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, 35 (48), 15934-47

CCL2 Mediates Neuron-Macrophage Interactions to Drive Proregenerative Macrophage Activation Following Preconditioning Injury

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CCL2 Mediates Neuron-Macrophage Interactions to Drive Proregenerative Macrophage Activation Following Preconditioning Injury

Min Jung Kwon et al. J Neurosci.

Abstract

CNS neurons in adult mammals do not spontaneously regenerate axons after spinal cord injury. Preconditioning peripheral nerve injury allows the dorsal root ganglia (DRG) sensory axons to regenerate beyond the injury site by promoting expression of regeneration-associated genes. We have previously shown that peripheral nerve injury increases the number of macrophages in the DRGs and that the activated macrophages are critical to the enhancement of intrinsic regeneration capacity. The present study identifies a novel chemokine signal mediated by CCL2 that links regenerating neurons with proregenerative macrophage activation. Neutralization of CCL2 abolished the neurite outgrowth activity of conditioned medium obtained from neuron-macrophage cocultures treated with cAMP. The neuron-macrophage interactions that produced outgrowth-promoting conditioned medium required CCL2 in neurons and CCR2/CCR4 in macrophages. The conditioning effects were abolished in CCL2-deficient mice at 3 and 7 d after sciatic nerve injury, but CCL2 was dispensable for the initial growth response and upregulation of GAP-43 at the 1 d time point. Intraganglionic injection of CCL2 mimicked conditioning injury by mobilizing M2-like macrophages. Finally, overexpression of CCL2 in DRGs promoted sensory axon regeneration in a rat spinal cord injury model without harmful side effects. Our data suggest that CCL2-mediated neuron-macrophage interaction plays a critical role for amplification and maintenance of enhanced regenerative capacity by preconditioning peripheral nerve injury. Manipulation of chemokine signaling mediating neuron-macrophage interactions may represent a novel therapeutic approach to promote axon regeneration after CNS injury.

