The primary macrophage chemokine, CCL2, is not necessary after a peripheral nerve injury for macrophage recruitment and activation or for conditioning lesion enhanced peripheral regeneration

Abstract

Background Peripheral nerve injuries stimulate the regenerative capacity of injured neurons through a neuroimmune phenomenon termed the conditioning lesion (CL) response. This response depends on macrophage accumulation in affected dorsal root ganglia (DRGs) and peripheral nerves. The macrophage chemokine CCL2 is upregulated after injury and is allegedly required for stimulating macrophage recruitment and pro-regenerative signaling through its receptor, CCR2. In these tissues, CCL2 is putatively produced by neurons in the DRG and Schwann cells in the distal nerve. Methods Ccl2fl/fl mice were crossed with Advillin-Cre, P0-Cre, or both to create conditional Ccl2 knockouts (CKOs) in sensory neurons, Schwann cells, or both to hypothetically remove CCL2 and macrophages from DRGs, nerves or both. CCL2 was localized using Ccl2–RFPfl/fl mice. CCL2–CCR2 signaling was further examined using global Ccl2 KOs and Ccr2gfp knock-in/knock-outs. Unilateral sciatic nerve transection was used as the injury model, and at various timepoints, chemokine expression, macrophage accumulation and function, and in vivo regeneration were examined using qPCR, immunohistochemistry, and luxol fast blue staining. Results Surprisingly, in all CKOs, DRG Ccl2 gene expression was decreased, while nerve Ccl2 was not. CCL2–RFP reporter mice revealed CCL2 expression in several cell types beyond the expected neurons and Schwann cells. Furthermore, macrophage accumulation, myelin clearance, and in vivo regeneration were unaffected in all CKOs, suggesting CCL2 may not be necessary for the CL response. Indeed, Ccl2 global knockout mice showed normal macrophage accumulation, myelin clearance, and in vivo regeneration, indicating these responses do not require CCL2. CCR2 ligands, Ccl7 and Ccl12, were upregulated after nerve injury and perhaps could compensate for the absence of Ccl2. Finally, Ccr2gfp knock-in/knock-out animals were used to differentiate resident and recruited macrophages in the injured tissues. Ccr2gfp/gfp KOs showed a 50% decrease in macrophages in the distal nerve compared to controls with a relative increase in resident macrophages. In the DRG there was a small but insignificant decrease in macrophages. Conclusions CCL2 is not necessary for macrophage accumulation, myelin clearance, and axon regeneration in the peripheral nervous system. Without CCL2, other CCR2 chemokines, resident macrophage proliferation, and CCR2-independent monocyte recruitment can compensate and allow for normal macrophage accumulation.

Modulation of central synapse remodeling after remote peripheral injuries by the CCL2-CCR2 axis and microglia

“…Although we cannot rule out the possibility that motoneuron- and microglia-derived CCL2 compensate for each other, the results agree with a recent communication reaching similar conclusions in DRGs and peripheral nerves after similar injuries. 24 The main difference we observed in mice with ccl2 removed from microglia was less variation in average number of cells per section. Coefficient of variations were relatively low for MG Δ ccl2 mice (19.6%) and larger for MN Δ ccl2 (47.5%) and ccl2 flx/flx (73.0%) mice.…”

“…However, an alternative view of CCL2 being dispensable for CCR2 + cell recruitment was recently proposed. 24 …”

Frataxin deficiency in proprioceptive neurons is causal to inflammatory and glial responses in dorsal root ganglia

“…However, upregulation of two specific genes retained our attention as possible markers for a new inflammatory response shift. First, upregulation of the macrophage Ccl12 gene (orthologous to Ccl2) encoding a chemoattractant cytokine very common in the recruitment of immune cells [126][127][128] . Second, upregulation of the specific SGC Gfap gene, suggesting that SGCs have entered into a reactivity state 24,[83][84][85]119 .…”

Neither injury induced macrophages within the nerve, nor the environment created by Wallerian degeneration is necessary for enhanced in vivo axon regeneration after peripheral nerve injury

“…Accordingly, loss of either CCR2 or CCL2 was reported to dramatically impair DN and DRG macrophage accumulation [ 18 , 19 , 33 , 34 ] and CL-enhanced regeneration in vitro [ 18 , 33 ], suggesting MDMs and DRG macrophages are necessary for the CL response. However, recent studies demonstrated that the DRG macrophage response is dominated by proliferation of a true resident population [ 16 , 17 ] independent of CCR2 signaling [ 35 ]. The nerve response is dominated by MDMs and thus is impaired in Ccr2 null animals [ 17 , 19 , 36 ], although resident compensation results in only a 50% macrophage reduction [ 35 ].…”