Regulatory role of cytosolic phospholipase A2 alpha in the induction of CD40 in microglia
© The Author(s). 2017
Received: 19 October 2016
Accepted: 2 February 2017
Published: 10 February 2017
The aberrant expression of CD40, a co-stimulatory receptor found on the antigen-presenting cells, is involved in the pathogenesis of various degenerative diseases. Our previous study demonstrated that the reduction of cytosolic phospholipase A2 alpha (cPLA2α) protein overexpression and activation in the spinal cord of a mouse model of ALS, hmSOD1 G93A, inhibited CD40 upregulation in microglia. The present study was designed to determine whether cPLA2α has a direct, participatory role in the molecular events leading to CD40 induction.
Cultures of primary mouse microglia or BV-2 microglia cell line exposed to lipopolysaccharide (LPS) or interferon gamma (IFNγ) for different periods of time, in order to study the role of cPLA2α in the events leading to CD40 protein induction.
Addition of LPS or IFNγ caused a significant upregulation of cPLA2α and of CD40, while prevention of cPLA2α upregulation by a specific oligonucleotide antisense (AS) prevented the induction of CD40, suggesting a role of cPLA2α in the induction of CD40. Addition of LPS to microglia caused an immediate activation of cPLA2α detected by its phosphorylated form, while addition of IFNγ induced cPLA2α activation at a later time scale (4 h). The activation of cPLA2α is mediated by ERK activity. Suppression of cPLA2α activity inhibited superoxide production by NOX2-NADPH oxidase and activation of NF-κB detected by the phosphorylation of p65 on serine 536 at 15 min by LPS and at 4 h by IFNγ. Inhibition of NOX2 prevented NF-κB activation and CD40 induction but did not affect cPLA2α activation, suggesting cPLA2α is located upstream to NOX2 and NF-κB. The activation of cPLA2 by LPS was mediated by both adaptor proteins downstream to LPS receptor; TRIF and MyD88, while the activation of cPLA2α by IFNγ was mediated by the secreted TNF-α at 4 h. The early activation of STAT1α (detected by phospho-serine727 and phoshpo-tyrosine701) by IFNγ and the late activation of STAT1α by LPS were not affected in the presence of cPLA2α inhibitors, indicating that STAT1α is not under cPLA2α regulation.
Our results show for the first time that cPLA2 upregulates CD40 protein expression induced by either LPS or IFNγ, and this regulatory effect is mediated via the activation of NOX2-NADPH oxidase and NF-κB. Cumulatively, our results indicate that cPLA2α may serve as a pivotal amplifier of the inflammatory response in the CNS.
KeywordsCytosolic phospholipase A2α CD40 Microglia Lipopolysaccharide Interferon gamma Nuclear factor-κB
The co-stimulatory receptor, CD40 molecule, is a 50-kDa type I member of the tumor necrosis factor receptor superfamily that is widely expressed by the various immune and non-immune cells [1–7]. The interaction between CD40 and its ligand, CD40L (CD154), is one of multiple signals necessary for a productive immune response [8–10]. The CD40-CD154 interaction promotes a wide spectrum of molecular and cellular processes including, immunoglobulin class switching, cell differentiation and maturation, B-cell growth, and expression of other co-stimulatory molecules such as MHC class II, ICAM-1, VCAM-1, E-selectin, LFA-3, B7.1, and B7.2) [11, 12]. In addition, CD40-CD154 interaction induces the production of cytotoxic radicals and of various pro-inflammatory cytokines (TNF-α, IL6, IL-8, and IL-12) and chemokines (CCL-2) [13, 14].
In the central nervous system (CNS), the microglial cells are constantly in motion, surveying their environment to protect the nervous system acting as debris scavengers, killers of pathogens, and regulators of innate and adaptive immune responses. The microglia cells express the key surface molecules for antigen presentation (CD40, MHC-II, and B7); therefore, they are considered the most potent endogenous antigen-presenting cells in the CNS . In a healthy nervous system, microglia constitutively expresses CD40 at a low level, which is enhanced under inflammatory conditions. Several studies show that the aberrant expression of CD40 is involved in the initiation and maintenance of various neurodegenerative diseases including multiple sclerosis, Alzheimer’s disease, HIV-1-associated dementia and cerebral ischemia [16–20], and other diseases as rheumatoid arthritis and atherosclerosis [18, 21, 22]. Blockade of CD40-CD40L signaling has been shown to provide a significant beneficial effect in a number of animal models of neurological human diseases [1, 18, 23–28].
