Background
Microglia are the resident immunocompetent cells of the central nervous system (CNS), responsible for mounting appropriate responses to injuries such as trauma, ischemia, brain tumors and neurodegenerative diseases that target the brain parenchyma [
1,
2]. Moreover, microglia display a wide range of receptors that enable the recognition of pathogens or cell damage-related antigens, thereby promoting phagocytosis and removal of cell debris [
3]. Phagocytosis is a coordinated process, triggered by environmental signals that requires a dynamic actin cytoskeleton rearrangement and a plethora of receptor signaling pathways [
4].
The role of microglia in inflammation has been experimentally dissected using lipopolysaccharide (LPS) stimulation, which mimics Gram-negative infection, through the activation of Toll-like receptor 4 (TLR4). While microglia randomly scan the healthy brain parenchyma, activated cells undergo significant morphological changes and an ensuing targeted movement toward the site of injury, where they release both neurotrophic and neurotoxic factors [
2,
5]. Amongst the inflammatory mediators initially secreted, interleukin-1β (IL-1β) is particularly relevant given its involvement in excitotoxicity, ischemia, brain trauma and cell death [
6‐
8]. Recently, we have described a chemokinetic effect of IL-1β on microglial motility, whereby IL-1β stimulates microglial motility with involvement of p38 MAPK signaling [
9].
Growing evidence supports involvement of neuropeptide Y (NPY) in the modulation of the immune system, with effects on macrophage, B and T cell function; as well as dendritic cell stimulatory ability [
10]. However, its role in phagocytosis remains controversial. Neuropeptide Y (NPY) is widely distributed within the peripheral and central nervous systems and has well defined physiological roles that include regulation of blood pressure, circadian rhythms, feeding behavior, memory processing and learning [
11].
In this context, our objective was to unravel the role of NPY in the modulation of Fc receptor-mediated phagocytosis (the best characterized phagocytic receptor) by activated microglial cells during inflammation. Microglial cells have specific signaling systems that regulate rapid rearrangement of the actin cytoskeleton enabling the cell to phagocytose when needed. Here, we report an inhibitory effect of NPY, acting via Y1 receptors, on IL-1β-stimulated phagocytosis, a process accompanied by p38 MAPK and HSP27 activation. Our results highlight the modulation of phagocytosis as part of the putative anti-inflammatory role of NPY, supporting the importance of this neuropeptide in the regulation of important microglial responses to danger signals in the brain.
Methods
Cell line cultures
A murine N9 microglia cell line (a kind gift from Professor Claudia Verderio, CNR Institute of Neuroscience, Cellular and Molecular Pharmacology, Milan, Italy) was grown in RPMI medium supplemented with 30 mM glucose (Sigma, St. Louis, MO, USA), 100 U/ml penicillin and 100 μg/ml streptomycin (GIBCO, Invitrogen, Barcelona, Spain). Cells were kept at 37°C in a 95% atmospheric air and 5% CO2 humidified atmosphere. Numbers of viable cells were evaluated by counting trypan blue-excluding cells that were then plated at a density of 2 × 104 cells per well in 24-well trays, or plated at a density of 5 × 105 cells per well in 6-well trays (for remaining experiments).
Cell treatment for phagocytosis studies included the following incubation setup: NPY (human, rat/amidated sequence) (1 μM) (Bachem, Bubendorf, Switzerland), LPS (100 ng/ml) (Sigma), ATP (1 mM) (Sigma), IL-1β (1.5 ng/ml) (R&D System, Minneapolis, MN, USA), IL-1ra (150 ng/ml) (R&D Systems), SB239063 (chemically synthesized) (20 μM) (Tocris, Bristol, UK), Y1 receptor agonist [Leu31, Pro34]NPY (porcine, amidated sequence) (1 μM) (Bachem), Y1 receptor antagonist BIBP3226 (1 μM, in water) (Bachem), Y2 receptor antagonist BIIE0246 (1 μM, in 0.06% DMSO) (Tocris) and Y5 receptor antagonist L-152,804 (1 μM, in 0.2% DMSO) (Tocris), for 6 hrs. ATP, SB239063 and all receptor antagonists were added 30 min prior to cell treatment and maintained during the course of experiments.
