Role of MCP-1 and CCR2 in ethanol-induced neuroinflammation and neurodegeneration in the developing brain
Neuroinflammation and microglial activation have been implicated in both alcohol use disorders (AUD) and fetal alcohol spectrum disorders (FASD). Chemokine monocyte chemoattractant protein 1 (MCP-1) and its receptor C-C chemokine receptor type 2 (CCR2) are critical mediators of neuroinflammation and microglial activation. FASD is the leading cause of mental retardation, and one of the most devastating outcomes of FASD is the loss of neurons in the central nervous system (CNS). The underlying molecular mechanisms, however, remain unclear. We hypothesize that MCP-1/CCR2 signaling mediates ethanol-induced neuroinflammation and microglial activation, which exacerbates neurodegeneration in the developing brain.
C57BL/6 mice and mice deficient of MCP-1 (MCP-1−/−) and CCR2 (CCR2−/−) were exposed to ethanol on postnatal day 4 (PD4). Neuroinflammation, and microglial activation, and neurodegeneration in the brain were evaluated by immunohistochemistry and immunoblotting. A neuronal and microglial co-culture system was used to evaluate the role of microglia and MCP-1/CCR2 signaling in ethanol-induced neurodegeneration. Specific inhibitors were employed to delineate the involved signaling pathways.
Ethanol-induced microglial activation, neuroinflammation, and a drastic increase in the mRNA and protein levels of MCP-1. Treatment of Bindarit (MCP-1 synthesis inhibitor) and RS504393 (CCR2 antagonist) significantly reduced ethanol-induced microglia activation/neuroinflammation, and neuroapoptosis in the developing brain. MCP-1−/− and CCR2−/− mice were more resistant to ethanol-induced neuroapoptosis. Moreover, ethanol plus MCP-1 caused more neuronal death in a neuron/microglia co-culture system than neuronal culture alone, and Bindarit and RS504393 attenuated ethanol-induced neuronal death in the co-culture system. Ethanol activated TLR4 and GSK3β, two key mediators of microglial activation in the brain and cultured microglial cells (SIM-A9). Blocking MCP-1/CCR2 signaling attenuated ethanol-induced activation of TLR4 and GSK3β.
MCP-1/CCR2 signaling played an important role in ethanol-induced microglial activation/neuroinflammation and neurodegeneration in the developing brain. The effects may be mediated by the interaction among MCP-1/CCR2 signaling, TLR4, and GSK3β.
KeywordsAlcohol abuse Apoptosis Chemokines Development Glia Neurodegeneration
Alcohol use disorders
Chemokine (C–C motif) receptor 2
Central nervous system
Fetal alcohol spectrum disorder
Glycogen synthase kinase 3 beta
Ionized calcium binding adaptor molecule 1
Monocyte chemoattractant protein-1
Toll-like receptor 4
Tumor necrosis factor-α
Fetal alcohol spectrum disorders (FASD) are a spectrum of defects that can present as physical, mental, and\or behavioral disabilities that stem from ethanol exposure during fetal development. FASD is estimated to affect as many as 2–5% of the population and costs the USA more than $5 billion annually . One of the most devastating effects of developmental exposure to ethanol is the permanent loss of neurons in the central nervous system (CNS) [2, 3]. Although significant progress has been achieved in this line of research, the underlying molecular mechanisms remain unclear. Ethanol-induced neuronal death is accompanied by microglial activation and neuroinflammation [4, 5, 6, 7]. Monocyte chemoattractant protein 1 (MCP-1), also called chemokine (CC motif) ligand 2 (CCL2), is a key chemokine involved in neuroinflammation [8, 9, 10]. In mouse and human brain, MCP-1 and its receptor CCR2 are primarily expressed by microglia . Although inflammatory response represents one of the first immune processes that protect an organism against diseases following injury, prolonged and sustained inflammation may have cytotoxic effects, aggravating the incidence and the severity of the disease. Increased MCP-1 expression and microglial activation have been observed in the brain of human alcoholics . It has been hypothesized that ethanol exposure may sensitize the immune system to subsequent insults and lead to excess neuroinflammation in alcohol use disorders (AUD) and FASD [12, 13]. It is also proposed that microglia primed by ethanol exposure may