Previous findings suggested that cPLA2α plays an important role in inflammation. cPLA2α specifically hydrolyzes phospholipids containing arachidonic acid at the sn-2 position [29, 30] and is generally thought to be the rate-limiting step in the generation of eicosanoids and platelet activating factor. These lipid mediators play critical roles in the initiation and modulation of inflammation and oxidative stress. cPLA2α is ubiquitous in the brain cells and is essential for their physiological regulation. However, elevated cPLA2α expression and activity were detected in the inflammatory sites in a vast array of inflammatory diseases , including neurodegenerative diseases such as Alzheimer’s disease, multiple sclerosis, and amyotrophic lateral sclerosis (ALS) [32–35]. Our previous study  in a mouse model of ALS, hmSOD1 G93A, demonstrated that the blunting cPLA2α protein expression and inhibition of its activity inhibited microglial-CD40 upregulation. This inhibitory effect could be a result of a direct regulatory role of cPLA2α on CD40 inductive process or an indirect effect due to damping of inflammation. The present study was designed to determine whether cPLA2α has a direct role in the events leading to CD40 protein induction. To this aim, we used mouse microglia cultures and two different stimuli, LPS and IFNγ that have been reported to induce CD40 upregulation. The signal transduction events leading to CD40 upregulation by both stimuli have been studied, and it was reported that they include two transcription factors NF-κB and STAT1α that are activated in different rank order and time scale by the two stimuli [37–39].
Glutamine, penicillin-streptomycin-nystatin, phosphate buffered saline (PBS) Dulbecco’s Modified Eagle’s Medium (DMEM), Hanks’ Balanced Salts Solution (HBSS), fetal bovine serum (FBS), HEPES, sodium pyruvate, Dulbecco’s Modified Eagle’s/F12 (HAM) medium (DMEM/F12) were from Beth Ha-Emek, Biological Industries, Israel.
Sodium azide, trypan blue, p-nitrophenylphosphate, phenylmethylsulfonyl fluoride, leupeptin, benzamidine, aprotinin, DMSO, Tween 20, Tris, 4,6-diamidino-2-phenylindole (DAPI), bovine serum albumin (BSA), Trypsin-EDTA, dihyroethidium (DHE), lipopolysaccharide (LPS), Skim Milk Powder, Poly-L-lysine, horseradish peroxidase (HRP), 1,2-Dioleoyl-sn-glycerol, Triton X-100, β-mercaptoethanol, Percoll, non-essential amino-acids, Diphenyliodonium chloride (DPI) were from Sigma Israel, Rehovot, Israel. Fetal calf serum was from GE Healthcare Life Sciences HyClone Laboratories, Inc., Logan Utah, USA. ECL detection kit for the immunoblot analysis was from PerkinElmer, MA, USA. Pyrrophenone was from Cayman Chemical, Michigan, USA. TNF-α-neutralizing antibody and U0126 (MEK1/2 inhibitor) were from Cell Signaling Technology, Danvers, MA, USA. Interleukin (IL)-4, IL-10, TNF-α, IFN-γ were from PeproTech Asia, NJ, USA.
