Journal of Neuroscience Research 93:1345–1352 (2015)
Dual Polarization of Microglia Isolated from Mixed Glial Cell Cultures Lili Ju,1 Hui Zeng,2,3 Yun Chen,1 Yanhong Wu,1 Beibei Wang,2,3* and Qunyuan Xu1* 1
Department of Neurobiology, Beijing Institute for Brain Disorders, Beijing Center of Neural Regeneration and Repair, Key Laboratory for Neurodegenerative Diseases of the Ministry of Education, Capital Medical University, Beijing, China 2 Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing, China 3 Beijing Key Laboratory of Emerging Infectious Diseases, Beijing, China
Microglia are versatile immune effector cells of the CNS and are sensitive to various stimuli. The different methods used to isolate microglia may affect some of their characteristics, such as their polarization state. The influence of cell sorting methods on the polarization state of microglia has never been studied. Mixed glial culture system (MGCS) and magnetic activated cell sorting (MACS) are two methods that are commonly used to purify microglia. This study compares the immunological states between microglia isolated by MGCS and microglia isolated by MACS. We show that microglia isolated by MGCS exhibit a stronger immune-activated state than microglia isolated by MACS. They present an elevated phagocytic ability and high levels of markers associated with classical activation (M1) and alternative activation (M2). In addition, high levels of M1-type and M2-type chemokine (C-C motif) ligand 2 and transforming growth factor-b1 were detected in the culture medium of mixed glial cells. Our results show that microglia isolated by MGCS are in an immune-activated state, whereas microglia isolated by MACS appear to be closer to their primary in vivo state. Therefore, the immune status of microglia, depending on the protocol used to purify them, should be carefully considered in neuropathology research. VC 2015 Wiley Periodicals, Inc. Key words: microglia; central nervous system; immune effector cells
Microglia are the resident macrophages of the CNS (Batchelor et al., 1999; Block et al., 2007) and account for approximately 12% of the total cellular population (Block et al., 2007) and 5–20% of total glial cells in the mammalian brain (Garden and Moller, 2006). They play a pivotal role in maintaining homeostasis in the CNS, including maturation, synaptic plasticity, clearance of debris, host defense, wound healing, and immune regulation (Remington et al., 2007). Because they are involved in nearly all pathological processes, microglia are attractive targets for the treatment of diseases of the CNS, such as multiple sclerosis, neurodegenerative disorders, demyelination, and spinal cord injury (McGeer et al., 1988; Colton et al., 2006; Garden and Moller, 2006; Remington et al., 2007). C 2015 Wiley Periodicals, Inc. V
Given the different adherence properties of glial cells, Giulian and Baker (1986) and McCarthy and de Vellis (1980) developed an in vitro mechanical shake-off method to isolate microglia from the mixed glial culture system (MGCS). Recently, magnetic activated cell sorting (MACS), a new, convenient and efficient method, has been described (de Haas et al., 2007; Marek et al., 2008). Because microglia are versatile immune effector cells, various stimuli can convert them from a quiescent state to an active state and induce variations in cell migration, cell proliferation, scavenging of debris or pathogens, antigen presentation, and secretion of biological factors (Gehrmann et al., 1995; Ransohoff and Cardona, 2010). Therefore, we suggest that the methods used to purify microglia can influence and alter their physiological states. Which of these physiological characteristics are affected by the purification process? Do the purification methods MGCS and MACS influence the physiological state of microglia in a different manner? These important questions must be answered to investigate microglial functions in the fields of neuroscience and immunology further. Peripheral macrophages exhibit two functionally different activation states: classically activated (M1) proinflammatory macrophages are characterized by strong antigen-presentation abilities and by the production of Contract grant sponsor: Beijing Nova Program; Contract grant number: 2009B05; Contract grant sponsor: Chinese National Key Developing Project of Basic Research (973); Contract grant number: 2007CB947704 *Correspondence to: Qunyuan Xu, Department of Neurobiology, Beijing Institute for Brain Disorders, Beijing Center of Neural Regeneration and Repair, Key Laboratory for Neurodegenerative Diseases of the Ministry of Education, Capital Medical University, Beijing 100069, China. E-mail: [email protected], or Beibei Wang, Beijing Key Laboratory of Emerging Infectious Diseases, Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing 100015, China. E-mail: [email protected] Q. Xu and B. Wang contributed equally to this work. Received 3 December 2014; Revised 2 January 2015; Accepted 5 January 2015 Published online 6 June 2015 in Wiley Online (wileyonlinelibrary.com). DOI: 10.1002/jnr.23563
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proinflammatory cytokines, whereas alternatively activated (M2) macrophages are characterized by secretion of anti-inflammatory cytokines (Mosser and Edwards, 2008; David and Kroner, 2011). Accordingly, as the counterpart to the peripheral macrophages, microglia can be classified into two activation states. Changes in physiological conditions can stimulate the conversion of microglia from one state to the other (Colton et al., 2006; Czeh et al., 2011; Selenica et al., 2013). Thus, a full characterization of the specific cellular changes induced by the purification processes is required to choose the appropriate approach for microglial purification. The present study shows that microglia isolated by MGCS have the morphological, phenotypical, and molecular characteristics of dual polarization, whereas most microglia isolated by MACS are in a quiescent state. MATERIALS AND METHODS Preparation of Brain Tissues Forty-eight-hour-old mice (C57BL/6) were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (Beijing, China), and handled according to the guidelines of the laboratory animal facility of Beijing as approved by the local animal experimental committee. Brains were isolated under sterile conditions, stripped of meninges and blood vessels in ice-cold phosphate-buffered saline (PBS), mechanically dissociated, and then passed through a 40mm cell strainer (BD Biosciences, San Jose, CA). The homogenate was centrifuged at 1,000 rpm for 10 min at 4 C, and the supernatant, containing debris, was discarded. The cell pellet was resuspended with PBS to obtain single-cell suspension for further processing. This study was carried out in strict accordance with the Guide for the care and use of laboratory animals of the National Institutes of Health. The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Capital Medical University. Purification of Microglia By MGCS Microglia were isolated and purified by MGCS as described elsewhere (McCarthy and de Vellis, 1980; Giulian and Baker, 1986). The whole-brain suspension was plated in a 75-cm2 flask coated with 0.01% poly-L-lysine and cultured in 10 ml Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. The medium was refreshed on day 3. On day 14 or 15, the mixed glial culture was stratified and shaken at 250 rpm for 3 hr at 37 C in a Forma orbital shaker (Thermo Fisher Scientific, Waltham, MA). Finally, the suspended cells were collected and filtered through a cell strainer. Purification of Microglia By MACS MACS was performed with CD11b MicroBeads (Miltenyi Biotec, Cologne, Germany) as described elsewhere, with minor modifications (de Haas et al., 2007; Marek et al., 2008). Briefly, cells were resuspended at a density of up to 1 3 107 cells/90 ml in a separation buffer (PBS containing 0.5% BSA and 2 mM EDTA, pH 7.2) and incubated with 10 ml of mono-