contribute to neurotoxicity observed in AUD and FASD [12, 13]. Elevated MCP-1 levels have been observed in brains which undergo various pathogenesis processes, such as multiple sclerosis [14, 15], stroke [16, 17], and Alzheimer’s disease patients [18, 19], and it is believed that the upregulation of MCP-1 is involved in the progression of these diseases [15, 16, 19]. Interestingly, the detrimental effects caused by overexpression of MCP-1 in those diseases were not observed in mice lacking CCR2 [20, 21, 22, 23]. Therefore, MCP-1/CCR2 signaling may play an important role in neuroinflammatory diseases. In this study, we sought to determine whether MCP-1/CCR2 signaling is involved in ethanol-induced microglial activation and neuroinflammation in the context of neurodegeneration in the developing brain. We used a well-established third trimester equivalent mouse model in which ethanol exposure caused wide spread neurodegeneration. We showed that inhibiting MCP-1/CCR2 signaling significantly reduced ethanol-induced microglial activation/neuroinflammation and ameliorated neurodegeneration in the developing brain. It appeared that ethanol-induced microglial activation/neuroinflammation was mediated by the interaction among MCP-1/CCR2 signaling, TLR4, and GSK3β.
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.
Animals and ethanol exposure
C57BL/6J mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA). C57BL/6 mice, MCP-1−/− (B6.129S4-Ccl2tm1Rol/J), and CCR2−/− (B6.129S4-Ccr2tm1Ifc/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).
Culture of microglial cells and neurons
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 . 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 . 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 . 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 .
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 . 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).
Quantitative real-time RT-PCR
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.
Immunohistochemistry and determination of activated microglia
The procedure for immunohistochemistry (IHC) has been previously described . 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 . 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 staining
Fluoro-Jade C is a sensitive fluorescent marker for degenerating neurons. The procedure for Fluoro-Jade C staining has been previously described in detail . 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).
The data were presented as mean ± SEM. Statistical significance was determined by the ANOVA followed by Tukey’s post hoc test. Differences in which p was < 0.05 were considered statistically significant.
Ethanol increases MCP-1 expression in the developing brain and cultured microglial cells
Bindarit and RS504393 block ethanol-induced caspase-3 activation in the developing brain
MCP-1−/− and CCR2−/− mice are more resistant to ethanol neurotoxicity
Bindarit and RS504393 alleviate ethanol-induced proinflammatory cytokines and microglial activation
Bindarit and RS504393 protect neurons against ethanol-induced neuronal death in co-cultures of primary neurons and microglia cells
TLR4 and GSK3β mediate ethanol-induced proinflammatory cytokines in microglial cells
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.
In this study, we showed that ethanol increased the expression of MCP-1 but not CCR2 in the brain of PD4 mice and microglia cells (SIM-A9). MCP-1 synthesis inhibitor Bindarit and CCR2 antagonist RS504393 inhibited ethanol-induced neuroapoptosis, microglial activation, and the expression of pro-inflammatory factors. Further studies using gene knock out mice confirmed that the deficiency in MCP-1 or CCR2 made mice more resistant to ethanol-induced neurodegeneration. Moreover, ethanol and MCP-1 caused more neuronal death in neuron/microglia co-cultures than neuronal culture alone. Blocking MCP-1/CCR2 signaling protected primary cortical and cerebellar neurons against ethanol-induced death in the neuron/microglia co-cultures. It appeared that TLR4 and GSK3β mediated ethanol-induced microglial activation and pro-inflammatory cytokines in cultured microglia cells, and there was considerable interaction among TLR4, GSK3β, and MCP-1/CCR2 signaling in response to ethanol exposure.
