Animals

Seven types of mice were used: (1) C57BL/6J mice (n = 374 and another 22 as donors of monocytes); (2) heterozygous mutant Cx₃cr1^GFP/+ mice (B6.129P- Cx₃cr1tm1Litt/J, in which one of the CX₃CR1 chemokine receptor alleles is replaced with a gene encoding GFP [green fluorescent protein] [38]; n = 12 as donors of monocytes, and another eight as donors of bone marrow [BM]); (3) CD45.1 mice (carrying an allotypic marker, CD45.1; n = 8, another 24 as donors of monocytes, and another three as donors of BM); (4) Cx₃cr1^GFP/+(CD45.1) transgenic mice (carrying both the Cx₃cr1^GFP locus and the allotypic marker, CD45.1; n = 8 as donors of monocytes, and another one as a donor of BM); (5) CD11c-DTR transgenic mice (B6.FVB-Tg Itgax-DTR/GFP 57Lan/J [39], carrying a transgene encoding a human diphtheria toxin receptor [DTR] under control of the murine CD11c promoter; n = 10 as donors of BM); (6) CD11c-DTR: Cx₃cr1^GFP/+ transgenic mice (heterozygous for both the Cx₃cr1^GFP locus and the CD11c-DTR transgene; n = 3 as donors of BM); and (7) IL-10 null mice (B6.129P2-Il10tm1Cgn/J [40]; n = 40 as donors of monocytes; a generous gift from Prof. Irun Cohen, Weizmann Institute of Science). In each case, adult males aged 8–10 wk were used. Animals were supplied by the Animal Breeding Center of The Weizmann Institute of Science. All animals were handled according to the regulations formulated by the Institutional Animal Care and Use Committee (IACUC).

BM Radiation Chimeras

[Cx₃cr1^GFP/+>wt], [CD11c-DTR>wt], [CD45.1>wt (CD45.2)], and [CD11c-DTR: Cx₃cr1^GFP/+>wt] BM chimeras were prepared by subjecting gender-matched recipient mice (8–10 wk old) to lethal whole-body irradiation (950 rad) while shielding the brain, as previously described [41]–[43]. Empirically, we found that this shielding prevented the massive infiltration of myeloid cells to noninjured spinal cords (Figure S1A, S1B). Following SCI, however, an intense infiltration of GFP⁺ cells was seen in the spinal cord of these head-shielded chimeras (Figure S1C, S1D). The mice were then reconstituted with 3–5×10⁶ BM cells harvested from the hind limbs (tibia and femur) and forelimbs (humerus) of the appropriate donor mice. BM cells were obtained by flushing the bones with Dulbecco's PBS under aseptic conditions, and then they were collected and washed by centrifugation (10 min, 1,000 rpm, 4°C). The extent of chimerism observed in these chimeras (68.5%±2.2%; [mean±standard error (SE)]) was comparable to that reported elsewhere [8],[44],[45]. To rule out the possibility that the observed homing of monocytes to the injured site of the chimeric mice was due to any side effect related to the process of the chimerism, the homing of grafted monocytes to the injured site in chimeric versus nonchimeric mice was compared; no differences were found (Figure S1E). Thus, the use of chimeras created with head shielding preserved the physiological recruitment and homing of MΦ following SCI. The chimeric mice were subjected to spinal cord contusion 8–10 wk after BM transplantation.

Antigens and Vaccination

A MOG-derived altered peptide ligand, MEVGWYRSPFDRVVHLYRNGK (MOG-45D) (an analog of pMOG 35–55), in which aspartic acid is substituted for serine [46], was prepared by the Peptide Synthesis Unit at the Weizmann Institute. 45D peptide is a weak agonist of the encephalitogenic MOG 35–55 peptide and does not induce autoimmune disease [46]. Ovalbumin (OVA) peptide was purchased from Sigma-Aldrich (Rehovot, Israel). Adult mice were vaccinated with 45D or OVA (100 µg) emulsified in an equal volume of complete Freund's adjuvant (CFA; Difco) containing Mycobacterium tuberculosis (2.5 mg/ml; Difco), as previously described [47],[48]. The emulsion (total volume 0.1 ml) was injected subcutaneously at one site in the flank, 7 d prior to the spinal cord injury.

Spinal Cord Injury

The spinal cords of deeply anesthetized mice were exposed by laminectomy at T12, and contusive (200 kdynes) centralized injury was performed using the Infinite Horizon spinal cord impactor (Precision Systems), as previously described [47],[48] causing bilateral degeneration without complete penetration of the spinal cord. The animals were maintained on twice-daily bladder expression. Animals that were contused in a nonsymmetrical manner were excluded from the experimental analysis.

Assessment of Functional Recovery from Spinal Cord Contusion

Recovery was evaluated by hind-limb locomotor performance, assessed according to the open-field Basso Mouse Scale (BMS) [49], with nonlinear scores ranging from 0 (complete paralysis) to 9 (normal mobility); each score represents a distinct motor functional state. We randomly separated the mice into groups without any preferences, while verifying that the average starting score was similar in all groups. Blind scoring ensured that observers were not aware of the treatment received by each mouse. Locomotor activity in an open field was monitored twice a week by placing the mouse for 4 min at the center of a circular enclosure (diameter 90 cm, wall height 7 cm) made of molded plastic with a smooth, nonslippery floor. Before each evaluation, the mice were carefully examined for peritoneal infection, wounds in the hind limbs, and tail and foot autophagia. Animals that showed a difference of more than 2 score points between their two hind limbs were excluded from the experimental analysis. The results showing functional outcomes presented in this study were, in each case, from a single experiment representative of several independent replicates, as indicated in the figure legends.

Diphtheria Toxin Administration

Diphtheria toxin (DTx; 8 ng/g body weight; Sigma) was injected intraperitoneally (IP), repeatedly at 1-d intervals, starting immediately after the injury, unless described otherwise. The efficiency of DTx treatment was routinely confirmed both in the periphery, by assessing the ablation of CD11c^hi cells in the spleen (Figure S2), and at the lesion site.

MC-21 Administration

MC-21 (an antibody to CCR2) [50] was injected IP starting immediately after the injury throughout the first week of recovery (d0, d1, d2, d4, and d6 postinjury).

