Key materials
Live Cell Imaging solution, Fluo4-AM, CellTracker Deep Red, pHrodo iFL Red STP Ester, NucBlue live cell nuclear stain, NucGreen dead cell nuclear stain, and LIVE/DEAD fixable aqua stain were obtained from Invitrogen. DAPI was from Sigma, and paraformaldehyde (4% in PBS) was obtained from Alfa Aesar. Human annexin V protein was from BD Biosciences, human C5a protein from Peprotech, human TGFβ1 from Miltenyi Biotec, and recombinant human IFNγ and recombinant truncated human vitronectin from Gibco. Cytochalasin D was from Cayman Chemicals, Bafilomycin A1 from Abcam, and Jasplakinolide from Santa Cruz. PSB0739, MRS2179, MRS2211, and cilengitide were all obtained from Tocris. ADP, BSA, and crystal violet were from Sigma.
iPSC culture
iPSC lines BIONi010-C (control, BioSample ID: SAMEA3158050, ECACC ID: 66540023), BIONi010-C-7 (R47H TREM2, BioSample ID: SAMEA4454010, ECACC ID: 66540369), and BIONi010-C-17 (TREM2 KO, BioSample ID: SAMEA104386270, ECACC ID: 66540632) were obtained from Bioneer and are available from the European Collection of Authenticated Cell Cultures (ECACC). The parent line BIONi010-C was re-programmed by Bioneer with a non-integrating episomal vector, using normal adult human skin fibroblasts sourced from Lonza (#CC-2511). For some supplementary data, pMac differentiated from SFC840-03-03 iPSC were used, a previously published line derived from dermal fibroblasts from a disease-free donor recruited through the Oxford Parkinson’s Disease Centre [32]. iPSC were cultured in mTeSR™1 media (STEMCELL Technologies), on hESC-qualified Geltrex-coated plates (Gibco), passaging as clumps using 0.5 mM EDTA in PBS. Large-scale SNP quality-controlled batches were frozen at p15–25 and used for experiments within a minimal number of passages post-thaw to ensure consistency. An Illumina Omniexpress 24 v1.2 SNP microarray analysis was performed to verify genomic integrity, as previously described in [30].
Human iPSC differentiation to pMac
iPSC were differentiated to primitive, tissue-type macrophages as previously described [29]. In brief, 4 × 106 iPSC were seeded into an Aggrewell-800 plate well (STEMCELL Technologies) to form embryoid bodies, in mTeSR™1 media (STEMCELL Technologies), and fed daily with medium plus growth factors: 50 ng/mL BMP4 (Peprotech), 50 ng/mL VEGF (Peprotech), and 20 ng/mL SCF (Miltenyi Biotec). In a modification to the previously published protocol, the embryoid bodies were cultured for 5–6 days in growth factors instead of 4 days, and after the first 2 days they were transferred into low-adherence 6-well plates. Embryoid bodies were then differentiated in T175 flasks (150 per flask), known as “differentiation factories”. iPSC-macrophage precursors (pMacpre), emerging into the supernatant after approximately 2–3 weeks, were harvested weekly, were plated in their final assay format, and were differentiated to pMac for 6–10 days at 37 °C/5% CO2, in X-VIVO15 with 100 ng/mL M-CSF, 2 mM Glutamax, 100 U/mL penicillin, and 100 μg/mL streptomycin.
DNA sequencing of R47H mutation
DNA was extracted from a cell pellet of iPSC using the DNeasy Blood & Tissue kit from QIAGEN. A PCR reaction in 25 μL was performed using Phusion HF buffer, 0.5 units Phusion HF DNA polymerase, 200 μM dNTPs, 500 nM forward primer (AAACACATGCTGTGCCATCC), 500 nM reverse primer (CACAGACGCCCAAAACATGAG), and genomic DNA (50–100 ng). PCR reaction: 1 × (98 °C, 30 s), 30 × (98 °C, 5 s; 60.7 °C, 10 s; 72 °C, 15 s), 1 × 72 °C, 5 min. PCR products were sent for sequencing (Eurofins) with the reverse primer: TGATGGCTGTGCTCCCATTC.
