Source and propagation of human glial primary cells and cell lines
Primary human astrocytes were purchased from ScienCell Research Laboratories (Carlsbad, CA). These cells were isolated from human cerebral cortex, characterized by the vendor by immunofluorescence for glial fibrillary acidic protein (GFAP), and cryopreserved at passage one. The immortalized human astrocytic cell line, U87-MG, was obtained from the American Type Culture Collection (ATCC; HTB-14). Cells were maintained in Eagle minimum essential media (EMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin–100 μg/ml streptomycin at 37 °C 5% CO2. A human microglia cell line (hμglia) was a generous gift from Dr. Jonathan Karn (Case Western Reserve University). These cells were derived from primary human cells transformed with lentiviral vectors expressing SV40 T antigen and human telomerase reverse transcriptase. The characterization and classification of this cell line has been previously described [10, 36, 37]. These cells are classified as microglia due to microglia-like morphology, expression of the microglia surface markers CD11B, TGFβR, and P2RY12, and their migratory and phagocytic activity. Cell were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% FBS and 100 U/ml penicillin–100 μg/ml streptomycin at 37 °C 5% CO2.
Murine glial cell isolation and culture
Primary murine glial cells were isolated as described previously by our laboratory [1, 6, 8, 38, 39]. Briefly, six to eight neonatal C57BL/6J mouse brains per preparation were dissected free of meninges and large blood vessels, minced using sterile surgical scissors, incubated with 0.25% trypsin 1 mM EDTA in serum-free RPMI 1640 medium for 5 min, and forced through a wire screen. The cell suspension was pelleted and suspended in RPMI 1640 containing 10% FBS and penicillin-streptomycin mix for 2 weeks. Astrocytes were isolated from mixed glial cultures by trypsinization (0.25% trypsin–1 mM EDTA for 20 min) in the absence of FBS [40]. The remaining intact layer of adherent cells was demonstrated to be > 98% microglia via immuno-histochemical staining for the microglial surface marker CD11b [40]. Isolated astrocytes were determined to be > 96% pure based on morphological characteristics and the expression of the astrocyte marker GFAP as determined by immunofluorescence microscopy [6]. Microglia were maintained in RPMI 1640 with 10% FBS and 20% conditioned medium from LADMAC cells (ATCC number CRL-2420), a murine monocyte-like cell line that secretes colony-stimulating factor-1 (CSF-1), for 24 h prior to experiments. It is important to note that prior to separation of mixed glial cultures, astrocytes produce the CSF-1 necessary to maintain microglial cells. Post separation, microglia were cultured in media containing the 20% conditioned medium from LADMAC cells to provide the necessary CSF-1. Astrocytes were maintained in RPMI 1640 containing 10% FBS for 24 h prior to experiments. All studies were performed in accordance with relevant federal guidelines and institutional policies regarding the use of animals for research purposes.
Bacterial propagation
Neisseria meningitidis strain MC58 (ATCC BAA-335) was grown on Columbia agar plates supplemented with 5% defibrinated sheep blood (BD, Franklin Lakes, NJ) and cultured in Columbia broth (BD Biosciences, San Jose, CA) on an orbital rocker at 37 °C with 5% CO2 overnight prior to in vitro challenge. Streptococcus pneumoniae strain CDC CS109 (ATCC 51915) and Salmonella enterica serovar Typhimurium SB300 (provided by Dr. Michael C. Hudson, formally of the University of North Carolina at Charlotte) were grown from frozen stock on commercially available trypticase soy agar with 5% sheep blood (BD Biosciences). Staphylococcus aureus strain UAMS-1 (ATCC 49230) was grown on lysogeny broth (LB) agar plates. S. pneumoniae, S. aureus, and S. typhimurium were cultured overnight in tryptic soy broth on an orbital rocker at 37 °C with 5% CO2 overnight prior to in vitro challenge. The number of colony forming units (CFU) for each bacterial species was determined by spectrophotometry using a Genespec3 spectrophotometer (MiraiBio Inc., Alameda CA). Bacterial DNA and RNA were isolated using the commercially available kits, GeneElute bacterial genomic DNA, and RNeasy protect bacteria mini kit (Sigma and QIAGEN).
Bacterial infection
Glial cells were infected with bacteria at multiplicities of infection (MOI) of 1, 10, or 50 bacteria to glia in antibiotic-free medium for 2 h at 37 °C with 5% CO2. These doses are based on bacterial numbers previously reported in the cerebral spinal fluid of children with bacterial meningitis [41]. After 2 h of infection, media containing penicillin-streptomycin (MilliporeSigma, St. Louis, MO) was added to kill extracellular bacteria. At the indicated time points following challenge, cell supernatants, whole cell protein lysates, and RNA were isolated for ELISAs, immunoblot analysis, and RT-PCR, respectively.
