Human post-mortem tissue
Post-mortem tissue of brain donors was provided by the Netherlands Brain Bank (NBB, Amsterdam, The Netherlands, www.brainbank.nl). All brain donors gave informed consent to perform autopsies and to use tissue, clinical and neuropathological information for research purposes, approved by the Ethics Committee of VU medical center (Amsterdam, The Netherlands).
Subcortical white matter (WM) tissue was collected from non-MS WM control donors (n = 8), and from MS donors we collected subcortical NAWM (n = 10) and subcortical WM lesions (n = 10). NAWM MS tissue was dissected on post-mortem magnetic resonance imaging (MRI) guidance during autopsy [14]. In addition, macroscopically visible MS lesions were dissected by a neuropathologist.
Neurological diagnoses were confirmed by a neuropathologist. Information on MS diagnosis and disease duration was obtained from clinical data, showing that all donors were diagnosed with progressive MS, 3 donors with primary progressive MS and 7 donors with secondary progressive MS. Donor characteristics and post-mortem variables are displayed in Additional file 1, Supplementary Table 1-3 and 6.
MS lesion characterization
From post-mortem tissue that was taken out for microglia isolation, a small part was snap-frozen in liquid nitrogen and stored in − 80 °C until further use. Frozen tissue sections (20 μm) of control WM, NAWM and MS lesions were cut and dried overnight. For immunohistochemistry, these sections were fixed for 15 min with 4% paraformaldehyde in phosphate buffered saline (PBS) pH 7.6, followed by endogenous peroxidase blocking in 1% H2O2 in PBS for 20 min. Sections were incubated with primary antibodies HLA-DR/DQ/DP (1:1000, M0775; Dako, Glostrup, Denmark) or PLP (1:3000, MCA839G; Serotec, Oxford, UK) in incubation buffer (0.5% Triton X-100 and 1% bovine serum albumin (BSA) in PBS) overnight at 4 °C. Secondary antibodies were incubated for 1 h at room temperature (RT); for HLA-DR biotinylated anti-mouse (1:400, BA-2001; Vector Laboratories, Burlingame, CA, USA) was diluted in incubation buffer, for PLP the HRP-labeled mouse antibody (K5007, Dako Real EnVision detection system; Dako) was used. Next, sections for HLA-DR staining were incubated for 45 min in avidin-biotin complex (1:800, PK-6100; Vector Laboratories) at RT, followed by 3,3′-diaminobenzidine (DAB) incubation (1:100, K5007; Dako) for 10 min at RT, for both HLA-DR and PLP stainings. Immunoreactivity was examined using an Axioskop980 microscope (Zeiss, Oberkochen, Germany) and Photomacroscope M420 (Wild Heerbrugg, Zwitserland) to characterize lesions based on HLA-DR presence and morphology of HLA-DR+ cells together with myelin intactness based on PLP staining [5].
Microglia isolation
Microglia were isolated from post-mortem WM tissue, as described previously [11, 15]. Briefly, post-mortem tissue that was collected during autopsy was stored in Hibernate-A medium (Invitrogen, Carlsbad, CA, USA) at 4 °C until further processing. Within 24 h, the tissue was homogenized for 5 min in Hibernate-A medium supplemented with DNAseI (10 mg/ml; Roche, Basel, Switzerland), using a tissue homogenizer (VWR, Radnor, PA, USA). Next, undiluted Percoll (density of 1.13 g/ml; GE Healthcare, Little Chalfont, UK) was added to form a single gradient for density centrifugation and the interlayer was collected for magnetic activated cell sorting (MACS; Miltenyi, Bergisch Gladbach, Germany) using CD11b magnetic beads (catalogue number #130–049-601, Miltenyi Biotech). Viable cells were counted using a hemocytometer (Optic Labor, Friedrichshof, Germany) or eFluor™ 506. Cells were then collected in beads buffer (0.5% BSA + 2 mM EDTA in PBS, pH 7.6) for flow cytometry analysis. Using this protocol, about 95% of viable cells were identified as myeloid cells (Supplementary Fig. 1). For CyTOF analysis, CD11b+ cells were incubated for 11 min in fixation/stabilization buffer (Smart Tube Inc., San Carlos, CA, USA) and stored in − 80 °C.