Adoptive Monocyte Transfer

Gr1⁺ monocytes were isolated as previously reported [51]. Briefly, BM cells were harvested from the femora and tibiae of naïve mice, and enriched for mononuclear cells on a Ficoll density gradient. The CD115⁺ BM monocyte population was isolated through MACS enrichment using biotinylated anti-CD115 antibodies and streptavidin-coupled magnetic beads (Miltenyi Biotec) according to the manufacturers' protocols. Following this procedure, monocytes (wt, Cx₃cr1^GFP/+ [CD45.1] or IL-10 deficient; purity 90%) were intravenously (IV) injected (3.5×10⁶ cells per mouse) during the first week of recovery (two or three injections, as indicated in the figures). This procedure theoretically enriched the subset of “Gr1⁺ monocytes” (commonly recruited to inflamed tissues) in the peripheral blood by 3-fold, as their total numbers in the circulation is approximately 10⁶ cells.

Immunohistochemistry

Due to technical limitations of some of the antibodies that were used, two different tissue preparation protocols (paraffin embedded and microtomed frozen sections) were applied, as previously described [41]. Whenever possible, the results were confirmed using both techniques. The following antibodies were used: rabbit anti-GFP (1∶100; MBL); rabbit anti- glial fibrillary acidic protein (GFAP; 1∶100; Dako Cytomation), goat anti-IL-10 (1∶20; R&D Systems), mouse anti-arginase I (1∶100; BD Biosciences), rat anti-Ly6C (1∶200; Abcam), and hamster anti-CD11c (1∶50; Chemicon). For microglial/ MΦ labeling, TRITC- or FITC-conjugated Bandeiraea simplicifolia isolectin B4 (IB-4; 1∶50; Sigma-Aldrich) was added for 1 h to the secondary antibody solution. Secondary antibodies used included: Cy2-conjugated donkey anti-rabbit antibody, Cy2/Cy5 conjugated donkey anti-mouse antibody, Cy3-conjugated donkey anti-mouse, Cy3-conjugated donkey anti-goat, and biotin goat anti-hamster (1∶200; all from Jackson Immuno Research). Cy3-streptavidin was used for CD11c staining. The slides were exposed to Hoechst stain (1∶4,000; Invitrogen Probes) for 1 min.

Myelin integrity was qualitatively examined on paraffin-embedded sections that were stained with Luxol fast blue for myelin, and with Nissl to identify the nuclei and the thin cytoplasmic layer around them. GFAP staining was used for demarcation of the lesion site.

Isolation of Spinal Cord Cells and Flow Cytometric Analysis

Mice subjected to spinal cord injury were killed by an overdose of anaesthetic and their spinal cords were prepared for flow cytometric analysis by perfusion with PBS via the left ventricle. Spinal cord sections were cut from individual mice, including the injured site and adjacent margins, or an area distal to it (4 mm long in each of the sections), and tissues were homogenized using a software controlled sealed homogenization system (Dispomix; http://www.biocellisolation.com). The following fluorochrome-labeled monoclonal antibodies were purchased from BD Pharmingen, BioLegend, or eBioscience and used according to the manufacturers' protocols: PE conjugated anti-CD11c, MHCII, Gr1(Ly6C), CD34, Mac3, CD11b, CD31, CD4, TCRβ, and CD115 antibodies; allophycocyanin-conjugated anti-CD45.1, Gr1 (Ly6C/G), and CD8 antibodies; AlexaFluor anti-CD19 antibodies; PerCP-conjugated anti-CD11b antibody; and biotin-conjugated anti-CD115 antibody. Cells were analyzed on a FACSCalibur cytometer (BD Biosciences) using CellQuest software (BD Biosciences). Isotype controls were routinely used in all the experiments. In addition, in each experiment, relevant negative control groups were used to determine the populations of interest and to exclude others.

Data Quantification

For microscopic analysis, a Nikon fluorescent microscope (Nikon E800) or Zeiss LSM 510 confocal laser scanning microscope were used. Longitudinal sections of the spinal cord were analyzed. Numbers of cells, immunoreactivity (density) and lesion size were determined automatically with Image-Pro Plus 4.5 software (Media Cybernetics). For quantification of number of cells, a unique quantification method was used, based on Image-Pro software, that takes into consideration the accumulation of cells in clusters. Manual quantification and flow-cytometric analysis were also performed to verify the results provided by the automated analysis. In order to avoid overestimation due to counting of partial cells that appeared within the section, we took special care to count only cells with intact morphology and a nucleus that was larger than 4 µm in diameter, both in the manual and software-automated counting. Throughout the study, the area counted was 1–2 mm². The number of cells per mm³ was calculated by considering the thickness of the sections. For quantification based on FACS analysis (n = 4–6 mice per group), positive events were counted in 25% of the sample, except for the adoptive transfer experiments in which the entire sample was analyzed.

Statistical Analysis

Data were analyzed using the Student's t-test to compare between two groups. One-way or two-way ANOVA was used to compare several groups; the Tukey's HSD procedure (p = 0.05) was used for follow-up pairwise comparison of groups. Repeated measures ANOVA was used in the functional BMS scoring with follow-up comparison of treatments for each day by contrast t-test and correction for multiple comparison by the Holm method (p = 0.05). The specific tests used to analyze each set of experiments are indicated in the figure legends. The results are presented as mean±SE. In the graphs, y-axis error bars represent SE.

Macrophages Derived from Infiltrating Monocytes Are Recruited to the Margins of the Lesion Site Following Spinal Cord Injury

As circulating precursor cells, monocytes are well known to infiltrate sites of injury outside of and within the CNS [9],[36],[37]. To test their involvement in the recovery from SCI, we first performed an adoptive transfer of labeled naïve monocytes to investigate features of their recruitment and fate. The monocyte graft was isolated from Cx₃cr1^GFP/+ mice [38] (C57BL/6, CD45.1) that carry a genetic reporter gene label (Cx₃cr1^GFP/+) and an allotypic marker (CD45.1), allowing detection of graft-derived cells in the recipient animals (C57BL/6, CD45.2) by immunohistochemistry and by flow cytometric analysis, respectively. Wild type (wt) C57BL/6 mice were subjected to a severe, well-calibrated contusive SCI at the level of T-12 [47]. Following injury, the animals were injected intravenously with labeled naïve monocytes (Cx₃cr1^GFP/+/CD45.1⁺). As we did not yet know the time frame following injury at which the monocyte-derived MΦ infiltrate the CNS, and whether and when they are needed, we repeatedly injected naïve monocytes (3.5×10⁶ cells) during the first week after the injury (d0, d3 postinjury), thereby trying to ensure continuous and stable elevated levels of graft-derived naïve monocytes in the blood. This procedure theoretically enriches the subset of “Gr1⁺ monocytes” (commonly recruited to inflamed tissues) in the peripheral blood by 3-fold, as their numbers in the circulation is approximately 10⁶ cells.

