In this study, we demonstrated that a population of MΦ derived from infiltrating monocytes located at the margins of the lesion site contribute to recovery following SCI by mediating an immunoregulatory role via the anti-inflammatory cytokine IL-10. These cells are pivotal and nonredundant players in the spontaneous recovery process following injury, as their CNS counterpart, the resident microglia, could not replace their functions. The physiological recruitment of the monocyte-derived MΦ to the injured spinal cord and their essential role in the recovery were demonstrated using a variety of independent techniques. We found that monocytes spontaneously infiltrate to the damaged CNS, and that their descendents preferentially accumulate at the margins of the lesion site. Augmenting the naïve monocyte pool by either adoptive transfer or CNS-specific vaccination resulted in improved recovery. By using antibody-mediated monocyte depletion in wt mice and conditional ablation of monocyte-derived MΦ in BM chimeras (resulting in the depletion of either naïve monocytes in the peripheral blood or their infiltrating descendents at the lesion margins, respectively), in a manner that spared the resident microglia, we demonstrated that monocyte-derived MΦ are pivotal for recovery. Reconstitution of the monocyte pool by wt monocyte transfer restored the improvement of motor function, whereas transfer of IL-10-deficient monocytes failed to do so.
Two recent publications have questioned the use of BM chimeras in CNS studies 
; irradiation, even in the absence of further injury, mobilized monocytes to the noninjured CNS. Our results are in line with these reports, though the numbers of these recruited cells were negligible relative to their massive infiltration following spinal cord injury. Nevertheless, the chimeras used throughout this study were prepared while shielding their heads during irradiation, a procedure that prevents any nonspecific infiltration of monocytes and therefore provides a reliable model for investigating monocyte recruitment following SCI. To unequivocally prove that this infiltration of monocytes resulted from the injury and not from the irradiation, we used passive transfer of naïve monocytes to nonchimeric wt mice, and demonstrated, in agreement with other studies 
, the spontaneous infiltration of monocytes under CNS pathological conditions.
Immune cells, and in particular MΦ/monocytes, have been recognized as a heterogeneous population in terms of their functional role, beyond host defense, in the healing of peripheral organs. However, in the CNS, which is considered an immune-privileged site, the low levels of spontaneous recovery have been attributed, at least in part, to a robust and detrimental local inflammatory response 
Our group demonstrated almost a decade ago that alternatively activated MΦ promote CNS recovery from injury; specifically, we showed that peripheral MΦ activated ex vivo by exposure to peripheral nerve tissue, when injected at a specific time frame after the injury to the margins of the lesion site, benefit repair 
. Our finding that such MΦ are needed for repair was unexpected, primarily due to the fact that the site of injury is already laden with microglia/MΦ, and it was not clear what further functionality could be provided by the additional cells that were introduced. At that early stage of our research, it was not clear whether beneficial MΦ could be induced only by local administration of autologous ex vivo–activated MΦ, or alternatively, whether protective MΦ are spontaneously recruited as part of the endogenous repair mechanism, but in numbers that are insufficient within the critical therapeutic time frame.
The present study highlights the fact that infiltrating monocyte-derived MΦ and resident microglia differ in their distribution and activities following SCI. The resident activated microglia were distributed throughout the epicenter of the lesion and at its margins. In contrast, monocyte-derived MΦ that contributed to the repair process were largely excluded from the lesion center and preferentially located at its margins. Selective ablation of monocyte-derived MΦ resulted in increased numbers of resident activated microglia and impaired recovery. Thus, the increased number of microglia failed to compensate for the loss of infiltrating monocyte-derived MΦ, suggesting that these recruited cells have a unique role in the recovery process that cannot be replaced by the resident microglia. The distinctive roles of activated microglia and this subset of monocyte-derived MΦ may be related to the activation state of these myeloid cells prior to encountering the damaged tissue; the resident microglia are embedded in the CNS prior to the injury and are immediately activated by the insult, whereas naïve infiltrating monocyte-derived MΦ do not encounter the injured CNS tissue prior to their delayed arrival to the damage site 
. Notably, although we followed the recruitment of naïve monocytes, our present study is consistent with the previous report of Rapalino et al. 
, which reported that preactivation of the transplanted MΦ is crucial for their beneficial effect; we demonstrate here that in order to be supportive, the infiltrating monocyte-derived MΦ are locally activated to acquire a nonclassical phenotype, probably en route to the site or within the local microenvironment of the lesion. Yet, the present study identifies the beneficial MΦ as part of the endogenous response to the insult, and also shows that these nonclassically activated MΦ serve an anti-inflammatory role.