Generation of an N9 murine microglia cell line stably expressing human FcγRIIA
The N9 microglia cell line expresses a variety of Fc receptors. To overcome this issue, and due to the absence of good antibodies against Fc receptors, we decided to constitutively express human FcγRIIA tagged with c-myc. The protocol for generation of N9 cells expressing FcγRIIA, retrovirus production, cell infection and selection was adopted from [
12,
13]. Briefly, human FcγRIIA tagged with C-terminal myc-His6 was subcloned into the retroviral vector pBABE-puro, which contains a resistance gene to puromycin. The viruses were produced by tranfecting the human Phoenix gag-pol packaging cell line with the retroviral plasmid together with incorporation of the vesicular stomatitis virus G protein. Because of the incorporation of the vesicular stomatitis virus G protein into the virus envelope, these viral particles are able to infect almost any dividing mammalian cell. For infection, 1.5 × 10
5 cells were incubated with 1 ml of retrovirus, pseudotyped with the VSV-G envelope protein, expressing the Fc-receptor in the presence of 4 μg/ml polybrene (Sigma), at 32°C. After 24 hrs of incubation, the medium was changed and the infection procedure repeated. Twenty-four hours later, cells were trypsinized and seeded into a 6-well tray in the presence of 7 μg/ml puromycin (Sigma). Selection was done for another 48 hrs.
Phagocytosis assay
Beads were opsonized with rabbit IgG (1 μg/ml) (Sigma) or with human IgG (0.5 μg/ml) (Sigma, St. Louis, MO, USA) (for the phagocytic cup studies) under constant agitation at 8 rpm, overnight at 4°C. Beads were then resuspended in modified RPMI medium, without NaHCO3, and distributed at a density of 1 × 105 beads per well. After 40 min of incubation (or 20 min, for phagocytic cup studies), cells were washed with PBS and fixed with 4% paraformaldehyde (PFA). Extracellular and/or adherent beads were labeled with secondary antibody Alexa Fluor 594 donkey anti-rabbit or with Cy5 donkey anti-human (Molecular Probes, Oregon, USA), 1: 500, in PBS. For each field, three photomicrographs were acquired in order to capture stained nuclei (in blue), extracellular and/or adherent beads (in red) and total number of beads (differential interference contrast image). The location of each bead was analyzed by comparing the three separate images simultaneously. Only beads without fluorescent labeling were considered as internalized particles. For nuclear labeling, cell preparations were stained with Hoechst 33342 (2 μg/ml) (Molecular Probes) in PBS, for 5 min at room temperature (RT). Coverslips were then mounted in Dako fluorescent medium (Dakocytomation Inc., California, USA). Fluorescent images were acquired using an Axiovert 200 M microscope, equipped with an AxiocamHRm and Plan-ApoChromat 40 ×/1.30 oil objective (Göttingen, Germany).
Immunocytochemistry
Cells were fixed with 4% PFA (Sigma) and unspecific binding was prevented by incubating cells in a 3% BSA and 0.3% Triton X-100 solution (all from Sigma) for 30 min, at RT. Cells were kept overnight at 4°C, in 0.3% BSA and 0.1% Triton X-100 primary antibody solution, then washed with PBS, and incubated for 1 hr at RT with the corresponding secondary antibody.
Antibodies used were: rabbit polyclonal anti-phosphorylated HSP27 (1:400) (Cell Signaling Tech, Beverly, MA, USA); mouse monoclonal anti-c-myc (1:2000) (Cell Signaling Tech,); rat monoclonal anti-CD11b (1:1000) (AbD Serotec, Oxford, UK); Alexa Fluor 594 goat anti-rabbit; Alexa Fluor 488 donkey anti-rabbit; Alexa Fluor 594 donkey anti-mouse; Alexa Fluor 488 goat anti-rat (all 1:200 in PBS, from Molecular Probes).
Membrane ruffling was observed using a marker for filamentous actin, phalloidin. Cells were incubated for 2 hrs in phalloidin-Alexa Fluor 594 conjugate, 1:100 (Molecular Probes) in PBS, at RT, protected from light.
For nuclear labeling, cell preparations were stained with Hoechst 33342 (2 μg/ml) (Molecular Probes, Eugene, Oregon, USA) in PBS, for 5 min at RT and mounted in Dakocytomation fluorescent medium (Dakocytomation Inc., California, USA). Fluorescent images were acquired using a confocal microscope, with a Plan-ApoChromat 63 ×/1.40 oil objective (LSM 510 Meta, Carl Zeiss, Göttingen, Germany).
