Ethics statement
The studies involving human peripheral blood mononuclear cells (PBMCs) were approved by the local ethics committee (Ethikkommission der Medizinischen Fakultät Bonn) according to the ICH-GCP guidelines. Written informed consent was provided by voluntary blood donors.
Primary cells and cell lines
Human PBMCs were isolated from whole human blood of healthy, voluntary donors by Ficoll-Hypaque density gradient centrifugation (Biochrom). PBMCs were cultured in RPMI-1640 medium supplemented with 10 % FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin. BLaER1 cells were kindly provided by Thomas Graf, Centre for Genomic Regulation, Barcelona, Spain. Monoclonal cell lines were generated by seeding as single-cell clones via limited dilution and expansion. BLaER1 and THP-1 cells were cultivated in RPMI-1640 medium supplemented with 10 % FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin. HEK-Blue IFNα/β cells, purchased from Invivogen, were cultured in DMEM supplemented with 10 % FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin.
Oligonucleotides
The RNA oligonucleotide 9.2s was ordered from Biomers. DNA oligonucleotides were ordered from Integrated DNA Technologies (IDT). Sequences are shown in Table 1.
Table 1. DNA and RNA oligonucleotide sequences
The sequences of DNA and RNA oligonucleotides used for qRT-PCR, cloning and cell stimulation are shown.
Name | Sequence (shown 5' to 3') |
qRT-PCR primers | |
ITGAM fwd | ACAACCCTAACCCAAGATCAC |
ITGAM rev | AAACAGCTCTCGTACCACTTT |
FCGR1B fwd | CCTTGAGGTGTCATGCGTG |
FCGR1B rev | AAGGCTTTGCCATTTCGATAGT |
CD14 fwd | GATTACATAAACTGTCAGAGGC |
CD14 rev | TCCATGGTCGATAAGTCTTC |
PIGH fwd1 | GCCATCTTCATCACCCTCTTAG |
PIGH rev1 | CTGAATGCCAAGGGAATCAATG |
PIGH fwd2 | GCTGGACTGCTTGATTGAAGTAT |
PIGH rev2 | AAGACAATGCTGGCCTTCTG |
PIGH fwd3 | AGTCATGGTGTCAAGCACATTA |
PIGH rev3 | TCCAATTTCTGTCTGGCCTTT |
PIGH fwd4 | GCCATTTACATGCAGAAGGT |
PIGH rev4 | GACTGTGTCCACCTGATGGT |
PIGH fwd5 | GAGCAGGTGACTGGAAATCTAA |
PIGH rev5 | CCGAAAGTGTTCTTTGCTGAC |
PIGH fwd6 | GCTAAGCATGTTCAGGTTTACTTT |
PIGH rev6 | CCAGTACTGAAGGGCTTGTT |
PIGH fwd7 | GGAGGATGAGCGGAGCTTT |
PIGH rev7 | AGCGAACGCAGCGAGAG |
PIGH fwd8 | GGCGTCATGGAGGATGAG |
PIGH rev8 | CAGAATTCCCGGCAGGAC |
PIGH fwd9 | GGTGATTTACTACCTCTGCATCTT |
PIGH rev9 | AAGACGGGTACTACTTGGGATA |
PIGH fwd10 | TTCCGTACTCCAAACCATCAG |
PIGH rev10 | ATTTCCAGTCACCTGCTCTATG |
PIGH fwd11 | GGGAGATGACTCTTAAGCCATAG |
PIGH rev11 | GCTCTAAGATGCACTACATCCATAA |
PIGH fwd12 | GCAGGTGACTGGAAATCTAAC |
PIGH rev12 | CTCCTTTGGTAAAGTAGGCAAA |
PIGH gDNA fwd | TACTGCGTTCAGCTGATTTAGG |
PIGH gDNA rev | CCTCCAGAGAGGCCTGTAATA |
Cloning primers | |
PIGH GA fwd | GAATTTCGACCCGGATCCGCGGCCGCGCCACCATGGAGGATGAGC |
PIGH/PIGA GA rev | CCCCTACCCGGTAGAATTCACGCGTTCACTTATCGTCGTCATCCTTGTAATC |
PIGA GA fwd | GAATTTCGACCCGGATCCGCGGCCGCGCCACCATGGCCTGTAGAG |
RNA stimuli | |
3pRNA, sense | ppp-GGCCGAGACCUCGAAGAGAACUCU |
3pRNA, antisense | ppp-AGAGUUCUCUUCGAGGUCUCGGCC |
IVT4 template for in vitro transcription, sense | TTGTAATACGACTCACTATAGGGACGCTGACCCAGAAGA TCTACTAGAAATAGTAGATCTTCTGGGTCAGCGTCCC |
IVT4 template for in vitro transcription, antisense | GGGACGCTGACCCAGAAGATCTACTATTTCTAGTAGATCT TCTGGGTCAGCGTCCCTATAGTGAGTCGTATTACAA |
9.2s | AGCUUAACCUGUCCUUCAA |
