Animals

MacGreen [22], Rs1h-/Y[19] and wild-type mice were all on a pure C57BL/ 6 J background. Animals were maintained in an air-conditioned environment on a 12-hour light–dark schedule at 22°C, and had free access to food and water. The health of the animals was regularly monitored, and all procedures complied with the German Law on Animal Protection and the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals, 2011.

Human tissue

Retinal samples of donors were obtained from the Eye Bank of the Center of Ophthalmology, University of Cologne, Germany. The donor age ranged between 54 and 72 years. Postmortem time ranged between 5 and 36 h. After dissection of the anterior segment, the remaining tissue included the posterior pole. The research followed the tenets of the Declaration of Helsinki.

Reagents

E. coli 0111:B4 lipopolysaccharide and aminoglutethimide were purchased from Sigma Aldrich (St. Louis, MO, USA). XBD173 (emapunil) was obtained by custom synthesis from APAC Pharmaceuticals (Ellicott City, MD, USA). XBD173 was dissolved in ethanol.

Cell culture and retinal explants

BV-2 microglia-like cells were cultured in RPMI/5% fetal calf serum (FCS) supplemented with 2 mM L-Glutamine and 195 nM β-mercaptoethanol. Isolation and culture of primary retinal microglia has been described previously [21]. BV-2 cells were stimulated with 50 ng/ml lipopolysaccharide (LPS) and various concentrations of XBD173 or ethanol as vehicle control. 661 W photoreceptor-like cells were a gift from Prof. Muayyad Al-Ubaidi (Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA) and the culture conditions have been described elsewhere [23]. Human microglial cell lines (iPSdM) were generated from induced pluripotent stem (iPS) cell lines obtained by reprogramming from skin fibroblasts as previously described [24, 25]. These cells proliferate without addition of growth factors and they were passaged 1:3 twice a week. The microglial phenotype was confirmed by flow cytometry (CD11b, CD16/32, CD36, CD45, CX3CR1). Retinas from MacGreen mice were rinsed in DMEM/Ham’s F12 medium supplemented with 1% FCS and placed on 25 mm circular Nucleopore filters (VWR, Darmstadt, Germany) with the photoreceptor side facing the membrane. After 24 h of in vitro culture with vehicle, 1 μg/ml LPS, 20 μM XBD173 or 1 μg/ml LPS + 20 μM XBD173, retinas were fixed and imaged in flat-mounts. Ramified and amoeboid microglial cells were directly imaged by green fluorescent protein (GFP) fluorescence using the Axioskop2 MOT Plus Apotome microscope (Carl Zeiss, Jena, Germany) and counted.

Scratch wound-healing assay

A total of 400,000 BV-2 microglial cells were grown in six-well plates as 80% confluent monolayers and were wounded with a sterile 100 μl pipette tip. Thereafter, the cells were stimulated with 50 ng/ml LPS, 50 μM XBD173, 50 ng/ml LPS + 50 μM XBD173, or ethanol as solvent control. Migration into the open scar was documented with microphotographs taken at different time points after wounding using a Nikon ECLIPSE TE2000 inverted microscope (Nikon, Tokyo, Japan). The number of migrating cells was quantified by counting all cells within a 0.4 mm2 region in the center of each scratch. The number of migrated cells was then normalized to the average cell density to account for changes in proliferation. A minimum of five individual cultures was used to calculate the mean migratory capacity of each cell culture condition.

Proliferation assay

For carboxyfluorescein diacetate succinimidyl ester (CFSE) proliferation assays, BV-2 microglial cells were labeled with 1 μM CFSE (e-Bioscience, San Diego, CA, USA) and cultured (1.5 × 105 per well) in a six-well plate. After 24 h of culture with vehicle, 100 ng/ml LPS, 50 μM XBD173 or 100 ng/ml LPS + 50 μM XBD173, cells were stained with a fixable viability dye (e-Bioscience), to exclude dead cells from the analysis. The fluorescence intensity of CFSE-labeled BV-2 cells was analyzed by flow cytometry (FACS Canto II). Analysis of cell division was performed using FlowJo software (Treestar Inc., Ashland, OR, USA).

shRNA knock-down of TSPO in BV-2 cells

For knockdown of endogenous TSPO in BV-2 cells, shRNA vectors were obtained from the RNAi consortium (TRC). Briefly, BV-2 cells were transfected with 2.5 μg vector DNA using TransIT®-LT1 transfection reagent (Mirus Bio LLC, Madison, WI, USA) to express TSPO-specific or scrambled shRNAs. Twenty-four hours after transfection cells were stimulated with vehicle, 50 ng/ml LPS, 20 μM XBD173 or 50 ng/ml LPS + 20 μM XBD173 for 12 hours before cells were harvested for RNA isolation and mRNA expression analysis.