Nuclear translocation
At the indicated time points, hμglia cells were suspended in a pH 7.9 lysis buffer containing 10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.05% NP40, and protease inhibitor cocktail for 10 min at 4 °C. The nuclei and other fragments were pelleted by centrifugation and supernatants were retained as cytoplasmic fractions. Nuclei were lysed by exposure to pH 7.9 high salt buffer containing 5 mM HEPES, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 26% glycerol, and 300 mM NaCl for 30 min at 4 °C. Samples were cleared of cellular debris by centrifugation, and supernatants containing the nuclear fraction were subjected to immunoblot analysis using a rabbit monoclonal antibody specific for total IRF-3 (Cell signaling).
Immunoblot analysis
Cell lysates were evaluated for the presence of RIG-I, RNA polymerase III subunit A, and phosphorylated IRF3 (pIRF3) by immunoblot analyses [9]. Blots were incubated with a rabbit polyclonal antibody against mouse and human RIG-I (Abgent, cat# AP1900a, 0.5 μg/ml), a rabbit monoclonal antibody specific for RNA polymerase III subunit A (Cell Signaling, cat # 12825S, 1:1000), a rabbit monoclonal antibody specific for IRF-3 phosphorylated at Ser396 (Cell Signaling, cat# 4947, 1:1000), or a rabbit monoclonal antibody for total IRF-3 (Cell Signaling, cat# 4302, 1:1000) overnight at 4 °C. Blots were then washed and incubated in the presence of a horseradish peroxidase (HRP)-conjugated secondary anti-rabbit antibody. Bound antibody was detected with WesternBright ECL kit (Advansta). Immunoblots were reprobed with a mouse monoclonal antibody against β-actin (Abcam, cat# 49900, 0.13 μg/ml) to assess total protein loading. Immunoblots shown are representative of at least three separate experiments and ImageLab software (BioRad) was used for densitometric analysis.
Quantification of cytokines in glial cell supernatants
To quantify human IL-6 and IFN-β production, specific capture ELISAs were performed. A rat anti-human IL-6 capture antibody (BD Pharmingen, cat# 554543; Clone Mq2-13A5, 2 μg/ml) and a biotinylated rat anti-human IL-6 detection antibody (BD Pharmingen, cat# 554546; Clone MQ2-39C3, 2 μg/ml) were used in IL-6 ELISAs. While, a polyclonal rabbit anti-human IFN-β capture antibody (Abcam, cat# ab186669, 0.25 μg/ml) and a biotinylated polyclonal rabbit anti-human IFN-β detection antibody (Abcam, cat# ab84258, 0.25 μg/ml) were used in IFN-β ELISAs. Bound antibody was detected using streptavidin-HRP (BD Biosciences) followed by the addition of tetramethylbenzidine substrate. H2SO4 was used to stop the reaction and absorbance was measured at 450 nm. A standard curve was generated using dilution of recombinant cytokines for IL-6 (BD Pharmingen) and IFN-β (Abcam). The cytokine concentration in cell supernatants was determined by extrapolation of absorbance to the standard curve.
Ligand stimulation
Glial cells were exposed to bacterial lipopolysaccharide (LPS) isolated from Escherichia coli (MilliporeSigma), Pam3Cys-Ser-(Lys)4 (Pam3Cys; InvivoGen, San Diego, CA), bacterial flagellin isolated from Salmonella typhimurium strain 14028 (Enzolife Sciences, Farmingdale, NY), or polyinosinic polycytidylic acid (polyI:C; MilliporeSigma). Additionally, glial cells were transfected with 5′ppp RNA (Invivogen), BDNA (dA:dT) (Invivogen), or RNA/DNA isolated from Neisseria meningitidis strain MC58 (ATCC BAA-335), Streptococcus pneumoniae strain CDC CS109 (ATCC 51915), and Staphylococcus aureus strain UAMS-1 (ATCC 49230) using an RNA isolation kit or genomic DNA isolation kit. Genomic DNA isolation included RNase treatment to remove contaminating RNA. Any potential DNA contamination was removed from isolated bacterial RNA using a DNase I kit (Sigma-Aldrich) and we confirmed that the hμglia human microglial cell line produces significant levels of IFN-β in response to transfection with DNase-treated N. meningitidis or S. aureus RNA (556 pg/ml and 679 pg/ml respectively).
