Background
Growing evidence supports that neuroinflammation is involved in pathogenesis of a variety of neurological disorders, including Alzheimer’s disease, Parkinson’s disease, and psychiatric diseases [
1,
2]. Therefore, dampening neuroinflammation has been considered one of the leading therapeutic strategies for such diseases. Microglia, the resident immune cells of the brain parenchyma, play critical roles in neuroinflammation [
3,
4]. At quiescent state, microglia display characteristically ramified morphology with numerous branching processes, which dynamically survey the brain microenvironment [
5]. In response to injury, microglia become activated with amoeboid morphology by retracting their ramified processes and produce inflammatory cytokines such as tumor necrosis factor α (TNFα), interleukin (IL) 6, and IL1β [
6]. However, the molecular mechanisms underlying microglial activation have not been fully clarified and need to be further explored.
P2X7 is an ATP-gated, nonselective cation channel allowing Ca
2+ and Na
+ influx and K
+ efflux [
7]. In the periphery, P2X7 is abundant in hematopoietic lineage cells, including mast cells, B and T lymphocytes, monocytes, and macrophages [
8]. In the brain, P2X7 is expressed abundantly in microglia, while its expression and function in neurons and astrocytes is debatable [
9‐
14]. Additionally, there is a conflict with the expression of P2X7 between quiescent and activated microglia. Choi et al. reported that exposure of cultured human microglia to lipopolysaccharide (LPS) increases the expression of P2X7 in a time-dependent manner [
15], whereas some microglial cell lines treated with LPS showed a decreased P2X7 level [
16]. Therefore, the detailed mechanism for regulating P2X7 expression in mouse primary microglia under pro-inflammatory and anti-inflammatory conditions remains uncertain.
Functionally, P2X7 is best known for promoting NLRP3 inflammasome assembly and caspase-1-dependent release of proinflammatory IL1β and IL18 from innate immune cells after exposure to LPS and ATP [
17‐
19]. In activated macrophages, beyond NLRP3-inflammasome induced IL1β and IL18 release, P2X7 also controls the release of other proteins, including TNFα and chemokine (C-C motif) ligand 2 (CCL2) [
20], which suggests that P2X7 is coupled to a secretome in macrophages. However, whether or not this is also the case in activated microglia has not been well characterized yet.
Another hallmark of P2X7 activation is the formation of plasma membrane pores permeable to molecules up to a molecular weight of 900 Da with prolonged exposure to ATP, which leads to dramatic elevation of intracellular Ca
2+, depletion of intracellular ions and metabolites, and ultimately cell death [
21]. P2X7-mediated cell death has been reported in several types of cells, such as macrophages [
22], leukemic cells [
23], and rat microglial cell line N9 and N13 [
24]. In contrast, recently, it has been shown that P2X7 plays a trophic role in supporting cell growth and proliferation. Overexpression of P2X7 evokes both microglia activation and proliferation [
25]. P2X7 is also required for embryonic microglial proliferation, because absence of P2X7 leads to a decreased microglia density [
26]. This contradiction highlights the need to systemically investigate P2X7 functions in primary microglia.
To this end, we examined the expression and modulation of P2X7 in mouse and human microglia by RNA-sequencing (RNA-seq) and quantitative real-time PCR. Using genetic and pharmacological approaches, we also systemically investigated the role of P2X7 in microglial survival and activation, as well as the underlying signaling cascades. The results would extend our understanding of P2X7 in microglia and provide new insights in the mechanisms of P2X7 in neuroinflammation.
Methods
Animals
Wild-type (WT) C57BL/6 pregnant mice were obtained from Charles River Laboratories, Inc. P2X7−/− mice used in this study were derived from Pfizer. P2X7−/− pregnant mice and their background and age-matched WT pregnant mice were received from The Jackson Laboratory. Mice were allowed to acclimate for 7 days after receipt. They were kept on a 12-h light/dark cycle and allowed free access to food and water. All animal care and use complied with the Guide for the Care and Use of Laboratory.
Reagents
3′-O-(4-Benzoyl) benzoyl adenosine 5′-triphosphate (BzATP) and LPS were purchased from Sigma-Aldrich. Interferon γ (IFNγ), TNFα, IL6, and IL1β were purchased from Biolegend. P2X7 antagonist A-804598, ERK inhibitor U0126, and AKT inhibitor LY294002 were obtained from Tocris.