Western blotting
pMacpre were seeded in either 12-well plates (8 × 105 pMac per well) or 6-well plates (1.5 × 106 pMac per well) and differentiated for a week in macrophage media. Stimulations with a TREM2-activating antibody (goat polyclonal antibody against human TREM2 AF1828 from R&D Systems) used a concentration of 2.4 μg/1 × 106 cells (3.84 μg/mL) for 10 min. Stimulations with dead SH-SY5Ys used a ratio of 3:1 SH-SY5Ys to pMac, with an initial 1 h incubation at 4 °C to allow cells to settle, followed by incubation at 37 °C for the time periods indicated. After washing with PBS, pMac were lysed directly in modified RIPA buffer (50 mM Tris-HCl (pH 7.4), 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 150 mM sodium chloride, 2 mM ethylenediaminetetraacetic acid, 50 mM sodium fluoride) with protease and phosphatase inhibitor cocktails (Roche). Lysates were sonicated for 30 s at medium power and centrifuged at 14000×g for 3 min to a pellet-insoluble material. Triton X-100-soluble proteins were boiled with 1× LDS sample buffer with reducing agent (Thermo Fisher), and samples were resolved by denaturing SDS-PAGE with 8–16% Tris-glycine gels (Thermo Fisher). Subsequently, the separated proteins were transferred to low-fluorescence 0.2 μM-pore PVDF membrane (Thermo Fisher) using a Pierce Power Blotter semi-dry transfer apparatus (Thermo Fisher). Membranes were blocked for 0.5–2 h with iBind FD solution (Thermo Fisher) and incubated with primary antibodies in iBind solution overnight at 4 °C. Primary antibodies for TREM2, phospho-SYK (Y525/Y526), total SYK, and GAPDH were purchased from commercial sources and are listed in Table 1. Membranes were washed 3 × 10 min with Tris-buffered saline and 0.05% Tween-20 (TBS-T) and probed for 1 h at room temperature (RT) with secondary antibodies in iBind FD solution. Membranes were washed 4–6 × 10 min with TBS-T and developed with an Odyssey imaging system (LI-COR®). Optical densities of immunoreactivity were quantified using ImageStudio™ Lite 5.2 software and normalised to the GAPDH control.
Flow cytometry for surface proteins
pMac were lifted from 6-well plates by incubation with StemPro Accutase (Gibco) for 10 min at 37 °C. The cells were washed with PBS and blocked in FACS buffer (PBS, 1% FCS, 10 μg/mL human IgG) for 10 min at RT. 2 × 105 cells per sample were stained with directly conjugated primary antibodies against CD11b, CD14, CD45, αVβ3, or αVβ5, for 30 min on ice. A viability stain was included with the αVβ3 and αVβ5 antibodies (LIVE/DEAD fixable aqua, Invitrogen). Cells were then washed 2–3 times with FACS buffer and fixed with 4% paraformaldehyde in PBS (Alfa Aesar) for 10 min at RT. Cells were spun down and re-suspended in PBS. CD11b, CD14, and CD45 staining was analysed with a FACSCalibur flow cytometer (BD Biosciences), whereas αVβ3 and αVβ5 were analysed with an LSRFortessa X20 flow cytometer (BD Biosciences). Fluorophore-conjugated isotype controls from the same manufacturers were used. The antibodies are listed in Table 1.
Cell surface protein purification
pMacpre were seeded at 2 × 106 cells/well in 6-well plates and differentiated for a week. Cell surface proteins were extracted using a Pierce Cell Surface Protein Isolation kit (Thermo Fisher). In brief, the plates of pMac were placed on ice and washed twice with cold PBS with Ca2+ and Mg2+. Cell surface proteins were biotinylated with 0.5 mg/well of sulfo-NHS-SS-biotin for 30 min on ice. Free biotin was quenched with Quenching Buffer (Thermo Fisher); the cells were gently scraped into tubes, spun down, and washed with Tris-buffered saline. The cell pellets were lysed in RIPA buffer (50 mM Tris-HCl (pH 7.4), 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 150 mM sodium chloride, 2 mM EDTA, 50 mM sodium fluoride) and briefly sonicated, and protein content was measured by Bradford protein assay (Bio-Rad). Total protein concentrations between samples were normalised, and an aliquot of normalised whole-cell lysate was saved for Western blotting. The remaining homogenates were incubated with twice the volume of Neutravidin beads (Thermo Fisher) overnight at 4 °C with rotation. Beads were washed three times by centrifugation and re-suspension in Wash Buffer (Thermo Fisher). Cell surface proteins were eluted from the beads by boiling for 15 min with 1× “LDS Sample buffer” (Thermo Fisher) with 50 mM DTT. Cell surface proteins and whole-cell lysates (containing proteins soluble in 1% Triton X-100) were analysed by Western blotting, including a negative control of non-biotinylated cells.
