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

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. SIGNIFICANCE STATEMENT CNS axons do not regenerate spontaneously after injury. However, preconditioning peripheral nerve injury enables dorsal root ganglia sensory neurons to regenerate central axons beyond spinal lesion. The exact mechanism by which the conditioning injury enhances axon regeneration capacity remains elusive. We report here that neuronal CCL2 induced by conditioning injury mediates neuron–macrophage interactions, resulting in accumulation of perineuronal macrophages with a proregenerative phenotype. Genetic or immunological inhibition of CCL2 abolished conditioning effects in vitro and in vivo, indicating that CCL2-mediated activation of proregenerative macrophages is essential in the conditioning injury-induced enhanced regenerative capacity. Intraganglionic CCL2 gene delivery recapitulates conditioning effects after spinal cord injury, suggesting that a chemokine signal mediating neuron–macrophage interaction may be a novel target for axon regeneration therapeutics.