clonal CD11b MicroBeads for 15 min at 4 C. After having been washed, centrifuged, and resuspended in 3 ml of separation buffer, the cells were applied to the LS column, which was placed in a magnetic field, and the labeled cells were captured in the column. The column was washed four times with 3 ml separation buffer (one more time than in the manufacturer’s instructions for a better removal of negative components, such as myelin debris). The column was removed from the magnetic field. Cells were flushed out with 5 ml buffer and collected. To improve the purity, the collected cells were subjected to a second CD11b-positive selection with the LS column. Wright-Giemsa Staining The cells were placed onto glass slides by cytospinning at 750 rpm for 5 min at room temperature, air dried, and stained with Wright-Giemsa staining kits (Baso Diagnostics, Zhuhai, China) according to the manufacturer’s instructions. Immunofluorescence Staining Cells were plated at a density of 105 cells/ml on slides coated with 0.01% poly-L-lysine and cultured for 48 hr. They were rinsed three times with PBS, fixed with 4% paraformaldehyde for 20 min at room temperature, treated with 0.3% Triton X-100 (v/v) and 10% heat-inactivated FBS (1 hr at room temperature), and incubated overnight at 4 C with monoclonal goat anti-Iba1 (Abcam, Cambridge, United Kingdom). Cells were then washed three times with PBS and incubated with rabbit anti-goat IgG conjugated with Alexa Fluor 488 (Invitrogen Life Technologies, Carlsbad, CA) for 30 min at 37 C. Again, cells were washed three times with PBS and counterstained with Hochest 33258 (Sigma-Aldrich, St. Louis, MO) for 10 min at room temperature for nucleus visualization. Images were captured from 10 random fields in each sample with a fluorescence microscope (Olympus, Tokyo, Japan). Cells incubated without primary antibodies served as negative controls. Flow Cytometry The following monoclonal antibodies were used for fluorescence-activated cell sorting (FACS) staining: fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD11b and Ly6C, phycoerythrin (PE)-conjugated anti-mouse CD11b and CD45, peridinin-chlorophyll protein (PerCP)-Cy5.5-conjugated anti-mouse major histocompatibility complex (MHC)-II, allophycocyanin (APC)-conjugated anti-mouse CD80 and CD86 (BD Biosciences), and biotin-conjugated anti-CD204 antibody (AbD Serotec, Kidlington, United Kingdom). Suitable isotype controls were also purchased from BD Pharmingen (San Diego, CA). Purified microglia were incubated with these antibodies for 15 min at 4 C. For CD204 staining, the cell suspension was further incubated with APC-conjugated antibiotin antibody (Miltenyi Biotec) for 15 min at 4 C. 7-Aminoactinomycin D was used to discriminate dead cells. Data were acquired with a FACSCalibur flow cytometer (BD Biosciences) and analyzed in FlowJo v5.7.2-2 software (Tree Star, Ashland, OR). Phagocytosis Assay Phagocytosis was determined by measuring the uptake of FITC-conjugated latex beads (Sigma-Aldrich) according to the Journal of Neuroscience Research
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sample was analyzed in triplicate. Each well contained 1 ml cDNA template, 10 ml Master Mix (Applied Biosystems), 1 ml probe (Applied Biosystems), and 8 ml RNase-free water, and the total volume was adjusted to 20 ml with the PCR system. Amplification products were detected with an ABI 7500 sequence detector with the following thermal cycling conditions: 50 C for 2 min, 95 C for 10 min, and 45 cycles of 95 C for 15 sec and 60 C for 1 min. The probes used were interleukin (IL)-1b, IL-4, IL-6, IL-10, IL-12, IL-13, CCL2, interferon (IFN)-g, tumor necrosis factor (TNF)-a, transforming growth factor (TGF)-b1, arginase (Arg) 1, inducible nitric oxide synthase (NOS) 2, and CD204 (scavenger receptor A). Detection of Cytokines and Chemokines Microglia isolated by MGCS or by MACS were plated at a density of 2 3 105/ml on slides coated with 0.01% poly-Llysine and cultured in 100 ml DMEM/F12 medium containing 2% or 10% FBS, respectively. After 3, 12, and 24 hr, the medium was collected for detection of TNF-a, IFN-g, CCL2, IL-6, IL-10, and IL-12 with the mouse inflammation kit Cytometric Bead Array (CBA; BD Pharmingen) according to the manufacturer’s instructions. The culture medium from MGCS was collected on days 0, 3, 6, 9, 12, and 15 after inoculation with brain cells and analyzed as described above. TGF-b1 was detected by ELISA according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN). Statistical Analysis All statistical analyses were performed in SPSS 11.5. Data are mean 6 SEM. Differences between microglia isolated by MACS and microglia isolated by MGCS were evaluated by an independent sample t-test. Differences were considered to be statistically significant at P < 0.05. Fig. 1. Assessment of microglia purified by MGCS and MACS. Microglia from whole-brain homogenate (A) or purified by MGCS (B) or by MACS (C) were stained with anti-CD11b antibody and detected by flow cytometry. D: Chart shows the statistical analysis of the percentage of CD11b1 cells (n 5 10; **P < 0.01). FACS staining of CD45 (E) and Ly-6C (F) on MACS microglia, MGCS microglia, and peripheral monocytes. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
manufacturer’s instructions. After having been rinsed twice with ice-cold PBS, the cells were resuspended in 100 ml buffer and incubated for 15 min at 4 C in the dark with the following antibodies for flow cytometry: PE-conjugated anti-mouse CD45, PerCP-Cy5.5-conjugated anti-mouse CD11b, and the corresponding isotype controls (BD Bioscience). Cells were detected by flow cytometry (see above). All experiments were repeated three times independently. Real-Time PCR Analysis of Cytokines and Chemokines Total RNA was isolated from microglia (5 3 105 cells) purified by MGCS or by MACS with the RNeasy Micro kit (Qiagen) according to the manufacturer’s instructions. cDNA were synthesized with the TaqMan High Capacity RNA-tocDNA kit (Applied Biosystems, Foster City, CA). Each cDNA Journal of Neuroscience Research
RESULTS Purity of Microglia To evaluate the purity of the harvested microglia, we performed flow cytometry with CD11b, CD45, and Ly-6C staining. The proportion of CD11b1 cells was 4.2% 6 0.1% of total brain cells obtained from newborn mice (Fig. 1A). The proportions of microglia isolated by MGCS and by MACS were 79% 6 2.1% and 95% 6 0.4%, respectively (P < 0.01; Fig. 1B–D). Because highly purified microglia were required for further experiments, we added a purification step consisting of CD11b1 magnetic bead cell sorting. By doing so, we increased the proportion of microglia isolated by MGCS to 98% of the total cells. Moreover, the majority of CD11b1 cells purified either by MACS or by MGCS had a CD45lowLy-6Clow phenotype (Fig. 1E,F), unlike the CD11b1CD45highLy-6Chigh phenotype of monocytes and macrophages (Fux et al., 2008; Fukuda et al., 2013). Differences of Morphology, Phagocytic Capacity, and Phenotype With Wright-Giemsa staining, Iba-1 fluorescence staining, and flow cytometry we found that only
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Fig. 2. Morphology, phagocytic capacity, and phenotype differences between MGCS and MACS microglia. A–J: Morphology of microglia. Forward scatter (FSC; A) and side scatter (SSC; F) were detected by FACS. Wright–Giemsa staining of MGCS (B) and MACS (G) microglia. Immunofluorescence microscopy shows Iba1 in MGCS (C–E) and MACS (H–J) microglia. K–M: Phagocytic function detection of microglial cells. The percentages of FITC-labeled latex beads phagocytosed by MGCS microglia (K) and MACS microglia (L) were detected by FACS. Column chart (M) shows the statistical results.
Values are mean 6 SEM. Results are representative of three independent experiments (n 5 10; **P < 0.01). N–Q: Phenotypic identification of microglia. Surface expression of MHC-II (N), CD86 (O), CD80 (P), and CD204 (Q) on individual microglia was analyzed by flow cytometry. Data are representative of five independent experiments (n 5 10). Scale bars 5 10 lm in B,G; 50 mm in C–E,H–J. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
microglia isolated by MGCS had a large, irregular shape with short and wide processes and an abundant cytoplasm with high granularity (Fig. 2A–J). The fluorescence intensity of Iba-1 in microglia isolated by MGCS (Fig. 2C–E) was stronger than that of microglia isolated by MACS (Fig. 2H–J). These morphological data suggest that microglia isolated by MGCS were in an immune-activated state (Ransohoff and Cardona, 2010).
