Cell culture and reagents
The THP-1 human monocytic cell line and Raw 264.7 murine macrophage cell line were obtained from the Bioresource Collection and Research Center, Taiwan. THP-1 cells were incubated with 30 ng/ml PMA (Sigma-Aldrich, St. Louis, USA) in RPMI for 3 days to induce their differentiation into THP-1-derived macrophages (TDMs). Raw 264.7 cells were cultured in Dulbecco’s modified Eagle medium (DMEM, HyClone) containing 10% fetal calf serum (FCS). Oxidized low-density lipoprotein (oxLDL) and 1,19-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil)-oxLDL were purchased from Kalen Biomedical (Germantown, MD, USA). LDL was obtained from Alfa (Thermo Fisher Scientific, Heysham, Lancashire, UK). Cholesterol crystals (CCs) were prepared according to a previous report . Human and mouse IFN-α and interferon gamma (IFN-γ) was obtained from PBL Biomedical Laboratories (Piscataway, NJ, USA). Several Toll-like receptor (TLR) agonists, lipopolysaccharide (LPS), Pam3CSK4, poly(I:C), CpG ODN1826, CpGODN1585, and the Janus kinase (JAK)1/2 inhibitor ruxolitinib were purchased from Invitrogen (Hong Kong Science Park, Pak Shek Kok, Hong Kong.). BMS-986165, a tyrosine kinase 2 (TYK2) inhibitor, was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Mitogen-activated protein kinase (MAPK) inhibitors, including PD98059, SP600125, and SB203580, were obtained from Calbiochem (Darmstadt, Germany), and AG490, a JAK1 inhibitor, was acquired from TOCRIS. Anti-CMPK2 and anti-TOMM20 antibodies were purchased from Abcam (Cambridge, UK). Anti-cleaved interleukin-1β (IL-1β) antibody was obtained from Cell Signaling (Beverly, MA, USA). A scavenger receptor class A (SR-A) Ab (anti-SR-A) was purchased from Santa Cruz (Santa Cruz, CA, USA). Unless specified, all other reagents were from Sigma Aldrich.
Preparation of human primary cells and mouse bone marrow-derived macrophages (BMDMs)
Peripheral blood mononuclear cells (PBMCs) were prepared from buffy coat (purchased from the Blood Bank, Taipei, Taiwan), and both CD14+ monocytes and CD4+ T lymphocytes were then positively selected from among the PBMCs of SLE patients or controls by using a MACS cell isolation column (Miltenyi Biotech, Auburn, USA) as described in our previous report . The diagnosis of SLE was based upon 1982 diagnostic criteria, and the use of human blood samples was approved by the IRB (no. 201509825A3) of Chang Gung Memorial Hospital, Linko, Taiwan. The preparation of mouse BMDMs was performed according to a published report . In brief, male C57BL/6 mice (6–12 weeks) were purchased from the National Laboratory Animal Breeding and Research Center (Taipei, Taiwan). All of the animal studies were conducted in accordance with the protocol approved by the Institutional Animal Care and Use Committee of the National Health Research Institute (NHRI) (approval number: NHRI-IACUC-107159-AC1). Bone marrow was flushed from the tibias and femurs of mouse hind legs with DMEM using a needle syringe. After washing and filtering the marrow through a 40-μm nylon cell strainer, bone marrow cells were cultured in DMEM containing 20 ng/mL macrophage colony-stimulating factor (PeproTech Inc., New Jersey, USA) for 6 days with the medium refreshed every 2–3 days. The purity of the macrophages was more than 99%, as measured by F4/80 and CD11b staining (BioLegend CNS, Inc., USA).
BMDMs were collected and resuspended at a concentration of 1 × 107 cells/ml in modified eagle’s minimum essential medium (opti-MEM, Invitrogen) containing 300 nM siRNA specifically designed for these experiments (Stealth RNAi™ siRNA, Invitrogen). Electroporation was performed using a BTX electroporator (San Diego, CA) operating with one 300 V pulse every 3 ms. The cells (2 × 106) were then seeded with macrophage-conditioned medium (Invitrogen, Carlsbad, CA, USA) containing 10% FBS for 24 h before subsequent treatment. TDMs and Raw 264.7 cells were transfected with 50 nM siRNA by using lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. Four hours after the cells were transfected, the culture medium was replaced with fresh complete medium for further experiments.