Primary microglial cell culture
Microglia were isolated from the brains of mice C57BL 1-day-old pups as previously described  with minor modifications. Briefly, the pups were decapitated and the brains were taken out. The tissues were digested by incubation with an enzymatic solution containing papain (116 mM NaCl, 5.4 mM KCl, 26 mM NaHCO3, 1 mM NaH2PO4, 1.5 mM CaCl2, 1 mM MgSO4, 0.5 mM EDTA, 25 mM glucose, 1 mM cysteine, and 20 U/ml papain) for 60 min at 37 °C, 5% CO2. The enzymatic solution was quenched with 20% FBS in HBSS and centrifuged for 4 min at ×200g. A second digestion procedure was performed by treating the brain tissues with 0.5 mg/ml DNase-I (Worthington Biochemical Corp., NJ, USA) for 5 min and gently passing it through a fire-polished Pasteur pipettes several times. Then, the digested tissues were filtered through a 70 micron cell strainer (Corning, NY, USA) and centrifuged at 200g for 4 min. The pellet was resuspended in 20% isotonic percoll in HBSS. Fresh HBSS was carefully added and then the tubes were centrifuged at ×200g for 20 min with slow acceleration and no brakes. The pellet containing the mixed glial cells were washed with HBSS, centrifuged at ×200g for 4 min and then suspended in DMEM-F12 medium (10% FCS, 1% non-essential amino-acids, 11.4 μm β-mercaptoethanol, 10 mM HEPES, 1 mM sodium pyruvate 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 12.5 U/ml nystatin). The cells were seeded into Poly-L-lysine coated flasks and kept at 37 °C in a humidified atmosphere of 5% CO2. The growth medium was replaced with a fresh after 4 days. After two weeks, the microglial cells were separated from the astroglial cell monolayer by shaking the flasks for 1 h at 120 rpm on a rotator shaker and subjected to mild trypsinization with DMEM containing 0.25% Trypsin-EDTA (1:3) for about 90 min at 37 °C and then exchange with fresh DMEM. Then, the isolated microglial cultures were treated with 0.25% Trypsin for approximately 15 min at 37 °C and carefully detached. The cells were suspended with DMEM-F12 (containing 2% FBS 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 12.5 U/ml Nystatin) and cultured (6 × 105 cells/ml) in 24 wells on cover-slips coated with Poly-L-lysine at 37 °C in a humidified atmosphere of 5% CO2 for a week before the experiment. The purity of microglial cell preparations was confirmed by testing their immunoreactivity to the Iba-1 (Wako Chemicals, Richmond, VA, USA) marker.
BV2 immortalized murine microglial cell line was a kind gift from Prof. Rosario Donato (Department of Biochemical Sciences, University of Perugia, Italy). The cells were maintained in DMEM containing 5% FBS 2 mM L-glutamine, 100u/ml penicillin, 100 μg/ml streptomycin, and 12.5 U/ml Nystatin at 37 °C and 5% CO2 until they reached confluence. The cells (3.5 × 105 cells/ml) were suspended in DMEM containing 2% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 12.5 U/ml Nystatin and seeded in plates of 24 or 6 wells at 37 °C in a humidified atmosphere of 5% CO2.
The microglial cells were suspended in PBS and counted by Trypan Blue. The cells were pre-incubated with rat anti-mouse Fc Blocker (BD Pharmingen, San Jose, CA) at 4 °C for 10 min. For detection of CD40, the cells were incubated with PE anti-mouse CD40 (BioLegend, San Diego, CA) for 2 h on ice in the presence of Fc Blocker. Next, the cells were washed three times with PBS and subjected to fluorescence-activated cell sorter (FACS FC 500, Switzerland, Beckman Coulter) analysis. The median (median of fluorescence intensity) was calculated by subtracting the non-specific fluorescence.
Microglia were suspended in DMEM (2% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 12.5 U/ml Nystatin) and seeded on cover slips. The cells were fixed with ice-cold methanol for 3 min and then washed with HBSS. For immunofluorescence detection, the fixed microglial cells were incubated with the first antibody 1:50 in 5% BSA/PBS (anti cPLA2α (Santa Cruz Biotechnology, CA, USA), anti CD40 (Serotec, Cambridge, UK), anti CD206 (R&D Systems, Minneapolis, USA) Serotec, Oxfordshire, UK) for 90 min at room temperature. The cells were washed three times in HBSS and incubated with Cy3 anti-rabbit, DyLight anti-rabbit, and Cy3 anti-goat (1:50 in 5% BSA/PBS; Jackson ImmunoResearch Laboratories, Inc., PA, USA) for 60 min at room temperature. The cells were washed three times in HBSS, and the nuclei were stained with DAPI. Then, final wash was performed and the cells were taken to fluorescence microscope analysis (Olympus, BX60, Hamburg, Germany).