When purified microglia were cultured with FITClabeled latex beads, microglia isolated by MGCS showed a significantly greater phagocytic ability than microglia isolated by MACS by taking up more fluorescent beads (26.91% 6 0.63% vs. 7.9% 6 0.19%, respectively; P < 0.01; Fig. 2K–M). We further compared the phenotypes of the purified microglia by flow cytometry. Microglia isolated by MGCS expressed the costimulatory factor CD80/B7.1 Journal of Neuroscience Research
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Fig. 3. Gene and protein expression profiles of MGCS and MACS microglia. A: Gene expression levels were detected with real-time PCR and normalized by MACS microglia. Values are mean 6 SEM. The experiment was repeated three times, and the values presented are from one representative experiment (n 5 10). B–G: Concentration of TNF-a, CCL2, and IL-6 released by MGCS and MACS microglia. Purified microglial cells were cultured in medium containing 10% (B–
D) and 2% (E–G) FBS. The culture medium was collected after 3, 12, and 24 hr, and the concentration of TNF-a, CCL2, and IL-6 in the culture medium was detected by CBA. Values are mean 6 SEM. The experiment was repeated three times independently (n 5 12; *P < 0.05, **P < 0.01). ND, not detected. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]
and had a higher level of the costimulatory factor CD86/ B7.2 than microglia isolated by MACS (Fig. 2O,P). As expected, the expression level of M2 polarization marker CD204 was much higher in microglia isolated by MGCS than in microglia isolated by MACS (Fig. 2Q). However, levels of MHC-II molecules were not significantly different between the two groups of microglia (Fig. 2N).
microglia isolated by MGCS than in microglia isolated by MACS, respectively. Levels of TGF-b1 were identical in both cell groups, whereas IL-4 and IL-13 transcripts could not be detected (Fig. 3A). It is of interest that microglia isolated by MGCS also expressed higher amounts of M1related molecules, including TNF-a, IL-1b, IL-6, IL-12, CCL2, and NOS2, than microglia isolated by MACS. Only IFN-g was undetectable (Fig. 3A). Thus, microglia isolated by MGCS exhibited mixed characteristics of M1 and M2 polarization. We then compared the concentrations of released cytokines and chemokines between the two cell groups. Microglia from both groups were cultured in DMEM/ F12 medium containing 10% FBS (Fig. 3B–D) or 2% FBS (Fig. 3E–G) and analyzed by CBA at different time points. We found that IFN-g, IL-10, and IL-12 were below the detection limit (10 pg/ml). The releases of
Expression Profiles of M1 and M2-Related Genes Because the CD204 was highly expressed in microglia isolated by MGCS, we assessed several gene transcripts, indicators of M1 or M2 activation states. The mRNA level of CD204 was 212-fold higher in microglia isolated by MGCS than in microglia isolated by MACS (Fig. 3A). Transcript levels of the two M2-related factors Arg1 and IL-10 were fourfold and 163-fold higher in Journal of Neuroscience Research
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Fig. 4. Concentration of TNF-a, CCL2, IL-6, and TGF-b1 in the mixed glial culture medium. The medium from MGCS culture was collected on days 0, 3, 6, 9, 12, and 15 after inoculation of the whole-brain cells. The levels of TNF-a (A), CCL2 (B), IL-6 (C), and TGF-b1 (D) were detected. Values are mean 6 SEM. The experiment was repeated three times (n 5 12).
TNF-a (338.9 6 6.37 to 718.6 6 8.74 pg/ml), CCL2 (28.1 6 1.14 to 672.1 6 15.75 pg/ml), and IL-6 (29.5 6 1.04 to 171.0 6 13.24 pg/ml) from microglia isolated by MCGS increased dramatically along with the incubation time and were significantly higher than those of microglia isolated by MACS, without regard to the FBS concentration. Concentrations of Inducers of M1 and M2 Polarization Concentrations of the inducers of M1 or M2 polarization in mixed glial cell culture medium were also eval-
uated by CBA at different time points. The classical M1 stimulator IFN-g and the M2 inducers IL-4, IL-10, and IL-13 were undetectable even during a long-term cell culture, and only low levels of TNF-a and IL-6 were detected (Fig. 4A,C). In contrast, very high levels of CCL2 and TGF-b1 were measured and increased over time, reaching a peak at day 15 (1,396.2 6 22.83 pg/ml and 1,220.1 6 32.23 pg/ml, respectively; Fig. 4B,D). DISCUSSION Microglia are extremely sensitive to small pathological changes in the CNS. Therefore, the methods used to Journal of Neuroscience Research
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purify microglia can influence their immune state in vitro (Marek et al., 2008; Moussaud and Draheim, 2010). The present study demonstrates that microglia isolated by MGCS present both proinflammatory and antiinflammatory polarization profiles in contrast to microglia isolated by MACS. Evidence for cell polarization includes changes in morphology (retraction of branches, abundant cytoplasm, and higher granularity), upregulated expression of Iba-1 and the costimulatory molecule CD86, elevated phagocytic capacity, and secretion of inflammatory mediators and recruitment molecules. The term “double-edged sword” describes microglia as multifunctional immune cells in the CNS, linking their neurotoxic to their neuroprotective effects (Nguyen et al., 2002; Czeh et al., 2011). Activated microglia initiate inflammatory processes and potentially play a role in CNS injury (Hirsch et al., 1998; Tansey et al., 2007) by releasing a broad spectrum of proinflammatory or cytotoxic factors, such as IL-1b, TNF-a, and CCL2. On the other hand, microglia protect neurons by producing immunosuppressive factors (IL-10 and TGF-b1; WyssCoray et al., 2001; Brionne et al., 2003; Xin et al., 2011) and neurotrophic factors (Batchelor et al., 1999; Nguyen et al., 2002; Brionne et al., 2003) to inhibit inflammation and promote neuronal survival and repair. The activation of microglia in the CNS is similar to the dual polarization of peripheral macrophages. Therefore, this study was able to use the macrophage polarization marker CD204 to distinguish the M1 polarized microglia from the M2 polarized microglia (Batchelor et al., 1999; Sasaki et al., 2013). Accordingly, we observed a mixed phenotype of M1 and M2 polarization in the two groups of microglia. However, the proportions of each polarization type differed between the two groups, i.e., microglia isolated by MACS were dominated by M1 polarized cells, whereas microglia isolated by MGCS presented characteristics of M2 polarization. Indeed, the latter group possessed high levels of both proinflammatory (TNF-a, IL-1b, IL-6, IL12, CCL2, and NOS2) and anti-inflammatory (CD204, Arg1, and IL-10) factors. The expression of antiinflammatory factors was significantly lower in microglia isolated by MACS than in microglia isolated by MGCS. Macrophages acquire a predominant M1 polarization state in the early stage of tissue injury. Over time and up to the late stage of tissue injury, the number of M2 polarized cells rises (David and Kroner, 2011). We suggest that microglia isolated by MACS and MGCS are comparable to immune effector cells at early and late stages, respectively, of a dynamic inflammatory process of the CNS. Moreover, the majority of microglia isolated by MGCS present both M1 and M2 phenotypes, which may reflect a specific stage in the dynamic inflammatory process and may be induced by the complex cell culture conditions. Indeed, multiple factors may alter microglia isolated by MGCS. First, to use astrocytic factors better, we changed the medium only once on the third day after plating the cells. This might have caused nutritional deprivation and increased apoptosis of specific cells, especially of fragile neurons. In addition, microglial phagocyJournal of Neuroscience Research