Flow cytometric and histological analysis 7 d after transfer revealed the presence of labeled injected cells (GFP⁺, CD45.1⁺) at the lesion site, but not distal to it (Figure 1A–1C). Interestingly, these GFP⁺ monocyte-derived MΦ were concentrated at the margins of the lesion, but were absent from the epicenter (Figure 1C).

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Figure 1. Monocyte-derived macrophages, spontaneously recruited to the injured spinal cord following the injury, promote functional recovery.

Wild-type mice were subjected to SCI and received passive transfer (injected intravenously) of monocytes (CD45.1⁺ or Cx₃cr1^GFP) during the first week of recovery. (A–C) Spinal cords were excised 7 d after the injury and analyzed for the presence of infiltrating monocyte-derived MΦ. Flow cytometric analysis of (A) lesion area (4 mm segment) of injured spinal cord from mice treated with and without (w/o) adoptive transfer of monocytes (CD45.1⁺/CD11b⁺), indicating the arrival of graft-derived MΦ to the lesion area. (B) Flow cytometric analysis of lesion and distal areas (4 mm segment each) from injured spinal cords of adoptively transferred mice indicating the accumulation of the graft-derived MΦ (CD45.1⁺/CD11b⁺) mainly at the lesion and not at the distal areas (2,259±431 engrafted cells per gram of tissue taken from lesion area [mean±SE]). (C) Immunohistochemical analysis showing the adoptively transferred cells (Cx₃cr1^GFP/+; green) restricted to the margins of the lesion site, delineated by GFAP expression (red, right frame) (scale bar = 100 µm). (D) Similarly treated animals were followed for locomotor activity assessed according to the BMS (repeated measures ANOVA; F[between groups]_1,18 = 16.7; p = 0.0007). y-Axis error bar represents SE. (E) Mean locomotor score (BMS) of individual mice on d28 after spinal cord injury (Student's t-test; t = −5.09; df = 15; p = 0.0001), suggesting that increasing the pool of naïve monocytes by IV injection of wt mice following SCI enhanced recovery beyond spontaneous levels. The assessment of the functional outcome presented here is from one experiment representative of three independent experiments performed.

https://doi.org/10.1371/journal.pmed.1000113.g001

We next examined whether the augmentation of the physiological pool of naïve monocytes in the peripheral blood following SCI, by adoptive transfer of naïve monocytes, could enhance motor function recovery beyond the spontaneous level. Recovery of motor function was evaluated using a scale for hind limb motor ability in an open field (BMS [49]), in which a score of 0 indicates complete paralysis, and a score of 9 represents complete mobility. The augmentation of the monocyte pool resulted in recovery that exceeded the spontaneous recovery levels (Figure 1D, 1E; BMS score [mean±SE]: 4.5±0.4 and 3.0±0.1, respectively). These results suggested that monocytes can home from the peripheral blood to the margins of the lesion site following injury, and that increasing their numbers leads to improved recovery. However, these results were not sufficient to determine when the monocytes are needed, why this improved recovery does not occur spontaneously, and what the unique role of the monocyte-derived MΦ is that could not be provided by the resident microglia pre-existing in the injured site. To address these questions, we could not use this experimental paradigm of adoptive transfer of labeled monocytes to spinally injured wt mice; while the graft-derived MΦ could be discriminated from activated microglia according to their label (Figure 1), unlabeled infiltrating endogenous host monocyte-derived MΦ are indistinguishable from the resident microglia by standard immunohistochemical techniques. Thus, to differentiate between the resident activated microglia and the endogenously infiltrating monocyte-derived MΦ, we used an established BM chimera approach based on the unique radioresistance of resident microglia [9],[15],[26]. CX₃CR1 is expressed on monocyte-derived MΦ and resident microglia, and therefore, in transgenic mice that carry a GFP gene under the control of the CX₃CR1 promoter, both of these populations are labeled [38],[52],[53]. However, in [Cx₃cr1^GFP/+>wt] BM chimeras, whose wt BM was replaced with Cx₃cr1^GFP/+ BM, the GFP-label is restricted to monocytes, thereby enabling infiltrating monocyte-derived MΦ to be distinguished from resident microglia [9]. Notably, preparation of the chimeras requires irradiation, which has been reported to induce immune cell recruitment to the CNS [8],[9]. Indeed, we observed in [Cx₃cr1^GFP/+>wt] BM chimeras monocyte infiltration to both the brain (Butovsky et al., personal communication) and spinal cord, even in the absence of any injury (Figure S1A, S1B). Therefore, all the chimeric mice that were used in our study were prepared in a way that prevents irradiation-induced monocyte infiltration to the spinal cord in the absent of an insult (see Methods).

Following the SCI, both resident microglia and the infiltrating monocyte-derived MΦ expressed the activation marker IB-4 (IB-4⁺/GFP⁻ versus IB-4⁺/GFP⁺ cells, respectively) (Figure 2A). Importantly, the spatial distribution of the infiltrating endogenous monocyte-derived MΦ in the parenchyma of the injured spinal cord of the chimeric mice was similar to that seen following the passive transfer of monocytes to nonchimeric injured mice (Figure 1C); monocyte-derived MΦ were restricted to the margins of the lesion site (demarcated by GFAP expression) and excluded from the epicenter (Figure 2A, 2B). These results supported the concept, demonstrated above (Figure S1), that the infiltration of monocyte-derived MΦ to the injured spinal cord in the chimeric mice is a physiological phenomenon, and enabled us to use this model for further investigation of the monocyte-derived MΦ distribution and contribution. Notably, monocyte-derived MΦ localization to the margins of the lesion appeared to be independent of the lesion severity (Figure S3). In addition, the chimeric mice enabled us to determine the spatial distribution of the resident microglia. Thus, the activated resident microglia (IB-4⁺/GFP⁻), unlike the infiltrating monocyte-derived MΦ, were ubiquitously distributed throughout the lesion center and at its margins (Figure 2A).

