Astrocyte-Derived CCL2 is Associated with M1 Activation and Recruitment of Cultured Microglial Cells Open Access

Neurodegenerative diseases are associated with chronic inflammation of the central nervous system [1]. In the brain, inflammation is primarily governed by microglia, a cell type of the monocyte-macrophage lineage that accounts for up to 20% of all brain cells [2]. The accumulation and activation of microglia are implicated in the pathogenesis of Alzheimer's disease (AD) [3], Parkinson's disease (PD) [4] and amyotrophic lateral sclerosis [5]. Activated microglia release a variety of inflammatory mediators, including cytokines, chemokines, prostaglandins, excitatory aminoacids, reactive oxygen intermediates and nitric oxide [6]. These inflammatory signals may enhance oxidative stress, activate cell death pathways, and promote neurodegeneration [7]. Microglia can be categorized according to their activation state, described as classical (M1) and alternative (M2) types of activation. Although this categorization might be an oversimplification, microglia can be polarized into an activation state that is intermediate between a neuro-harmful and a protective state. M1 microglia express pro-inflammatory molecules that include tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and nitric oxide (NO), as well as cell surface markers, such as CD86 [8], while M2 microglia express different molecules, such as IL-4, arignase1, Ym1, CD206, and IL-10 [9,10].

The chemokine C-C motif ligand 2 (CCL2), also known as monocyte chemoattractant protein-1 (MCP-1), is an important mediator of inflammation following damage to the central nervous system (CNS) that acts as the main endogenous agonist of the chemokine receptor type 2 (CCR2) [11,12,13,14]. CCL2 has been shown to play a major role in the recruitment of inflammatory cells to sites of tissue injury [15,16], and the expression of CCR2 has been demonstrated in microglia [17]. Recently it was reported that astrocytes are the main source of CCL2 in neuroinflammation [13,18]. Mayo et al. found that CCL2 from astrocytes could control the recruitment and activation of microglia in chronic experimental autoimmune encephalomyelitis (EAE) [18]. Kim et al. also demonstrated that CCL2 deletionfrom astrocytes result in less activation of microglia during EAE [19]. However, the mechanisms for the control effects of astrocyte CCL2 on microglial activation have not been elucidated in vitro. The purpose of the current study was to understand the role of astrocyte-derived CCL2 in microglial activation and to elucidate the underlying mechanism(s).

In the present study, we investigate the selectivity of CCL2 from astrocytes in the induction of microglial polarization in vitro. CCL2 from astrocyte selectively induced the microglial polarization into the M1 state in vitro.

Dulbecco's modified Eagle's medium (DMEM), 0.25% Trypsin-EDTA solution, fetal calf serum (FCS), penicillin/streptomycin and poly-D-lysine were purchased from Gibco-BRL (Grand Island, NY, USA). RS102895 was purchased from Sigma-Aldrich (St. Louis, MO, USA). RIPA buffer and the BCA kit were purchased from Beyotime (Shanghai, China). Fluoroshieldmounting medium with 4, 6-diamidino-2-phenylindole (DAPI) and mouse anti-OX42 monoclonal antibody were purchased from Abcam (Hongkong, China). Mouse CCL-2 ELISA kit and TNF-α were obtained from R&D Systems, Inc. (Minneapolis, MN, USA). Rabbit anti-Iba1 monoclonal antibodies and anti-GFAP polyclonal antibodies were purchased from Abcam (Hongkong, China). A FITC-conjugated goat anti-rabbit IgG antibody was purchased from Santa Cruz (Santa Cruz Biotechnology, USA). Rat anti-mouse CD86 was purchased from BD Bioscience (San Jose, CA, USA). Aleva Fluor 488-conjugated donkey anti-rat IgG was purchased from Invitrogen (Carlsbad, CA, USA).

Primary mouse microglial cells and astrocytes were prepared as previously described [20] with minimal modification. Briefly, whole brains were isolated from C57BL/6J mice at postnatal day one or two. The meninges and blood vessels were removed completely in cold D-Hank's buffered saline. Brain tissue was minced with sterile scissors and digested with 0.25% Trypsin-EDTA solution for 10 min at 37°C. Trypsinization was stopped by adding an equal volume of culture medium, which was high-glucose DMEM containing 10% FBS and penicillin (100 U/ml)/streptomycin (100 µg/ml). Dissociated cells were passed through a 100 µm pore mesh, pelleted at 1500 rpm for 5 min, and resuspended in culture medium. Cells were seeded on poly-D-lysine precoated cell culture flasks and cultured at 37 °C in a humidified atmosphere of 5% CO₂/95% air. The medium was replaced every three to four days after seeding. After the glial cells formed a confluent monolayer (10 - 14 days), microglial cells were separated from the astrocytes shaking for 5 h at 150 rpm. After an overnight of incubation at 4ºC, the cells were subjected to experimental treatments. The purity of the microglia was > 98% as determined by OX-42 (CD11b)-IR, and the purity of astrocytes was > 95 %, as determined by immunostaining glial fibrillary acidic protein (GFAP) antibody.