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tosis of the debris generated by apoptosis might have affected cell phenotypes and cellular functions (Mosser and Edwards, 2008), and might have led to an elevated phagocytic capacity in microglia. Second, the presence of injured neurons in mixed cultures might have activated astrocytes and further stimulated microglia by secreting soluble factors and nucleotides, as reported by Ovanesov and colleagues (2008). In addition, some of the pro- and anti-inflammatory factors released by microglia and astrocytes can also induce macrophage polarization. High levels of CCL2 and TGF-b1 were detected in the culture medium of mixed glial cells. CCL2 was first described as an inducer of M1 polarization, whereas TGF-b1 induced IL4-mediated M2 activation and inhibited M1 activation (Zhou et al., 2012). A recent study demonstrated that CCL2 could promote both pro- and anti-inflammatory activation of microglia (Selenica et al., 2013). Thus, the mixed phenotype of microglia isolated by MGCS may be partially explained by the coexistence of CCL2 and TGFb1. Third, microglia isolated by MGCS proliferate actively, whereas the majority of microglia (in vivo or under physiological conditions) proliferate slowly (Gehrmann et al., 1995; Remington et al., 2007). Some studies have suggested that microglia should be cultured with a low serum concentration (e.g., 2%) to prevent any influence of the mixed culture system and to maximize the inflammatory response in further experiments (Deierborg, 2013). The current study, however, found that the ability of microglia isolated by MGCS to release cytokines and chemokines was much higher than that of microglia isolated by MACS, independently of the serum concentration (10% or 2% FBS). Indeed, the levels of TNF-a, CCL2, and IL-6 released by microglia isolated by MACS were close to the detection limit, especially immediately after purification. Therefore, we concluded that microglia isolated by MGCS were in an immuneactivated state, whereas microglia freshly purified by MACS were closer to their in vivo state. In summary, the current study shows that microglia purified by MGCS present phenotypes of dual spectra of polarization, leading to an immune-activated state in vitro. In contrast, microglia purified by MACS are closer to their in vivo state. Therefore, the immune status of microglia, influenced by the method of purification, should be carefully considered in neuropathological research. REFERENCES Batchelor PE, Liberatore GT, Wong JY, Porritt MJ, Frerichs F, Donnan GA, Howells DW. 1999. Activated macrophages and microglia induce dopaminergic sprouting in the injured striatum and express brainderived neurotrophic factor and glial cell line-derived neurotrophic factor. J Neurosci 19:1708–1716. Block ML, Zecca L, Hong JS. 2007. Microglia-mediated neurotoxicity: uncovering the molecular mechanisms. Nat Rev Neurosci 8:57–69. Brionne TC, Tesseur I, Masliah E, Wyss-Coray T. 2003. Loss of TGFbeta 1 leads to increased neuronal cell death and microgliosis in mouse brain. Neuron 40:1133–1145.
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Colton CA, Mott RT, Sharpe H, Xu Q, Van Nostrand WE, Vitek MP. 2006. Expression profiles for macrophage alternative activation genes in AD and in mouse models of AD. J Neuroinflammation 3:27. Czeh M, Gressens P, Kaindl AM. 2011. The yin and yang of microglia. Dev Neurosci 33:199–209. David S, Kroner A. 2011. Repertoire of microglial and macrophage responses after spinal cord injury. Nat Rev Neurosci 12:388–399. de Haas AH, Boddeke HW, Brouwer N, Biber K. 2007. Optimized isolation enables ex vivo analysis of microglia from various central nervous system regions. Glia 55:1374–1384. Deierborg T. 2013. Preparation of primary microglia cultures from postnatal mouse and rat brains. Methods Mol Biol 1041:25–31. Fukuda S, Nagano M, Yamashita T, Kimura K, Tsuboi I, Salazar G, Ueno S, Kondo M, Kunath T, Oshika T, Ohneda O. 2013. Functional endothelial progenitor cells selectively recruit neurovascular protective monocyte-derived F4/80/Ly6c macrophages in a mouse model of retinal degeneration. Stem Cells 31:2149–2161. Fux M, van Rooijen N, Owens T. 2008. Macrophage-independent Tcell infiltration to the site of injury-induced brain inflammation. J Neuroimmunol 203:64–72. Garden GA, M€ oller T. 2006. Microglia biology in health and disease. J Neuroimmune Pharmacol 1:127–137. Gehrmann J, Matsumoto Y, Kreutzberg GW. 1995. Microglia: intrinsic immuneffector cell of the brain. Brain Res Brain Res Rev 20:269–287. Giulian D, Baker TJ. 1986. Characterization of ameboid microglia isolated from developing mammalian brain. J Neurosci 6:2163–2178. Hirsch EC, Hunot S, Damier P, Faucheux B. 1998. Glial cells and inflammation in Parkinson’s disease: a role in neurodegeneration? Ann Neurol 44:S115–S120. Marek R, Caruso M, Rostami A, Grinspan JB, Das Sarma J. 2008. Magnetic cell sorting: a fast and effective method of concurrent isolation of high purity viable astrocytes and microglia from neonatal mouse brain tissue. J Neurosci Methods 175:108–118. McCarthy KD, de Vellis J. 1980. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol 85: 890–902. McGeer PL, Itagaki S, Boyes BE, McGeer EG. 1988. Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson’s and Alzheimer’s disease brains. Neurology 38:1285–1291.
Mosser DM, Edwards JP. 2008. Exploring the full spectrum of macrophage activation. Nat Rev Immunol 8:958–969. Moussaud S, Draheim HJ. 2010. A new method to isolate microglia from adult mice and culture them for an extended period of time. J Neurosci Methods 187:243–253. Nguyen MD, Julien JP, Rivest S. 2002. Innate immunity: the missing link in neuroprotection and neurodegeneration? Nat Rev Neurosci 3: 216–227. Ovanesov MV, Ayhan Y, Wolbert C, Moldovan K, Sauder C, Pletnikov MV. 2008. Astrocytes play a key role in activation of microglia by persistent Borna disease virus infection. J Neuroinflammation 5:50. Ransohoff RM, Cardona AE. 2010. The myeloid cells of the central nervous system parenchyma. Nature 468:253–262. Remington LT, Babcock AA, Zehntner SP, Owens T. 2007. Microglial recruitment, activation, and proliferation in response to primary demyelination. Am J Pathol 170:1713–1724. Sasaki A, Yokoo H, Tanaka Y, Homma T, Nakazato Y, Ohgaki H. 2013. Characterization of microglia/macrophages in gliomas developed in S-100beta–v-erbB transgenic rats. Neuropathology 33:505–514. Selenica ML, Alvarez JA, Nash KR, Lee DC, Cao C, Lin X, Reid P, Mouton PR, Morgan D, Gordon MN. 2013. Diverse activation of microglia by chemokine (C-C motif) ligand 2 overexpression in brain. J Neuroinflammation 10:86. Tansey MG, McCoy MK, Frank-Cannon TC. 2007. Neuroinflammatory mechanisms in Parkinson’s disease: potential environmental triggers, pathways, and targets for early therapeutic intervention. Exp Neurol 208:1–25. Wyss-Coray T, Lin C, Yan F, Yu GQ, Rohde M, McConlogue L, Masliah E, Mucke L. 2001. TGF-beta1 promotes microglial amyloidbeta clearance and reduces plaque burden in transgenic mice. Nat Med 7:612–618. Xin J, Wainwright DA, Mesnard NA, Serpe CJ, Sanders VM, Jones KJ. 2011. IL-10 within the CNS is necessary for CD41 T cells to mediate neuroprotection. Brain Behav Immun 25:820–829. Zhou X, Spittau B, Krieglstein K. 2012. TGFbeta signalling plays an important role in IL4-induced alternative activation of microglia. J Neuroinflammation 9:210.