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Figure 2. Monocyte-derived macrophages acquire a unique phenotype in close proximity to the lesion site.

Chimeric mice were subjected to SCI and analyzed a week later for homing of cells. (A) Cells labeled for IB-4 (red), and GFP (green) at the lesion site of [Cx₃cr1^GFP/+>wt] BM chimeric mice. (B) Cells labeled for GFAP (red) and GFP (green), demonstrating that the infiltrating myeloid cells barely penetrate the lesion epicenter. (C) Representative flow cytometric analysis showing the extent of expression of various markers by the infiltrating myeloid cells (CD11b⁺/ Cx₃cr1^GFP/+/CD45.1⁺) in the injured spinal cords of [Cx₃cr1^GFP/+ (CD45.1)>wt (CD45.2)] BM chimeras. The numbers above the bars refer to the percentage of the cells positive for the indicated marker out of the R1×R2 population (representing the infiltrating monocytes). The bars point to cells positive for the indicated marker (isotype control, gray line). (D) Spatial distribution map of monocyte-derived MΦ (GFP⁺) at specific distances relative to the epicenter of the lesion site in [Cx₃cr1^GFP/+>wt] BM chimeras based on immunohistochemical analysis. (E) High magnification of GFP⁺ cells (green) from distal and marginal areas of the lesion, demonstrating morphological differences. (F) Representative confocal micrograph of longitudinal sections from injured spinal cord of [Cx₃cr1^GFP/+>wt] BM chimeras, labeled for Ly6C (red), and infiltrating monocyte-derived MΦ by GFP (green). Lower panel: z-axis projection of a single cell. (G) Representative confocal micrograph of longitudinal sections of injured spinal cord, labeled for monocyte-derived MΦ by GFP (green) and CD11c (red). Lower panel: z-axis projection of single cell. (H) Flow cytometric analysis of distal and lesion spinal cord samples for CD11c expression on MΦ (CD11b+ cells) in [Cx₃cr1^GFP/+(CD45.1)>wt(CD45.2)] BM chimeras. Note the higher incidence of CD11c^high cells in the lesion sample. The histogram to the right is gated on CD11b⁺/CD11c^high cells at the lesion area, showing that both resident (CD45.1⁻) and infiltrating (CD45.1⁺) cells express CD11c. The dashed line demarcates the lesion site in (A), (B), (F), and (G), as determined by GFAP immunoreactivity. Scale bar in (B) and (G), represents 250 µm; in (A) and (F), 100 µm; in (E), and lower panels of (F) and (G), 10 µm. Five mice were analyzed in each group.

https://doi.org/10.1371/journal.pmed.1000113.g002

To gain further insight to the features of the infiltrating cells, we performed a comprehensive flow cytometric analysis. Because of the scarcity of the infiltrating cells, we adopted a new protocol that enabled efficient collection of the cells (see Methods for details). The bulk of infiltrating cells represented myeloid cells expressing CD11b, CD115, F4/80, Mac3, and Gr1 (Figure 2C), but lacking lymphoid or progenitor markers (Figure S4). Moreover, the cells displayed activation markers [54] including CD11a, CD11c, and MHC II (Figure 2C). Based on immunohistochemical analysis, we found that most of the GFP⁺ infiltrating cells were concentrated within the margins of the lesion (Figure 2D). These marginal infiltrating monocyte-derived MΦ acquired an activated morphology manifested by a large cell body with almost no processes, whereas the distal infiltrating cells demonstrated a multi-process morphology with a small cell body (Figure 2E). Notably, the infiltrating cells at the margins were Gr1⁺(Ly6C⁺), whereas the distal ones were Gr1⁻ (Ly6C⁻) (Figure 2F). Moreover, monocyte-derived MΦ at the margins of the lesion, unlike infiltrating cells (GFP⁺) distal to it, expressed CD11c (Figure 2G), a β-integrin expressed by activated MΦ at inflammatory lesions in the spinal cord [55]. Flow cytometric analysis of dissected spinal cord tissues confirmed the expression of CD11c by MΦ (CD11b⁺), including both infiltrating MΦ (CD45.1⁺) and activated resident microglia (CD45.1⁻), at the lesion area, but not distal to it (Figure 2H). However, the localization of the CD11c⁺ microglia changed as recovery progressed; at early time points, CD11c expressing microglia were confined to the margins (Figure 2G), whereas later (from d14 onward) these cells were also found at the epicenter of the lesion (Figure S5).

Macrophages Derived from Infiltrating Monocytes Are Involved in the Process of Recovery from Spinal Cord Injury

We next wished to investigate the role of these infiltrating cells in functional recovery. Since the spontaneously recruited monocyte-derived MΦ, located at the margins of the lesion site, expressed CD11c, we implemented a conditional in vivo cell ablation strategy targeting cells by virtue of their CD11c promoter activity. Specifically, we generated [CD11c-DTR: Cx₃cr1^GFP/+>wt] BM chimeras whose wt BM was replaced by BM of mice that carry both the GFP insertion in the Cx₃cr1 locus, and a DTR transgene under control of the CD11c promoter [39]. In the resulting mice, GFP expression could be used to trace the CNS infiltrates, and administration of DTx allowed the elimination of only the infiltrating monocyte-derived MΦ that expressed CD11c while sparing CD11c⁺ resident microglia. [CD11c-DTR: Cx₃cr1^GFP/+>wt] BM chimeras were subjected to the SCI protocol 2 mo after BM transplantation. The DTx was given immediately following the injury every other day to ensure depletion of all BM-derived cells that could be potentially recruited to the injury site. We confirmed that DTx treatment resulted in specific depletion of infiltrating monocyte-derived cells (CD11c⁺) at the injured cord (Figure 3A, 3B), but spared the resident CD11c⁺ microglia (GFP⁻) (Figure 3C). To assess recovery in the absence of CD11c⁺ monocyte-derived MΦ, we performed a similar experiment in which half of the mice received serial IP injections of DTx during the entire recovery period. Although under our experimental conditions of severe SCI spontaneous recovery was very limited, treatment with DTx further diminished this limited recovery of hind limb motor function (Figure 3D, 3E). As a corollary, depletion of the monocyte-derived MΦ resulted in a greater spread of damage, manifested by larger lesion size as determined by Luxol-Nissl staining (Figure 3F, 3G). Taken together, the results presented in Figure 1, showing that increasing the peripheral pool of naïve monocytes enhanced recovery, with the impaired spontaneous recovery following depletion of CD11c-expressing MΦ derived from infiltrating peripheral monocytes, suggest that CD11c⁺ descendants of spontaneously infiltrating monocytes contribute to the recovery process following SCI. These findings raised the further question as to why the spontaneous recovery is limited: is it due to the suboptimal number, to timing, or to activity of the infiltrating monocyte-derived MΦ?