CMPK2-knockout THP-1 cells
CMPK2 sgRNA was designed and constructed into a CRISPR/Cas9 lentivector by the National RNAi Core Facility (RNA technology platform and gene manipulation core, Academia Sinica, Taiwan). CMPK2 sgRNA lentivirus was produced after confirming the sequence and knockout (KO) efficiency. THP-1 cells were transduced by lentivirus with CMPK2 sgRNA in the presence of polybrene (4 μg/ml) for 48 h. Then, puromycin (5 μg/ml) was added into the culture medium for 10 days with regular replacement of the medium to remove untransduced cells. Subsequently, a single clone in the 96-well plates of CMPK2-KO THP-1 cells was selected after two rounds of serial dilutions. Successful KO was confirmed by Western blotting and DNA sequencing.
Overexpression of CMPK2 in THP-1 cells
The human CMPK2-GFP gene was a kind gift from Dr. Chang, Zee-Fen, professor in National Taiwan University. Expression of CMPK2-GFP and GFP plasmids in THP-1 cells was done by lipofectamine 3000 transfection according to the manufacture’s instruction.
Oil red O staining and Dil-oxLDL uptake measurement
Oil red O staining and Dil-oxLDL uptake measurements were performed according to our previous report . After treatment, the cells were washed with PBS and then fixed with 10% formalin. After formalin was removed, the cells were pretreated with 60% isopropyl alcohol and stained with 0.2% oil red O solution (Sigma-Aldrich) in 60% isopropyl alcohol. The cells were examined by light microscopy (× 400), and the percentages of oil red O positive cells in 5 microscopic fields for each independent experiment were counted and calculated. For measuring Dil-oxLDL uptake, the cells were incubated with Dil-OxLDL at a concentration of 10 μg/ml at 37 °C for 24 h and then analyzed by flow cytometry.
For intracellular staining of neutral lipid proteins, the collected cells were washed twice with cold PBS before being fixed in 4% paraformaldehyde. After 24 h of incubation at 4 °C, the cells were stained with the 0.5 μM fluorescent neutral lipid dye 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503) (Molecular Probes, Eugene, OR, USA) and incubated for 20 min at 37 °C. After washing with PBS and detaching the cells with 0.5 mM EDTA (Invitrogen), the level of neutral lipids in the cells was quantified by flow cytometry analysis.
Quantitative RT/PCR (qRT-PCR)
Total RNA was isolated with TRIzol reagent (Invitrogen) as described in our previous report . RNA concentrations were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcription was performed in a 20-μl mixture containing 2 μg of total RNA, random hexamers, a mixture containing 10× reverse transcription buffer, dNTPs, magnesium chloride, dithiothreitol, and Moloney murine leukemia virus reverse transcriptase (Invitrogen). Then, 20 ng of cDNA was amplified in a total mixture volume of 20 μl consisting of 1× KAPA SYBR FAST qPCR Master Mix (KAPA Biosystems, Boston, MA, USA) and the appropriate gene-specific primers at a final concentration of 200 nM. The primers used are shown in Supplementary Table 1. The amplification reactions were performed on a LightCycler 480 (Roche). The changes in gene expression induced by the designated stimulation in the presence or absence of inhibitors or siRNA were calculated with the following formula: fold change = 2−Δ (ΔC t), where ΔCt = Ct of target gene − Ct of GAPDH, and Δ(ΔCt) = ΔCt stimulated − ΔCt control.
Enhanced chemiluminescence Western blotting (Amersham, GE Healthcare Life Science, Uppsala, Sweden) was performed as previously described . Briefly, proteins were separated on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. For immunoblotting, the nitrocellulose membrane was incubated with TBS-T containing 5% nonfat milk for 1 h and then incubated overnight with Abs against individual proteins. After washing with TBS-T, the membrane was incubated with secondary Ab conjugated to horseradish peroxidase for 1 h. The membrane was then incubated with the substrate and exposed to X-ray film. After scanning, the intensities of bands in the Western blots were compared using ImageJ software.