IRF8+ nuclei isolation and sorting
IRF8+ nuclei were isolated and sorted as described previously [11]. Briefly, frozen tissue from MS donors, NAWM tissue (n = 7) and tissue containing MS lesions (n = 5), matched for age, was provided by the NBB. For each tissue block, the first and last section were double stained for HLA-DR/PLP to determine microglia activation and myelin integrity. MS lesions were characterized as previously described by Luchetti and colleagues [5].
From each tissue block, 10–12 sections of 50 μm thickness were cut and homogenized in 1 ml homogenization buffer (1 μm DTT (Thermo Fischer Scientific), 1x protease inhibitor (Roche), 80 U/ml RNAseIN (Promega, Madison, WI, USA) and 1% Triton X-100) with nuclei isolation medium #1 (NIM #1; 250 mM sucrose, 25 mM KCL, 5 mM MgCl2, 10 mM Tris buffer pH 8 diluted in nuclease free water) filtered through a 30-μm cell strainer. The amount of nuclei was counted using a hemocytometer (Optic Labor) and nuclei were incubated with Hoechst (#H3570, 1:1000; Invitrogen) and IRF8 antibody (#566373, PE-labeled, 1:50, clone U31–644; BD Biosciences, San Diego, CA, USA) in staining buffer (0.5% RNAse free BSA, 1% normal human serum and 0.2 U/μl RNAseIn in RNAse-free PBS, pH 7.4) for 1 h at 4 °C. Isotype control antibody IgG-PE (#12–4714-42, clone P3.6.8.1, 1:25; Invitrogen) was used to determine background staining.
Stained nuclei were sorted using a Sony SH800S cell sorter (Sony Biotechnology, San Jose, CA, USA). The Hoechst and IRF8 double positive nuclei fractions was collected and lysed in RNA lysis buffer (RNeasy Isolation mini kit; Qiagen, Hilden, Germany).
RNA isolation
RNA from sorted IRF8+ nuclei was isolated using the RNeasy Mini kit (Qiagen), according to the manufacturer’s protocol. Lysed samples were mixed with 70% ethanol and transferred to a mini spin column. After washing steps, elution was collected in 20 μl deionized water.
DNA synthesis and quantitative real-time PCR
The Quantitect Reverse Transcription Kit (Qiagen) was used for cDNA synthesis. According to manufacturer’s protocol, isolated RNA (25 ng) from sorted IRF8+ nuclei was mixed with gDNA wipeout buffer, incubated for 2 min at 42 °C and put on ice. Next, Quantiscript RT buffer, RT primer mix and Quantiscript Reverse Transcriptase were mixed and incubated with RNA sample at 42 °C for 30 min, followed by 3 min incubation at 95 °C.
For RT-qPCR, 0.6 ng cDNA was mixed with 17 μl SYBR Green PCR master mix (Applied Biosystems, Foster City, CA, USA) and 2 μl primer pairs. Samples were measured and analyzed using 7300 RT-PCR machine and software (Applied Biosystems).
Primer pairs were designed at the Integrated DNA Technologies website (eu.ifdna.com), using the PrimerQuest tool. For primer design the following criteria were used: same Tm, 50% GC content, amplicon size between 80 and 140 base pairs and exclude primers that span introns, to detect unspliced nuclear DNA. Primer pairs were checked for specificity using cDNA derived from pooled MS and control donor brain tissue. Optimal primers (Additional file 1: Supplementary Table 4) were selected based on dissociation curve, and 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel was used to detect PCR product and exclude primer pairs that can form dimers. Gene expression was normalized to the mean of 2 housekeeping genes, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and elongation factor-1 alpha (EEF1A1). Target gene expression values were calculated using the 2- ΔΔCT method.