DNA stimuli | |
G3-YSD, sense | GGGAAACTCCAGCAGGACCATTAGGG |
G3-YSD, antisense | GGGTAATGGTCCTGCTGGAGTTTGGG |
Stimulatory nucleic acids
9.2s RNA was synthesized by Biomers. IVT4 was generated by in vitro transcription (IVT) with a commercial In vitro T7 Transcription Kit (Thermo Fisher Scientific) using annealed DNA oligonucleotides as a template (see Table 1), as described before21. 3pRNA was chemically synthesized as described before22. For hybridization of G3YSD, DNA was heated in NEB2 buffer to 95°C for 5 min and was then cooled to RT at a cooling rate of 1°C/min. pDNA (pBluescript) was isolated from transformed E. coli K12 with a PureLink HiPure Plasmid Filter Midiprep Kit (Life Technologies).
Plasmids
For lentiviral overexpression, human PIGH and PIGA with C-terminal Flag tags were subcloned into a pLVX-puro-EF1α vector via Gibson assembly.
Transdifferentiation of BLaER1 cells
BLaER1 cells (3x105 per well of a 24-well plate) were seeded in 900 µl medium containing 100 nM 17estradiol (Sigma Aldrich), 10 ng/ml human IL-3 (Peprotech), and 10 ng/ml human MCSF (Peprotech) and incubated for five days.
Stimulation of cells
Cells were stimulated in duplicate with TLR ligands or oligonucleotides. Human PBMCs, transdifferentiated BLaER1 cells, or THP-1 cells (2x105, 1.5x105, or 6x104 per well of a 96-well plate, respectively) were seeded in 150 µl RPMI-1640 supplemented with 10 % FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin. LPS-EK ultrapure, R848, TL8-506, CL264, or Pam3CSK4 (all from InvivoGen) were diluted in 50 µl medium per well and added to the cells. Final concentrations are indicated in the figures. RIG-I (3pRNA, IVT4) and cGAS agonists (pDNA, G3-YSD) were transfected with Lipofectamine 2000 according to the manufacturer’s instructions, giving final stimuli concentrations between 1 ng/ml and 500 ng/ml, as indicated in the figures. Synthetic RNA ligand 9.2s was complexed with poly-L-Arginine of 5–15 kDa (pArg, SigmaAldrich, P4663) as delivery agent (1.8 µg per µg RNA). Per well of a 96-well plate, pArg and RNA were diluted in 25 µl PBS and incubated for 10 min before being added to the cells at a final concentration of 1 or 5 µg/ml RNA.
Cytokine detection
Sixteen hours after stimulation, cell-free supernatant was collected and IL-6, TNFα, and IL-1β were measured by ELISA (BD Biosciences, 555220, 555212, 557953) according to the manufacturer’s instructions.
HEK-Blue assay
HEK-Blue IFN-α/β reporter cells (InvivoGen) featuring type I IFN-inducible secreted embryonic alkaline phosphatase (SEAP) were seeded (6x104 per well of a 96-well plate) in 180 µl DMEM supplemented with 10 % FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin. 20 µl of diluted or undiluted BLaER1 cell supernatant harvested 16 h after stimulation or 20 µl of medium containing IFN-α2a (Miltenyi Biotec, 130-093-874, standard ranging from 4000 U/ml to 62.5 U/ml) were added to HEK-Blue IFN-α/β cells. After incubation for 24 h at 37°C, the supernatant was harvested and incubated with 1 volume 10 mg/ml p-nitrophenyl phosphate (pNPP, Sigma-Aldrich, 71768) in pNPP buffer (100 mM Tris, 100 mM NaCl, 5 mM MgCl2, pH 9.5). Absorption at 405 nm was measured to determine the formation of p-nitrophenol by SEAP activity.