661 W photoreceptor apoptosis assay

To test microglial neurotoxicity, a culture system of 661 W photoreceptors with microglia-conditioned medium was established. 661 W photoreceptor cells were incubated for 48 h either in their own medium or with culture supernatants from unstimulated, 50 ng/ml LPS, 50 μM XBD173 or 50 ng/ml LPS + 50 μM XBD173 treated microglial cells. The 661 W cell morphology was assessed by phase contrast microscopy and apoptotic cell death was determined with the Caspase-Glo® 3/7 Assay (Promega GmbH, Mannheim, Germany). Cells were lysed and incubated with a luminogenic caspase-3/7 substrate, which contains the tetrapeptide sequence DEVD. Luminescence was then generated by addition of recombinant luciferase and was proportional to the amount of caspase activity present. The luminescent signal was read on an Infinite F200 pro plate reader (Tecan, Crailsheim, Germany). A blank reaction was used to measure background luminescence associated with the cell culture system and Caspase-Glo® 3/7 Reagent (Promega). The value for the blank reaction was subtracted from all experimental values. Negative control reactions were performed to determine the basal caspase activity of 661 W cells. Relative luciferase units (RLU) reflect the level of apoptotic cell death in the different 661 W cell cultures.

Nitrite measurement

Nitric oxide concentrations were determined by measuring the amount of nitrite produced by BV-2 microglial cells into the culture medium using the Griess reagent system (Promega). A 50 μl cell culture supernatant was collected and an equal volume of Griess reagent was added to each well. After incubation for 15 minutes at room temperature, the absorbance was read at 540 nm on an Infinite F200 pro plate reader (Tecan). The concentration of nitrite for each sample was calculated from a sodium nitrite standard curve.

Phagocytosis assays

BV-2 microglial cells were pre-treated for 2 h with compounds before 4 μl latex bead solution (Polystyrene microparticles, Sigma Aldrich, St. Louis, MO, USA) was added to the wells. Cells were incubated for 6 h and five micrographs per well were taken using an AxioVert.A1 inverted microscope (Carl Zeiss). The phagocytic activity was determined by calculating the number of cells which phagocytosed 10 or more latex beads compared to all cells per field. The conditions for human microglial cells (iPSdM) were the same with the modification that cells were pre-treated for 24 h, the incubation time with beads was 24 h and only fully saturated cells were counted as positive. To study the microglial uptake of apoptotic photoreceptor cell material, 661 W photoreceptor cells were starved with serum deprivation, harvested and fluorescently labeled using CellTracker CM-DiI (Invitrogen, Carlsbad, CA, USA). For phagocytosis, BV-2 microglial cells were pre-treated for 2 h with compounds before 400 μl stained apoptotic 661 W solution was added for further 6 h. iPSdM cells were pre-treated for 24 h before 400 μl stained apoptotic 661 W solution was added for further 24 h. Cells were then fixed and nuclei were stained with 4′,6-diamidino-2-phenylindole. Fluorescence micrographs were taken and ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to determine the ratio of phagocytosed apoptotic photoreceptor fragments (red signal) relative to the total microglia cell number (DAPI signal).

Phalloidin staining

BV-2 microglial cells or human microglial cells (iPSdM) were grown on cover slips in six-well plates and the indicated compounds were added for 24 h. Thereafter, the cells were fixed, permeabilized with 0.1% Triton X-100 and f-actin was fluorescently labeled using 0.1 μg/ml Phalloidin-TRITC (Sigma). The nuclei were stained using 4′,6-diamidino-2-phenylindole and photomicrographs were taken with an Axioskop2 MOT Plus Apotome microscope (Carl Zeiss).

Immunohistochemistry

Immunohistochemical analyses were performed on 10 μm retinal sections embedded in optimal cutting temperature (OCT) compound (Hartenstein, Würzburg, Germany) or on retinal flat mounts. Samples were fixed in 4% paraformaldehyde, rinsed and rehydrated with PBS. Sections were blocked with a dried milk solution followed by an overnight incubation with primary antibodies at 4°C. Antibodies included rabbit anti-Iba1 antibody (Wako Chemicals, Neuss, Germany), rabbit anti-TSPO antibody (Abcam, Cambridge, UK), goat anti-MAP2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and goat anti-GFAP antibody (Santa Cruz Biotechnology). After washing, samples were labeled with a secondary antibody conjugated to Alexa488 (green) or Alexa594 (red) (Jackson Immuno-Research, West Grove, PA, USA) and counter-stained with DAPI. Sections and flat-mounts were mounted in DAKO fluorescent mounting medium (Dako Deutschland GmbH, Hamburg, Germany) and viewed with an Axioskop2 MOT Plus Apotome microscope (Carl Zeiss).