Flow cytometric analysis
Isolated microglia from MS donors (n = 7) were incubated for 15 min in FcR-blocking buffer (1:5; Miltenyi Biotec), to block unspecific binding of antibodies to Fc-receptors. Next, microglia were incubated with conjugated primary antibodies (Additional file 1: Supplementary Table 5) diluted in beads buffer (0.5% BSA and 2 mM EDTA in PBS, pH 7.6) for 30 min at 4 °C. To determine viability, cells were incubated with viability dye efluor506 (Additional file 1: Supplementary Table 5).
To assess minimal phenotyping of isolated microglia, CD45 and CD11b expression was determined. In addition, expression of homeostatic microglia receptors, P2Y12, CX3CR1 and GPR56 was measured. To exclude infiltrating leukocytes in the samples collected from MS lesion tissue, CD3, CD19, CD56 and CD66b were included.
Surface protein expression was detected on a 3-laser BD FACSCanto II machine (BD Biosciences) with software BD DIVA version 8.1. FlowJo software version 10.1 (Ashland, OR, USA) was used to determine median fluorescence intensity.
Immunohistochemical quantification
Paraffin tissue blocks from age-matched control (n = 5) and MS (n = 11) donors (Additional file 1: Supplementary Tables 1 and 6) were cut into 8 μm-thick sections. Tissue sections were deparaffinized with xylene and rehydrated in ethanol series, followed by antigen retrieval with citrate buffer pH 6 for 20 min in a steamer. Sections were blocked in 10% normal horse serum/normal donkey serum for 30 min and incubated with P2Y12 antibody and either CD68 (DAKO, # M0814) or HLA-DR (DAKO, #M0775) antibodies diluted in incubation buffer (0.5% Triton-X100 and 0.25% gelatin in tris-buffered saline (TBS, pH 7.6) and incubated overnight at 4 °C. After overnight incubation with primary antibody, samples were incubated for 2 h at RT with Alexa Fluor 568 and Alexa Fluor 488-conjugated secondary antibody. Nuclei were stained with DAPI. All images were acquired in a Leica TCS SP5 microscope (Leica microsystems). P2Y12+DAPI+ and P2Y12+CD68+DAPI+ or P2Y12+HLA-DR+DAPI+ cells were counted using IMARIS software. All image processing for visualization was performed with ImageJ software.
Intracellular barcoding for mass cytometry
Percoll-isolated myeloid cells were fixed with fixation/stabilization buffer (SmartTube) [16] and frozen at − 80 °C until analysis by mass cytometry. Cell were thawed and subsequently stained with premade combinations of six different palladium isotopes: 102Pd, 104Pd, 105Pd, 106Pd, 108Pd and 110Pd (Cell-ID 20-plex Pd Barcoding Kit, Fluidigm). This multiplexing kit applies a 6-choose-3 barcoding scheme that results in 20 different combinations of three Pd isotopes. After 30 min staining (at room temperature), individual samples were washed twice with cell staining buffer (0.5% bovine serum albumin in PBS, containing 2 mM EDTA). All samples were pooled together, washed and further stained with antibodies.
Antibodies
Anti-human antibodies (Additional file 1: Supplementary Tables 7 and 8) were purchased either pre-conjugated to metal isotopes (Fluidigm) or from commercial suppliers in purified form and conjugated in house using the MaxPar X8 kit (Fluidigm) according to the manufacturer’s protocol. Using different cell types from different body compartments, each antibody was titrated and validated as into the working panels prior to use to ensure that the resulted signals were informative [16, 17].