The method for determining the expression of cell surface markers has been previously described . The cells were collected and washed twice with cold PBS and then stained for 1 h with immunofluorescence-conjugated Abs against the designated cell surface markers at 4 °C. The cells were then analyzed and quantified using flow cytometry. The data were processed and analyzed with CellQuest software (BD Biosciences).
Preparation of mitochondrial fraction and measurement of mitochondrial ROS
A Mitochondria/Cytosol Fractionation Kit from Abcam (Cambridge, UK) was used to extract mitochondrial and cytosolic fractions. In brief, 2 × 107 cells were resuspended in 0.5 ml of 1X Cytosol Extraction Buffer Mix containing DTT and protease inhibitors. After incubation on ice for 10 min, cells were homogenized in an ice-cold Dounce tissue grinder (80–100 passes with the grinder). The homogenate was centrifuged at 700×g in a microcentrifuge for 10 min at 4 °C and the supernatant was then centrifuged at 10,000×g in a microcentrifuge for 30 min at 4 °C. After this, the supernatant was collected (cytosolic fraction) and the pellet (intact mitochondria) was re-suspended in 50 μl of the Mitochondrial Extraction Buffer Mix containing DTT and protease inhibitors (mitochondrial fraction). For mitochondrial reactive oxygen species (mtROS) measurement, cells were incubated with 5 μM MitoSOX™ red (Invitrogen) in culture medium for 30 min at 37 °C. After washing with PBS, the cells were detached by trypsin treatment and analyzed with flow cytometry .
CCK-8 cell viability measurement assay
A Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories, Kumamoto, Japan) was used to measure cell viability according to our previous report . In brief, BMDMs were seeded in 96-well plates and cultured overnight, and then, the medium was replaced with fresh culture medium containing various inhibitors and incubated for 24 h. CCK-8 reagents were then added and incubated for 2 h at 37 °C. The OD values for each well were read at a wavelength of 450 nm on a microplate reader. The viability of the cells (%) was determined by subtracting the OD of experiments from the OD of the blank group and dividing this value by the OD of the control group.
Immunofluorescence staining and confocal imaging
Cells were collected and washed two times with PBS before being fixed with 4% paraformaldehyde on ice for 20 min. The fixed cells were washed and resuspended homogenously in PBS with 0.05% Triton X-100 on ice for 5 min to permeabilize the cells. The cells were washed again with PBS and blocked with PBS containing 1% mouse FcR Blocking Reagent (Miltenyi Biotech, Bergisch Gladbach, Germany) for 30 min. Primary antibodies were added and incubated with the cells for 2 h at room temperature. After removing unbound antibodies by washing, secondary antibodies conjugated with an Alexa Fluor fluorescent dye were added and incubated in the dark for 1 h at room temperature. Cell nuclei were counterstained with 1 mg/ml DAPI (4′, 6-diamidino-2-phenylindole; Sigma) at a 1:5000 dilution. Finally, 2 × 103 cells were seeded on slides, air dried in the dark, and mounted with mounting reagent (FluorSave™ Reagent, EMD Millipore Calbiochem, San Diego, CA, USA) for subsequent confocal microscopy analysis. Samples were examined with a Leica TCS SP5 confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany) equipped with an HCX PL APO 3×/1.4–0.6 oil objective (Leica). Image processing and colocalization analysis were performed with Leica LAS AF software . In some, cells were fixed after incubation with MitoSOX™ red. Cell nuclei were stained with Hoechst 33528 and analyzed by confocal microscopy.
All determinants were performed in triplicate. The data from pooled donors are expressed as the means ± SEM. Statistical comparisons were performed using Student’s t test or one-way analysis of variance (ANOVA). When ANOVA revealed significant differences between groups, Bonferroni’s post hoc test was used to determine the specific pairs of groups that significantly differed. A P value < 0.05 was considered to indicate statistical significance. Asterisks indicate values that are significantly different from the relevant control (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).