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Figure 3. Infiltrating monocyte-derived macrophages are involved in the spontaneous process of functional recovery from spinal cord injury.

[CD11c-DTR: Cx₃cr1^GFP/+>wt] BM chimeras were generated by reconstitution of irradiated C57BL/6J mice with CD11c-DTR: Cx₃cr1^GFP/+ BM. All mice were subjected to SCI, and half of them received IP injections of DTx starting immediately after the injury, then every other day. (A) GFP staining of the injury site to detect the presence of monocyte-derived MΦ, without (w/o; left panel) or with (right panel) DTx ablation (scale bar = 250 µm). (B) Quantitative analysis of monocyte-derived MΦ (number of GFP⁺ cells) at the injured site. Treatment with DTx significantly reduced the number of monocyte-derived MΦ (Student's t-test; t = −7.39; df = 5; p = 0.0007). (C) Representative micrograph of injured spinal cord sections, labeled for monocyte-derived MΦ by GFP (green) and for CD11c (red). The sections were taken from DTx-treated animals, showing that the remaining GFP⁺ cells following ablation are CD11c negative, whereas the remaining CD11c⁺ cells are GFP⁻, representing the resident microglia (scale bar = 100 µm). (D) Hind-limb locomotor performance was assessed according to the BMS (repeated measures ANOVA; F[between groups]_1,15 = 4.88; p = 0.04). (E) Mean locomotor score (BMS) of individual mice on d28 after SCI (Student's t-test; t = −2.9; df = 5; p = 0.01). (F) Staining for myelin by Luxol (blue), and nuclei by Nissl (pink) both in the absence (w/o; upper panel) or in the presence (lower panel) of DTx (scale bar = 250 µm). (G) Quantitative analysis of the size of the injury site as a function of treatment with DTx, determined by Luxol and Nissl staining. Ablation of infiltrating CD11c⁺ monocyte-derived MΦ resulted in increased lesion size following SCI (Student's t-test; t = 3.6; df = 3.82; p = 0.02). y-Axis error bar represents SE. The assessment of the functional outcome is from one experiment, representative of two independent experiments performed.

https://doi.org/10.1371/journal.pmed.1000113.g003

Vaccination Augments Spontaneous Monocyte Infiltration to the Injured Spinal Cord

To address the factors involved in the limited spontaneous recovery, we chose an experimental paradigm involving T cell–based vaccination with a myelin-derived peptide, which has been shown to significantly enhance recovery from SCI, while avoiding the induction of experimental autoimmune encephalomyelitis [46],[56],[57]; this vaccination procedure is associated with the activation of microglia/MΦ at the injured site [47],[48],[58],[59]. We hypothesized that this vaccination protocol (using MOG-derived altered peptide [35]–[55; MOG–45D]) might affect monocyte infiltration, and thus would allow us to test whether one of the limiting factors in the spontaneous recovery is related to the process of monocyte-derived MΦ recruitment. To that end, we analyzed infiltration of monocytes in spinally injured chimeric mice that were vaccinated 1 wk before the injury. The analysis of SCI lesions of [Cx₃cr1^GFP/+>wt] BM chimeras revealed that the number of IB-4⁺ cells increased during the period of assessment in both the vaccinated and untreated mice (Figure 4A). Analysis for the presence of the GFP⁺ cellular infiltrate revealed that from d4 onward, there was a higher level of monocyte-derived MΦ in the immunized group compared to the control (Figure 4B, 4C). The effect of the vaccination on monocyte-derived MΦ recruitment was confirmed by flow cytometric analysis of [CD45.1>CD45.2] BM chimeras (1,698±361 versus 3,046±471 CD45.1⁺/CD11b⁺ infiltrating cells/mg tissue [mean±SE], in control and vaccinated groups respectively; Student's t-test p<0.05). Furthermore, the vaccination not only increased the total number of infiltrating monocyte-derived MΦ but also the abundance of these cells that expressed CD11c (Figure 4D). In accordance with the previously reported effects on recovery [47],[48], the enhanced monocyte-derived MΦ recruitment was specific for vaccination with a CNS-related antigen (45D), and was not seen following immunization with an irrelevant antigen (OVA), or challenge with CFA or PBS alone (Figure S6).

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Figure 4. Vaccination results in enhanced infiltrat? ion of monocyte-derived macrophages to the injured spinal cord.

[Cx₃cr1^GFP/+>wt] BM chimeric mice were generated by reconstitution of irradiated C57BL/6J mice with bone marrow cells from Cx₃cr1^GFP/+-transgenic mice. Half of these chimeras were vaccinated with myelin-derived altered peptide ligand (45D) 7 d before the SCI. Spinal cord sections were prepared from mice killed 1, 4, 7, 14, and 21 d after injury. (A) Quantitative analysis of cells labeled with IB-4, to identify both resident microglia and monocyte-derived MΦ, at different time points after the contusion, in either vaccinated (gray bar) or unvaccinated (black bar) mice (ANOVA; F_9,20 = 16.7, p = 0.0001). (B) Kinetics of monocyte-derived MΦ infiltration to the injured site, measured by the number of GFP⁺ cells in both vaccinated (gray bar) and unvaccinated (black bar) mice. Vaccination significantly enhanced the monocyte-derived MΦ infiltration to the injured site (ANOVA; F_9,37 = 52.12; p = 0.0001). (C) Sections from [Cx₃cr1^GFP/+>wt] BM chimeras labeled for GFP (representing infiltrating monocyte-derived MΦ; green) in unvaccinated (upper panel) and vaccinated mice (lower panel) (scale bar 250 µm). (D) Quantitative analysis of GFP⁺/CD11c⁺ cells at the injury site revealing increased numbers of infiltrating CD11c⁺ monocyte-derived MΦ following vaccination (Student's t-test; p<0.001). In the kinetic analysis, asterisks indicate significant differences between vaccinated and unvaccinated groups. Significant differences were also found between different time points. y-Axis error bar represents SE. The dashed line in (C) that demarcates the lesion site was determined according to GFAP immunoreactivity.