Immunocytochemistry
pMacpre were seeded at 4 × 104 cell/well in optically clear bottom CellCarrier 96-well plates (Perkin Elmer) and differentiated in macrophage media for a week. For cell surface staining of live pMac: cells were washed with cold PBS and incubated on ice for 20 min with primary antibody in a live-cell blocking buffer (PBS with 5% BSA and 10 μg/mL human IgG). Cells were washed three times for 5 min with cold PBS, on ice, and incubated with secondary antibody in live-cell blocking buffer on ice for 15 min. After three washes with cold PBS, cells were fixed with 2% paraformaldehyde in PBS for 20 min at RT, then washed with PBS. The cells were permeabilized and counterstained with DAPI as below. For total staining of permeabilized cells: cells were fixed in 2% paraformaldehyde in PBS (Alfa Aesar) for 20 min at RT, washed with PBS, permeabilized with 0.1% Triton X-100 in PBS for 10 min at RT, and blocked overnight at 4 °C in blocking buffer (PBS, 0.05% Triton-X100, 10% normal donkey serum, 5% BSA, 0.01% NaN3). Cells were incubated with primary antibodies for 1 h at RT, washed 3 × 15 min with 0.3% Triton X-100 in PBS, and incubated with secondary antibodies for 1 h at RT. Primary antibody for TREM2 (AF1828, 1:30, R&D Systems) was used in combination with either of the following primary antibodies for subcellular markers: TGN-46, calnexin, RAB11, LAMP1, TUJ1, and RAB9. Secondary antibodies were anti-goat IgG-Alexa Fluor 488 for TREM2, anti-rabbit IgG-Alexa Fluor 568 for TGN-46, calnexin, RAB11 and LAMP1, anti-mouse IgG-Alexa Fluor 647 for TUJ1, and anti-rabbit IgG-Alexa Fluor 647 for RAB9. The antibodies used are listed in Table 1. A DAPI nuclear counterstain (1:2000, Sigma) was incubated for 15 min at RT, and the cells washed 4 × 15 min. Images were acquired with an Opera Phenix High Content Screen System (Perkin Elmer) with a 63x water objective, Z-stacks of 9 fields per well, and 3 wells per condition. Columbus 2.7 software (Perkin Elmer) was used to generate a co-localization index for co-localization of TREM2 with intracellular markers. The analysis pipeline defined intracellular regions as “ER”, or “TGN”, or “lysosome” based upon the intensity and pattern of subcellular marker staining: “ER” is a tubular pattern of calnexin, “TGN” is a single large focal structure of TGN46 per cell, and “lysosome” is a punctate pattern of LAMP1. Calculated within these defined regions, the co-localization index used is (intensity of TREM2)/ (intensity of subcellular marker).
ELISAs
For TGFβ1, pMacpre were seeded at 1.2 × 106 cell/well in 6-well plates in full macrophage media, and the media were replaced after 7 days with 1 mL full media ± 2 μM OXSI-2 for a further 24 h incubation at 37 °C/ 5% CO2. Supernatants were spun down and stored in aliquots at − 80 °C. TGFβ1 was quantified using a Human/Mouse TGF beta-1 Uncoated ELISA Kit (Invitrogen), in accordance with the manufacturer’s protocol.
