Macrophage-based delivery of interleukin-13 improves functional and histopathological outcomes following spinal cord injury

Spinal cord injury (SCI) elicits a robust neuroinflammatory reaction which, in turn, exacerbates the initial mechanical damage. Pivotal players orchestrating this response are macrophages (Mφs) and microglia. After SCI, the inflammatory environment is dominated by pro-inflammatory Mφs/microglia, which contribute to secondary cell death and prevent regeneration. Therefore, reprogramming Mφ/microglia towards a more anti-inflammatory and potentially neuroprotective phenotype has gained substantial therapeutic interest in recent years. Interleukin-13 (IL-13) is a potent inducer of such an anti-inflammatory phenotype. In this study, we used genetically modified Mφs as carriers to continuously secrete IL-13 (IL-13 Mφs) at the lesion site. Mφs were genetically modified to secrete IL-13 (IL-13 Mφs) and were phenotypically characterized using qPCR, western blot, and ELISA. To analyze the therapeutic potential, the IL-13 Mφs were intraspinally injected at the perilesional area after hemisection SCI in female mice. Functional recovery and histopathological improvements were evaluated using the Basso Mouse Scale score and immunohistochemistry. Neuroprotective effects of IL-13 were investigated using different cell viability assays in murine and human neuroblastoma cell lines, human neurospheroids, as well as murine organotypic brain slice cultures. In contrast to Mφs prestimulated with recombinant IL-13, perilesional transplantation of IL-13 Mφs promoted functional recovery following SCI in mice. This improvement was accompanied by reduced lesion size and demyelinated area. The local anti-inflammatory shift induced by IL-13 Mφs resulted in reduced neuronal death and fewer contacts between dystrophic axons and Mφs/microglia, suggesting suppression of axonal dieback. Using IL-4Rα-deficient mice, we show that IL-13 signaling is required for these beneficial effects. Whereas direct neuroprotective effects of IL-13 on murine and human neuroblastoma cell lines or human neurospheroid cultures were absent, IL-13 rescued murine organotypic brain slices from cell death, probably by indirectly modulating the Mφ/microglia responses. Collectively, our data suggest that the IL-13-induced anti-inflammatory Mφ/microglia phenotype can preserve neuronal tissue and ameliorate axonal dieback, thereby promoting recovery after SCI.


Background
Following spinal cord injury (SCI), a wide variety of secondary injury processes are provoked, which significantly worsens patients' outcomes [1]. Among these, the most complex and important one is the neuroinflammatory response that contributes to progressive neurodegeneration. Macrophages (Mφs) and microglia are one of the key players, with a predominant role for Mφs, which accumulate specifically at the lesion epicenter [1,2]. At the injury site, local factors (e.g., cytokines, myelin debris, and apoptotic cells) determine the phenotypic properties of these cells, rendering them either detrimental or beneficial for disease pathology [1,2]. A general pragmatic classification divides them into (i) classically activated M1 Mφs, which secrete pro-inflammatory factors and induce axonal dieback, and (ii) alternatively activated M2 Mφs, which display anti-inflammatory properties and support axonal regrowth [3,4]. The M1/M2 dichotomy is an oversimplified model as Mφs are very heterogenic with remarkable functional plasticity [5]. Nevertheless, the M1/M2 paradigm helped to identify significant functional characteristics of selected Mφ populations. In particular, M2a Mɸs have been identified as essential players in neuroregeneration by counteracting inflammation and promoting wound healing [6,7]. This subset is characterized by upregulation of the signature molecules Arginase-1 (Arg1), found in inflammatory zone 1 (FIZZ1), chitinase-3-like protein 3 (Chi3l3, Ym1), and mannose receptor (CD206) [8,9]. This M2 Mφ subpopulation arrives first at the lesion site in the acute phase after injury but is only transiently detectable [3]. After SCI, the perilesional milieu favors M1 polarization, resulting in a persistently hostile environment for repair [1,3]. As this is a major contributor to poor regeneration, reducing the M1 dominance and promoting an M2 presence has become an attractive therapeutic approach for SCI.
In this study, the goal was to modulate perilesional Mφs/microglia and specifically exploit their M2a phenotype to influence motor recovery beneficially. For this aim, we used the canonical T-helper cell type 2 cytokine interleukin-13 (IL-13), which is one of the main inducers of this M2a subset [9]. Following SCI, IL-13 level dramatically decreases in the spinal cord tissue [10,11]. This drop suggests that the application of IL-13 in the acute phase after SCI may have therapeutic potential by altering Mφ/microglia polarization. Previous animal studies in the context of central nervous system (CNS) trauma revealed that IL-13 administration, either directly or via cell therapy, exerted anti-inflammatory effects and thereby provided neuroprotection [12][13][14]. Our prior study demonstrated that cell-based delivery of IL-13 via mesenchymal stem cells (MSCs) enhanced functional recovery after SCI. This improvement was associated with an increased perilesional presence of Arg1 + Mφs/ microglia. Thus, it was tempting to speculate that accumulated M2a Mφs were critical for functional repair [15]. Therefore, in this study, we used Mφs to continuously deliver IL-13 as (i) Mφs have a high migratory capacity towards inflammatory sites after trauma and (ii) IL-13 can introduce additional anti-inflammatory M2a Mφs at the lesion site [16].
We show for the first time that the use of genetically modified Mφs as carriers of IL-13 is a potent strategy to improve histopathological and functional recovery after SCI. Using IL-4Rα-deficient mice, we demonstrate that IL-13 signaling is essential to reduce neuronal death and suppress axonal dieback by promoting an anti-inflammatory phenotypic shift of perilesional Mφs/microglia. Overall, these findings underline the therapeutic potential of IL-13 and Mφ/microglia modulation in regeneration after CNS trauma.