Intracellular superoxide anion assay
O2 − production was measured using dihyroethidium (DHE). The cells were incubated in a 24-well plate on cover slips for 24 h at 37 °C. The next day the medium was replaced with heated HBSS containing 10 μm DHE, and the cells were incubated for 45 min at 37 °C. Then, the cells were stimulated with IFN-γ or LPS for 15 min. Then, the cells were stained with DAPI, washed, and fixed with ice-cold methanol for 3 min. the fluorescence intensity was measured by fluorescence microscope (Olympus, BX60, Hamburg, Germany).
Inhibition of cPLA2α expression using antisense oligonucleotides
An oligodeoxy-nucleotide antisense (tcaaaggtctcattccaca) and its corresponding sense with phosphorothioate modifications on the last three bases at both 5′ and 3′ ends were used as described in our previous article . The specificity to cPLA2α was analyzed by blast search program and was demonstrated in our previous study .
Microglial cell lysates were prepared using lysis buffer containing: 2% Triton X-100, 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 10 μm MgCl2, 10 μg/ml leupeptin, 1 mM phenylmethylsulphonylfluoride, 10 μg/ml aprotonin, 1 mM benzamidine, 20 mM para-nitrophenyl phosphate, 5 mM sodium orthovanadate, 10 mM sodium fluoride, and 50 mM β-glycerophosphate). Cell lysates were analyzed by SDS-PAGE on 9% gels. The amount of protein in each sample was quantified with the Pierce BCA Proteins Assay using BSA standards. The resolved proteins were transferred to nitrocellulose and blocked in 5% BSA in TBS-T (10 mM Tris, 135 mM NaCl, pH 7.4, 0.1% Tween 20). The blots were incubated overnight at 4 °C with primary antibodies (anti-cPLA2α and anti-phospho-(serine-505)-cPLA2α from Sigma, anti-NF-κB p65, anti-phospho-(serine-536)-NF-κB p65, anti phopho-p44/42 ERK1/2 (Thr202/Tyr204), anti-p44/42 ERK1/2, anti-STAT1α, anti-phospho-(serine-727)-STAT1α, anti-phospho-(Thr-701)-STAT1α from Cell Signaling, MA, USA; washed and incubated with peroxidase-conjugated secondary antibodies (Amersham Pharmacia Biotech, NJ, USA) for 1.5 h at room temperature. Detection of immunoreactive bands was carried out using enhanced chemiluminescence. Changes in protein expression or phosphorylation were quantified by densitometry using ImageJ program. The intensity of each band was divided by the intensity of each total protein band and expressed as arbitrary units. The quantitative measurements are adequate to determine the changes of each protein in the same immunoblot.
Separation of plasma membranes and immunoprecipitation
Plasma membranes were separated as described before (). Cell 108/ml suspended in relaxation buffer (100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2, 1.25 mM EGTA, 1 mM ATP, 10 mM PIPES, pH7.4) containing 1 mM PMSF and 100 μm leupeptin at 4 °C and sonicated , resulting in 95% cell breakage. After centrifugation (5 min; ×15,600g) to remove the granules, nuclei, and unbroken cells, the supernatant was centrifuge in a Beckman Airfuge (Beckman Instrument, Fulletron, CA) 30 min; ×134,000g to obtain cell membrane pellet and cytosol supernatant. The membranes were suspended at 109 cell equivalent/ml in 0.34 sucrose/half-strength relaxation buffer. The microglial cell membranes subjected to immunoprecipitation with goat anti-serum raised against recombinant p47phox (gift from Dr. T Leto, NIAID, NIH, Bethesda, USA). Immunoprecipitation was at a final volume of 0.5 ml at 4 °C. Recombinant protein A–Sepharose beads (Zymed Laboratories Inc., CA, USA) were added to each sample, and the samples were tumbled end-over-end for 1 h. The beads were then washed six times with lysis buffer boiled in lamely buffer and subjected to SDS-PAGE analysis.
TNF-a detection–using mouse TNF-α high sensitivity ELISA, eBioscience, Vienna, Austria.
