence microscopy

Cells were grown onto round glass coverslips. After treatments, cells were fixed with 4% paraformaldehyde in PBS for 20 min. For the immunostaining, cells were permeabilized and blocked with 2% BSA in PBS and 0.1% Triton X-100 for 45 min at room temperature followed by incubation for 1 h at room temperature with a 1:50 dilution (PBS, 2% BSA, and 0.1% Triton X-100) of the appropriate primary antibody: Glial Fibrillary Acidic Protein (GFAP, #3670S, Cell Signaling Technology, USA), p65 (sc-8008, Santa Cruz Biotechnology, USA), S100 calcium-binding protein β (S100β, # z0311, DAKO, Glostrup, Denmark), ionized calcium-binding adaptor molecule 1 (IBA-1, #17198, Cell Signaling Technology, USA), COX-2 (sc-376861, Santa Cruz Biotechnology, USA). After washing with PBS, cells were incubated for 1 h with a 1:150 dilutions (PBS and 2% BSA) of the corresponding fluorescent secondary antibody: Cy2-conjugated anti-rabbit (#111-225-144, Jackson ImmunoResearch Laboratories Inc., USA), Cy3-conjugated anti-mouse (#115-165-003, Jackson ImmunoResearch Laboratories Inc., USA). DAPI (1:10000 in PBS) was used for nuclear staining for 4 min. The coverslips were mounted to be analyzed with a Nikon Eclipse E600 microscope. Fluorescence intensity was measured with FIJI [19].

Gene expression by real-time quantitative PCR (RT-qPCR)

Total RNA was extracted by BIOZOL reagent (#RA02) following the manufacturer’s protocol. Retrotranscription was performed on 1 µg of total RNA using M-MLV reverse transcriptase (#EC0701; 200U/µl), hexamers (#OA0201, 100 µM), and dNTP set I (#K0101) from PB-L (Buenos Aires, Argentina). qPCR was executed using Sso Advanced Universal SYBR Green Supermix (#1725271, Bio-Rad Laboratories, USA) and 0.2 µM of each primer pair in a Rotor-Gene 6000 (Corbett Research, Australia). The relative expression of genes was assessed in triplicate according to the 2−ΔΔCt method using Gapdh as a housekeeping gene [20]. Gene-specific primer sequences are shown in Supplementary Table 1.

Western blot analysis

Cells were seeded in 60 mm dishes, and, after treatments, rinsed with PBS, scraped, and centrifuged. The pellet was homogenized with 60–80 µL of a lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 1% NP-40, 2 mM EDTA, 2 mM EGTA, 50 mM NaF, 2 mM β-glycerophosphate, 1 mM Na3VO4, 10 µg/mL leupeptin, 5 µg/mL aprotinin, 1 µg/mL pepstatin, 0.5 mM PMSF, and 0.5 mM DTT). After protein extraction for 1 h at 4 °C, homogenized samples were centrifuged at 15000×g for 15 min. The protein content was measured in aliquots from the supernatants by the Bradford method [21]. Samples were denatured with Laemmli’s buffer at 100 °C for 5 min. Protein extracts were separated by SDS-PAGE and then transferred onto PVDF membranes (Millipore, USA). After blocking with 5% non-fat milk in TBS-T buffer (20 mM Tris-HCl (pH 7.4), 100 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature, membranes were incubated overnight at 4 °C with the primary antibody (proliferating cell nuclear antigen (PCNA, ab2426-1, Abcam, UK); extracellular-signal-regulated kinase (ERK, cs-9102, Cell Signaling Technology, USA); p-ERK (cs-9101, Cell Signaling Technology, USA); TUBULIN (NB-690, Novus Biologicals LLC, USA); GFAP; IBA-1. Then, membranes were incubated with the corresponding HRP-conjugated secondary antibody (anti-rabbit NA934-IML; anti-mouse NA931-IML; Amersham Biosciences, USA) for 2 h at room temperature. Immunoreactive bands were detected by enhanced chemiluminescence (ECL; GE Healthcare Bio-Sciences, Argentina) using Syngene™ Gbox Chemi XRQ documentation system (Fisher Scientific, USA). The quantification of immunoreactive bands was performed using the FIJI platform [19].

Lipid extraction from secretomes for GC-MS and phospholipid phosphorus measurement

Culture media were collected from three 60 mm-dishes per condition in triplicate, then centrifuged at 800 x g for 5 min to precipitate cell debris. Supernatants were ultracentrifuged at 100,000 x g for 3 h to obtain a pellet containing the membrane fraction. Lipids from pellets were extracted by the Bligh & Dyer method [16]. To separate phospholipids by TLC, silica gel G plates were used with a mobile phase consisting of hexane: ethyl ether: acetic acid (50:50:2.6, v/v). After separation, spots corresponding to the total phospholipid fraction were scraped off for phosphorus measurement using the method described by Rouser and collaborators [22].

Statistical analysis

Quantitative results were expressed as the mean ± standard deviation (SD) of the n samples indicated in each figure. Statistical analysis of the data was performed using Student’s t-test, one-way ANOVA accompanied by Tukey’s test, and two-way ANOVA when appropriate. P values less than 0.05 were considered statistically significant.