Western blotting
Cells were incubated with lysis cocktail solution (137 mM NaCl, 20 mM Tris-HCl, 1% Triton X-100, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 1 μg/ml leupeptin, 0.5 mM sodium vanadate (all from Sigma), pH 8.0). After gentle homogenization, the total amount of protein was quantified using the BCA method (Thermo Scientific, Rockford, USA). Afterwards, samples were loaded onto 10% acrylamide/bisacrilamide gels (BioRad, Hercules, CA, USA). Proteins were separated by SDS-PAGE using a bicine/SDS (Sigma) electrophoresis buffer (pH 8.3) and then transferred to PVDF membranes (Millipore) with a 0.45-μm pore size, under the following conditions: 300 mA, 90 min at 4°C in a solution containing 10 mM CAPS (Sigma) and 10% methanol (VWR International S.A.S. France), pH 11.0) (protocol adapted from [
14]). For detection of phosphorylated proteins, membranes were blocked in Tris-buffer saline (TBS) containing 5% BSA, 0.1% Tween
® 20 (Sigma) for 1 hr, at RT, and then incubated overnight at 4°C with the primary antibody diluted in blocking solution.
The following primary antibodies were used: rabbit polyclonal anti-phosphorylated HSP27 (1:1000), rabbit polyclonal anti-HSP27 (1:1000) (both from Cell Signaling) and mouse monoclonal anti-c-myc (1:600) (Santa Cruz Biotechnology Inc.). After rinsing three times with TBS-T, membranes were incubated for 1 hr at RT with an alkaline phosphatase-linked secondary antibody anti-rabbit IgG 1:20,000 or anti-mouse IgG 1:10,000 in blocking solution (GE Healthcare UK Limited, Buckinghamshire, UK). Protein immunoreactive bands were visualized in a Versa-Doc Imaging System (Model 3000, BioRad Laboratories, CA, USA), after incubation of the membranes with enhanced chemofluorescence reagent (GE Healthcare UK Limited) for 5 min.
Data analysis
Statistical analysis was performed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA). Statistical significance was considered relevant for p values < 0.05 using one-way analysis of variance followed by Bonferroni post hoc test for comparison among experimental settings and Dunnett post hoc test for comparison with control condition. Data are presented as mean ± standard error of mean (SEM). For every immunocytochemical analysis, 5 independent microscopy fields were acquired per coverslip (about 50 cells per field). Every experimental condition was tested in three sets of independent experiments (n), unless stated otherwise, and performed in duplicate.
Discussion
Microglial cells play a pivotal role in immunosurveillance in the CNS, acting as resident macrophages in the brain parenchyma [
1,
7,
26,
27]. In recent years, increasing evidence has suggested that NPY is an important regulator of immune system function, with different results obtained concerning the role of this peptide in phagocytosis [
10,
28‐
32]. In our study, we used an experimental model of microglial phagocytic activity induced by lipopolysaccharide (LPS) to evaluate the role of NPY on Fc receptor-mediated phagocytosis. Microglial cells are constantly prowling the brain environment and are able to efficiently identify invading pathogens by expressing a vast array of pattern recognition receptors, such as Toll-like receptors (TLRs) [
1,
2]. Among the different members of the TLR family, TLR4 is the best characterized. TLR4 recognizes LPS, a component of the outer membrane of Gram-negative bacteria [
33]. Importantly, LPS-induced phagocytosis in macrophages requires TLR4 signaling since downregulation of TLR4 expression by RNAi significantly compromises the phagocytic activity of these cells [
34]. Accordingly, we show that LPS significantly enhances microglial Fc receptor-mediated phagocytosis while NPY,
via Y
1 receptor activation, inhibits this effect. Moreover, we observed that LPS-induced phagocytosis of IgG-opsonized beads is a process that occurs with the involvement of IL-1β and downstream activation of the p38 mitogen-activated protein kinase (MAPK) pathway.
In light of our previous results, showing involvement of interleukin-1β (IL-1β) in LPS-stimulated microglial cell activation and motility, we proposed to uncover a role for IL-1β in LPS-stimulated phagocytosis. Accordingly, LPS and ATP co-administration stimulated phagocytosis and this effect was abolished by IL-1ra treatment, suggesting that the effect of LPS is, at least in part, mediated by IL-1β. Interestingly, we would like to emphasize that IL-1β is vital for resolving bacterial infection caused by various pathogens [
35,
36]. Bacterial infection results in an increased release of IL-1β, which enhances phagocytic cell recruitment to infection sites [
36‐
38]. Moreover, during LPS-induced phagocytosis, the production of the endogenous anti-inflammatory cytokine IL-1ra is increased to balance possible cytotoxic effects of IL-1β on neighboring tissues [
39].