Significant differences between the parameters evaluated were determined by ANOVA using GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA) followed by multiple comparisons Bonferroni post hoc correction. p value less than 0.05 were considered statistically significant.
cPLA2α upregulation regulates the overexpression of CD40 in microglia
The location of cPLA2α in the signal transduction leading to CD40 upregulation by LPS
Next, we determined whether MyD88- or TRIF- pathways in the signal events induced by LPS are capable of activating cPLA2α. We used specific peptide inhibitors for either MyD88 signaling by inhibiting its homodimerization or TRIF signaling by interfering with TLR-TRIF interaction. As shown in the immunoblot, each peptide inhibitor inhibited the activation of cPLA2α detected by its phosphorylated form induced by LPS, to the levels detected in the unstimulated cells (Fig. 3i). Moreover, either of the inhibitors totally prevented the induction of CD40 induced by LPS, suggesting that both pathways participate in cPLA2α activation and in CD40 upregulation (Fig. 3j).
The location of cPLA2α in the signal transduction leading to CD40 upregulation by IFNγ
TNF-α secretion induced by INFγ
IFNγ treated (4 h)
0.06 ± 0.004
2.7 ± 0.05
Cells were treated with 10 ng/ml IFNγ
The present study shows that cPLA2α is involved in the induction of CD40 by either LPS or IFNγ. Reduction of cPLA2α upregulation by a specific antisense or inhibition of cPLA2α activity by a specific inhibitor prevented the induction of CD40 protein expression by either LPS or IFNγ. The results suggest that cPLA2α has a direct role in CD40 upregulation, a feature of the pro-inflammatory M1-phenotype. In accordance with this view, the regulatory role of cPLA2α in the induction of several characters of M1 phenotype in microglia and macrophages, such as iNOS, COX2, NOX2-NADPH oxidase as well as production of eicosanoids and pro-inflammatory mediators, was reported by us and others [40, 44]. cPLA2α, however, is not involved in the transformation to M2-phenotype, as its protein level was not elevated by addition of IL4 + IL10, and the presence of AS did not affect the significant induction of CD206 or of arginase 1 in microglia. In accordance with our results, it was reported that the antiinflammatory cytokines IL4 or IL10 by themselves did not affect cPLA2α activation or biosynthesis [45, 46], further supporting the role of cPLA2α in inflammatory processes.
The results of the present study show that superoxides generated by NOX2-NADPH oxidase participate in upregulation of CD40 expression induced by LPS or IFNγ in microglia, since inhibition of NOX-2 NADPH oxidase prevented the induction of CD40. We show here that in BV-2 microglia cell line, inhibition of the activation of cPLA2α induced by either LPS or IFNγ, as demonstrated by the use of antisense against cPLA2α or the specific inhibitor of cPLA2α activity, pyrrophenone, inhibited the production of superoxides by the NOX2-NADPH oxidase. Inhibition of the oxidase activity did not affect cPLA2α activation detected by its phosphorylated form. These results suggest that the NOX2-NADPH oxidase is regulated by cPLA2α in microglia stimulated with either LPS or IFNγ, that is similar to ours and other studies related to the various phagocytic cells stimulated with a variety of agonists [31, 40, 44, 47–50]. We show here that phoshpo-cPLA2α translocated to the cell membranes of activated microglia, where it binds p47phox subunit of NOX2-NADPH oxidase, in accordance with our previous studies in other phagocytic cells as well as in primary rat microglia [40, 43, 48, 50]. The binding between p-cPLA2α and 47phox was detected at 15 min when the microglia cells were stimulated with LPS and at 4 h when stimulated with IFNγ in correlation with the detection of superoxide production and the kinetic of cPLA2α phosphorylation by the two stimuli. Our previous study  demonstrated that arachidonic acid activated the assembled oxidase in activated cPLA2α-deficient cells, although the precise mechanism is not known. The restoration of CD40 upregulation in the activated cells that were pretreated with AS against cPLA2α by addition of arachidonic acid is probably due to the activation of the NOX2-NADPH oxidase. The activation of cPLA2α at 15 min by LPS and at 4 h by IFNγ was mediated by ERK activation since the presence of MEK inhibitor inhibited cPLA2α activation in accordance with ours and other earlier studies [44, 51].