For sTREM2/IL-6/TNF, pMacpre were seeded at 4 × 104 cells/well in optically clear bottom CellCarrier 96-well plates (PE) and differentiated in macrophage media for a week. In triplicate wells, cells were stimulated for 24 h ± 100 ng/mL IFNγ. All wells were given a full media change to 70 μL macrophage media ± E. coli lipopolysaccharide (LPS) and incubated for 4 h at 37 °C/ 5% CO2. Supernatants were spun down and stored in aliquots at − 80 °C. For cell counting, cells were stained with NucBlue Live ReadyProbes Reagent (Invitrogen), and nuclei counting was performed using an EVOS FL Auto automated microscope (Thermo Fisher) at 4x objective, 4 fields per well, and CellProfiler 2.2 software [33]. Supernatant TNF was quantified using a TNFα Human Uncoated ELISA Kit (Invitrogen), in accordance with the manufacturer’s protocol. An in-house ELISA was used to measure IL-6 and sTREM2, from the same supernatants measured for TNF, except with triplicate wells pooled. For in-house IL-6/TREM2 ELISA procedure, Greiner high-bind 96-well plates (Sigma) were coated with appropriate capture antibody (detailed in Table 1 for each ELISA) overnight at 4 °C. Plates were washed with PBS + 0.05% Tween20 and incubated with blocking buffer (PBS, 0.05% Tween20, and 1% BSA) to block non-specific binding sites. A standard curve was generated using human recombinant IL-6 or TREM2 (Sino Biologicals). Standard and diluted supernatants were incubated for 2 h at RT. After washing, plates were incubated with the appropriate biotinylated detection antibody (Table 1) for 1 h at RT. Plates were washed 3x in PBS-T, then incubated with HRP-conjugated streptavidin (Thermo Fisher Scientific) for 1 h at RT. Plates were washed and incubated with 1-Step Ultra TMB ELISA substrate solution (Thermo Fisher Scientific). The reaction was stopped with 2 N H2SO4, and the chemiluminescent signal was measured on a plate reader at 450 nm. Data from each well was normalised to the average cell count for that condition, and further normalised to the average pg/mL/cell for the whole ELISA plate. The in-house ELISA antibodies are listed in Table 1.
Calcium assay
pMacpre were seeded at 1 × 104 cells/well in optically clear bottom CellCarrier 384-well plates (Perkin Elmer) and differentiated in macrophage medium for 7 days. The stimuli used were 0.5 mM ATP (Sigma) and 10 μg/mL TREM2 antibody (R&D Systems). A 384-well plate containing stimuli was prepared for transfer onto the pMac. pMac were loaded with 25 μL of 4 μM calcium-sensitive dye Fluo4-AM (Thermo Fisher Scientific) in the presence of 0.5% pluronic acid (Life technologies) diluted in HBTS buffer (HEPES Buffered Tyrode’s Solution: NaCl 135 mM, KCl 5 mM, MgCl2 1.2 mM. CaCl2 2.5 mM, HEPES 10 mM, glucose 11 mM, pH 7.2) for 1 h at RT. pMac were washed with HBTS before the plates of pMac, and stimuli were loaded onto the FLIPR Tetra (Molecular Devices), a high-throughput cell-based screening system with a robotic pipettor. Each condition was run in quadruplicate. Relative fluorescent units (RFU) of the assay plate were read with the excitation/emission pairs 470–495 nm LEDs and 515–575 nm emission filters. Settings were adjusted in order to have values of ~ 1000 RFUs at baseline. Basal fluorescence was measured for 1 min, and following injection of stimuli, the response was recorded for 5 min at reading intervals of 5 s using the ScreenWorks software. Data was exported as maximum-minimum signal and RFU normalised to baseline values set to 100%.
Generation of dead SH-SY5Ys
SH-SY5Ys (ATCC) were cultured in T75 flasks with DMEM/F12 media (Gibco) with 10% FBS (Sigma) and penicillin/streptomycin (Invitrogen), and maintained at 37 °C/5% CO2. Cells were harvested with TrypLE Express (Gibco), washed with Hank’s Balanced Salt Solution (HBSS, Gibco), centrifuged at 400×g for 5 min, and re-suspended in 2 mL Live Cell Imaging Solution (LCIS, Invitrogen). Paraformaldehyde (Alfa Aesar) was added to a final concentration of 2%, and the cells were fixed for 10 min at RT. The cells were washed again with HBSS and centrifuged at 1200×g for 7 min.