The expression of CCL-2 was measured with an ELISA kit from R&D Systems (Minneapolis, MN, USA).

To assess microglial activation, cells were fixed via incubation in 4% paraformaldehyde solution for 30 minutes. Non-specific binding was blocked by incubating cells in a 5% BSA and 0.1% Triton X-100 solution for 1 hour at room temperature. Microglial cells were incubated with the following primary antibodies: rabbit anti-Iba1 (1:500), and rat anti-mouse CD86 (1:200) in the blocking solution overnight at 4 °C. After three washes with PBS, cells were incubated with the corresponding FITC-conjugated goat anti-rabbit IgG and Aleva Fluor 488-conjugated donkey anti-rat IgG for 2 hours at room temperature, and the nuclei were stained with DAPI. Fluorescence images were acquired using a Leica TCS SP2 (Leica Microsystems, Buffalo Grove, IL, USA) laser scanning spectral confocal microscope. Quantification was performed using the associated Leica LCS software by placing a rectangular region of interest (ROI) across the full image and within the ROI, for every image, mean fluorescence intensity (MFI) was measured and the values were plotted.

Cellular proteins were extracted from the primary microglial cells using RIPA buffer. The homogenates were centrifuged for 15 min at 12,000 g at 4°C. The quantity of protein in each supernatant was determined using a BCA protein assay kit. Proteins (60 µg) were denatured with sodium dodecyl sulfate (SDS) sample buffer and separated using 10% SDS-polyacrylamide gel electrophoresis (PAGE). The proteins were transferred to a polyvinylidene fluoride (PVDF) microporous membrane (Millipore, Bedford, MA), which was then blocked with 5% skim milk for one hour at room temperature. The membrane was incubated with primary antibody (rabbit anti-Iba1 and/or anti-GAPDH) overnight at 4°C. After addition of the anti-rabbit secondary antibody for 1 hour, the protein bands on the membranes were detected with ECL kits (Thermo Fisher Scientific, Rockford, IL, USA). The relative density of the protein bands was evaluated by densitometry using Image Lab software (Bio-Rad, Richmond, CA, USA), and quantified by NIH ImageJ software (Bethesda, MD, USA).

Total RNA was extracted from primary microglial cells using RNAiso Plus (TaKaRa Bio, Tokyo, Japan) according to the manufacturer's protocol. cDNA was prepared from 1 µg of total RNA by using a PrimeScript RT Master Mix kit (TaKaRa Bio, Tokyo, Japan) following the standard protocols. Quantitative PCR was performed on a StepOnePlus Real-Time PCR System (ABI) using the synthetic primers and SYBR Green (TaKaRa Bio, Tokyo, Japan). Samples were subjected to 40 cycles of amplification at 95°C for 5 s and 60°C for 30 s, after holding at 95°C for 30 s. Relative expression was calculated using the 2-^{(ct experimental sample-Ct internal control sample (GAPDH))} method. Primer sequences are listed in Table 1.

Transwell migration assays were performed using 8-µm pore diameter inserts (Corning, Lowell, MA). Briefly, 2 × 10⁴ microglial cells were plated in the upper chamber with 200 µL serum-free medium. This upper chamber was then placed within the bottom wells containing 600 µL astrocyte conditioned medium. Following incubation at 37°C for 24 h, non-migrating cells on the upper surface of the membrane were carefully removed with a cotton swab. Cells on the lower surface of the membrane were first fixed in 4% paraformaldehyde for 30 min, followed by staining with 0.2% crystal violet for 1 h. For quantification, six randomly chosen fields on the lower membrane surface were imaged using computer-assisted microscopy.

All experimental results were performed in triplicate. Statistical analyses were performed using GraphPad Prism 5 software (version 5.01, GraphPad Software, San Diego, CA). The results are expressed as the mean ± s.e.m. Data were analyzed with one-way ANOVA followed by Newman-Keuls post-hoc test wherever appropriate. A p-value of < 0.05 was considered as statistically significant.

