Role of MCP-1 and CCR2 in ethanol-induced neuroinflammation and neurodegeneration in the developing brain

Bindarit, an inhibitor of MCP-1 synthesis, was purchased from Cayman chemical (Ann Arbor, MI, USA). CCR2 antagonist RS504393 was purchased from TOCRIS, Inc. (Minneapolis, MN, USA). TLR4 inhibitor TAK242 was purchased from EMD Millipore, Inc. (Burlington, MA, USA). GSK3β inhibitor SB-216763 was purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Anti-MCP-1 antibody was purchased from Bio-Rad AbD Serotec, Inc. (Raleigh, NC, USA). Anti-CCR-2 antibody was purchased from BioVision, Inc. (Milpitas, CA, USA). Anti-Iba-1 antibody was purchased from Wako Chemicals USA, Inc. (Richmond, VA, USA). All other antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Other chemicals/reagents used in this project were obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA) unless stated otherwise.

C57BL/6J mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA). C57BL/6 mice, MCP-1^−/− (B6.129S4-Ccl2^tm1Rol/J), and CCR2^−/− (B6.129S4-Ccr2^tm1Ifc/J) mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA). All animals were housed in a specific pathogen-free room within the animal facilities at the University of Kentucky, and handled according to the Institutional Animal Care and Use Committee (protocol #: 2008-0401).

Postnatal day 4 (PD4) mouse pups of either sex from six litters were weighed, and randomly assigned to one of six groups: control, ethanol (EtOH), Bindarit, RS504393, EtOH plus Bindarit, and EtOH plus RS504393. Pups received a total of 5 g/kg ethanol in two subcutaneous (SC) injections which were 2 h apart; each contained ethanol (2.5 g/kg, 20% solution in saline) [24, 25]. We have previously determined that the blood alcohol concentration (BAC) was 338 mg/dl 8 h after the first injection [25]. BACs greater than 300 mg/dl are not uncommon in human alcoholics [26‐29]. Some alcoholics with a BAC > 400 mg/dl were coherent and able to drive [29]. BACs> 500 mg/dl have been reported in heavy drinkers [27, 28, 30]. Bindarit is an inhibitor of MCP-1 synthesis and RS504393 is an antagonist for CCR2; they have been extensively used to block MCP-1/CCR2 signaling in vitro and in vivo [31, 32]. Bindarit (100 mg/kg) or RS504393 (1 mg/kg) were administered by two subcutaneous (SC) injections at 24 and 0.5 h prior to ethanol exposure. The concentrations of Bindarit and RS504393 used in this study were based on previous publications [31, 32]. The control pups received an injection of equal volume of saline or DMSO, depending on whether it was an inhibitor or ethanol exposure. At 8 h after the first ethanol injection, pups were sacrificed and the brains were dissected and processed for further analysis. A diagram showing the paradigm of ethanol and inhibitor administration is demonstrated (Fig. 1).

Immortalized mouse microglia cells (SIM-A9) were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). SIM-A9 cells are a spontaneously immortalized microglial cell line that exhibits key characteristics of cultured primary microglia [33]. SIM-A9 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium supplemented with 5% horse serum, 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in 5% CO₂ in a humidified atmosphere. The culture medium was replaced with DMEM/F12 medium without FBS at 50–60% confluence overnight before indicated treatment. The cultures were exposed to ethanol (0.2, 0.4, or 0.8%) in sealed containers. The containers were placed in a humidified environment and maintained at 37 °C with 5% CO₂. Using this method, the ethanol concentration in the culture medium can be accurately maintained [34]. To block MCP-1/CCR2 signaling, SIM-A9 cells were exposed to Bindarit (300 μM) or RS504393 (100 μM) for 12 h prior to ethanol treatment. The concentrations of Bindarit and RS504393 were based on previous publications [35, 36]. To block TLR4 or GSK3β signaling, SIM-A9 cells were exposed to TAK242 (1 μM) or SB 216763 (10 μM) for 12 h prior to ethanol treatment. The concentrations of TAK242 and SB 216763 were based on previous studies [37, 38]. Primary cortical and cerebellar neurons were generated from the brain of C57BL6 mice on postnatal day 1. The methods for the isolation and culture of primary cortical and cerebellar neurons have been previously described in detail [39, 40]. Briefly, the pups were decapitated, and the brain immediately transferred into dissection medium (97.5% Hank’s balanced salt solution, 0.11 mg/ml sodium pyruvate, 0.1% glucose, 10 mM HEPES, 100 U/ml penicillin, and100 μg/ml streptomycin). The meninges were removed, and the cerebral cortices and cerebella dissected. Cerebral cortical tissues and cerebella were dissociated with 0.25% trypsin followed by 0.1% DNase treatment. Then, tissues were carefully triturated, and the cell suspension was mixed with 4% bovine serum albumin and centrifuged. The cell pellet was resuspended in Neurobasal/B27 medium containing B27 (2%), glutamine (1 mM/L), penicillin (100 U/ml), and streptomycin (100 μg/ml). Cells were plated onto poly-D-lysine (50 μg/ml)-coated cell culture plates. The co-cultures of microglia cells and primary cortical neurons or cerebellar neurons were established in 24-well cell culture plates with inserts as described previously [41]. The cortical or cerebellar neurons were maintained in the cell culture plate containing 500 μl of medium at a density of 4 × 10⁶ cells/well. SIM-A9 cells were grown in a Falcon™ Cell Culture Insert (1 μm pore size, Cat#: C353104) in a separate plate at a density of 1 × 10⁶ cells/per insert in 200 μl of medium overnight at 37 °C and 5% CO₂, then treated with Bindarit (300 μM) or RS504393 (100 μM) for 12 h. After that, the inserts containing SIM-A9 cells were placed into the wells containing the cortical neurons or cerebellar neurons. The co-cultures were then treated with ethanol for 48 h. The viability of neurons was determined by a 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described [40].