Generation of rat cortical synaptosomes
Two wildtype female ex-breeder Sprague-Dawley rats (Charles River) were sacrificed using a CO2 procedure, in accordance with the approved humane killing protocols detailed in Schedule 1 of the Animals in Scientific Procedures Act, 1986, and the brain cortices dissected. Synaptosomes were purified from the fresh cortices using a previously described method of Percoll gradient fractionation, with four Percoll gradients per rat [34]. An aliquot of the purified synaptosomes was dissolved in 1% NP-40 and the protein concentration determined by Bradford assay. Accordingly, the synaptosomes were diluted to 1 mg/mL of their total protein content with HEPES-buffered media (pH 7.4, 140 mM NaCl (VWR), 5 mM KCl (VWR), 5 mM NaHCO3 (Sigma), 1.2 mM NaH2PO4 (Sigma), 1 mM MgCl2.6H2O (Sigma), 10 mM glucose (VWR), 1 mg/ml BSA (Sigma), 10 mM HEPES (Sigma)) with 5% (v/v) DMSO and frozen in single-use aliquots at − 80 °C. Thawed synaptosomes were characterised by negative staining transmission electron microscopy, performed by the Sir William Dunn School of Pathology Electron Microscopy Facility. Upon thawing, synaptosomes were centrifuged at 3000×g for 10 min at 4 °C and washed once with Live Cell Imaging Solution (Invitrogen), to remove residual BSA before pHrodo-labelling.
Annexin V-FITC staining for phosphatidylserine
Phosphatidylserine exposure of phagocytic cargo was visualised using an annexin V-FITC Apoptosis Detection Kit (Abcam). One day prior to staining, SH-SY5Ys were seeded to 50% confluence in a 24-well plate. Synaptosomes were thawed, washed, and re-suspended in annexin binding buffer, and approximately 0.3 μg per well was added to empty wells of the plate, allowing an hour to settle at 37 °C/5% CO2. SH-SY5Ys were washed with HBSS and some wells fixed with 2% paraformaldehyde for 10 min, before another wash, replacing with annexin binding buffer containing NucBlue. Both SH-SY5Ys and synaptosomes, except for unstained controls, were stained with 1:70 annexin V-FITC and 1:70 propidium iodide for 5 min. Propidium iodide stains nuclei of permeable cells, controlling for annexin V staining of the plasma membrane inner leaflet. The plate was imaged at 37 °C/5% CO2 using an EVOS FL Auto automated microscope (Thermo Fisher) with on-stage incubator at 40x objective with phase and using the DAPI, GFP, and Texas Red light cubes.
Phagocytosis assays
pMacpre were seeded at 2 × 104 cells/well in optically clear bottom CellCarrier 96-well plates (Perkin Elmer) and differentiated in macrophage media for a week. Cells were stained for 45 min at 37 °C/5% CO2 with 1 μM CellTracker Deep Red (Invitrogen) and 1 drop/mL NucBlue Live ReadyProbes Reagent (Invitrogen). Cells were washed with PBS, and then incubated for 1 h at 37 °C/5% CO2 with 100 μL of Live Cell Imaging Solution (LCIS, Invitrogen) ± phagocytosis inhibitors, before addition of phagocytic cargo. Phagocytosis inhibitors used for validation were 10 μM cytochalasin D (Cayman), 1 μM bafilomycin A1 (Abcam), 1 μM jasplakinolide (Santa Cruz), and 2 μg unlabelled human recombinant annexin V (BD Biosciences). Annexin V was added to well immediately prior to addition of phagocytic cargo. The phagocytic cargo- synaptosomes or dead SH-SY5Ys- were stained with pHrodo iFL Red STP Ester (Invitrogen), using 20 μg of dye per 1 mg synaptosomes, or 12.5 μg of dye per 1 × 106 SH-SY5Ys, aiming for a final concentration of 40 μg/mL. pHrodo-labelling was performed for 30 min at RT, protected from the light, in a low protein-binding tube. Cargo was washed twice with HBSS, (centrifugation: 3000×g synaptosomes, 1200×g dead SH-SY5Ys) and re-suspended in LCIS to a concentration of 0.6 μg/μL synaptosomes or 8 × 105 cells/mL SH-SY5Ys, and 50 μL/well added to the pMac. Phagocytosis was performed at 37 °C/5% CO2 for 0.5–5 h, in triplicate wells. Cells were fixed with 2% paraformaldehyde in PBS (Alfa Aesar) for 15 min at RT and washed with PBS before imaging. For staining TREM2, TUJ1, and RAB9a, immunocytochemistry was performed on permeabilized cells as described above. Images were taken with an INCell Analyzer 6000 high-content imaging system (GE Healthcare Life Sciences) with a 40x objective, 9 fields/well on a single plane. Images were quantified with Columbus 2.7 software (Perkin Elmer). The parameters measured for each field were average number of spots/cell, the sum of the spot areas, and the % spot-positive cells. Data was averaged for the technical replicates and normalised to the overall plate average, to adjust for differences between plates.