Journal of Neuroscience Research
Dual Polarization of Microglia Isolated from Mixed Glial Cell Cultures Lili Ju,1 Hui Zeng,2,3 Yun Chen,1 Yanhong Wu,1 Beibei Wang,2,3* and Qunyuan Xu1* 1
Department of Neurobiology, Beijing Institute for Brain Disorders, Beijing Center of Neural Regeneration and Repair, Key Laboratory for Neurodegenerative Diseases of the Ministry of Education, Capital Medical University, Beijing, China 2 Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing, China 3 Beijing Key Laboratory of Emerging Infectious Diseases, Beijing, China
Microglia are versatile immune effector cells of the CNS and are sensitive to various stimuli. The different methods used to isolate microglia may affect some of their characteristics, such as their polarization state. The influence of cell sorting methods on the polarization state of microglia has never been studied. Mixed glial culture system (MGCS) and magnetic activated cell sorting (MACS) are two methods that are commonly used to purify microglia. This study compares the immunological states between microglia isolated by MGCS and microglia isolated by MACS. We show that microglia isolated by MGCS exhibit a stronger immune-activated state than microglia isolated by MACS. They present an elevated phagocytic ability and high levels of markers associated with classical activation (M1) and alternative activation (M2). In addition, high levels of M1-type and M2-type chemokine (C-C motif) ligand 2 and transforming growth factor-b1 were detected in the culture medium of mixed glial cells. Our results show that microglia isolated by MGCS are in an immune-activated state, whereas microglia isolated by MACS appear to be closer to their primary in vivo state. Therefore, the immune status of microglia, depending on the protocol used to purify them, should be carefully considered in neuropathology research. VC 2015 Wiley Periodicals, Inc. Key words: microglia; central nervous system; immune effector cells
Microglia are the resident macrophages of the CNS (Batchelor et al., 1999; Block et al., 2007) and account for approximately 12% of the total cellular population (Block et al., 2007) and 5–20% of total glial cells in the mammalian brain (Garden and Moller, 2006). They play a pivotal role in maintaining homeostasis in the CNS, including maturation, synaptic plasticity, clearance of debris, host defense, wound healing, and immune regulation (Remington et al., 2007). Because they are involved in nearly all pathological processes, microglia are attractive targets for the treatment of diseases of the CNS, such as multiple sclerosis, neurodegenerative disorders, demyelination, and spinal cord injury (McGeer et al., 1988; Colton et al., 2006; Garden and Moller, 2006; Remington et al., 2007). C 2015 Wiley Periodicals, Inc. V
Given the different adherence properties of glial cells, Giulian and Baker (1986) and McCarthy and de Vellis (1980) developed an in vitro mechanical shake-off method to isolate microglia from the mixed glial culture system (MGCS). Recently, magnetic activated cell sorting (MACS), a new, convenient and efficient method, has been described (de Haas et al., 2007; Marek et al., 2008). Because microglia are versatile immune effector cells, various stimuli can convert them from a quiescent state to an active state and induce variations in cell migration, cell proliferation, scavenging of debris or pathogens, antigen presentation, and secretion of biological factors (Gehrmann et al., 1995; Ransohoff and Cardona, 2010). Therefore, we suggest that the methods used to purify microglia can influence and alter their physiological states. Which of these physiological characteristics are affected by the purification process? Do the purification methods MGCS and MACS influence the physiological state of microglia in a different manner? These important questions must be answered to investigate microglial functions in the fields of neuroscience and immunology further. Peripheral macrophages exhibit two functionally different activation states: classically activated (M1) proinflammatory macrophages are characterized by strong antigen-presentation abilities and by the production of Contract grant sponsor: Beijing Nova Program; Contract grant number: 2009B05; Contract grant sponsor: Chinese National Key Developing Project of Basic Research (973); Contract grant number: 2007CB947704 *Correspondence to: Qunyuan Xu, Department of Neurobiology, Beijing Institute for Brain Disorders, Beijing Center of Neural Regeneration and Repair, Key Laboratory for Neurodegenerative Diseases of the Ministry of Education, Capital Medical University, Beijing 100069, China. E-mail: [email protected], or Beibei Wang, Beijing Key Laboratory of Emerging Infectious Diseases, Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing 100015, China. E-mail: [email protected] Q. Xu and B. Wang contributed equally to this work. Received 3 December 2014; Revised 2 January 2015; Accepted 5 January 2015 Published online 6 June 2015 in Wiley Online (wileyonlinelibrary.com). DOI: 10.1002/jnr.23563
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proinflammatory cytokines, whereas alternatively activated (M2) macrophages are characterized by secretion of anti-inflammatory cytokines (Mosser and Edwards, 2008; David and Kroner, 2011). Accordingly, as the counterpart to the peripheral macrophages, microglia can be classified into two activation states. Changes in physiological conditions can stimulate the conversion of microglia from one state to the other (Colton et al., 2006; Czeh et al., 2011; Selenica et al., 2013). Thus, a full characterization of the specific cellular changes induced by the purification processes is required to choose the appropriate approach for microglial purification. The present study shows that microglia isolated by MGCS have the morphological, phenotypical, and molecular characteristics of dual polarization, whereas most microglia isolated by MACS are in a quiescent state. MATERIALS AND METHODS Preparation of Brain Tissues Forty-eight-hour-old mice (C57BL/6) were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (Beijing, China), and handled according to the guidelines of the laboratory animal facility of Beijing as approved by the local animal experimental committee. Brains were isolated under sterile conditions, stripped of meninges and blood vessels in ice-cold phosphate-buffered saline (PBS), mechanically dissociated, and then passed through a 40mm cell strainer (BD Biosciences, San Jose, CA). The homogenate was centrifuged at 1,000 rpm for 10 min at 4 C, and the supernatant, containing debris, was discarded. The cell pellet was resuspended with PBS to obtain single-cell suspension for further processing. This study was carried out in strict accordance with the Guide for the care and use of laboratory animals of the National Institutes of Health. The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Capital Medical University. Purification of Microglia By MGCS Microglia were isolated and purified by MGCS as described elsewhere (McCarthy and de Vellis, 1980; Giulian and Baker, 1986). The whole-brain suspension was plated in a 75-cm2 flask coated with 0.01% poly-L-lysine and cultured in 10 ml Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. The medium was refreshed on day 3. On day 14 or 15, the mixed glial culture was stratified and shaken at 250 rpm for 3 hr at 37 C in a Forma orbital shaker (Thermo Fisher Scientific, Waltham, MA). Finally, the suspended cells were collected and filtered through a cell strainer. Purification of Microglia By MACS MACS was performed with CD11b MicroBeads (Miltenyi Biotec, Cologne, Germany) as described elsewhere, with minor modifications (de Haas et al., 2007; Marek et al., 2008). Briefly, cells were resuspended at a density of up to 1 3 107 cells/90 ml in a separation buffer (PBS containing 0.5% BSA and 2 mM EDTA, pH 7.2) and incubated with 10 ml of mono-