Keywords: CCL2; axon regeneration; cAMP; chemokine; macrophage; neuron–macrophage interaction.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1.
Figure 1.
Identification of CCL2 as a potential chemokine in the DRGs after SNI. A, Color-coded heat maps of expression levels measured by chemokine PCR array. Fold changes in chemokine expression level relative to that in the control group were log2-transformed and color-coded based on the color scale shown at the top. The chemokines and chemotaxis-related molecules shown here belonged to the cluster of genes whose expression was already elevated 1 d after SNI. The signals from three independent arrays were averaged to obtain the expression level at each time point. B, A quantification graph of real-time RT-PCR results for CCL2 gene expression in DRG samples obtained before (0 d) or 1, 3, and 7 d after SNI. N = 3 animals for each time point. ***p < 0.001 compared with control (0 d) values by one-way ANOVA followed by Tukey's post hoc analysis. C–F, Representative immunofluorescence images of L5 DRG tissue sections from an uninjured control (CTL) animal (C) or obtained 1 d after SNI (D), dorsal column injury (DCI; E), or rhizotomy (F). Sections were stained with a CCL2 antibody (red) and a NeuN antibody (green). Arrows indicate cells positive for both markers. Scale bars, 50 μm.
Figure 2.
Figure 2.
CCL2 plays an essential role in neuron–macrophage interactions in vitro. A, A quantification graph of real-time RT-PCR results for CCL2 expression in cultured DRG neurons treated with db-cAMP (cAMP) or PBS. N = 3 independent cultures for each condition. ***p < 0.001 compared with PBS by unpaired t tests. B, ELISA measurement of the CCL2 concentration in the cell culture media obtained from different culture conditions. N + M, Neuron–macrophage cocultures; N, neuron-only cultures; M, macrophage-only cultures. N = 3 independent cultures for each condition. ***p < 0.001 by unpaired t test. C, Representative images of β III tubulin-positive cultured DRG neurons grown for 15 h with CM obtained from neuron–macrophage cocultures. To obtain the CM, neuron–macrophage cocultures were treated first for 24 h with PBS or cAMP together with control IgG or CCL2-neutralizing antibodies (αCCL2) and then the IgG or CCL2-neutralizing antibodies were maintained in the cultures during the subsequent 72 h CM collection period (CM). In other outgrowth assays, CCL2-neutralizing antibodies were added directly to CM obtained from neuron–macrophage cocultures treated with cAMP so that the antibodies were present only during the neurite outgrowth assay (NOA). D, E, Quantification graphs of neurite length in the experiments in which the antibodies were present during the neuron–macrophage cocultures (CM collection period; D) or only during the NOA (E). N = 4 independent cultures using independent CMs for each condition. ***p < 0.001 compared with PBS values by one-way ANOVA followed by Tukey's post hoc analysis. Scale bars, 100 μm.
Figure 3.
Figure 3.
CCL2 in neurons and CCR2/CCR4 in macrophages are required for in vitro neuron–macrophage interactions to produce proregenerative activity. A, C, E, G, I, Representative images of neurite outgrowth in DRG neuron cultures treated with CM obtained from neuron–macrophage cocultures using WT, CCL2-deficient (CCL2−/−), or CCR2-deficient (CCR2−/−) neurons (N) or macrophages (M). Genotype conditions for the cocultures to obtain CM were indicated at the left of the representative images. All cocultures were treated with either PBS or db-cAMP (cAMP), and the CMs were collected for 72 h. I, C 021, CCR4 antagonist, was added at a concentration of 0.1 μm during the coculture period. WT neurons were used for the neurite outgrowth assays for all conditions. DRG neurons and their neurites were visualized by immunofluorescence staining for β III tubulin. Scale bars, 100 μm. B, D, F, H, J, Quantification graphs of neurite outgrowth in the presence of CM from cultures of the different neuron–macrophage genotype combinations treated with PBS or cAMP. N = 4 independent cultures using independent CMs for each condition. ***p < 0.001 and **p < 0.01 compared with PBS values by unpaired t test.
Figure 4.
Figure 4.
Changes in the number of macrophages in DRG, axon growth capacity, and expression of RAGs in CCL2-deficient mice. A, B, Representative images of Iba1-positive macrophages in DRGSs of WT (A) and CCL2−/− (B) mice at different time points after SNI. C, Quantification of the number of macrophages in WT and CCL2−/− mice 0 (CTL), 1, 3, and 7 d after SNI. N = 3 animals for each condition. Scale bars, 50 μm. D, E, Representative images of neurite outgrowth of DRG neurons taken from WT (D) and CCL2−/− (E) mice at different time points after SNI. Neurons from the L4, L5, and L6 DRGs were cultured for 15 h before being fixed for the immunofluorescent visualization of neurites with anti-β III tubulin. Scale bar, 100 μm. F, Comparison of the mean neurite length between cultures from WT and CCL2−/− mice 0 (CTL), 1, 3, and 7 d after SNI. N = 4 animals for each condition. ***p < 0.001 compared with WT values by unpaired t test. G, H, J, K, Representative immunofluorescence images of GAP-43 (G, H) or c-Jun (J, K) staining in L5 DRG sections obtained from WT (G, J) and CCL2−/− (H, K) mice at different time points after SNI. Scale bars, 50 μm. I, L, Quantification graphs of the percentage of GAP-43-positive (F) or c-Jun-positive (I) cells in WT and CCL2−/− mice 0 (CTL), 1, 3, and 7 d after SNI. N = 3 animals for each condition. ***p < 0.001 compared with WT values by one-way ANOVA followed by Tukey's post hoc analysis.
Figure 5.
Figure 5.
Intraganglionic injection of CCL2 neutralizing antibodies abolishes conditioning effects. A, Representative images of Iba1 staining in L5 DRGs obtained 7 d after SNI of animals with intraganglionic injection of IgG or CCL2-neutralizing antibody (αCCL2). The injection was performed immediately after SNI. Scale bars, 50 μm. B, Comparison of the number of macrophages. N = 3 animals per group. ***p < 0.001. C, Representative images of neurons cultured from L5 DRGs with intraganglionic injection of IgG or αCCL2 of animals subjected 7 d previously to SNI. Scale bars, 100 μm. D, Quantification of neurite outgrowth. N = 3 animals per group. ***p < 0.001.
Figure 6.
Figure 6.