https://doi.org/10.1371/journal.pmed.1000113.g004

Enhanced Recovery, Achieved by Vaccination, Is Dependent on Augmentation of Spontaneous Monocyte Infiltration to the Injured Spinal Cord

Having demonstrated that vaccination induced earlier and augmented infiltration to the injured spinal cord of monocyte-derived MΦ expressing CD11c, we next used this paradigm to test whether this earlier increase of infiltration could be responsible for the observed enhanced recovery. We therefore adopted again the conditional in vivo cell ablation strategy, targeting cells by virtue of CD11c promoter activity. We vaccinated [CD11c-DTR: Cx₃cr1^GFP/+>wt] BM chimeric mice and 1 wk later subjected all mice to SCI. The animals were then separated into two groups (see Methods for details); one group received treatment with DTx along the entire recovery period and one group remained untreated. The efficiency of the ablation and its specificity for CD11c⁺/GFP⁺ monocyte-derived MΦ, but not activated CD11c⁺/GFP⁻ microglia, were confirmed by flow cytometry (Figure 5A; 6% versus 1.5% CD11c⁺/GFP⁺ cells out of CD11b⁺ cells without versus with DTx treatment, respectively) and histology (Figure S7A). The specific depletion of the CD11c⁺/GFP⁺ monocyte-derived MΦ resulted in increased numbers of CD11c⁺ resident microglia (Figure 5A; 4.6% versus 8.5% CD11c⁺/GFP⁻ cells out of CD11b⁺ cells without versus with DTx treatment, respectively), suggesting that the infiltrating monocyte-derived MΦ regulated activated resident microglia. We did not observe any significant effect on other inflammatory cell populations by the DTx treatment (Figure S7B, S7C). Importantly, in accordance with our previous studies, the vaccination resulted in enhanced recovery (Figure 3D, dark blue, versus Figure 5B, green), which was completely abrogated when the activated monocyte-derived CD11c⁺ MΦ were ablated (Figure 5B, 5C). To substantiate the functional BMS results, we analyzed the size of the lesions 21 days after injury by Luxol-Nissl staining. Spinal cords of vaccinated, DTx-treated mice showed significantly enlarged lesions as compared to injured chimeric controls (Figure 5D, 5E). Interestingly, impairment of recovery was evident earlier in the vaccinated mice than in animals that were allowed to recover spontaneously (Figure 5B versus Figure 3D), in correlation with the earlier monocyte infiltration and appearance of CD11c⁺/GFP⁺ cells in close proximity to the injury site (Figure 4B, 4D). Notably, the vaccinated mice showed improvement in motor function similar to that of the wt mice that were adoptively transferred with naïve monocytes (Figure 1D, 1E). To exclude a general toxic effect of the DTx that could directly cause a decline in motor function, we repeated the experiment using wt C57BL/6J mice that are resistant to DTx. Repeated injections of DTx starting immediately after SCI had no effect on functional motor recovery in these animals (Figure S8), supporting the reliability and specificity of this ablation procedure. The fact that both recovery and monocyte recruitment were augmented using the vaccination paradigm encouraged us to use this paradigm as a more sensitive system to further study the involvement of infiltrating monocytes in the recovery process. Therefore, MOG-45D vaccination was used in all further experiments.

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Figure 5. Conditional ablation of monocyte-derived macrophages at the injured site or antibody-mediated monocyte-depletion in the peripheral blood abolishes the augmented recovery in a paradigm of vaccination.

(A–E) [CD11c-DTR: Cx₃cr1^GFP/+>wt] BM chimeras were vaccinated 7 d before SCI and divided into two groups; one group was left untreated and one group was injected IP with DTx every other day starting immediately after the injury. (A) Flow cytometric analysis of lesion sites in injured DTx-treated and non-treated [CD11c-DTR>wt] BM chimeras. Note specific ablation of CD11c⁺ infiltrating monocyte-derived MΦ in DTx-treated mice, but persistence of CD11c⁺ resident microglia (GFP⁻). The cells in the dot plots to the right are gated according to CD11b expression, as indicated. (B) Hind-limb locomotor performance was assessed according to the BMS (repeated measures ANOVA; F[between groups]_1,23 = 144; p = 0.0001). (C) Mean locomotor score (BMS) of individual mice on d28 after spinal cord injury (Student's t-test; t = 8.6; df = 12.21; p<0.0001). (D) Staining for myelin by Luxol (blue), and nuclei by Nissl (pink) in the absence (w/o; upper panel) or in the presence (lower panel) of DTx (scale bar = 250 µm). (E) Quantitative analysis of the size of the injury site as a function of treatment with DTx, determined by Luxol and Nissl staining. Ablation of infiltrating CD11c⁺ monocyte-derived MΦ resulted in significantly increased lesion size following SCI (Student's t-test; t = −6.5; df = 8; p = 0.0002). (F–I) Depletion of Ly6C(Gr1⁺)CCR2⁺ monocytes in the blood, using MC-21 antibody (F), resulted in decreased recruitment of infiltrating monocytes to the injured cord (G), worsening of recovery (H; repeated measures ANOVA; F[between groups]_1,11 = 73.623; p = 0.0001), and larger lesion size compared to control group (I; Student's t-test; t = −3.77; df = 4.2; p = 0.017). Asterisks denote statistically significant differences between the indicated groups in (C), (E), and (I), and compared to the relevant controls in (B) and (H). y-Axis error bar represents SE. The assessment of the functional outcome presented is from one experiment representative of four independent experiments performed.

https://doi.org/10.1371/journal.pmed.1000113.g005

To provide additional independent evidence for the critical role of infiltrating monocyte-derived MΦ in the recovery from SCI, we took advantage of the anti-CCR2 antibody MC-21, which selectively depletes from the peripheral blood the Ly6C⁺(Gr1⁺)CCR2⁺ monocyte subset that can potentially infiltrate to inflamed tissues [9],[50]. Wild-type mice were vaccinated with the 45D peptide 1 wk before the injury, and then half of the mice received the MC-21 treatment at d0, d1, d2, d4, and d6 postinjury. The treatment of 45D-immunized C57BL/6 wt mice with MC-21, which ablated their Ly6C⁺ monocytes in the circulation (Figure 5F), resulted in decreased monocyte recruitment to the lesion site (Figure 5G; 3,090±581 versus 293±108 CD45.1⁺/CD11b⁺ infiltrating monocyte-derived MΦ [mean±SE] without versus with MC-21 treatment, respectively; Student's t-test; t = 5.6; df = 5; p = 0.0026), without significantly affecting the infiltration of other inflammatory cell types (Figure S9). This treatment abrogated the beneficial effect of the vaccination, as it resulted in impaired motor function (Figure 5H) and increased lesion size (Figure 5I) compared to the control group. Collectively, the data from these two independent depletion experiments established the critical contribution of monocyte-derived CD11c-expressing MΦ in the recovery process following SCI.