The mechanisms underlying ethanol-induced neurodegeneration in the developing brain are complex. Multiple mechanisms may be involved; these include oxidative stress [63, 64], endoplasmic reticulum (ER) stress , and interference of signaling by neurotrophic factors and disruption of microRNAs . Recent evidence indicates that neuroinflammation plays an important role in the pathogenesis of FASD and AUD [12, 66, 67]. Ethanol-induced neurodegeneration in both adult and developing brain is accompanied by microglial activation and neuroinflammation [12, 66, 68, 69]. We have recently shown that inhibition of microglial activation and neuroinflammation by minocycline offered protection against ethanol-induced neurodegeneration in the developing brain , suggesting that microglial activation and neuroinflammation may contribute to ethanol neurotoxicity in the immature CNS.
MCP-1 is a key chemokine involved in neuroinflammation. In mouse and human brain, MCP-1 and its receptor CCR2 are primarily expressed by microglia . MCP-1/CCR2 signaling is involved in numerous neuroinflammatory diseases, such as multiple sclerosis, stroke, and Alzheimer’s disease [14, 15, 16, 17, 18, 19]. Chronic ethanol exposure induces MCP-1 expression in adult human and mice brain [24, 25]. We have recently shown that ethanol stimulated MCP-1/CCR-2 signaling in the spinal cord of early postnatal mice, and knocking out MCP-1 or CCR2 made mice resistant to ethanol-induced apoptosis of spinal cord neurons , indicating the involvement of MCP-1/CCR2 signaling in ethanol-induced neurodegeneration in the developing spinal cord.
The current study showed that ethanol upregulated MCP-1 at both mRNA and protein levels but not CCR2 expression in the developing brain. Ethanol stimulation of MCP-1/CCR2 signaling was confirmed by an upregulation of the downstream effector, MCPIP (Fig. 2). Disrupting MCP-1/CCR2 signaling by either MCP-1 synthesis inhibitor or CCR2 antagonist made mice less susceptible to ethanol-induced apoptotic cell death, microglial activation, and the expression of pro-inflammatory factors (Figs. 3, 4, 5, and 6). Gene deletion of MCP-1 or CCR2 also offered protection against ethanol-induced neurodegeneration in the developing brain (Fig. 4). It appears that MCP-1 deletion provided better protection than CCR2 deletion (Fig. 4b). The underlying mechanisms are currently unknown. It is possible that MCP-1 may interact with other chemokine receptors than CCR2 in microglia. It is reported that MCP-1 binds to CCR5 in osteoblastic cells . MCP-1/CCR2 signaling plays an important role in microglial activation and neuroinflammation [8, 9, 10, 72]. Therefore, the neuroprotection caused by the disruption of MCP-1/CCR2 signaling may result from suppression of microglial activation and neuroinflammation. Using a neuron/microglia co-culture system, we demonstrated that the presence of microglial cells indeed exacerbated ethanol-induced neuronal death; while blocking MCP-1/CCR2 signaling attenuated enhanced ethanol neurotoxicity in this neuron/microglia co-culture system (Fig. 7). The result further supported the hypothesis that the activation of microglia contributed to ethanol neurotoxicity. Although our findings suggest that MCP-1/CCR2-mediated microglial activation and neuroinflammation is involved in ethanol neurotoxicity, we could not rule out the possibility of ethanol directly killing the neurons. In primary neuronal cultures alone, ethanol also reduced neuronal viability but to a lesser extent, compared to neuron/microglia co-cultures (Fig. 7). It is possible that in the brain, ethanol initially causes neuronal damage, triggering MCP-1/CCR2-mediated microglial activation and neuroinflammation, which further exacerbates neurodegeneration.
Although MCP-1 and CCR2 are primarily expressed by microglia in mouse and human brain, they are also expressed in other cell types in the brain, such as astrocytes and neurons, but to a lesser extent [8, 73, 74]. Therefore, the situation in vivo is complex and it is difficult to separate the effects of microglia-derived MCP-1 from that deprived from other cell types unless microglia-specific MCP-1 or CCR2 knock out mice are available.