clonal CD11b MicroBeads for 15 min at 4 C. After having been washed, centrifuged, and resuspended in 3 ml of separation buffer, the cells were applied to the LS column, which was placed in a magnetic field, and the labeled cells were captured in the column. The column was washed four times with 3 ml separation buffer (one more time than in the manufacturer’s instructions for a better removal of negative components, such as myelin debris). The column was removed from the magnetic field. Cells were flushed out with 5 ml buffer and collected. To improve the purity, the collected cells were subjected to a second CD11b-positive selection with the LS column. Wright-Giemsa Staining The cells were placed onto glass slides by cytospinning at 750 rpm for 5 min at room temperature, air dried, and stained with Wright-Giemsa staining kits (Baso Diagnostics, Zhuhai, China) according to the manufacturer’s instructions. Immunofluorescence Staining Cells were plated at a density of 105 cells/ml on slides coated with 0.01% poly-L-lysine and cultured for 48 hr. They were rinsed three times with PBS, fixed with 4% paraformaldehyde for 20 min at room temperature, treated with 0.3% Triton X-100 (v/v) and 10% heat-inactivated FBS (1 hr at room temperature), and incubated overnight at 4 C with monoclonal goat anti-Iba1 (Abcam, Cambridge, United Kingdom). Cells were then washed three times with PBS and incubated with rabbit anti-goat IgG conjugated with Alexa Fluor 488 (Invitrogen Life Technologies, Carlsbad, CA) for 30 min at 37 C. Again, cells were washed three times with PBS and counterstained with Hochest 33258 (Sigma-Aldrich, St. Louis, MO) for 10 min at room temperature for nucleus visualization. Images were captured from 10 random fields in each sample with a fluorescence microscope (Olympus, Tokyo, Japan). Cells incubated without primary antibodies served as negative controls. Flow Cytometry The following monoclonal antibodies were used for fluorescence-activated cell sorting (FACS) staining: fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD11b and Ly6C, phycoerythrin (PE)-conjugated anti-mouse CD11b and CD45, peridinin-chlorophyll protein (PerCP)-Cy5.5-conjugated anti-mouse major histocompatibility complex (MHC)-II, allophycocyanin (APC)-conjugated anti-mouse CD80 and CD86 (BD Biosciences), and biotin-conjugated anti-CD204 antibody (AbD Serotec, Kidlington, United Kingdom). Suitable isotype controls were also purchased from BD Pharmingen (San Diego, CA). Purified microglia were incubated with these antibodies for 15 min at 4 C. For CD204 staining, the cell suspension was further incubated with APC-conjugated antibiotin antibody (Miltenyi Biotec) for 15 min at 4 C. 7-Aminoactinomycin D was used to discriminate dead cells. Data were acquired with a FACSCalibur flow cytometer (BD Biosciences) and analyzed in FlowJo v5.7.2-2 software (Tree Star, Ashland, OR). Phagocytosis Assay Phagocytosis was determined by measuring the uptake of FITC-conjugated latex beads (Sigma-Aldrich) according to the Journal of Neuroscience Research
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sample was analyzed in triplicate. Each well contained 1 ml cDNA template, 10 ml Master Mix (Applied Biosystems), 1 ml probe (Applied Biosystems), and 8 ml RNase-free water, and the total volume was adjusted to 20 ml with the PCR system. Amplification products were detected with an ABI 7500 sequence detector with the following thermal cycling conditions: 50 C for 2 min, 95 C for 10 min, and 45 cycles of 95 C for 15 sec and 60 C for 1 min. The probes used were interleukin (IL)-1b, IL-4, IL-6, IL-10, IL-12, IL-13, CCL2, interferon (IFN)-g, tumor necrosis factor (TNF)-a, transforming growth factor (TGF)-b1, arginase (Arg) 1, inducible nitric oxide synthase (NOS) 2, and CD204 (scavenger receptor A). Detection of Cytokines and Chemokines Microglia isolated by MGCS or by MACS were plated at a density of 2 3 105/ml on slides coated with 0.01% poly-Llysine and cultured in 100 ml DMEM/F12 medium containing 2% or 10% FBS, respectively. After 3, 12, and 24 hr, the medium was collected for detection of TNF-a, IFN-g, CCL2, IL-6, IL-10, and IL-12 with the mouse inflammation kit Cytometric Bead Array (CBA; BD Pharmingen) according to the manufacturer’s instructions. The culture medium from MGCS was collected on days 0, 3, 6, 9, 12, and 15 after inoculation with brain cells and analyzed as described above. TGF-b1 was detected by ELISA according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN). Statistical Analysis All statistical analyses were performed in SPSS 11.5. Data are mean 6 SEM. Differences between microglia isolated by MACS and microglia isolated by MGCS were evaluated by an independent sample t-test. Differences were considered to be statistically significant at P < 0.05. Fig. 1. Assessment of microglia purified by MGCS and MACS. Microglia from whole-brain homogenate (A) or purified by MGCS (B) or by MACS (C) were stained with anti-CD11b antibody and detected by flow cytometry. D: Chart shows the statistical analysis of the percentage of CD11b1 cells (n 5 10; **P < 0.01). FACS staining of CD45 (E) and Ly-6C (F) on MACS microglia, MGCS microglia, and peripheral monocytes. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
manufacturer’s instructions. After having been rinsed twice with ice-cold PBS, the cells were resuspended in 100 ml buffer and incubated for 15 min at 4 C in the dark with the following antibodies for flow cytometry: PE-conjugated anti-mouse CD45, PerCP-Cy5.5-conjugated anti-mouse CD11b, and the corresponding isotype controls (BD Bioscience). Cells were detected by flow cytometry (see above). All experiments were repeated three times independently. Real-Time PCR Analysis of Cytokines and Chemokines Total RNA was isolated from microglia (5 3 105 cells) purified by MGCS or by MACS with the RNeasy Micro kit (Qiagen) according to the manufacturer’s instructions. cDNA were synthesized with the TaqMan High Capacity RNA-tocDNA kit (Applied Biosystems, Foster City, CA). Each cDNA Journal of Neuroscience Research
RESULTS Purity of Microglia To evaluate the purity of the harvested microglia, we performed flow cytometry with CD11b, CD45, and Ly-6C staining. The proportion of CD11b1 cells was 4.2% 6 0.1% of total brain cells obtained from newborn mice (Fig. 1A). The proportions of microglia isolated by MGCS and by MACS were 79% 6 2.1% and 95% 6 0.4%, respectively (P < 0.01; Fig. 1B–D). Because highly purified microglia were required for further experiments, we added a purification step consisting of CD11b1 magnetic bead cell sorting. By doing so, we increased the proportion of microglia isolated by MGCS to 98% of the total cells. Moreover, the majority of CD11b1 cells purified either by MACS or by MGCS had a CD45lowLy-6Clow phenotype (Fig. 1E,F), unlike the CD11b1CD45highLy-6Chigh phenotype of monocytes and macrophages (Fux et al., 2008; Fukuda et al., 2013). Differences of Morphology, Phagocytic Capacity, and Phenotype With Wright-Giemsa staining, Iba-1 fluorescence staining, and flow cytometry we found that only
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Fig. 2. Morphology, phagocytic capacity, and phenotype differences between MGCS and MACS microglia. A–J: Morphology of microglia. Forward scatter (FSC; A) and side scatter (SSC; F) were detected by FACS. Wright–Giemsa staining of MGCS (B) and MACS (G) microglia. Immunofluorescence microscopy shows Iba1 in MGCS (C–E) and MACS (H–J) microglia. K–M: Phagocytic function detection of microglial cells. The percentages of FITC-labeled latex beads phagocytosed by MGCS microglia (K) and MACS microglia (L) were detected by FACS. Column chart (M) shows the statistical results.
Values are mean 6 SEM. Results are representative of three independent experiments (n 5 10; **P < 0.01). N–Q: Phenotypic identification of microglia. Surface expression of MHC-II (N), CD86 (O), CD80 (P), and CD204 (Q) on individual microglia was analyzed by flow cytometry. Data are representative of five independent experiments (n 5 10). Scale bars 5 10 lm in B,G; 50 mm in C–E,H–J. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
microglia isolated by MGCS had a large, irregular shape with short and wide processes and an abundant cytoplasm with high granularity (Fig. 2A–J). The fluorescence intensity of Iba-1 in microglia isolated by MGCS (Fig. 2C–E) was stronger than that of microglia isolated by MACS (Fig. 2H–J). These morphological data suggest that microglia isolated by MGCS were in an immune-activated state (Ransohoff and Cardona, 2010).