CCL2 is sufficient for enhancement of axon regenerative capacity by mobilizing M2-like macrophages. A, Representative images of Iba1 staining in L5 DRG sections obtained 7 d after intraganglionic injection of PBS or CCL2, CXCL1, and CCL3. Scale bars, 50 μm. B, Representative images of neurons cultured from L5 DRGs dissected from animals with intraganglionic injection of PBS, or CCL2, CXCL1, and CCL3. Scale bars, 100 μm. C, Quantitative comparison of the number of Iba1-positive macrophages in DRGs. N = 3 animals per group. ***p < 0.001. D, Quantitation of neurite outgrowth. The culture period was 15 h and DRG neurons and their neurites were visualized by immunofluorescence staining for β III tubulin. N = 3–4 animals per group. ***p < 0.001. E, Real-time RT-PCR results for M1 and M2 markers in cultured macrophages treated with CCL2, CX3CL1, or CCL3 for 24 h. N = 3 independent cultures per group. ***p < 0.001 compared with the control (untreated) condition. F, Real-time RT-PCR results for of M1 and M2 marker gene expression in MACS-separated (using CD68 antibody) macrophages from the L4 and L5 DRGs at the indicated time points after SNI (CD68-positive fraction). N = 4 animals per group. **p < 0.01 and ***p < 0.001 compared with control values, respectively, by one-way ANOVA followed by Tukey's post hoc analysis.
Figure 7.
Figure 7.
Intraganglionic gene delivery by AAV5. A–C, Confocal images of GFP-positive and NeuN-stained cells in the L5 DRG 7 d (A), 14 d (B), and 28 d (C) after intraganglionic injection of AAV5-GFP. Arrows indicate GFP+/NeuN+ cells. Scale bars, 100 μm. D–F, Confocal images of DRG sections double stained for CCL2 (green) and NeuN (red) 7 d (D), 14 d (E), and 28 d (F) after intraganglionic injection of AAV5-CCL2. Arrows indicate CCL2+/NeuN+ cells. Scale bars, 100 μm.
Figure 8.
Figure 8.
Intraganglionic AAV5-CCL2 injection increases the number of macrophages in the DRGs and enhances neurite outgrowth. A, B, Representative images of Iba1 staining in L5 DRG sections obtained 7, 14, and 28 d after intraganglionic injection of AAV5-GFP (A) or AAV5-CCL2 (B). Scale bars, 50 μm. C, A quantification graph comparing the mean number of Iba1-positive macrophages in DRGs injected with AAV5-GFP or AAV5-CCL2. N = 4 animals per group. *p < 0.05 and ***p < 0.001, respectively, by unpaired t test. D, Representative images of DRG sections stained with neurofilament (NF; green) and Iba1 (red) 28 d after intraganglionic injection of AAV5-CCL2. Scale bars, 50 μm. E, Representative images of neurons cultured from L5 DRGs freshly dissected from animals subjected 28 d previously to intraganglionic injection of AAV5-GFP or AAV5-CCL2. The culture period was 15 h and DRG neurons and their neurites were visualized by immunofluorescence staining for β III tubulin. Scale bars, 100 μm. F, A quantification graph comparing the mean neurite length. ***p < 0.001 by unpaired t test.
Figure 9.
Figure 9.
CCL2 overexpression promotes axon regeneration in a spinal cord injury model. A–D, Representative images of CTB-labeled axons (red) and GFAP-immunostained spinal cord sections (green) from animals injected with AAV5-GFP 7 d before injury (A), with AAV5-CCL2 7 d before injury (B, −7D), or with AAV5-CCL2 1 d after injury (C, +1D), and those subjected to preconditioning SNI before creating the spinal lesion (D). Dashed lines indicate caudal lesion borders as determined by GFAP immunostaining. The boxed regions in A–D are magnified in the center panels. Scale bars, 100 μm. A'–D', Axons regenerating beyond the caudal lesion border were reconstructed using Photoshop software from six consecutive parasagittal sections collected at a 60-μm intersection interval. The virtual section image uses different colors to distinguish the different sections. Scale bars, 100 μm. E, A quantification graph comparing the mean total axon length beyond the caudal lesion border measured in the composite images. ***p < 0.001 compared with the AAV5-GFP control value by one-way ANOVA followed by Tukey's post hoc analysis. F, A quantification graph of the mean longest distance of axon regeneration beyond the caudal lesion border. ***p < 0.001 compared with the AAV5-GFP control value by one-way ANOVA followed by Tukey's post hoc analysis. G, A quantification graph comparing the number of CTB-positive axons at different distances from the lesion epicenter. Negative values for distance indicate areas caudal from the epicenter. *p < 0.05 and ***p < 0.001 between animals injected with AAV5-GFP and those with AAV5-CCL2 7 d before the lesion (−7D); ##p < 0.01 and ###p < 0.001 between animals with AAV5-GFP and those with AAV5-CCL2 1 d after the lesion (+1D); §§p < 0.01 and §§§p < 0.001 between animals with AAV5-GFP and those subjected to SNI 7 d before the spinal lesion, by one-way ANOVA followed by Tukey's post hoc analysis. N = 8, 8, 9, and 6 animals for the AAV5-GFP, AAV5-CCL2 (−7D), AAV5-CCL2 (+1D), and SNI groups respectively.
Figure 10.
Figure 10.
Overexpression of CCL2 did not induce neuropathic pain. A, Representative images of CGRP-immunostained lumbar spinal cord sections obtained from animals subjected to intraganglionic injection of AAV5-GFP or AAV5-CCL2 7 d before or 1 d after dorsal hemisection spinal cord injury. Scale bars, 200 μm. B, Graphs showing the withdrawal response latency (in seconds) to nociceptive heat stimulation 14 and 28 d after spinal cord injury in animals with intraganglionic injection of AAV5-GFP or AAV5-CCL2 7 d before or 1 d after the dorsal hemisection spinal injury. C, Graphs showing the flexor reflex withdrawal threshold (in grams) after stimulation of the plantar surface with a series of Von Frey hairs in the same animals. N = 8, 8, and 9 animals for the AAV5-GFP, AAV5-CCL2 (−7D), and AAV5-CCL2 (+1D) groups, respectively.
Figure 11.
Figure 11.
A diagram of the proposed model for the role of CCL2-mediated macrophage activation following preconditioning SNI. A, An axotomy at the sciatic nerve produces injury signals that are retrogradely transmitted to neurons in the DRGs. B, The injury signals induce initial activation (dotted black arrows) of multiple RAGs. Activation of a particular RAG (RAGX) may be specifically linked to the production of CCL2 by DRG neurons (dotted blue arrows). C, Activated macrophages (M2-polarized) expressing CCR2 provide proregenerative factors resulting in the amplification and maintenance (thick red arrows) of RAGs. Changes in the tip morphology of central axons reflect the enhanced regenerative capacity of DRG neurons.

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