Transfection
Transfection of hμglia cells was conducted using lipofectamine 2000 (L2K, Invitrogen) according to the manufacturer’s guidelines. Ligands were incubated for 30 min with lipofectamine 2000 prior to transfection of hμglia with 0.1 μg/ml BDNA, 1 μg/ml 5′pppRNA, 0.5 μg/ml bacterial gDNA, 1 μg/ml bacterial RNA, or 5 nM nucleic acid nanoparticles for 4 h in DMEM supplement with 5% FBS. Cell culture media was subsequently changed to media additionally supplemented with 100 U/ml penicillin–100 μg/ml streptomycin at 4 h post transfection. Cell lysates and supernatants were collected for analysis at the indicated time points.
BX795 treatment
In some experiments, microglia were untreated or treated with 1 μM BX795 (Invivogen) in DMEM supplemented with 5% FBS and 100 U/ml penicillin–100 μg/ml streptomycin at 37 °C 5% CO2 for 3 h prior to transfection with bacterial nucleic acids or nucleic acid nanoparticles as described above. BX795 blocks the phosphorylation of TANK-binding kinase 1 (TBK1)/IkappaB kinase-ε (IKKε) which inhibits the catalytic activity of these proteins. These signaling molecules are downstream of RIG-I ligand binding and are required for IRF3 phosphorylation and nuclear translocation. Cell lysates and supernatants were collected for analysis at the indicated time points.
siRNA knockdown
Microglia were transfected with 5 nM control siRNA (silencer select negative control number 1 siRNA ThermoFisher Scientific), siRNA targeted against RIG-I (αRIG-I) (ThermoFisher Scientific assay identification number s223615), or siRNA targeting RNA polymerase III subunit A (Thermo Fisher Scientific assay identification number s21945), 48 h prior to transfection with bacterial nucleic acids or nucleic acid nanoparticles as described above. Silencer select siRNA was transfected according to the manufacture’s guidelines using RNAimax (ThermoFisher Scientific). Cells were placed in fresh media for 24 h prior to transfection with bacterial nucleic acids or nucleic acid nanoparticles. Cell lysates and supernatants were collected for analysis at the indicated time points.
Nucleic acid-based nanoparticles assembly
All individual DNA oligonucleotides were purchased from Integrated DNA technologies and dissolved in Hyclone HyPure water cell culture grade (GE Healthcare Life Sciences). RNA strands were synthesized by run-off transcription of PCR-amplified DNA templates carrying the T7 RNA polymerase promoter region and amplified DNA products were subjected to an in vitro transcription with T7 RNA polymerase [42]. Transcribed RNAs were purified by denaturing gel electrophoresis (8% acrylamide, 29:1 acrylamide:bis-acrylamide, 8 M urea) and extracted from excised gel slices using 0.5 ml of a buffer containing 89 mM tris-borate, pH 8.2, 1 mM EDTA, 0.3 M of sodium chloride, with overnight shaking at 4 °C. RNAs were ethanol precipitated (3:1 volume ratio), rinsed twice with cold 90% ethanol, dried, and re-dissolved in molecular grade water. To assemble RNA and DNA triangles, individual strands were mixed at equimolar concentrations (at 1 or 5 μM final) in assembly buffer (89 mM tris-borate pH = 8.2, 2 mM MgCl2, 50 mM KCl), heated to 80 °C for 5 min, and slow cooled to 4 °C over a 1-h period (Supplemental 1). The assembly of triangles was confirmed by 6% native-PAGE with subsequent staining with ethidium bromide and visualization using the BioRad Gel Doc system.
Atomic force microscopy imaging
Thirty microliters of assembled triangles at a concentration of 5 μM were subjected to an 8% non-denaturing PAGE. The gel was run for 50 min at a constant voltage of 90 and triangles were visualized using UV shadowing, then cut, and eluted from the gel using 500 μl of assembly buffer (89 mM tris-borate pH = 8.0, 2 mM MgCl2, 50 mM KCl) overnight. Triangles were then precipitated using three volumes of cold ethanol, washed twice with 80% ethanol, dried, and redissolved in 30 μl of the assembly buffer. A freshly cleaved muscovite mica surface (Tedd Pella, Inc.) was treated with 20 mM NiCl2 for 2 min and washed with 100 μl of ddH2O. Purified triangles (2 nM final) were applied to the mica and incubated for 10 min, washed with 100 μl of ddH2O, and dried under a stream of compressed air. Atomic force microscopy (AFM) imaging of the triangles was performed using a 5500 AFM (Keysight Technologies) in alternate contact mode and the images were recorded with a 2 Hz scanning rate using a PPP-NCHR-50 probe from NanoAndMore USA Corp.
Statistical analysis
Data is presented as the mean ± standard error of the mean (SEM). Statistical analyses were performed using Student’s t test or two-way analysis of variance (ANOVA) with Dunnet’s post hoc test as appropriate using commercially available software (GraphPad Prism, GraphPad Software, La Jolla, CA). In all experiments, results were considered statistically significant when a P value of less than 0.05 was obtained.