When purified microglia were cultured with FITClabeled latex beads, microglia isolated by MGCS showed a significantly greater phagocytic ability than microglia isolated by MACS by taking up more fluorescent beads (26.91% 6 0.63% vs. 7.9% 6 0.19%, respectively; P < 0.01; Fig. 2K–M). We further compared the phenotypes of the purified microglia by flow cytometry. Microglia isolated by MGCS expressed the costimulatory factor CD80/B7.1 Journal of Neuroscience Research
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Fig. 3. Gene and protein expression profiles of MGCS and MACS microglia. A: Gene expression levels were detected with real-time PCR and normalized by MACS microglia. Values are mean 6 SEM. The experiment was repeated three times, and the values presented are from one representative experiment (n 5 10). B–G: Concentration of TNF-a, CCL2, and IL-6 released by MGCS and MACS microglia. Purified microglial cells were cultured in medium containing 10% (B–
D) and 2% (E–G) FBS. The culture medium was collected after 3, 12, and 24 hr, and the concentration of TNF-a, CCL2, and IL-6 in the culture medium was detected by CBA. Values are mean 6 SEM. The experiment was repeated three times independently (n 5 12; *P < 0.05, **P < 0.01). ND, not detected. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]
and had a higher level of the costimulatory factor CD86/ B7.2 than microglia isolated by MACS (Fig. 2O,P). As expected, the expression level of M2 polarization marker CD204 was much higher in microglia isolated by MGCS than in microglia isolated by MACS (Fig. 2Q). However, levels of MHC-II molecules were not significantly different between the two groups of microglia (Fig. 2N).
microglia isolated by MGCS than in microglia isolated by MACS, respectively. Levels of TGF-b1 were identical in both cell groups, whereas IL-4 and IL-13 transcripts could not be detected (Fig. 3A). It is of interest that microglia isolated by MGCS also expressed higher amounts of M1related molecules, including TNF-a, IL-1b, IL-6, IL-12, CCL2, and NOS2, than microglia isolated by MACS. Only IFN-g was undetectable (Fig. 3A). Thus, microglia isolated by MGCS exhibited mixed characteristics of M1 and M2 polarization. We then compared the concentrations of released cytokines and chemokines between the two cell groups. Microglia from both groups were cultured in DMEM/ F12 medium containing 10% FBS (Fig. 3B–D) or 2% FBS (Fig. 3E–G) and analyzed by CBA at different time points. We found that IFN-g, IL-10, and IL-12 were below the detection limit (10 pg/ml). The releases of
Expression Profiles of M1 and M2-Related Genes Because the CD204 was highly expressed in microglia isolated by MGCS, we assessed several gene transcripts, indicators of M1 or M2 activation states. The mRNA level of CD204 was 212-fold higher in microglia isolated by MGCS than in microglia isolated by MACS (Fig. 3A). Transcript levels of the two M2-related factors Arg1 and IL-10 were fourfold and 163-fold higher in Journal of Neuroscience Research
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Fig. 4. Concentration of TNF-a, CCL2, IL-6, and TGF-b1 in the mixed glial culture medium. The medium from MGCS culture was collected on days 0, 3, 6, 9, 12, and 15 after inoculation of the whole-brain cells. The levels of TNF-a (A), CCL2 (B), IL-6 (C), and TGF-b1 (D) were detected. Values are mean 6 SEM. The experiment was repeated three times (n 5 12).
TNF-a (338.9 6 6.37 to 718.6 6 8.74 pg/ml), CCL2 (28.1 6 1.14 to 672.1 6 15.75 pg/ml), and IL-6 (29.5 6 1.04 to 171.0 6 13.24 pg/ml) from microglia isolated by MCGS increased dramatically along with the incubation time and were significantly higher than those of microglia isolated by MACS, without regard to the FBS concentration. Concentrations of Inducers of M1 and M2 Polarization Concentrations of the inducers of M1 or M2 polarization in mixed glial cell culture medium were also eval-
uated by CBA at different time points. The classical M1 stimulator IFN-g and the M2 inducers IL-4, IL-10, and IL-13 were undetectable even during a long-term cell culture, and only low levels of TNF-a and IL-6 were detected (Fig. 4A,C). In contrast, very high levels of CCL2 and TGF-b1 were measured and increased over time, reaching a peak at day 15 (1,396.2 6 22.83 pg/ml and 1,220.1 6 32.23 pg/ml, respectively; Fig. 4B,D). DISCUSSION Microglia are extremely sensitive to small pathological changes in the CNS. Therefore, the methods used to Journal of Neuroscience Research
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purify microglia can influence their immune state in vitro (Marek et al., 2008; Moussaud and Draheim, 2010). The present study demonstrates that microglia isolated by MGCS present both proinflammatory and antiinflammatory polarization profiles in contrast to microglia isolated by MACS. Evidence for cell polarization includes changes in morphology (retraction of branches, abundant cytoplasm, and higher granularity), upregulated expression of Iba-1 and the costimulatory molecule CD86, elevated phagocytic capacity, and secretion of inflammatory mediators and recruitment molecules. The term “double-edged sword” describes microglia as multifunctional immune cells in the CNS, linking their neurotoxic to their neuroprotective effects (Nguyen et al., 2002; Czeh et al., 2011). Activated microglia initiate inflammatory processes and potentially play a role in CNS injury (Hirsch et al., 1998; Tansey et al., 2007) by releasing a broad spectrum of proinflammatory or cytotoxic factors, such as IL-1b, TNF-a, and CCL2. On the other hand, microglia protect neurons by producing immunosuppressive factors (IL-10 and TGF-b1; WyssCoray et al., 2001; Brionne et al., 2003; Xin et al., 2011) and neurotrophic factors (Batchelor et al., 1999; Nguyen et al., 2002; Brionne et al., 2003) to inhibit inflammation and promote neuronal survival and repair. The activation of microglia in the CNS is similar to the dual polarization of peripheral macrophages. Therefore, this study was able to use the macrophage polarization marker CD204 to distinguish the M1 polarized microglia from the M2 polarized microglia (Batchelor et al., 1999; Sasaki et al., 2013). Accordingly, we observed a mixed phenotype of M1 and M2 polarization in the two groups of microglia. However, the proportions of each polarization type differed between the two groups, i.e., microglia isolated by MACS were dominated by M1 polarized cells, whereas microglia isolated by MGCS presented characteristics of M2 polarization. Indeed, the latter group possessed high levels of both proinflammatory (TNF-a, IL-1b, IL-6, IL12, CCL2, and NOS2) and anti-inflammatory (CD204, Arg1, and IL-10) factors. The expression of antiinflammatory factors was significantly lower in microglia isolated by MACS than in microglia isolated by MGCS. Macrophages acquire a predominant M1 polarization state in the early stage of tissue injury. Over time and up to the late stage of tissue injury, the number of M2 polarized cells rises (David and Kroner, 2011). We suggest that microglia isolated by MACS and MGCS are comparable to immune effector cells at early and late stages, respectively, of a dynamic inflammatory process of the CNS. Moreover, the majority of microglia isolated by MGCS present both M1 and M2 phenotypes, which may reflect a specific stage in the dynamic inflammatory process and may be induced by the complex cell culture conditions. Indeed, multiple factors may alter microglia isolated by MGCS. First, to use astrocytic factors better, we changed the medium only once on the third day after plating the cells. This might have caused nutritional deprivation and increased apoptosis of specific cells, especially of fragile neurons. In addition, microglial phagocyJournal of Neuroscience Research