After treatment, mice were anesthetized by intraperitoneal injection of ketamine/xylazine, and the cerebral cortices and cerebella were immediately dissected. The tissue was frozen in liquid nitrogen and stored at − 80 °C. The protein from brain tissue or SIM-A9 cells were extracted and processed for immunoblotting (IB) as previously described [40]. Briefly, tissues or cells were homogenized in an ice cold lysis buffer containing 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM EGTA, 0.5% NP-40, 0.25% SDS, 1 mM PMSF, 5 μg/ml leupeptin, and 5 μg/ml aprotinin. Homogenates were centrifuged at 20,000 g for 30 min at 4 °C, and the supernatant fraction was collected. Aliquots of the protein samples were separated on a SDS-polyacrylamide gel by electrophoresis. The separated proteins were transferred to nitrocellulose membranes. The membranes were probed with primary antibodies overnight at 4 °C. The immune complexes were detected by the enhanced chemiluminescence substrate (GE Healthcare, Chalfont, Buckinghamshire, UK). The density of immunoblotting was quantified with the software of Image lab 5.2 (Bio-Rad Laboratories, Hercules, CA, USA).

After ethanol treatment, mice were decapitated and the cortex was quickly frozen in liquid nitrogen and then stored in − 80 °C until use. Total RNA was isolated using TRIZOL reagent (Invitrogen) according to the manufacturer’s instructions. 1 μg of total RNA was used for first strand cDNA synthesis (Promega, A3500). Quantitative real-time RT-PCR was performed on a Lightcycler 480 system (Roche) using a Power SYBR Green PCR Master kit (Invitrogen, 4368706) with cDNA and primers (1 μM) according to the manufacturer’s recommendation. The primers used for analysis were as follows: CCR2 forward 5′-GGTCATGATCCCTATGTGG-3′. CCR2 reverse 5′-CTGGGCACCTGATTTAAAGG-3′. MCP-1 forward 5′-CTTCTGGGCCTGCTGTTCA-3′. MCP-1 Reverse 5′-CAGCCTACTCATTGGGATCA-3′. After finishing the last cycle, a melting curve analysis was performed. Standard −ΔΔCt method was used for determining the gene expression.

The procedure for immunohistochemistry (IHC) has been previously described [42]. Briefly, the pups were decapitated and the brain tissues were removed, post fixed in 4% paraformaldehyde for 24 h and then transferred to 30% sucrose in PBS until the brain sunk to the bottom. Sagittal brain sections (20–40 μm) were cut on a freezing microtome. Floating sections were permeabilized with 0.1% Triton X-100 in PBS and incubated in 0.3% H₂O₂/50% methanol in PBS and then mounted on slides and dried. The slides were blocked with 5% normal goat serum containing 0.5% Triton X-100 in PBS at room temperature, and then incubated with primary antibodies (diluted in PBS with 1% BSA) overnight at 4 °C. The dilution for the primary antibodies was anti-cleaved caspase-3 antibody, 1:600; anti-Iba1 antibody, 1:600. After washing with PBS, slides were incubated with a biotin-conjugated goat anti-rabbit secondary antibody (1:1000) for 1 h at room temperature and then washed with PBS. Avidin-biotin-peroxidase complex was prepared according to the manufacturer’s instructions. The slides were incubated in the complex for 1 h at room temperature. After rinsing, the slides were developed in 0.05% 3, 3′-diaminobenzidine (DAB) containing 0.003% H₂O₂ in PBS.