Transwell assays
pMacpre were seeded at 1.2 × 106 cells/well in 6-well plates and differentiated in macrophage media for a week. Cells were dissociated with StemPro Accutase (Gibco) for 10 min at 37 °C and washed with PBS. pMac were re-suspended in cold macrophage media, and 100 μL with 5.5 × 104 cells was pipetted onto transwells (PET with 5 μm pores, Sarstedt) suspended over wells of an empty 24-well plate. In total, 600 μL of macrophage media ± inhibitors was added beneath the transwells and incubated 30 min at RT, before the chemotactic stimulus was added: 30 μM ADP (Sigma) or 3 nM human recombinant C5a (Peprotech). The 24-well plates were incubated for 6 h to allow cell migration. After cell migration, the transwells were gently rinsed with PBS, transferred to a fresh 24-well plate, and fixed with 4% paraformaldehyde (Alfa Aesar) for 20 min at RT. Cells were stained with NucBlue (Invitrogen) nuclear stain and imaged with an EVOS FL Auto automated microscope (Thermo Fisher), 4× with DAPI light cube, set up to scan each transwell in full and generate a knitted single image. The transwells were swabbed with a cotton wool bud to remove cells on the top surface, leaving behind only migrated cells, transferred into a fresh plate with fresh PBS, and imaged again with the same settings. Nuclei counting was performed with CellProfiler2.2 software [33], and for each transwell, the % migration was calculated: (no. cells in second scan) ÷ (no. cells in first scan) × 100. Treatments were performed in duplicate and duplicate wells were averaged, then normalised to the average % migration for the entire plate, to control for age-dependent differences in cell speed.
Survival assay
pMacpre were seeded at 4 × 104 cells/well in three optically clear bottom black 96-well plates (Costar) and differentiated in macrophage media. After 7 days, a full media change was performed to 100 μL macrophage media ± M-CSF, triplicate wells for each condition on each plate. Plates were incubated at 37 °C/ 5% CO2 for 3, 7, or 10 days, and the 10-day plate received a 50% media change at 7 days. At the end of each incubation, cells were stained 20 min (37 °C/5% CO2) with the ReadyProbes Cell Viability Imaging Kit (Invitrogen). Nuclei counting was performed using an EVOS FL Auto automated microscope (Thermo Fisher), 4x objective with DAPI and GFP light cubes, 4 fields/well, and CellProfiler 2.2 software [33]. Data was presented as (mean number of dead cells/mean number of total cells) × 100 for each condition.
RNA-seq sample and library preparation
pMacpre were seeded at 1.5 × 106 cells/well in 6-well plates at 7, 8, and 9 weeks after setting up differentiation factories, and they were differentiated in macrophage media for 7 days. Cells were washed once with PBS, aspirated thoroughly, and lysed by addition of 350 μL Buffer RLT (QIAGEN) with 1% (v/v) 2-mercaptoethanol. Plates were stored at − 80 °C until all samples were collected, and then lysates were passed through QIAshredder columns (QIAGEN) and the total RNA extracted using a QIAGEN RNeasy Mini kit, according to the manufacturer’s protocol using on-column DNase treatment (QIAGEN). RNA samples were eluted in 30 μL of RNase-free water. The quantity and quality of RNA was measured by Nanodrop and RNA Tapestation (Agilent), with measured RNA integrity (RIN) values ≥ 8.4. Poly-A library preparation and sequencing (HiSeq 4000, 75 bp paired-end reads), and basic data processing, was conducted at the Oxford Genomics Centre, Wellcome Centre for Human Genetics.