To provide further confirmation that the depletion of monocyte-derived MΦ was the reason for the impaired recovery following DTx administration, we examined whether restoration of the monocyte pool would reverse the functional loss that resulted from this depletion. We therefore tested whether complementation of the DTR transgenic monocytes with wt monocytes that do not harbor the CD11c-DTR transgene, and whose descendants would hence be resistant to the toxin treatment, could reverse the DTx-induced loss of recovery. Since we found that the depletion of monocyte-derived MΦ starting on the second week and onward did not impair recovery (Figure S10), we injected the [CD11c-DTR>wt] BM chimeras with DTx and transferred the resistant Cx₃cr1^GFP/+ (CD45.1) monocytes (Figure 6A) in this experiment only during the time frame of the first week (DTx was injected on d0, d2, d4, and d7; monocytes were administrated on d0, d3, and d7 post injury). In this experiment, all animals were vaccinated 1 wk before the SCI and then divided into three groups; one group was left untreated and served as a control, and the two others were subjected to DTx-mediated ablation. One of the DTx-treated groups received a passive transfer of wt (DTx-resistant) monocytes. Flow cytometric analysis on d7 revealed the accumulation of graft-derived MΦ that expressed CD11c at the lesion area, but not distal to it (Figure 6B). Immunohistochemical analysis showed the localization of these cells at the lesion margins; similar to the endogenous infiltrating myeloid cells, the graft-derived GFP⁺ MΦ hardly penetrated the epicenter of the lesion site and acquired distinct morphologies depending on their location (Figure 6C, 6D). Follow-up of locomotion activity of animals that were similarly treated revealed that the adoptively transferred wt monocytes restored the lost functional recovery of DTx-treated [CD11c-DTR>wt] BM chimeras (Figure 6E). The BMS results correlated with histological data showing significantly smaller lesions in DTx-treated mice that had received monocytes, as compared to mice that received DTx but were not replenished with monocytes (Figure 6F, 6G). Of note, these results provide further evidence against a general toxic mechanism as a cause for the functional loss induced by the depletion procedure.

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Figure 6. Replenishment of the monocyte pool restores functional recovery from spinal cord injury.

Vaccinated [CD11c-DTR>wt] BM chimeras were subjected to SCI and subsequently divided into three groups; one group remained untreated and served as a control. The two others were treated with DTx during the first week after injury with or without replenishment of their monocyte pool with Cx₃cr1^GFP/+ monocytes (CD45.1). (A) Flow cytometric analysis of the naïve monocyte graft, isolated from the BM, prior to its injection to the injured mice. Note that the vast majority of cells represent Cx₃cr1^GFP/+/CD115⁺ monocytes. (B) Flow cytometric analysis of spinal cord samples to identify descendents of grafted monocytes (CD45.1⁺). Note specific homing of transferred monocytes to the lesion area. The dot plot to the right is gated on CD11b⁺ MΦ indicating CD11c expression on both grafted and endogenous cells. (C) Immunohistological analysis of engrafted cells (GFP⁺; green), demonstrating their preferential localization to the margins of the lesion site demarcated by GFAP (red). (D) Morphological differences between marginal (right) and distal (left) engrafted cells (GFP⁺; green). (E) Mean locomotor score (BMS) for each group as a function of time postinjury. Adoptive transfer of monocytes resistant to ablation administered in parallel to DTx treatment during the first week restored recovery (repeated measures ANOVA; F[between groups]_2,29 = 15.97; p = 0.0001). (F) Staining for myelin by Luxol (blue), and nuclei by Nissl (pink) of sections taken from DTx-treated mice with or without adoptive transfer of monocytes. (G) Quantitative analysis of the size of the injury site as a function of monocyte transfer, determined by Luxol and Nissl staining. Adoptive transfer of monocytes significantly reduced the size of the lesion site compared to the group that was DTx-ablated and did not receive monocytes (ANOVA; F_2,9 = 42.17, p = 0.0001). Asterisks indicate significant differences compared to the relevant controls in (E) and between the indicated groups in (G). y-Axis error bar represents SE. Scale bar representation: (C) 100 µm; (D) 10 µm; and (F) 200 µm. The dashed line that demarcates the lesion site was determined according to GFAP immunoreactivity. The assessment of the functional outcome presented is from one experiment representative of two independent experiments performed.

https://doi.org/10.1371/journal.pmed.1000113.g006

Macrophages Derived from Infiltrating Monocytes Display Immunoregulatory Features

To gain insight into the mechanism by which the infiltrating monocyte-derived MΦ, located at the margins of the lesion site, contribute to recovery, we examined whether and how their ablation affected the level of activated resident microglia. Interestingly, both the DTx-mediated ablation of monocyte-derived MΦ and the depletion of Ly6C⁺(Gr1⁺) monocytes from the peripheral blood caused an overall increase in the number of IB-4⁺ cells, indicating enhanced activation of the resident microglia in the absence of infiltrating monocyte-derived MΦ (Figure 7A–7C). Furthermore, transfer of wt monocytes following ablation restored the regulation of the local immune response, as the numbers of activated resident microglia were decreased to levels similar to those found in mice that were not treated with DTx (Figure 7D). These results suggested that the beneficial effect of the infiltrating monocyte-derived MΦ might be due to a regulatory role in controlling the local inflammation, induced following SCI.

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Figure 7. Infiltrating monocyte-derived macrophages exhibit an immunoregulatory phenotype.