Cell-surface and intracellular staining
After cell barcoding, washing and pelleting, the combined samples were stained and processed as described previously [16, 17]. Briefly, cells were re-suspended in 100 μl of antibody cocktail directed against cell surface markers (Additional file 1: Supplementary Tables 7 and 8) and incubated for 30 min at 4 °C. Then, cells were washed twice with cell staining buffer (PBS containing 0.5% BSA and 2 mM EDTA). For intracellular staining, the stained (non-stimulated) cells were then incubated in fixation/permeabilization buffer (Fix/Perm Buffer, eBioscience) for 60 min at 4 °C. Cells were then wash twice with permeabilization buffer (eBioscience). The samples were then stained with antibody cocktails directed against intracellular molecules (Additional file 1: Supplementary Tables 7 and 8) in permeabilization buffer for 1 h at 4 °C. Cells were subsequently washed twice with permeabilization buffer and incubated overnight in 4% methanol-free formaldehyde solution. The fixed cells were then washed and re-suspended in 1 ml iridium intercalator solution (Fluidigm) for 1 h at RT, followed by two washes with cell staining buffer and two washes with ddH2O (Fluidigm). Finally, cells were pelleted and kept at 4 °C until CyTOF measurement.
Bead staining
For the bead-based compensation of the signal spillover, AbC total antibody compensation beads (Thermo Fisher Scientific) were single stained with each of the antibodies used in all three antibody panels according to manufacturer’s instructions. Stained beads were then measured with CyTOF and the compensation matrix was then generated [17, 18].
CyTOF measurement
Cells were analysed using a CyTOF2 upgraded to Helios specifications, with software version 6.7.1014 [16, 17], using a narrow bore injector. The instrument was tuned according to the manufacturer’s instructions with tuning solution (Fluidigm) and measurement of EQ four element calibration beads (Fluidigm) containing 140/142Ce, 151/153Eu, 165Ho and 175/176Lu served as a quality control for sensitivity and recovery.
Directly prior to analysis cells were re-suspended in ddH2O, filtered through a 20-μm cell strainer (Celltrics, Sysmex), counted and adjusted to 5–8 × 105 cells/ml. EQ four element calibration beads were added at a final concentration of 1:10 v/v of the sample volume to be able to normalize the data to compensate for signal drift and day-to-day changes in instrument sensitivity.
Samples were acquired with a flow rate of 300–400 events/s. The lower convolution threshold was set to 400, with noise reduction mode turned on and cell definition parameters set at event duration of 10–150 pushes (push = 13 μs). The resulting flow cytometry standard (FCS) files were normalized and randomized using the CyTOF software’s internal FCS-Processing module on the non-randomized (‘original’) data. The default settings in the software were used with time interval normalization (100 s/minimum of 50 beads) and passport version 2. Intervals with less than 50 beads per 100 s were excluded from the resulting FCS file.
Mass cytometry data processing and analysis
Following the workflow from our previous study [16, 17], Cytobank (www.cytobank.org) was used for initial manual gating on live single cells and Boolean gating for de-barcoding. Nucleated single intact cells were manually gated according to DNA intercalators 191Ir/193Ir signals and event length. For de-barcoding, Boolean gating was used to deconvolute individual sample according to the barcode combination. Prior to data analysis, each FCS file was compensated for signal spillover using R package CATALYST [18]. For dimensionality reduction, visualization and further exploration, (2D) tSNE maps were generated according to the expression levels of all markers in each panel. For embedding, we set hyperparameters to perplexity of 30, theta of 0.5, and iterations of 1000 per 100,000 analysed cells. To visualize marker expression arcsinh transformation was applied to the data. All FCS files were then loaded into R and further data analysis was performed with an in-house written script based on the workflow proposed by M. Nowicka and colleages [19]. Briefly, for unsupervised cell population identification we performed cell clustering with the FlowSOM [20] and ConsensusClusterPlus [21] packages using all markers (Exp-I) or TYPE markers (Exp-II and -III). We then performed visual inspection of cluster-coloured tSNE plots and phenotypic heatmaps for a more detailed profile of each cluster and determined the number of meta-clusters on the basis of delta area under cumulative distribution function (CDF) curve and k value of the clustering analysis and the consistency of phenotypes for statistical test. For detection of differential abundance of clusters between conditions we used generalized linear mixed models (GLMM) performed with the diffcyt package [17], with a false discovery rate (FDR) adjustment (Benjamini-Hochberg (BH) procedure) for multiple hypothesis testing. A P value < 0.05 (unadjusted) and < 0.05 (FDR-BH adjusted) was considered statistically significant.