Flow cytometry
Differentiated or undifferentiated BLaER1 cells (3x105) were washed with FACS buffer (2 % FCS, 2 mM EDTA, PBS) once and incubated for 15 min in 25 µl Fc Receptor Blocking Solution (BioLegend, 422302, 1:100 in FACS buffer) at 4°C. 25 µl of isotype control (BioLegend, 400220 or 400120, 1:50) or specific antibody for CD14, CD48, CD55, CD11b, CD45, CD64 (BioLegend, 301808, 336713, 311311, 301309, 368511, 305013, all 1:50), CD36, CD163, or CD191 (Miltenyi Biotec, 130-100-307, 130-100-612, 130-100-360, all 1:50) was added and incubated for 15 min at 4°C. Cells were washed twice with FACS buffer and fixation was performed for 20 min in 4 % paraformaldehyde/PBS at RT. After two further washings in FACS buffer, samples were measured on an LSRII flow cytometer (BD Biosciences).
Data analysis was performed using Flowjo (Treestar). After doublet exclusion, live cells were identified by GFP expression. The percentage of cells expressing the protein of interest (CD14, CD48, CD55, CD11b, CD45, CD64, CD36, CD163, or CD191) among GFP-positive cells was determined using matched isotype controls for gating.
qRT-PCR
BLaER1 cells (1x106) were lysed in 350 µl RLT buffer (Qiagen) and frozen at -80°C for at least 5 min. After thawing, 1 volume of 70 % ethanol was added and the sample was loaded onto a Zymo III column (Zymo). The column was washed sequentially with 1 volume RW1 buffer (Qiagen) and 1 volume RNA wash buffer (Zymo). The column was dried by centrifugation at maximum speed for 2 min and the RNA was eluted with RNase-free distilled H2O. Residual genomic DNA was digested by DNase I, and reverse transcription was performed with random hexamer primers and RevertAid Reverse Transcriptase (all from Thermo Fisher Scientific) according to the manufacturer’s instructions. For qRT-PCR, 5x EvaGreen QPCR Mix II (ROX) (Bio-Budget) was used. The reaction was performed in a QuantStudio 5 Real-Time PCR cycler (Thermo Fisher Scientific). Primers were tested for efficiency using cDNA dilution.
Immunoblotting
After washing once with PBS, cells were resuspended in RIPA lysis buffer supplemented with cOmplete Protease Inhibitor (Roche) and incubated on ice for 30 min. Lysates were cleared by centrifugation at 21,000 x g for 10 min at 4°C. The protein concentration was determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions and 10–25 µg of protein were loaded on an SDSPAGE gel. Gel electrophoresis was performed for 90 min at 110 V. Proteins were blotted onto a nitrocellulose membrane for 90 min at 450 mA. CD14 and GFP were detected with primary antibodies D7A2T and 4B10 (Cell Signaling Technology, 1:1000), respectively. The housekeeping protein β-actin was stained with primary antibody 926-42212 (LI-COR Biosciences, 1:3000). PIGH and PIGA were detected with a Flag-tag primary antibody (Genscript, A00187, 1:1000). As secondary antibodies, goat anti-rabbit IRDye800CW, goat anti-mouse IRDye800CW, and goat anti-mouse IRDye680 (LI-COR Biosciences, 1:10,000) were used. Blots were recorded on an Odyssey FC Dual imaging system (LI-COR Biosciences).
3' mRNA sequencing
Undifferentiated BLaER1 cells (1x106) were resuspended in 500 µl TRIzol Reagent (Thermo Fisher Scientific) and their total RNA was purified according to the manufacturer’s instructions. Libraries of sequences close to the 3' end of polyadenylated RNA were generated with a QuantSeq 3' mRNA-Seq Library Prep Kit FWD (Lexogen) and sequenced on an Illumina HiSeq1500 device.