Animals
In vitro experiments were done using female 8-12-week old wild type (WT) BALB/c (Envigo) or C57BL/6J mice (Janvier Labs). CX3CR1 + /GFP mice were kindly provided by prof. Brône (Uhasselt). In vivo experiments were performed with either female 10-14-week old WT C57BL/6j mice (Janvier Labs) or IL-4Rα control and knockout (KO) BALB/cJ mice (#000651, #003514, The Jackson Laboratory) [17]. As male mice have a substantially higher dropout rate after SCI due to wounding and higher risk of bladder or other infections, only female mice were used in this study. Mice were housed at the conventional animal facility of Hasselt University under stable conditions (temperature-controlled room, 12 h light/dark cycle, food, and water ad libitum). Experiments and sample size calculations were approved by the local ethical committee and were performed according to the guidelines of Directive 2010/63/EU on the protection of animals used for scientific purposes.

Conclusions:
Collectively, our data suggest that the IL-13-induced anti-inflammatory Mφ/microglia phenotype can preserve neuronal tissue and ameliorate axonal dieback, thereby promoting recovery after SCI.

Lentiviral transduction
To generate IL-13 overexpressing and Arg1 overexpressing Mɸs, naive Mɸs were transduced with pIRES-mIL-13-puro or pIRES-Arg1-puro lentiviral particles provided by the Laboratory for Viral Vector Technology & Gene Therapy (Leuven Viral Vector Core of the KU Leuven) according to previously optimized protocol with minor adaptations [15]. Mɸs were seeded in a 24-well plate at 1.5 × 10 5 cells/well in standard medium. Positive selection of the transduced cells was made using puromycin (2.25 μg/ml, InvivoGen) for 72 h. CM was collected after puromycin selection.
qPCR Cells were lysed using QIAzol (Qiagen), and RNA was extracted using the RNeasy mini kit (Qiagen), according to the manufacturer's instructions. Conversion to cDNA was done with qScript cDNA supermix (Quanta Biosciences). Quantitative PCR was performed on a Ste-pOnePlus detection system (Applied Biosciences) under universal cycle conditions. Primer sequences can be found in the Additional information (Additional file 1: Table S1). The comparative Ct method was used to determine relative gene expression. Data were normalized to the most stable housekeeping genes (CYCA, GADPH, HMBS, HPRT, YHWAZ) determined by GeNorm.

Western blot
Cells were lysed using RIPA buffer supplemented with protease and phosphatase inhibitors (Sigma-Aldrich).
The protein concentration was determined by the Pierce ™ BCA assay kit (ThermoFisher Scientific), according to the manufacturer's guidelines. Samples (4-10 µg) were separated on 7.5% or 12% (dependent on size target) sodium dodecyl sulfate-polyacrylamide gels by

ELISA
IL-4 and IL-13 secretion in the supernatants of cell cultures was measured using ELISA kits (ThermoFisher Scientific), according to the manufacturer's instructions.

Griess assay
Nitrite secretion in the supernatants of cell cultures was measured using the Griess reagent system kit (Promega), according to the manufacturer's instruction.