Western blot analysis

Mouse retinal tissue was homogenized in cold RIPA buffer (20 mM Na-phosphate buffer, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100 and protease inhibitors) using a TissueLyser LT (Qiagen, Hilden, Germany). Insoluble debris was removed by centrifugation for 15 minutes at 16,000 g. LPS-treated and control BV-2 microglia were directly lysed in RIPA buffer. Protein concentrations were determined by Bradford assay (Roti-quant, Roth, Karlsruhe, Germany). A total of 10 μg of microglial or 30 μg of total-retina proteins were separated by SDS-PAGE on 15% gels with PageRuler pre-stained protein ladder (Thermo Scientific, Waltham, MA, USA). Proteins were then transferred to 0.45 μm nitrocellulose membranes (Biorad, Munich, Germany). After blocking in TBS-T containing 5% nonfat dry milk, membranes were incubated with primary antibodies against TSPO (ab109497, Abcam,) or Actin (sc-1616, Santa Cruz Biotechnology). Blots were then incubated with secondary goat anti-rabbit IgG-HRP or rabbit anti-goat IgG-HRP antibodies (sc-2004, sc-2768, Santa Cruz Biotechnology). Enhanced chemiluminescence signals were then visualized and imaged with the MultiImage II system (Alpha Innotech, Santa Clara, CA, USA).

RNA isolation and reverse transcription

Total RNA was extracted from total retina, BV-2 microglial cells or isolated retinal microglia according to the manufacturer’s instructions using the RNeasy Mini Kit (Qiagen). Purity and integrity of the RNA was assessed on the Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip® reagent set (Agilent Technologies, Santa Clara, CA, USA). The RNA was quantified spectrophotometrically and then stored at −80°C. First-strand cDNA synthesis was performed with the RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas, Schwerte, Germany).

Quantitative real-time RT-PCR

Amplifications of 50 ng cDNA were performed with an ABI7900HT machine (Applied Biosystems, Carlsbad, CA, USA) in 10 μl reaction mixtures containing 1 × TaqMan Universal PCR Master Mix (Applied Biosystems), 200 nM of primers and 0.25 μl of dual-labeled probe (Roche ProbeLibrary, Roche Applied Science, Basel, Switzerland). The reaction parameters were as follows: 2 minutes 50°C hold, 30 minutes 60°C hold and 5 minutes 95°C hold, followed by 45 cycles of 20 s 94°C melt and 1 minute 60°C anneal/extension. Primer sequences and Roche Library Probe numbers were as follows: CCL2, forward primer 5′-catccacgtgttggctca-3′, reverse primer 5′-gatcatcttgctggtgaatgagt-3′, probe #62; IL6, forward primer 5′-gatggatgctaccaaactggat-3′, reverse primer 5′-ccaggtagctatggtactccaga-3′, probe #6; iNOS, forward primer 5′-ctttgccacggacgagac-3′, reverse primer 5′- tcattgtactctgagggctga-3′, probe #13. Measurements were performed in triplicates and results were analyzed with an ABI sequence detector software version 2.3 using the ΔΔCt method for relative quantification.

Pregnenolone ELISA

BV-2 cells were seeded on 24-well plates in 1 ml/well of RPMI/5% FCS supplemented with 2 mM L-Glutamine and 195 nM β-mercaptoethanol. After cells had attached after 6 h, 50 ng/ml LPS and/or 20 μM XBD173 were added to each well. After 21 h, cells were washed twice with HEPES assay buffer (140 mM NaCl, 5 mM KCl, 1.8 mM CaCl 2, 1 mM MgSO 4, 10 mM glucose, 10 mM HEPES/NaOH, pH 7.4) as described previously [26]. Then 1 ml of HEPES assay buffer supplemented with BSA (0.1%) and Trilostane (25 μM) (Sigma-Aldrich) was added to each well. Again 50 ng/ml LPS and/or 20 μM XBD173 were added. After 3 h the supernatants were removed to perform pregnenolone ELISA according to the manufacturer’s recommendations (IBL International, Hamburg, Germany). In brief, 50 μl of each sample were pipetted into a rabbit anti-pregnenolone antibody coated 96-microwell Plate. A total of 100 μl of pregnenolone-HRP conjugate was then added. Ready-to-use-calibrators were provided by IBL international. After 1 h of incubation and washing 150 μl of tetramethylbenzidine/hydrogen peroxide (TMB) substrate was added. Assays were read with a Tecan Spectra at 450 nm. Data were analyzed by Magellan Data Analysis Software (Tecan).

Statistical analyses

Real-time quantitative RT-PCR data were analyzed with the ΔΔCt method using an unpaired Student’s t-test. Assays for nitrite secretion, microglial migration and pregnenolone ELISAs were analyzed with an unpaired Student’s t test. Caspase 3/7 assays and phagocytosis assays were analyzed with a Mann–Whitney U-test. P <0.05 was considered as statistically significant.