Sequencing reads were aligned to the human reference genome using STAR23. The transcripts were quantified with HTSeq24. Differential expression analysis was performed using edgeR25.
Sanger sequencing of PIGH cDNA
After reverse transcription of RNA from undifferentiated BLaER1 cells, PIGH cDNA was amplified in a PCR reaction with gene-specific primers (PIGH fwd7, PIGH rev12) using 5x EvaGreen QPCR Mix II (ROX) (Bio-Budget). Sanger sequencing of the PCR products from GPI-positive and GPI-negative monoclonal cell lines was performed by Microsynth Seqlab GmbH (Göttingen, Germany) using the PIGH fwd7 primer.
Sanger sequencing of PIGH genomic DNA
BLaER1 cells (2x105) were pelleted and resuspended in 40 µl PBND buffer (50 mM KCl, 10 mM Tris-HCl, 2.5 mM MgCl2, 0.1 mg/ml gelatin, 0.45 % v/v IGEPAL, 0.45 % v/v Tween-20) supplemented with Proteinase K. Genomic DNA (gDNA) was obtained by incubation at 55°C for 60 min, followed by heat inactivation of the enzyme at 95°C for 10 min. PIGH DNA was amplified in a DreamTaq PCR reaction according to the manufacturer’s instructions (Thermo Fisher Scientific) using gene-specific primers (PIGH gDNA fwd, PIGH gDNA rev). Sanger sequencing of PCR products from GPI-positive and GPI-negative monoclonal cell lines was performed by Microsynth Seqlab GmbH (Göttingen, Germany) using the PIGH gDNA fwd primer.
Sequence analysis
Alignment of sequences obtained by Sanger sequencing to the Reference Sequence (RefSeq) and open reading frame (ORF) prediction were performed with ApE. Poly Peak Parser was used for peak parsing and deconvolution26.
Treatment of cells
Undifferentiated BLaER1 cells (5x105 per well of a 24-well plate) were seeded in 1 ml medium (untreated), or 1 ml medium containing 0.4 µM or 2 µM 5-aza-2’-deoxycytidine (Abcam, 2353335). On days 2 and 4, cells were subcultured with fresh medium (with or without 5-aza-2’-deoxycytidine). CD48 and CD55 surface expression were assessed by flow cytometry 2, 4, and 7 days after the initial treatment.
Lentiviral overexpression
Lentiviral vectors were produced by transfection of HEK 293FT cells with lentivector (1.6 µg of pLVX-puro-EF1α-PIGH-Flag, -PIGA-Flag or -GFP) and packaging plasmids (0.4 µg, 0.6 µg, and 1.1 µg of pRSVrev, pMD2.G, and pMDL.g, respectively) using calcium phosphate transfection. Viral supernatants were harvested 72 h post transfection. BLaER1 cells were resuspended in virus supernatant and spin infection was performed (centrifugation at 600 x g for 60 min at 32°C). The next day, the medium was replaced by Puromycin-containing medium (2.5 µg/ml) to select for cells containing the construct of interest. After three days, the medium was exchanged to Puromycin-free medium.
Statistical analysis
Statistical analysis was performed in GraphPad Prism 7. To compare cells with high responsiveness to LPS (GPI-positive) to cells with lower responsiveness to LPS (GPI-negative), data from up to three monoclonal cell lines were pooled. As such pooled data cannot be assumed normally distributed, non-parametric Mann-Whitney tests were performed to compare both groups. Two-way ANOVA followed by Dunnett’s multiple comparisons test was used to statistically analyze differences between control cells and cells transduced with GFP, PIGH-Flag, or PIGA-Flag. Stimulation experiments with GFP-transduced and PIGH-Flag-transduced cells were statistically analyzed by performing unpaired t tests. * indicates a p value ≤ 0.05, ** a p value ≤ 0.01, *** a p value ≤ 0.001 and **** a p value ≤ 0.0001. Repetition and error bases are defined in the respective figure legends.
Data availability
3' mRNA sequencing data generated in this study are uploaded at GEO (GSE169623).