[CD11c-DTR: Cx₃cr1^GFP/+>wt] BM chimeras were treated and analyzed as follows. (A) Labeling of spinal cord sections with IB-4 (red) to detect the presence of local inflammation, in DTx untreated (w/o), and treated mice. (B, C) Quantitation of IB-4⁺ cells representing local inflammation, in mice treated with DTx along the entire recovery process, only during the second week, or untreated (B; ANOVA; F_2,11 = 35.69; p = 0.0001); or with and without treatment with MC-21 (C; Student's t-test; t = −4.9; df = 8; p = 0.001). (D) Quantification of the number of IB-4⁺ cells in injured mice, with and without adoptive transfer of DTx-resistant monocytes given in parallel with the DTx treatment (ANOVA; F_2,8 = 6.1; p = 0.025). (E) Arginase I–expressing cells (ARG; red) are restricted to the margins of the lesion site demarcated by GFAP (green). (F) In [Cx₃cr1^GFP/+>wt] BM chimeras, infiltrating monocyte-derived MΦ (GFP⁺; green) expressed arginase I (ARG; red), arrows indicate double-labeled cells. (G) Interleukin 10 (IL-10; red) expression is confined to the margins of the lesion site demarcated by GFAP (green). (H) In [Cx₃cr1^GFP/+>wt] BM chimeras, monocyte-derived MΦ (GFP⁺; green) expressed IL-10 (red) only in close proximity to the lesion site. Right panel represents z-axis projection of individual cell. (I) In [CD11c-GFP>wt] BM chimeras, monocyte-derived MΦ that expressed CD11c (GFP⁺; green) coexpressed IL-10 (red). Right panel represents z-axis projection of individual cell. (J) Graft-derived MΦ (GFP⁺) expressing interleukin 10 (IL-10; red) at the margins of the lesion site following adoptive transfer to DTx-treated mice. Asterisks denote statistically significant differences between the indicated groups. y-Axis error bar represents SE. Scale bar in (A) represents 250 µm; in (E), (G), (H), and (I), 100 µm; in (J), 50 µm; and in (F) and right panels of (H) and (I), 10 µm. The dashed line that demarcates the lesion site was determined according to GFAP immunoreactivity.

https://doi.org/10.1371/journal.pmed.1000113.g007

To investigate the factors mediating this regulatory effect, we tested whether these cells exhibited characteristics associated with immune-regulating activities [60],[61]. Interestingly, analysis of lesion sites revealed that the monocyte-derived MΦ that were located at the margins of the lesion site, but not the distal ones, expressed arginase-I (Figure 7E, 7F) and the anti-inflammatory cytokine IL-10 (Figure 7G–7I). Moreover, ablation of CD11c-expressing monocyte-derived MΦ resulted in an overall decreased level of IL-10 at the injury site of [CD11c-DTR>wt] BM chimeras (Figure S11). Furthermore, adoptively transferred naïve monocytes that were injected to the DTx-treated mice, and were shown to restore recovery, also expressed similar immunoregulatory features upon encountering the lesion margins (Figure 7J).

The Contribution of Infiltrating Monocyte-Derived Macrophages to the Recovery Process Is Crucially Dependent upon Their Expression of IL-10

To investigate the specific contribution of IL-10 to the recovery-promoting activity of monocyte-derived MΦ, we compared the functional recovery of DTx-treated mice that were engrafted with wt monocytes to that of mice that were engrafted with monocytes derived from IL-10–deficient mice. [CD11c-DTR>wt] BM chimeric mice were immunized with 45D and subjected a week later to SCI. After the injury, the mice were separated into four groups: one group was left untreated, one group was treated with DTx, and the other two groups received DTx and a passive transfer of DTx-resistant monocytes either from wt or from IL-10 deficient mice. The analysis of DTx-treated [CD11c-DTR (CD45.1)>wt (CD45.1)] BM chimeras that had received an adoptive transfer of CD45.2⁺ monocytes (IL-10 deficient or wt) established that graft-derived IL-10–deficient MΦ arrived at the lesion area and expressed CD11c, similarly to their wt counterparts (Figure 8A). However, in contrast to the engraftment with wt monocytes, transfer of IL-10-deficient monocytes failed to restore recovery, and resulted in similar motor function as that observed in the DTx-treated mice that did not receive monocytes (Figure 8B). Moreover, replenishment of IL-10–deficient monocytes did not reduce the lesion size nor restrict the activation of resident microglia (Figure 8C, 8D). Furthermore, the absence of IL-10 production by monocytes had no significant effect on the composition of other inflammatory infiltrates (Figure S12). These results establish that expression of IL-10 by monocyte-derived MΦ is a critical factor determining their ability to control the local immune response and to contribute to the motor function recovery.

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Figure 8. The contribution of monocyte-derived macrophages to the healing process is crucially dependent upon their expression of IL-10.

[CD11c-DTR (CD45.1)>CD45.1] BM mice were vaccinated with 45D, a week later subjected to SCI, and then divided into four groups. One group remained untreated and served as a control. The three other groups were treated during the first week, with DTx and with or without administration of DTx-resistant monocytes isolated from either wt or IL-10–deficient mice. (A) Flow cytometric analysis of engrafted wt or IL-10– deficient (IL-10D) monocytes (CD45.2) found at the lesion area of vaccinated [CD11c-DTR (CD45.1)>CD45.1] BM chimeras treated with DTx. The cells in the dot plots to the right were gated according to CD45.2 expression, as indicated. MΦ derived from wt and IL-10–deficient monocytes were located at the lesion area, and expressed CD11c to a similar extent (284.4±45.9 versus 301.4±10.8 CD11b⁺/CD45.2⁺ [mean±SE] engrafted IL-10 deficient and wt MΦ, respectively; Student's t-test; t = 0.36; df = 4.4; p = 0.73). (B) Mean locomotor score (BMS) for each group as a function of time post injury (repeated measures ANOVA; F[between groups]_3,33 = 23.4; p = 0.0001). (C) Quantitative analysis of the size of the injury site, determined according to Luxol-Nissl staining, as a function of the IL-10 expression by the transferred monocytes (wt; IL-10D) (ANOVA; F_3,29 = 8.32, p = 0.0004). (D) Quantification of the number of IB-4⁺ cells in the lesion area of the different groups (ANOVA; F_3,24 = 21.05; p = 0.0001). Asterisks denote statistically significant differences between the indicated groups in (C) and (D), and compared to the relevant controls in (B). y-Axis error bar represents SE. The assessment of the functional outcome presented is from one experiment representative of two independent experiments performed.

https://doi.org/10.1371/journal.pmed.1000113.g008