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tosis of the debris generated by apoptosis might have affected cell phenotypes and cellular functions (Mosser and Edwards, 2008), and might have led to an elevated phagocytic capacity in microglia. Second, the presence of injured neurons in mixed cultures might have activated astrocytes and further stimulated microglia by secreting soluble factors and nucleotides, as reported by Ovanesov and colleagues (2008). In addition, some of the pro- and anti-inflammatory factors released by microglia and astrocytes can also induce macrophage polarization. High levels of CCL2 and TGF-b1 were detected in the culture medium of mixed glial cells. CCL2 was first described as an inducer of M1 polarization, whereas TGF-b1 induced IL4-mediated M2 activation and inhibited M1 activation (Zhou et al., 2012). A recent study demonstrated that CCL2 could promote both pro- and anti-inflammatory activation of microglia (Selenica et al., 2013). Thus, the mixed phenotype of microglia isolated by MGCS may be partially explained by the coexistence of CCL2 and TGFb1. Third, microglia isolated by MGCS proliferate actively, whereas the majority of microglia (in vivo or under physiological conditions) proliferate slowly (Gehrmann et al., 1995; Remington et al., 2007). Some studies have suggested that microglia should be cultured with a low serum concentration (e.g., 2%) to prevent any influence of the mixed culture system and to maximize the inflammatory response in further experiments (Deierborg, 2013). The current study, however, found that the ability of microglia isolated by MGCS to release cytokines and chemokines was much higher than that of microglia isolated by MACS, independently of the serum concentration (10% or 2% FBS). Indeed, the levels of TNF-a, CCL2, and IL-6 released by microglia isolated by MACS were close to the detection limit, especially immediately after purification. Therefore, we concluded that microglia isolated by MGCS were in an immuneactivated state, whereas microglia freshly purified by MACS were closer to their in vivo state. In summary, the current study shows that microglia purified by MGCS present phenotypes of dual spectra of polarization, leading to an immune-activated state in vitro. In contrast, microglia purified by MACS are closer to their in vivo state. Therefore, the immune status of microglia, influenced by the method of purification, should be carefully considered in neuropathological research. REFERENCES Batchelor PE, Liberatore GT, Wong JY, Porritt MJ, Frerichs F, Donnan GA, Howells DW. 1999. Activated macrophages and microglia induce dopaminergic sprouting in the injured striatum and express brainderived neurotrophic factor and glial cell line-derived neurotrophic factor. J Neurosci 19:1708–1716. Block ML, Zecca L, Hong JS. 2007. Microglia-mediated neurotoxicity: uncovering the molecular mechanisms. Nat Rev Neurosci 8:57–69. Brionne TC, Tesseur I, Masliah E, Wyss-Coray T. 2003. Loss of TGFbeta 1 leads to increased neuronal cell death and microgliosis in mouse brain. Neuron 40:1133–1145.
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Colton CA, Mott RT, Sharpe H, Xu Q, Van Nostrand WE, Vitek MP. 2006. Expression profiles for macrophage alternative activation genes in AD and in mouse models of AD. J Neuroinflammation 3:27. Czeh M, Gressens P, Kaindl AM. 2011. The yin and yang of microglia. Dev Neurosci 33:199–209. David S, Kroner A. 2011. Repertoire of microglial and macrophage responses after spinal cord injury. Nat Rev Neurosci 12:388–399. de Haas AH, Boddeke HW, Brouwer N, Biber K. 2007. Optimized isolation enables ex vivo analysis of microglia from various central nervous system regions. Glia 55:1374–1384. Deierborg T. 2013. Preparation of primary microglia cultures from postnatal mouse and rat brains. Methods Mol Biol 1041:25–31. Fukuda S, Nagano M, Yamashita T, Kimura K, Tsuboi I, Salazar G, Ueno S, Kondo M, Kunath T, Oshika T, Ohneda O. 2013. Functional endothelial progenitor cells selectively recruit neurovascular protective monocyte-derived F4/80/Ly6c macrophages in a mouse model of retinal degeneration. Stem Cells 31:2149–2161. Fux M, van Rooijen N, Owens T. 2008. Macrophage-independent Tcell infiltration to the site of injury-induced brain inflammation. J Neuroimmunol 203:64–72. Garden GA, M€ oller T. 2006. Microglia biology in health and disease. J Neuroimmune Pharmacol 1:127–137. Gehrmann J, Matsumoto Y, Kreutzberg GW. 1995. Microglia: intrinsic immuneffector cell of the brain. Brain Res Brain Res Rev 20:269–287. Giulian D, Baker TJ. 1986. Characterization of ameboid microglia isolated from developing mammalian brain. J Neurosci 6:2163–2178. Hirsch EC, Hunot S, Damier P, Faucheux B. 1998. Glial cells and inflammation in Parkinson’s disease: a role in neurodegeneration? Ann Neurol 44:S115–S120. Marek R, Caruso M, Rostami A, Grinspan JB, Das Sarma J. 2008. Magnetic cell sorting: a fast and effective method of concurrent isolation of high purity viable astrocytes and microglia from neonatal mouse brain tissue. J Neurosci Methods 175:108–118. McCarthy KD, de Vellis J. 1980. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol 85: 890–902. McGeer PL, Itagaki S, Boyes BE, McGeer EG. 1988. Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson’s and Alzheimer’s disease brains. Neurology 38:1285–1291.
Mosser DM, Edwards JP. 2008. Exploring the full spectrum of macrophage activation. Nat Rev Immunol 8:958–969. Moussaud S, Draheim HJ. 2010. A new method to isolate microglia from adult mice and culture them for an extended period of time. J Neurosci Methods 187:243–253. Nguyen MD, Julien JP, Rivest S. 2002. Innate immunity: the missing link in neuroprotection and neurodegeneration? Nat Rev Neurosci 3: 216–227. Ovanesov MV, Ayhan Y, Wolbert C, Moldovan K, Sauder C, Pletnikov MV. 2008. Astrocytes play a key role in activation of microglia by persistent Borna disease virus infection. J Neuroinflammation 5:50. Ransohoff RM, Cardona AE. 2010. The myeloid cells of the central nervous system parenchyma. Nature 468:253–262. Remington LT, Babcock AA, Zehntner SP, Owens T. 2007. Microglial recruitment, activation, and proliferation in response to primary demyelination. Am J Pathol 170:1713–1724. Sasaki A, Yokoo H, Tanaka Y, Homma T, Nakazato Y, Ohgaki H. 2013. Characterization of microglia/macrophages in gliomas developed in S-100beta–v-erbB transgenic rats. Neuropathology 33:505–514. Selenica ML, Alvarez JA, Nash KR, Lee DC, Cao C, Lin X, Reid P, Mouton PR, Morgan D, Gordon MN. 2013. Diverse activation of microglia by chemokine (C-C motif) ligand 2 overexpression in brain. J Neuroinflammation 10:86. Tansey MG, McCoy MK, Frank-Cannon TC. 2007. Neuroinflammatory mechanisms in Parkinson’s disease: potential environmental triggers, pathways, and targets for early therapeutic intervention. Exp Neurol 208:1–25. Wyss-Coray T, Lin C, Yan F, Yu GQ, Rohde M, McConlogue L, Masliah E, Mucke L. 2001. TGF-beta1 promotes microglial amyloidbeta clearance and reduces plaque burden in transgenic mice. Nat Med 7:612–618. Xin J, Wainwright DA, Mesnard NA, Serpe CJ, Sanders VM, Jones KJ. 2011. IL-10 within the CNS is necessary for CD41 T cells to mediate neuroprotection. Brain Behav Immun 25:820–829. Zhou X, Spittau B, Krieglstein K. 2012. TGFbeta signalling plays an important role in IL4-induced alternative activation of microglia. J Neuroinflammation 9:210.
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