Cell culture
Cortices from P0-2 C57BL/6 mouse pups were dissected and stripped of meninges and mechanically dissociated with a hand homogenizer and a 25-gauge needle. The cell suspension was seeded into poly-l-lysine-coated (Sigma-Aldrich) T150 tissue culture flasks and maintained in DMEM/F12 with 10% FBS and 1% penicillin-streptomycin for 10–14 days to grow a confluent mixed astrocyte/microglia population. We collected and applied the cells to an antigen-antibody-mediated magnetic cell-sorting (MACS, Miltenyi Biotech) assay to positively select microglia. The mixed glial population was re-suspended in MACS buffer (Miltenyi Biotech) and incubated with CD11b MicroBeads (Miltenyi Biotech). The cell suspension was then applied to LS separation column (Miltenyi Biotech) fitted into a QuadroMACS cell separator (Miltenyi Biotech). Unlabeled cells were allowed to pass through the column while labeled cells remained captured in the magnetic field. After washing the column with MACS buffer, the column was then removed from the magnetic separator and flushed with MACS buffer to collect the purified microglia population. For an increased level of purity, the eluted microglia population was passed through a new LS separation column a second time. The purity of microglia used in our study was more than 95% assessed by immunocytochemistry (data not shown).
Human primary microglia (Catalog #1900) and astrocytes (Catalog #1800) were obtained from ScienCell Research Laboratories, Inc., and cultured as instructed.
Microglia or brain tissues were homogenized, and total RNA was extracted using RNeasy plus mini kit (Qiagen). Total RNA concentrations were measured using NanoDrop ND-1000 spectrophotometer. For RNA-seq, RNA quality was assessed by using Agilent RNA 6000 Nano Kit and Agilent 2100 Bioanalyzer according to the manufacturer’s instructions before sequencing by BGI, a fee-for-service provider. For other experiments, RNA was reverse-transcribed into cDNA using Superscript III reverse transcriptase (Invitrogen) with random hexamer primers. Transcript abundance was determined by quantitative PCR using SYBR Green PCR mix (Applied Biosystems), with primer pairs against
P2rx7 and
Gapdh. Three
P2rx7 spliced variants were amplified by PCR with corresponding primers, and the PCR products were separated by electrophoresis on a 1.5% agarose gel. The following primer pairs were used for quantitative real-time PCR:
Primers for reverse transcription PCR:
-
P2rx7a: 5′ TCAGTAGGGATACTTGAAGCC 3′ (R)
-
P2rx7b: 5′ TCTGTGAGAAACAAGTATCTAGGTTGG 3′ (R)
-
P2rx7c: 5′ TCAGGTGCGCATACATACATG 3′ (R)
-
Gapdh: 5′ TCCACCCATGGCAAATTCCATG 3′ (F) and 5′ TGGACTCCACGACGTACTCAGC 3′ (R)
Forward primer shared by P2rx7 variants is 5′ TGCTCTTCTGACCGGCGTTG 3′ (F)
Immunocytochemistry and immunohistochemstry
Immunocytochemistry was performed as described previously [
27]. Briefly, cells were fixed with 4% paraformaldehyde and permeabilized by 0.1% Triton X-100. After blocking with 10% donkey serum, fixed cells were incubated with primary antibodies (Iba1, 1:1,000, WAKO Chemicals; GFAP, 1:1,000, Abcam) for 2 h followed by fluorochrome-conjugated secondary antibodies (Alexa Fluor 488 and 555, 1:200, Molecular Probes, respectively). Nuclei were counterstained with DAPI. Fluorescence images were acquired using a confocal-laser microscope (LSM 700; Carl Zeiss MicroImaging) with a multi-track configuration.
For immunohistochemistry, WT and P2X7−/− aged matched mice were perfused. Brains were dissected out, cryo-protected, and cut. Brain sections were stained with primary antibodies (P2X7, 1:500, Sigma; Iba1, 1:500, Abcam; GFAP, 1:500, Abcam) for 48 h at 4 °C followed by fluorochrome-conjugated secondary antibodies (Alexa Fluor 488, 647, and Cy3, 1:500, Jackson Laboratory, respectively). Nuclei were counterstained with Hoechst. Images were acquired using a confocal-laser microscope (LSM 700; Carl Zeiss MicroImaging) and displayed with maximum projection of z-stacks.