Analysis of RNA-seq data
Quantified transcript abundance counts were obtained with the tool Salmon (v0.12.0)(PMID: 28263959) using mapping-based mode with default parameters, automatic library type-inference, and the additional command line arguments “validatemappings”, “seqBias”, “posBias”, and “gcBias”, in order to enable selective alignment of the sequencing reads, and account for sequence-specific biases, fragment-level GC biases, and 5′ or 3′ positional biases in the data. The paired-end sequencing reads for each sample were mapped to the human reference transcriptome (GRCh38; Ensembl release 95), which combined cDNA and ncRNA. The transcript abundances were imported and summarised to gene-level counts using the R library “tximport” (PMID: 26925227).
Only the protein-coding genes were used, and data were filtered to include genes (n = 15,341) with > 10 counts across all samples. Differential gene expression between the TREM2 KO vs WT and TREM2 R47H vs WT was performed on genes expressed across all samples with the R library “DESeq2” (PMID: 25516281) using the Wald test, and LFC shrinkage was performed using the “ashr” method (PMID: 27756721). We considered a gene to be differentially expressed at FDR < 0.05. Principal component analysis was performed using the “prcomp” function in R, on variance-stabilising transformed data for all genes. PC1 was added as a covariate to the design model, to remove the influence of the covariate “differentiation age” on the differential expression. Gene ontology enrichment analysis was performed with the R library ClusterProfiler (PMID: 22455463) and adjusted p values for multiple testing following a Benjamini-Hochberg correction, and terms were filtered out if they were associated with fewer than 10 differentially expressed genes. All plotting was performed with multiple libraries in R.
Gene functional network and clustering method
A combined protein-protein interaction (PPI) network was created based on diverse resources: BioGRID 3.4 (accessed on September 2017) [35], HitPredict (accessed on September 2017) [36], IntAct (accessed on September 2017) [37], STRING (accessed on September 2017, restricted to Homo sapiens and experimental scores higher than zero) [38], CORUM (accessed on September 2017) [39], and Reactome (accessed on September 2017) [40]. The combined network consisted of a total of 20,591 genes and 1,973,967 interactions. A gene functional network was built for the differentially expressed genes between TREM2 KO and WT samples, by using the top 500 K PPI interactions. To identify modules of highly interconnected genes in the network, we employed “cluster_louvain” function in “igraph” R library [41], where proteins in clusters with fewer than 30 members were grouped into a cluster with the label “0”.
Randomisation for PPI network analysis
The randomisation approach takes into account both coding sequence length and the number of PPIs of the corresponding proteins. For every input gene, a random gene with similar coding sequence length and similar connectivity of its corresponding protein was selected, by applying a binning approach (PMID: 25319962). A total of 10,000 random gene sets, of the same size as the number of differentially expressed genes, were tested for their connectivity within the combined PPI network, and the number of connections were compared to the one obtained using the list of differentially expressed genes for TREM2 KO vs WT samples. The PPI analysis was performed on the basis of pairs of genes (using ensembl gene IDs annotated in combined PPI network), which means that an interaction between two genes is reported and counted if any of the possible gene products show an interaction.
External dataset comparison
Processed FPKM counts for RNA-seq data from Abud et al. [42] were downloaded from GEO: GSE89189 and were filtered to include only protein-coding genes that were common with the RNA-seq data in this study. The study batch-effect of the combined log2-transformed FPKM gene expression levels was removed by using the “ComBat” function in the “sva” R library (PMID: 22257669) and hierarchical clustering of the Euclidean distances of the samples was performed using the “ward. D2” method, and the dendrogram visualised with the “dendextend” R library. A scatter plot was made using R library “ggplot2” of significant (FDR < 0.05) differentially expressed genes for TREM2 KO vs WT from the Claes et al. RNA-seq study [27], against the significant results for TREM2 KO vs WT from the current study.