Spinal cord injury model
A T-cut spinal cord hemisection injury was performed as previously described [15,18]. Briefly, mice were anesthetized with 2-3% isoflurane (IsofFlo, Abbot Animal Health) and underwent a partial laminectomy at thoracic level 8. Iridectomy scissors were used to transect left and right dorsal funiculi, the dorsal horns, and the ventral funiculus, resulting in a T-cut hemisection injury. The back muscles were sutured, and the skin was closed with wound clips (Autoclip, Clay-Adams Co. Inc.). Postoperative treatment included blood-loss compensation by glucose and pain relief by buprenorphine (0.1 mg/kg bodyweight, Temgesic). Mice were placed in a recovery chamber (33 °C) until they regained consciousness. Bladders were voided daily until animals were able to urinate independently.

Cell transplantation
For in vivo transplantation, Mφs were isolated from the same mouse strain as the recipient mice in the in vivo experiment, being either C57BL/6J or BALB/c. Mφs were cultured using Nunc ™ Dishes with UpCell ™ Surface (ThermoFisher Scientific) to avoid enzymatic harvesting. Immediately after SCI induction, mice were randomized and received an intraspinal injection of vehicle or (GFP + ) Mφs. As previously described, a motorized stereotaxic injector pump (Stoelting) with a 10 µl Hamilton syringe (34-gauge needle) was positioned 1-3 mm rostral to the lesion [19]. The needle was stereotactically inserted into the spinal cord at a depth of 1 mm. Either vehicle (PBS or RMPI) or cells (1 × 10 4 in 2.5 µl) were injected over a 4 min period, after which the needle was kept in place for again 4 min to allow pressure equilibration and prevent backflow. Investigators were blinded to the treatment groups during the complete experiment.

Locomotion testing
Functional recovery was assessed using the standardized Basso Mouse Scale (BMS) for locomotion (0 = complete hind limb paralysis, 9 = normal motor function) [20]. Scores are based on hind limb movements in an open field during a 4 min interval. At the day of operation, animals were checked for locomotion, and no abnormalities were observed (BMS score = 9). The evaluation was done by an investigator blinded to the experimental groups and was performed daily from 1 until 7 days post-injury (dpi), followed by a scoring every 2nd or 3rd day. For the analysis, the mean BMS score of the left and right hind limb was used for each animal. Mice who did not display any increase in BMS score were excluded.
To investigate lipid load, an oil red O (ORO) staining was performed. Sections were incubated with a 0.33% ORO solution (Sigma-Aldrich) for 15 min, followed by a 5 min incubation with 60% isopropanol. To wash away any unbound particles, the sections were rinsed with distilled water. Pictures were taken using the Leica DM 2000 LED microscope (Leica Microsystems).

Immunohistochemical quantification
Quantification was carried out on the original, unedited pictures. Pictures displayed in the figures were digitally enhanced to improve visibility. Quantification of histopathological parameters was performed as previously described and was done by investigators blinded to experimental groups [15,18]. To investigate cell survival, the number of GFP + cells at the injection, perilesional, and lesion site were counted. To quantify lesion size (GFAP − area) and demyelinated area (MBP − area), 5-7 sections per animal containing lesion center and consecutive rostral and caudal area were analyzed. The proximal and distal end of the lesion was detected by identifying the area, where the integrity of the dorsal side of the spinal cord tissue is disrupted. As no intensity was measured, contrast was altered differently among groups. To determine astrogliosis (GFAP expression) and Mφ/ microglia presence (Iba-1 expression or Iba-1 + cells), an intensity analysis was performed, or the number of positive cells was counted using ImageJ [15]. To identify alternatively and classically activated phagocytes, Arg1 + or MHCII + cells were counted caudally and rostrally from the lesion site. Quantification of foam cell presence was carried out using ImageJ software according to the method previously described by Deutsch et al. with slight adaptations [21]. To determine neuronal cell death at the lesion site, the number of cleaved caspase 3 + NeuN + cells was counted at both rostral and caudal areas from the lesion epicenter and normalized to the amount of NeuN + cells. To quantify the interaction between axons and Mφ/microglia, the number of contacts between NF + dystrophic axons and Iba-1 + cells was counted in the perilesional environment. Of note, dystrophic axons were identified by their globular and bulbus morphology extending from an axon fiber [22,23]. A contact was detected when a cell-cell interaction between a dystrophic axonal bulb and an Iba-1 + cell contained a DAPI nucleus. For analysis, two standardized areas, rostral and caudal, from the lesion epicenter were used. The mean number of contacts in these two areas per animal was quantified.