Imaging mass cytometry
Paraffin tissue microarray (TMA) blocks containing samples from control, NAWM and lesion were cut into 5 μm-thick sections. Sections were deparaffinized with xylene and rehydrated in ethanol series, followed by heat-induced antigen retrieval in Tris-EDTA buffer (pH = 9.0) for 20 min at 95 °C in a steamer. The sections were then blocked with 3% purified BSA in 0.1% Triton-X PBS for 1 h at RT. Sections were incubated overnight at 4 °C with anti-P2Y12 conjugated with biotin. After washing, all sections were incubated with metal-conjugated antibodies (Additional file 1: Supplementary Table 9) overnight at 4 °C. Nuclei were detected using an Ir-Intercalator (1:500). Samples were then dried and stored at RT until measurement.
Imaging mass cytometry acquisition and data analysis
Imaging mass cytometry was performed on a CyTOF2/upgraded to Helios specifications coupled to a Hyperion Tissue Imager (Fluidigm), using CyTOF software version 6.7.1014. Prior to ablation the instrument was tuned according to the manufactures instructions, using the 3-Element Full Coverage Tuning Slide (Fluidigm). The dried slide was loaded into the imaging module and regions of interest were selected for each sample of the TMA on a preview (panorama). Optimal laser power was determined for each sample to obtain complete ablation of the tissue. Laser ablation was performed at a resolution of 1 μm and a frequency of 200 Hz. Data were stored as MCD files as well as txt files. Original files were opened with MCD viewer and single 16-bit images were extracted as. TIFF files. For visualization only, images were transferred to ImageJ and the different channels were merged. A Gaussian blurr (kernel width, 0.70 pixels) was used for noise reduction.
For single-cell analysis, we first processed images from each sample using Ilastik [22], an open-source program that uses interactive machine learning to separate single cells from background. The program was trained to identify DNA iridium-intercalator as nuclei and P2Y12-Ho165 as cell membrane, and the pixel classificator was then applied to all images. As a result, a binary mask delimiting each single-cell was obtained and transferred on to CellProfiler [23]. We applied a set of modules to create single-cell masks, the modules included filters for cell size, negative selection for cells on the border of the image or exclusion of cytoplasm signal with no nuclei, thus generating 16-bit .tiff single-cell masks with only full cells for each image. Each of the .tiff files and single-cell masks were then transferred to histoCAT [24] for further analysis. In histoCAT, we ran a dimensionality reduction tSNE algorithm to visualize single cell data from all samples. We then ran a Phenograph analysis in which cells were clustered according to their marker expression (for markers CD11c, CD44, CD45, CD68, HLA-DR, P2Y12 and TNF, using k = 50 nearest neighbours). The mean expression and cell frequencies per sample/cluster where then extracted using R.
Statistical analysis
No randomization and blinding strategies were applied in this study. However, data processing and analysis, as well as statistical testing were carried out in an unsupervised manner. No priori statistical methods were used to predetermine sample sizes due to sample accessibility and insufficient previous data to enable this. However, sample sizes were chosen based on estimates of anticipated variability through previous studies on scRNA-Seq analysis of microglia in acute lesion MS [12]. Dichotomous variables of the sample cohort were analysed with Fisher’s exact test (GraphPad Prism). Quantitative data are shown as independent data points with median or Box-Whisker. Unless otherwise stated, analyses of statistical significance were performed by computational analysis using generalized linear mixed-effects model (GLMM) available through R package diffcyt and false discovery rate (FDR) adjustment (using Benjamini-Hochberg procedure) for multiple hypothesis testing. A p-value < 0.05 (FDR-adjusted) was considered statistically significant.