To explore possible mechanisms underlying astrocyte CCL2-induced microglial activation, CCL2 expression and release was examined in cultures using an experimental protocol designed to mimic in vivo conditions, illustrated in Fig. 1a. Incubation of astrocytes with TNF-α (10 ng/ml) for 15 min resulted in increased CCL2 expression. Interestingly, even after removal of TNF-α by three washes with PBS, CCL2 expression continued to increase 3 h after initial TNF-α stimulation (Fig. 1b). Brief application of TNF-α (15 min) also resulted in increased CCL2 release in culture medium. As long as 3 h after removal of TNF-α and replacement of medium, CCL2 release increased 10-fold (Fig. 1a and 1b).

To further confirm the role of astrocyte-derived CCL2 release in inducing microglial activation, cultured astrocytes were treated with a specific interfering RNA (siRNA) targeting CCL2. Astrocytes were incubated with CCL2 siRNA for 24 h followed by TNF-α stimulation for 15 min, and the medium and cell lysates were collected 3 h later. CCL2 siRNA treatment inhibited TNF-α-induced CCL2 expression and release in a dose-dependent manner (Fig. 2a and 2b). At a concentration of 1 and 2 µg/ml, CCL2 siRNA inhibited CCL2 expression by 41% and 83% and CCL2 release by 51% and 80%, respectively compared with non-treated astrocytes. Non-targeting siRNA demonstrated no effect on CCL2 expression and release.

After confirmation of successful silencing of CCL2 expression and release in treated astrocytes, the effect of this knockdown on microglial activation was assessed. Following incubation with CCL2 siRNA (2 µg/ml, 24h), astrocytes were stimulated with TNF-α for 15 min and washed with PBS. The culture medium was collected 3 h later for subsequent addition to microglial cultures. Anti-Iba1 antibody was used to evaluate the activation level of microglia. As evident from immunofluorescence images (Fig. 2c and 2d), medium from TNF-α-stimulated astrocytes resulted in increased expression of the Iba1 protein in microglia in comparison to the control group. However, CM from TNF-α-stimulated astrocytes pre-treated with CCL2 siRNA inhibited the expression of Iba1 protein in microglia as compared to treatment with CM from TNF-α-stimulated astrocyte group (Fig. 2c and 2d). These immunofluorescence findings were confirmed by Western blot (Fig. 2e). The increase of Iba1 protein in the microglia treated with CM from TNF-α-stimulated astrocyte was lower when compared with the microglia treated CM from TNF-α-stimulated astrocyte pretreated with CCL2 siRNA (Fig. 2e). These results indicate that astrocyte-derived CCL2 induces microglial activation.

Transwell assays were performed to determine the effects of astrocyte-derived CCL2 on the migration of microglial cells. Microglial cells were seeded into the upper chamber, which was then placed into bottom wells containing CM from astrocyte, and the migration of microglial cells from the upper to the lower surface of the membrane was assayed. As shown in Fig. 3a and 3b, the number of migrating cells was increased after incubation with CM from TNF-α-stimulated astrocyte in comparison to the number of control group. However, CM from TNF-α-stimulated astrocyte pre-treated with CCL2 siRNA reduced the number of migration cells when compared with the CM from TNF-α-stimulated astrocyte group (Fig. 3a and 3b). These results suggest that astrocyte CCL2 may lead to recruitment of microglial cells.

The effect of astrocyte-derived CCL2 on microglial activation prompted us to examine M1 marker expression in microglia. As demonstrated by immunofluorescence imaging, CM from TNF-α-stimulated astrocyte led to a higher expression of the CD86 in microglia in comparison to the control group (Fig. 4a and 4b). However, CM from TNF-α-stimulated astrocyte pretreated with CCL2 siRNA inhibited the expression of CD86 in microglia when compared with the CM from TNF-α-stimulated astrocyte group (Fig. 4a and 4b).

The findings were further confirmed by flow cytometric analysis (Fig. 4c and 4d). The expression of CD86 the microglia treated with CM from TNF-α-stimulated astrocyte was higher when compared with to microglia treated CM from TNF-α-stimulated astrocyte pre-treated with CCL2 siRNA (Fig. 4c and 4d). These results demonstrate that astrocyte CCL2 is associated with increased expression of CD86 and, therefore, M1 activation of microglia.

To investigate the effect of astrocyte CCL2 on M1 polarization, M1 and M2 markers expression in microglia were assessed by qRT-PCR. The expression of M1 markers (TNF-α, IL-1β, CD86 and iNOS) was significantly increased by CM from TNF-α-stimulated astrocyte, and this upregulation was attenuated by the CM from TNF-α-stimulated astrocyte pretreated with CCL2 siRNA (Fig. 5a-d). In contrast, there was no observable effect of CM fromTNF-α-stimulated astrocytes on the expression of M2 markers (IL-4, arginase1 and CD206), however, the expression of IL-10, one of the M2 markers was increased, while CM from TNF-α-stimulated astrocytes pre-treated with CCL2 siRNA did not demonstrate a similar decrease in IL-10 expression (Fig. 5e-h). These results indicate that astrocyte CCL2 could induce M1 polarization of microglia.