Iba-1 IHC has been extensively used to identify microglia. Activated microglia were determined morphologically. The resting microglia exhibit long branching processes and a small cellular body, while the activated microglia have fewer but thicker processes with a larger cell body [43]. The average numbers of activated Iba-1-positive cells and all Iba-1-positive cells were calculated from three randomly selected microscopic fields, and three consecutive sections were analyzed for each brain.

Fluoro-Jade C is a sensitive fluorescent marker for degenerating neurons. The procedure for Fluoro-Jade C staining has been previously described in detail [44]. Briefly, frozen slides of brain tissue were prepared at a thickness of 10 μm and first immersed in a basic alcohol solution consisting of 1% sodium hydroxide in 80% ethanol. They were then rinsed in 70% ethanol and incubated in 0.06% potassium permanganate solution. Slides were then transferred to a 0.0001% solution of Fluoro-Jade C (Chemicon, Temecula, CA, USA), dissolved in 0.1% acetic acid vehicle. The slides were then rinsed with distilled water. The air-dried slides were then cleared in xylene for at least 1 min and then cover slipped with DPX (Sigma) non-fluorescent mounting media. The slides were examined and recorded with a fluorescent microscope (IX81, Olympus).

Ethanol exposure increases the expression MCP-1 in the adult brain of mice and human alcoholics [11, 45]. To evaluate the role of MCP-1/CCR2 in ethanol-induced damage in the developing brain, we first sought to determine the effect of ethanol on MCP-1/CCR2 expression in the developing brain. Using a well-established mouse model of postnatal ethanol exposure [2, 24, 25], we showed that ethanol increased MCP-1 expression but not CCR2 in the brain of postnatal day 4 (PD4) mice (Fig. 2a). Ethanol also upregulated MCPIP, a down-stream effector of MCP-1/CCR2 signaling. Consistently, ethanol increased mRNA levels of MCP-1 but not CCR2 (Fig. 2b). Since microglia are the major source of MCP-1, we examined the effect of ethanol on a cultured microglial cell line (SIM-A9). Ethanol drastically increased the expression of MCP-1 but not CCR2 in SIM-A9 cells (Fig. 2c).

In this model of postnatal ethanol exposure, ethanol caused a wide-spread neuroapoptosis in the developing brain, which was indicated by the expression of cleaved caspase-3 [2, 24, 25]. As shown in Fig. 3, a significant alteration in cleaved caspase-3 was observed in the cortex [F(5,30) = 59.56; p ≤ 0.05] and cerebellum [F(5,30) = 48.18; p ≤ 0.05]. Ethanol increased the expression of cleaved caspase-3 in the cerebral cortex and cerebellum. Bindarit or RS504393 treatment significantly decreased ethanol-induced caspase-3 activation. There was little caspase-3 activation in the saline-injected controls and in Bindarit- or RS504393-treated groups.

To verify the study using MCP-1 and CCR2 inhibitors, we compared ethanol-induced neuroapoptosis in the cortex and cerebellum among wild-type (WT), MCP-1^−/− mice, and CCR2^−/− mice. As shown in Fig. 4, ethanol increased the expression of cleaved caspase-3 in the cortex and cerebellum of WT mice; ethanol also increased the expression of cleaved caspase-3 in MCP-1^−/− mice and CCR2^−/− mice, but to a much lesser extent (Fig. 4a). It appeared that deletion of MCP-1 was more effective than CCR2 in terms of protective effects (Fig. 4b). The results from IB analysis confirmed that MCP-1^−/− and CCR2^−/− mice were less affected by ethanol-induced caspase 3 activation (Fig. 4b); F(2,33) = 16.57, p ≤ 0.05 for the cortex, and F(2,33) = 9.04, p ≤ 0.05, for the cerebellum. It appeared that CCR2 deletion did not offer protection in the cerebellum. MCP-1 and CCR2 deficiency-mediated protection against ethanol-induced neurodegeneration was confirmed by a decrease in Fluoro-Jade C-positive cells in MCP-1^−/− and CCR2^−/− mice, compared to WT mice following ethanol exposure (Fig. 4c).

Ethanol-induced neuronal death is accompanied by neuroinflammation which may play a role in CNS damage. We sought to determine whether Bindarit or RS504393 can reduce ethanol-induced neuroinflammation. Interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα) are major pro-inflammatory cytokines that are involved in injuries in the CNS [46, 47]. Ethanol increased the expression of IL-6 and TNFα in the brain of PD4 mice and cultured SIM-A9 cells; Bindarit and RS504393 inhibited ethanol-induced increase of IL-6 [F(3,20) = 40.12; p ≤ 0.05], and TNFα [F(3,20) = 31.21; p ≤ 0.05] in the developing brain (Fig. 5a) and in cultured SIM-A9 cells [IL6, F(3,8) = 29.31; p ≤ 0.05; TNFα, F(3,8) = 30.54; p ≤ 0.05] (Fig. 5b). Pathological activation of microglia is involved in neuroinflammation and neurodegeneration in various CNS disorders [48‐51]. We therefore examined the effect of Bindarit or RS504393 on ethanol-induced microglia activation. Microglial activation was assessed morphologically by IHC of Iba-1. Ethanol significantly increased the number of active microglia; Bindarit and RS504393 inhibited ethanol-induced activation of microglia [F(5,12) = 200.63; p ≤ 0.05] (Fig. 6).