Enzyme-linked immunosorbent assay (ELISA) and secretome analysis
ELISA kits for mouse IL1β and IL18 (R&D systems) were used for quantification of IL1β and IL18 in cell culture supernatants following the manufacturer’s instruction.
The relative concentrations of secreted molecules in cell supernatants were measured using antibody-based 38-plex immunoassays (Luminex, R&D systems). The 38 secreted proteins we measured were as follows: CCL2/JE/MCP1, CCL3/MIP1α, CCL4/MIP1β, CCL5/RANTES, CCL20/MIP3α, CXCL1/KC, CXCL2/MIP2, CXCL10/IP10/CRG2, CXCL12/SDF1α, FGFb, FGF21, GCSF, GMCSF, IFNγ, IGFI, IL1α, IL1β, IL2, IL4, IL5, IL6, IL10, IL12 p70, IL13, IL17A, IL23 p19, IL33, LIX, MCSF, MMP9, Resistin, TNFα, VEGF, CCL11/Eotaxin, CCL22/MDC, CXCL9/MIG, IL9, and RAGE. To generate proteomic heat maps, we normalized immunoassay measurements of the listed proteins and clustered them using an unsupervised clustering algorithm (Array Studio). Any undetectable proteins for a sample were removed from the analysis.
Cytotoxicity assay
Cell viability was determined by cell counting kit-8 (CCK-8, Dojindo), which measures mitochondrial dehydrogenase activity inside the cells. Briefly, 10 μl of CCK-8 solution was added to 100 μl of media in each well of the plate. After incubating the plate for 2–4 h at 37 °C, the absorbance at 450 nm was measured using the Bio-Rad microplate reader.
Western blots
Cells were homogenized and lysed using RIPA buffer (Amresco) with protease and phosphatase inhibitors (Sigma and Roche, respectively). After centrifugation at 13,000g, protein concentrations were measured using the BCA protein assay kit (Pierce) and lysates were separated on a 4–12% Bis-Tris gels (Invitrogen) using MOPS sodium dodecyl sulfate running buffer (Invitrogen). Proteins were transferred with the iBlot system onto nitrocellulose membranes (Novex) and incubated with antibodies p-AKT (1:1000, Cell Signaling Technology), p-ERK (1:1000, Cell Signaling Technology), AKT (1:1000, Cell Signaling Technology), ERK (1:1000, Cell Signaling Technology), and GAPDH (1:1000, Millipore). Signal intensities were detected using ECL western blotting detection reagents (Amersham Biosciences) and evaluated by ImageJ.
Statistical analysis
Data were statistically compared using one-way or two-way ANOVA followed by Tukey’s or Dunnett’s post hoc test among multiple groups using GraphPad Prism 6 (GraphPad Software, Inc.). P < 0.05 was considered statistically significant.
Discussion
In the current study, both genetic and pharmacological approaches demonstrate that P2X7 mediates BzATP-induced cell death and secretion of IL1 family cytokines in mouse microglia. First, we showed constitutive expression of P2X7 in mouse and human microglia. The expression of P2X7 was decreased in microglia following LPS priming at all tested time points; in the presence of LPS and IFNγ, we observed a transient increase followed by a decrease of P2rx7 mRNA level. Moreover, BzATP stimulation led to cell death and robust release of IL1β and IL18 in WT microglia, while this effect was inhibited in P2X7−/− microglia. To validate that P2X7 mediates microglial cell death and cytokine release, the highly selective P2X7 antagonist A-804598 was used to block BzATP-induced cell death and secretion of IL1β and IL1α in microglia. Last, we verified P2X7 activation is linked to AKT and ERK pathways which contribute to cell death but not the production of IL1 family cytokines via P2X7 in microglia.
P2X7 was expressed in microglia (Fig.
1), and its mRNA level was modestly regulated in inflammatory microglia (Fig.
2). However, it is the functional changes of P2X7 activation that plays a critical role during neuroinflammation. Under normal physiology, P2X7 is not functionally engaged since it takes high micromolar concentrations of ATP to activate P2X7, and extracellular ATP is rapidly degraded [
7]. On the other hand, during neuroinflammation under cellular stress and necrosis, intracellular ATP (often millimolar) is dumped in the extracellular space that is sufficient to activate P2X7. Thus, functional consequences of P2X7 activation more than expression changes are important pathological drives of disease process.