To confirm the role of the CCL2 receptor, CCR2, in astrocyte-induced microglial activation and M1 polarization, microglial cells were treated with a specific CCR2 inhibitor, RS102895. RS102895 attenuated CM from TNF-α-stimulated astrocyte induced microglial activation (Fig. 6a, 6b, 6d and 6e). Additionally, RS102895 was able to attenuate the increases in the number of migration cells induced by CM from TNF-α-stimulated astrocyte (Fig. 6c and 6f).

To investigate the role of CCR2 in M1 polarization, the expression of M1 and M2 markers were examined in RS102895-treated microglia. RS102895 significantly inhibited the expression of M1 markers (TNF-α, IL-1β, CD86 and iNOS) induced by CM from TNF-α-stimulated astrocyte but had no effect on the expression of M2 markers (IL-4, arginase1 CD206) (Fig. 6g and 6h). Furthermore, RS102895 substantially inhibited and expression of CD86 induced by CM from TNF-α-stimulated astrocyte (Fig. 6a and 6d).

In the current study, we demonstrate the pro-inflammatory properties of CCL2-CCR2 pathway in cell culture assays. TNF-α served as the stimulator of astrocyte, which induced the sustained release of CCL2. Additionally, as evidenced by siRNA experiments, CCL2 from astrocytes was associated with increased activation and enhanced migratory ability of primary microglia. To elucidate the underlying mechanism, we investigated whether CCL2 from astrocyte could induce M1 polarization in microglia. After treatment with CCL2-siRNA, the primary microglial cells cultured in astrocyte-CM showed a significantly decrease in migratory ability and M1 polarization. As CCR2 is the receptor of CCL2, an inhibitor of CCR2 was able to suppress the observed increased migratory ability and M1 polarization of microglia. These results demonstrate the ability of the CCL2-CCR2 pathway to induce migratory ability and M1 polarization of microglia.

Microglia play an important role in the pathogenesis of many neurological disorders, such as cerebral ischemia, traumatic brain injury and neuropathic pain [17,21,22,23,24,25]. Numerous studies have shown that microglia are activated in response to CNS injury and disease [26]. Activated microglia show M1 polarization, a state in which they can produce a large amount of inflammatory cytokines, leading to inflammatory tissue damage. Pharmacotherapeutic attenuation of M1 microglia could have a therapeutic consequence for reducing neuronal damage and degeneration. The migration of microglia to the site of damage/injury is a central consequence of inflammation in the brain [27]. There is increasing evidence that microglia respond to focal cerebral ischemic insults by migrating rapidly toward the lesion sites. Besides releasing pro-inflammatory mediators, microglia also respond to pro-inflammatory signals released from other cells. In this context, CCL2 may be a key contributor of this response. A growing body of evidence has demonstrated that CCL2 is associated with microglial activation in vivo. Spinal CCL2 expression and microglial activation has been found to coincide in time and space after nerve injury [28]. Administration of anti-CCL2 neutralizing antibodies, as well as CCR2 antagonists, could prevent the microglial activation cause by nerve damage and bone cancer [17,29,30,31]. Exogenous CCL2 was induced by an increase in the size of microglial cell bodies in intact wild-type mice, and these changes are abolished in CCR2-deficient mice [17]. The in vitro studies described in the current study offer further proof that CCL2 stimulates microglial activation by inducing microglial M1 polarization through its receptor, CCR2.

Reactive astrocytosis with production of NFkappaB (NFκB)-dependentpro-inflammatory molecules (including CCL2) are thought to play a role in continued inflammation and secondary injury process not only in EAE and MS, but also in other neurological disorders. Astrocyte-CCL2-CKO miceshowed a significant reduction in ramified/activated microglia as compared to WT littermates during progressive EAE [19]. CCL2 from astrocytes could increase the number of recruitment and activation of microglia in EAE [18]. In the present study, we demonstrated that knockdown the expression of CCL2 from astrocyte or addition of a CCR2 inhibitor could suppress microglial activation and M1 polarzation and migration ability in vitro. These results are in consistent with the reports above.

In summary, the present study showed thatthe CCL2/CCR2 pathway mediating astrocyte stimulated microglial activation by inducing microglial M1 polarization and migration ability, indicating that they could be used as a potential target to ameliorate neuroinflammation.

This project was sponsored by the National Natural Science Foundation of China (No. 81400889), and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

The authors declare that this article content has no conflicts of interest.

M. He and H. Dong contributed equally to this work.