In vitro studies have described a role for NPY in the modulation of various functions of macrophages, such as adherence, chemotaxis, phagocytosis and superoxide anion production [
32,
40‐
43]. Furthermore, we have also identified a role for NPY in inhibition of nitric oxide production and motility/migration by microglial cells [
9,
16]. In the present study, we show that NPY inhibits IL-1β-induced phagocytosis
via Y
1 receptor activation. In agreement with our results, NPY has been shown to decrease phagocytosis in older mice. Interestingly, in these animals the release of IL-1β from peritoneal macrophages was higher than that observed in younger animals, and NPY also inhibited this effect [
32]. The role of NPY in regulation of phagocytosis seems to depend also upon the particular pathogen studied and their mechanisms of replication.
In vitro studies have shown that NPY inhibits engulfment of
Leishmania major by a monocyte/macrophage murine cell line [
43]. Since infection of phagocytes is a crucial step for replication of
Leishmania major, inhibiting phagocytosis results in a protective action [
44]. Moreover, the effect of NPY may vary according to other parameters (e. g. concentration, target cell, stimulus) and depends on interactions between different cell types [
30‐
32,
45,
46]. Recognition of pathogen-associated molecular patterns triggers TLR signaling through myeloid differentiation primary response gene 88 (MyD88) adapter molecule and activation of mitogen-activated protein kinases (MAPKs) and NF-κB [
47]. Furthermore, p38 MAPK signaling has been implicated in phagocytosis carried out by murine macrophages [
48] and Drosophila hemocytes [
49]. Since cytoskeleton remodeling is vital for cell phagocytosis, p38 could be a putative molecular target to discern which signaling pathways are involved in this process. There are several reports implicating p38 activation in phagocytosis, either as a consequence of phagocytosis or as a necessary step to initiate this process [
48‐
50]. In fact, Blander and colleagues have shown that the use of selective p38 inhibitors impairs the ability of macrophages to phagocyte
E. coli [
51]. Moreover, upon activation, phosphorylated p38 translocates to the nucleus and phosphorylates MAPK-activated protein kinase 2 (MK2) [
52]. Cells deficient in MK2 are unable to regulate actin reorganization and, therefore, to form membrane protrusions [
53]. MK2 modulation of phagocytosis may occur through the small heat shock protein HSP25/27, since it regulates actin polymerization [
54].
Phagocytosis can be triggered by different signals that are recognized by different receptors, triggering different signaling cascades. In our study, we focused on the role of NPY on Fc receptor-mediated phagocytosis, the main phagocytic process occurring in macrophages, a cell type that shares close functional and morphological resemblances to microglia. In fact, NPY reduced expression of Fc receptor on the cell surface of stimulated microglia, thereby restraining the phagocytic capacity of these cells under inflammatory challenge. In this regard, NPY may modulate the phagocytic process in order to maintain microglial activity close to a physiological resting/surveying state, even in the presence of a stimulatory/activating agent.
However, we should emphasize that our findings are limited to the modulation of Fc receptor-mediated phagocytosis. Nevertheless, our data further highlight the putative anti-inflammatory and anti-phagocytic role of NPY, and broaden our knowledge of the therapeutic value of this neuropeptide in the treatment of CNS disorders.
Acknowledgements
The authors wish to thank Prof. Claudia Verderio, from National Research Council, Institute of Neuroscience and Department of Medical Pharmacology, Milan, Italy, for her generous gift of murine N9 microglial cell line, Prof. Paulo Santos from Center for Neuroscience and Cell Biology, Department of Zoology, University of Coimbra, Portugal, and Carla Cardoso from Crioestaminal, Biocant, Cantanhede, Portugal, for her kind and constructive remarks. Myc-tag antibody was kindly provided by Dr. Ana Luísa Carvalho from Center for Neuroscience and Cell Biology, Portugal. Work was supported by FCT Portugal and FEDER, PTDC/BIA-BCM/112138/2009, POCTI/SAU-NEU/68465/2006, PTDC/SAU-NEU/104415/2008, PTDC/SAU-OSN/101469/2008, PTDC/SAU-NEU/101783/2008 and SFRH/BD/23595/2005.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
RF carried the phagocytosis assays, western blotting and immunocytochemistry studies, performed the statistical analysis and wrote the manuscript. TS participated in the phagocytosis assays and western blotting studies. MV participated in the phagocytic cup assays. LC participated in the acquisition of confocal microscopy images. LB participated in the design and coordination of the study and provided financial support. JM and OV conceived the study, participated in its design, provided financial support and coordinated the project. All authors read and approved the manuscript.