Since inhibition of MCP-1/CCR2 signaling alleviated ethanol-induced neuroapoptosis and microglial activation in the developing brain, we hypothesized that MCP-1/CCR2-regulated microglial activation may contribute to neuronal death. To test this hypothesis, we used a co-culture system in which primary neurons (mouse cortical neurons or cerebellar neurons) and microglial cells (SIM-A9 cells) were cultured together, allowing communication without direct contact. As shown in Fig. 7a, ethanol- and MCP-1-induced neuronal death was greater in microglia/neuron co-cultures than in cerebellar neuron cultures [F(6,14) = 34.18, p ≤ 0.05] or cortical neuron cultures [F(6,14) = 32.45, p ≤ 0.05] alone. Bindarit and RS504393 significantly ameliorated ethanol-induced neuronal death in the microglia/cerebellar neuron co-cultures [F(6,14) = 28.71, p ≤ 0.05], and microglia/cortical neuron co-cultures [F(6,14) = 26.19, p ≤ 0.05] (Fig. 7b).

It is well established that TLR4 and its downstream adaptors TRIF and MyD88 regulate microglial activation and production of inflammatory mediators [52‐54]. GSK3β is also a key mediator of microglial activation and neuroinflammation [55‐57]. MCP-1 was shown to activate GSK3β signaling in human breast carcinoma cells [58]. We hypothesized that the interaction among MCP-1/CCR2, TLR4, and GSK3β may mediate ethanol-induced microglial activation and expression of proinflammatory factors. We first determined whether TLR4 and GSK3β were involved in ethanol-induced expression of proinflammatory cytokines. We pre-treated SIM-A9 cells with either TLR4 inhibitor TAK242 or GSK3β inhibitor SB216763 to block TLR4 and GSK3β signaling prior to ethanol exposure. As shown in Fig. 8, TAK242 and SB216763 drastically attenuated ethanol-induced increase in the expression of Iba-1 [F(3,8) = 40.13, p ≤ 0.05], IL-6 [F(3,8) = 56.78, p ≤ 0.05], and TNF-α [F(3,8) = 65.17, p ≤ 0.05]. We next sought to determine whether MCP-1/CCR2 signaling was involved in ethanol-activated GSK3β and TLR4. The activity of GSK3β is mainly regulated by the phosphorylation at serine 9 which results in the inhibition of GSK3β [59‐61], conversely, the phosphorylation at tyrosine 216 positively regulated its activity [62]. As shown in Fig. 9a, ethanol activated GSK3β by reducing the phosphorylation at Ser9 in SIM-A9 cells; blocking MCP-1/CCR2 signaling by Bindarit or RS504393 attenuated ethanol-induced dephosphorylation of GSK3β (Ser9) [F(3,8) = 50.87, p ≤ 0.05]. Blocking MCP-1/CCR2 signaling also inhibited ethanol-induced TLR4 (F(3,8) = 35.27; p < 0.05). Animal studies confirmed these findings and showed that Bindarit or RS504393 treatment attenuated ethanol-induced dephosphorylation of GSK3β (Ser9) [F(3,8) = 59.54; p ≤ 0.05] and upregulation of TLR4 [F(3,8) = 52.18; p ≤ 0.05], MyD88 [F(3,8) = 57.15, p ≤ 0.05], and TRIF [F(3,8) = 63.17; p ≤ 0.05] in the brain of PD4 mice (Fig. 9b).

We then investigated the interaction between TLR4 and GSK3β in response to ethanol exposure. Inhibition of TLR4 signaling by TAK242 attenuated ethanol-induced dephosphorylation of GSK3β (Ser9) [F(3,8) = 19.98; p ≤ 0.05] and upregulation of MCP-1 [F(3,8) = 56.78; p ≤ 0.05] in SIM-A9 cells (Fig. 9c). On the other hand, blocking GSK3β activation by SB216763 inhibited ethanol-induced increase of MCP-1 [F(3,8) = 70.35; p ≤ 0.05] and TLR4 [F(3,8) = 42.36; p ≤ 0.05](Fig. 9d). The results indicated that there was considerable interaction among MCP-1/CCR2 signaling, TLR4, and GSK3β in response to ethanol exposure.