Ingenuity pathway analysis
For each of the PPI modules of differentially expressed genes, we identified upstream regulators using Ingenuity Pathway Analysis software [43]. The algorithm takes into account the observed gene expression changes, and the manually curated Ingenuity Knowledge Base, to infer the “activated” or “inhibited” state of upstream regulators (including transcription factors, small molecules, microRNA, and other genes). The “Regulators Effect” algorithm takes into account each identified upstream regulator and links it through the Ingenuity Knowledge Base to functions, phenotypes, and disease. Briefly, it takes the best matching pair (regulator and downstream effect networks) and tests for overlapping sets with a Fisher exact test and then iteratively groups regulators to both increase consistency and reduce redundancy of the network.
qRT-PCR
pMacpre were seeded at 0.8 × 106 cells/well in 12-well plates, differentiated in macrophage media for 7 days, and stimulated ± 50 ng/mL TGFβ1 (Miltenyi Biotec) for 24 h. Cells were washed once with PBS, aspirated thoroughly, and lysed by addition of 350 μL Buffer RLT (QIAGEN) with 1% (v/v) 2-mercaptoethanol. Plates were stored at − 80 °C, and RNA extracted as detailed for the RNA-seq sample preparation above. Reverse transcription was performed using a High-Capacity RNA-to-cDNA kit (Applied Biosystems), with 400 ng RNA input per reaction, following the manufacturer’s protocol. qRT-PCR was performed using TaqMan probes and TaqMan Gene Expression Master-mix (Applied Biosystems) in a 384-well PCR plate, 2 μL cDNA in a final volume of 6 μL per well, on a QuantStudio 5 qRT-PCR machine (Applied Biosystems). Taqman probes were CHCHD2 (Hs00855326_g1), ITGAV (Hs00233808_m1), ITGB3 (Hs01001469_m1), ITGB5 (Hs00174435_m1), FN1 (Hs01549976_m1), LAMB2 (Hs00158642_m1), SDC4 (Hs01120908_m1), GPC4 (Hs00155059_m1), and TBP (Hs00427620_m1). Samples were run in triplicate wells. ΔΔCt values were calculated using the average Ct for each triplicate: ΔCt was generated by subtraction of the average Ct for reference gene TBP, and then the ΔCt was normalised to the average ΔCt for all WT unstimulated samples (by subtraction).
Adhesion assay
pMacpre were seeded at 1.2 × 106 cell/well in 6-well plates in full macrophage media, and the media replaced after 6 days with 1 mL full media ± 50 ng/mL TGFβ1 (Miltenyi Biotec) for a further 24 h incubation at 37 °C/ 5% CO2. Wells of a clear 96-well flat-bottomed plate were coated with 0.5 μg/well (1.56 μg/cm2) truncated vitronectin (Gibco) in PBS, by incubation at room temperature for 1 h. The vitronectin was aspirated, and remaining non-specific binding sites were blocked with a solution of 10 mg/mL denatured bovine serum albumin (BSA) in PBS for 1 h. Wells were washed once with PBS, and 50 μL of Live Cell Imaging Solution (LCIS; Invitrogen) ± cilengitide added (10 μM final well concentration). pMac dissociated from the 6-well plates by StemPro Accutase (Gibco) were pelleted and re-suspended in LCIS, and 50 μL added to the vitronectin-coated wells at a density of 5 × 104 cells/well. Cells were also added to no-vitronectin BSA-blocked wells as a control for non-specific binding. Adhesion was performed for 3 h at 37 °C/5% CO2, and then the plate was washed once with PBS and cells fixed for 10 min in 4% paraformaldehyde at RT. Following two PBS washes, the cells were stained with crystal violet solution (0.1% (w/v) in 50 mM MES, 150 mM NaCl, pH 6) for 1 h at RT. Wells were washed 3 times with ddH2O, and the cells and dye were solubilised with 50 μL of 10% (v/v) acetic acid. Absorbance was measured at 570 nm on a microplate spectrometer, normalised to the non-specific binding controls.
Statistical analysis
Statistical analysis of the data was performed in GraphPad Prism software (version 7 for Windows), GraphPad Software, La Jolla, CA, USA, www.graphpad.com. All “n” numbers represent independent biological replicates, i.e., separate harvests of the differentiation cultures, with experiments performed independently on different weeks. Means were obtained from three or more independent repeats, and paired t-tests and one-way or two-way ANOVAs performed where appropriate, with Bonferroni, Sidak, or Dunnett corrections for multiple comparison. P values < 0.05 were considered to be significant and are indicated as follows: *p < 0.05, **p < 0.01, *** p < 0.001, **** p < 0.0001.