TLR4, GSK3β, and p38 MAPK are important regulators of microglial activation and neuroinflammation [44, 52, 54, 75, 76]. Since ethanol had little effect on p38 MAPK in the developing brain , we focused on TLR4 and GSK3β in this study. Indeed, TLR4 and GSK3β were activated by ethanol and involved in ethanol-induced microglial activation and upregulation of proinflammatory cytokines (Figs. 8 and 9). Ethanol activated GSK3β by inducing dephosphorylation of GSK3β at Ser9 in vitro and in vivo (Fig. 9a, b). This is consistent with our previous findings that ethanol decreased the phosphorylation of GSK3β (Ser9) in the developing brain and cultured neuronal cells [60, 77]. Blocking MCP-1/CCR2 signaling partially mitigated ethanol-induced dephosphorylation of GSK3β (Ser9), therefore alleviating ethanol-mediated activation of GSK3β. Similarly, blocking MCP-1/CCR2 signaling effectively inhibited ethanol-induced upregulation of TLR4 in vitro and in vivo (Fig. 9a, b). These results suggest that MCP-1/CCR2 signaling was involved in ethanol-induced activation of GSK3β and TLR4.
There are several important implications from this study. The current finding establishes an important role of GSK3β in microglial activation and neuroinflammation. We have previously demonstrated that ethanol-induced activation of GSK3β in neurons may cause neurodegeneration in the developing brain and in vitro . Our recent study showed that minocycline inhibited GSK3β and offered protection against ethanol neurotoxicity in the developing brain . Together, these findings suggest that the activation of GSK3β in both neurons and microglia contributes to ethanol neurotoxicity, although it may operate by different mechanisms. It further supports that both neuronal and microglial GSK3β are valid therapeutic targets for the treatment of ethanol neurotoxicity in the developing CNS.
In addition to the microglial activation and neuroinflammation, stimulation of MCP-1/CCR2 signaling may cause neurotoxicity through other mechanisms. For example, the activation of MCP-1/CCR2 signaling is reported to cause ER stress through the upregulation of MCPIP in cardiomyocytes and osteoclasts . Kim et al. showed that CCR2 inhibitor attenuated ER stress and decreased the expression of inflammatory cytokines in the liver of type 2 diabetic mice . ER stress has recently been proposed as an important mechanism for ethanol-induced damage to the CNS [25, 81]. Ethanol-induced ER stress in the developing spinal cord is significantly reduced in MCP-1−/− and CCR2−/−mice . Therefore, it is also likely that ethanol-induced upregulation of MCP-1 may cause ER stress which exacerbates neurodegeneration in the developing CNS.
MCP-1/CCR2 signaling has been implicated in AUD. For example, it is shown that MCP-1/CCR2 signaling regulates voluntary ethanol consumption. MCP-1 is also involved in ethanol-induced anxiety-like behavior in adolescent rats . The current finding along with our recent study  add that MCP-1/CCR2-mediated neuroinflammation and microglial activation contribute to ethanol neurotoxicity in the developing CNS. Thus, MCP-1/CCR2 signaling is likely also involved neurological deficits in associated with FASD.
In conclusion, MCP-1/CCR2 signaling played an important role in ethanol-induced microglial activation/neuroinflammation and neurodegeneration in the developing brain. The findings offer a potential new therapeutic avenue for FASD by targeting MCP-1/CCR2 signaling pathway.
This research is supported by grants from the National Institutes of Health (NIH) (AA017226 and AA015407). It is also supported in part by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development [Biomedical Laboratory Research and Development: Merit Review (BX001721)].
Availability of data and materials
The authors agree to share all data generated from this study.
KZ performed most experiments in vitro and in vivo, and analyzed/compiled the data. HW and MX performed qPCR, IHC, and IB analyses in MCP-1−/− and CCR2−/− mice. KZ and JL contributed to the experimental design and writing of the manuscript. JAF contributed to writing and editing the manuscript. All authors read and approved the final manuscript.
All procedures were performed according to the Institutional Animal Care and Use Committee (protocol #: 2008-0401).
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The authors declare that they have no competing interests.
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