Our present results suggest that the recovery following spinal injury involves monocyte-derived MΦ, yet these cells, at their spontaneous levels and activation state, are not sufficient. Increasing the naïve-monocyte pool by either adoptive transfer or CNS-specific vaccination resulted in a higher number of spontaneously recruited cells and improved recovery. This suggests that at least one of the limiting factors in the beneficial involvement of innate immune cells following CNS injury is the availability and/or the extent of the spontaneous recruitment of monocytes from the circulation. Importantly, although our current study identifies monocytes as key players in the recovery process, our findings do not contradict the established contribution of other immune components 
. Furthermore, the use of T cell–based vaccination allowed us to link monocyte involvement in the recovery process, identified in the current study, with the beneficial contribution of adaptive immunity that was previously established by our group 
. However, it remains to be shown how the stimulation of the CNS antigen-reactive T cells in the vaccination protocol used, contributes to the enhanced monocyte recruitment, be it via local modifications at the lesion site or through systemic effects.
Previous studies, which reported enhanced recovery following depletion of MΦ, mediated by injection of liposome-encapsulated clodronate 
or by blockage of their recruitment using anti-integrin antibodies 
or chemokine antagonists 
, suggested that myeloid cells are detrimental to tissue recovery. The techniques used in those studies resulted in nonselective depletion or prevented recruitment of all MΦ/myeloid cells, regardless of their phenotype, activation, location, and, most importantly, origin. However, in the present study, we ablated only activated CD11c expressing monocyte-derived MΦ, but spared the resident microglia.
Interestingly, the monocytic infiltrate, whether spontaneous or enhanced by vaccination, was not detected immediately after injury. Moreover, ablation of monocyte-derived MΦ from the second week onward had no effect on functional recovery. Taken together, these two observations suggest that the essential effect of these cells is restricted to the first week following injury, and probably between d4 and d7 postinjury. Thus, our results do not refute the potential deleterious effect of other blood-derived cells or even of other subsets of infiltrating monocytes following SCI, but rather establish that assuming all MΦ population to be destructive at all time points is an inaccurate generalization.
Importantly, our work does not contradict other studies demonstrating the benefit attained by the restriction of local inflammation at a certain time point and by a specific subpopulation of immune cells 
. Our results, however, attribute a novel anti-inflammatory function to CD11c+
monocyte-derived MΦ, which benefit the injured CNS by controlling the local immune response, rather than harm it by adding to the already-detrimental inflammation. Such regulation depends on timing and location. Thus, our study further substantiates the notion that while the global suppression of the immune system denies its potential benefit, a controlled immune response is pivotal for preventing the spread of damage following CNS insult.
Histological examination of the SCI lesion sites showed that the monocyte-derived MΦ located at the margins of the lesion site expressed immunoregulatory factors, such as arginase-I and IL-10. Notably, they share these features with the so-called myeloid-derived suppressor cells (MDSC) 
, which were originally identified in the context of tumor immune escape mechanisms 
, and more recently it has been suggested that they have a role in non-CNS tissue repair 
and in the resolution of autoimmune disease of the CNS 
. Importantly, our adoptive transfer experiments showed that IL-10-deficient monocyte-derived MΦ failed to promote recovery. This established that the anti-inflammatory cytokine IL-10, which dictates MDSC activities outside the CNS 
, is a critical factor determining the beneficial function of the monocyte-derived MΦ in CNS recovery. Further studies are needed to establish whether these infiltrating monocyte-derived MΦ, identified here as essential for CNS recovery, are related to the MDSC population. Interestingly, ablation of the monocyte-derived MΦ seemed to affect the resident microglia. Thus, the SCI lesions of DTx-treated mice displayed a consistently significant increase in IB-4+
cells in the histological examinations. Moreover, following this ablation, flow cytometric analysis showed a higher percentage of activated CD11c+
resident microglia at the lesion area. Although further experiments are required to explore this crosstalk between the MΦ infiltrate and the resident microglia, this study argues against the general assumption that any increased infiltration of immune cells to the CNS will necessarily lead to enhanced destructive inflammation, but rather reveals that the infiltrating CD11c+
MΦ locally exhibit a phenotype that mediates the down-regulation of the local immune response. It is possible that other beneficial properties of infiltrating MΦ contribute to the repair process; MΦ are capable of secreting growth and neurotrophic factors 
, phagocytosis and clearance of cell debris 
, degradation of growth-limiting scar tissue 
, and promotion of remyelination 
, all of which may lead to regeneration which is the most efficient repair process in the CNS.
Altogether, our study brings a new insight into the long-standing debate regarding the contribution of MΦ to CNS recovery. We defined a critical nonredundant role of a unique subset of infiltrating monocyte-derived MΦ that, at a specific location and time frame, mediate their beneficial activity through secretion of the immunoregulatory cytokine IL-10. This new understanding of the differential role of activated resident microglia and infiltrating monocyte-derived MΦ and their mutual relationship following CNS insult might enable the development of novel approaches to manipulate and refine the endogenous repair mechanisms, and thereby improve currently available therapies for CNS injuries.