P2X7 is expressed predominantly in cells of hematopoietic lineage, and its function is mixed by the presence of N- and C-terminal splice variants. There are several strains of P2X7
−/− mice generated by inserting a lacZ and a neomycin cassette into exon 1 (Glaxo) [
31], or by inserting a neomycin cassette into exon 13 (Pfizer) [
18,
32], or by knocking-in of human
P2rx7 cDNA to the mouse
P2rx7 locus [
14], or by inserting shRNA vectors [
33]. In the current study, we used microglia from Pfizer P2X7
−/− mice, which showed low levels of C-terminal truncated splice variants as detected by reverse transcription PCR using primers specific for various
P2rx7 transcripts (Fig.
3a). This finding is reminiscent of Masin’s report [
28]. The reduced function of C-terminal truncated splice variants in P2X7
−/− mouse may partly explain the difference in cytotoxicity and cytokine secretion profiles between P2X7
−/− microglia and microglia exposed to P2X7 antagonist A-804598.
P2X7 is mainly responsible for ATP-induced cell death not only in immune cells such as macrophage [
22], dendritic cells [
34], mast cells, and lymphocytes [
23] but also in cancer cells [
35] and neural stem cells [
36]. Our data are in line with these reports and expand the role of P2X7 in microglial cell death induced by BzATP, a more potent agonist for P2X7 than ATP (Figs.
4 and
6). In addition to quiescent microglia, BzATP also caused cell death in LPS-primed microglia, suggesting that P2X7 in activated microglia is still sufficient to function upon BzATP. Compared with knockout of P2X7, A-804598 did not completely abrogate BzATP-induced cell death. This may be explained by the reversibility of A-804598 binding to P2X7 [
37]. Moreover, P2X7 is known to mediate cell death through either apoptosis [
34] or necrosis [
23]. Nevertheless, whether apoptosis or necrosis is the predominant mechanism for P2X7-dependent microglial cell death remains to be determined.
Secretion of IL1 cytokine family needs two steps which includes activation of both toll-like receptors and inflammasome. P2X7 is one of the most potent activators of NLRP3 and makes NLRP3 inflammasome sensitive to extracellular ATP. In our study, we confirmed P2X7-dependent release of IL1β and IL18 in pro-inflammatory microglia responding to BzATP (Fig.
5). Furthermore, our 38-cytokine multiplex results exhibited that only IL1β and IL1α were suppressed in pro-inflammatory microglia with BzATP stimulation when exposed to A-804598 (Fig.
7), which supports the major player of P2X7 in the production of IL1 family cytokines. Interestingly, we found that LPS alone induced release of IL1β in WT microglia which was not P2X7 dependent as a similar release was observed in the P2X7
−/− microglia (Fig.
5a, b). Contrary to this, IL18 release was entirely dependent of P2X7 activation because knockout of P2X7 completely blocked LPS plus BzATP-induced IL18 release (Fig.
5c, d). More intriguingly, we observed that LPS plus IFNγ-primed WT cells did not elicit IL1β or IL18 release as high as LPS alone-primed WT cells (Fig.
5), indicating that the P2X7 signaling arm may be modified by priming with different cytokine stimuli.
AKT and ERK pathways are involved in a variety of biological events, such as cell differentiation, cell survival, cell cycle, and protein synthesis. P2X7 activation induces AKT phosphorylation in rat cortical astrocytes [
29], while ERK cascade has been identified as a key signaling pathway for P2X7-induced death of renal fibroblasts [
30]. In contrast, we found de-phosphorylation of ERK and AKT upon P2X7 activation in response to BzATP in microglia (Fig.
8), which may be due to the context-dependent manner of these pathways such as in different cell types [
38]. Furthermore, we verified that the ERK and AKT pathways mediated BzATP-induced microglia cell death but not IL1β release, confirming that P2X7 is coupled to AKT and ERK activation.
Primary cultures provide sound in vitro models for studying molecular mechanism and directly testing the effect of compounds on microglial activation and function. However, as any in vitro models, culture conditions may not reflect exactly the in vivo environment, which possibly alter astrocyte and microglial gene expression as compared them to their naïve counterparts in the brain [
39]. These changes are most likely attributed to the exposure to serum with different components and concentrations from those in the brain, the absence of neurons and other cell types in culture, as well as the altered cell type ratios from which they are exposed in vivo. Therefore, gene expression and phenotypes of microglia and astrocytes from in vitro studies should be carefully considered when translated to an in vivo setting. As such, further studies for P2X7 function in microglia in vivo are critical for the development of effective therapies for neurological diseases.