The involvement of two transcription factors, NF-κB and STAT1α, was reported in the signal transduction pathways leading to induction of CD40 by either LPS or IFNγ [37–39]. While NF-κB was shown to be rapidly activated by LPS, it was activated only at 4 h following exposure to IFNγ. In contrast, STAT1α was rapidly activated by IFNγ and only at 4 h by LPS. Time-dependent activation of cPLA2α detected by its phosphorylation on serine 505 revealed that cPLA2α is rapidly activated by LPS and only considerably later (4 h) by IFNγ, that is in accordance with a previous report . We show in the present study that the kinetic of activation of cPLA2α coincided with the kinetic of NF-kB activation and that the activation of cPLA2α is required for the activation of NF-κB in BV-2 microglia cell line, a finding consonant with our earlier study in microglia activated with amyloid beta . While superoxide production by NOX2-NADPH oxidase is extremely important for killing invading pathogens, it is also an important activator of diverse cell signaling pathways such as mitogen activated protein kinase and NF-κB to regulate the expression of genes encoding a variety of pro-inflammatory factors [40, 52, 53]. The activation of NF-κB by either LPS or IFNγ shown in the present study detected by the phosphorylation of its p-65 subunit on serine 536 is probably mediated by superoxides produced by the NOX2-NADPH oxidase since the inhibition of the oxidase activity prevented NF-kB action. In line with this suggestion, the phosphorylation of p65 NF-kB RelA on Ser-536 is known to be redox-sensitive . The activation of NF-kB by NOX2-NADPH oxidase activity is consistent with our previous studies in microglia and macrophages [40, 55] and with other in various systems and by various agonist [56, 57].
It was reported that the activation of NF-kB under IFNγ stimulation is mediated by an autocrine effect of released TNF-α from the stimulated cells . Consistent with this observation, we show here that the activation of cPLA2α and of NF-kB and the induction of CD40 by IFNγ are mediated by an autocrine effect of TNF-α, since TNF-α secretion from the activated cells was detected and the levels of secreted TNF-α activated cPLA2α and NF-kB. In addition, the presence of antibodies against TNF-α in microglia stimulated with IFNγ of all three processes were inhibited, suggesting that cPLA2α activation by TNF-α regulates the induction of CD40 via NF-kB activation. The activation of cPLA2α by TNF-α coincided with other reports in microglia and macrophages [46, 58]. However, addition TNF-α is not sufficient to induce CD40, although it activates cPLA2α, probably since it stimulates the activation of NF-κB but not the activation of STAT1α that is also required for CD40 induction.
The activation of cPLA2α and NF-κB in the signals leading to CD40 upregulation by LPS is mediated by both MyD88 and TRIF pathways, since inhibition of each pathway inhibited cPLA2α and NF-κB activation and abolished CD40 induction. In accordance with our results, the activation of cPLA2α by MyD88 and by TRIFF adaptive protein was shown in macrophages stimulated by LPS . The activation of NF-κB leading to CD40 upregulation by LPS was suggested to be mediated only by MyD88 adaptive protein in macrophages . However, several studies reported, similar to our results, that both pathways are mediating NF-κB by TLR4 receptor in macrophages and other cell types [59–61].
Antisense oligonucleotide against cPLA2α
Cytosolic phospholipase A2 alpha
Inducible nitric oxide synthase
We thank Dr. Sergio Lamprecht for assistance in editing the English text. This research was supported by a grant from the Israel Sciences Foundation founded by the Israel Academy of Sciences and Humanities 1012/09.
This research was supported by a grant from the Israel Sciences Foundation founded by the Israel Academy of Sciences and Humanities 1012/09 for Prof. R. Levy. She designed the study, directed the study, and wrote the manuscript.
Availability of data and materials
Information about experimental methods and data described in this paper are available to scientific and medical communities for review, verification, and research studies.
YPME designed and carried out all the experiments, researched the data, prepared the figures, and participated in writing the manuscript. NH participated in the design of the study and guided some methodologies. RL designed the study, directed the study, and wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
The study was approved by the Ben-Gurion University Institutional Animal Care and Use Committee (IL-37-05-2012).
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