Neuroblastoma cell culture
The murine Neuro2A cell line (CCL-131 ™ , ATCC ® ) was cultured in RPMI 1640 medium supplemented with 10% FCS and 1% penicillin/streptomycin. The human SH-SY5Y cell line (Sigma-Aldrich) was cultured in DMEM (Gibco) supplemented with 10% FCS and 1% penicillin/ streptomycin. Cells were maintained at 37 °C and 5% CO 2 . For immunocytochemistry, cells were seeded in a 24-well plate at 3 × 10 4 cells/well for 48 h. To perform cell viability assay, cells were seeded in a 96-well plate at 5 × 10 3 cells/well and left untreated for 24 h to attach.

Immunocytochemistry
Cells were fixed with 4% PFA and blocked for 1 h at RT with 10% protein block in PBS. Anti-IL-13Rα1 and anti-IL-4Rα (1:250, ab79277, Abcam, and 1:50, NBP1-00884, Novus Biologicals) antibodies were diluted in PBS with 1% protein block and were incubated overnight at 4 °C. Following washing, secondary antibody (donkey antirabbit IgG 555, 1:500) incubation was done for 1 h at RT. The specificity of the secondary antibody was tested by omitting the primary antibody. Counterstaining with DAPI was performed for 10 min. Pictures were taken using a LEICA DM4000 B LED microscope and LAS X software (Leica Microsystems).

Neurospheroid culture and staining
Neurospheroids were generated by seeding humaninduced pluripotent stem cell-derived neural stem cells (hiPSC-NSCs, 1.6 × 10 4 cells/well), stably expressing luciferase in an ultra-low attachment 96-well plate. Half medium changes with cNEM (1:1 advanced DMEM/F12: neurobasal medium, 1× neural induction supplement, Gibco) were performed every other day. Cultures were kept under constant orbital shaking (88 rpm). After 4 weeks, neurospheroids were fixed with 4% PFA, and 10 μm thick slices were made for staining. The slices were permeabilized using 0.1% Triton-X-100 in TBS for 30 min before blocking the sections for 1 h with 20% goat serum in TBS. The primary antibody (mouse anti-Tuj1, 1:250, MAB1195, R&D Systems) was diluted in 10% milk in TBS and was applied overnight at 4 °C. Following washing, secondary antibody (goat anti-mouse IgG AF555, 1:200, A21425, Invitrogen) incubation was done for 1 h at RT. Counterstaining with DAPI was performed for 10 min. Pictures were taken using a BX51 fluorescence microscope equipped with an Olympus DP71 digital camera (Olympus).

Statistics
Data analysis was performed using GraphPad Prism version 7 (GraphPad Software). D' Agostino and Pearson omnibus test was used to assess normality. The BMS results were analyzed using a two-way ANOVA for repeated measurements with a Bonferroni post hoc test. GFAP and Iba-1 intensity were analyzed using a twoway ANOVA with a Bonferroni post hoc test. All other data were analyzed using either a one-way ANOVA with a Bonferroni post hoc test or with a non-parametric Kruskal Wallis test with a Dunn's multiple comparison test. Data are presented as mean ± standard error of the mean (SEM). Differences with P values < 0.05 were considered significant (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

IL-13 secreting Mφs express receptors for lesion-associated chemokines and have an anti-inflammatory phenotype
Mφs are reported to migrate towards inflammatory sites after CNS trauma [16]. Therefore, they were selected as carriers of IL-13 to provide a continuous and stable secretion of this anti-inflammatory cytokine locally at the lesion site. IL-13 secretion by IL-13 Mφs, generated by molecular cloning, was confirmed via ELISA. IL-13 Mφs secreted significantly more IL-13 compared to M0, M1 (LPS-stimulated), and M2 (rIL-13-stimulated) Mφs (Fig. 1a). To address the homing potential of IL-13 Mφs, the gene expression of different receptors for lesionassociated chemokines was analyzed [25,26]. No upregulation of C5aR was observed, whereas IL-13 Mφs had increased CCR2 (C-C chemokine receptor 2) and CCR5 gene expression compared to M0 Mφs (Fig. 1b-d). Notably, the expression of these receptors by IL-13 Mφs was also significantly higher compared to M2 Mφs (Fig. 1c,  d).
Next, an extensive phenotypical analysis of typical antiand pro-inflammatory markers expressed by the IL-13 Mφs was performed before in vivo transplantation. Gene data analysis showed that expression of Arg1, FIZZ1, and Ym1 was significantly increased in IL-13 Mφs, as well as in M2 Mφs, compared to M0 Mφs ( Fig. 2a and Additional file 2: Fig. S1a, b). Notably, IL-13 Mφs also showed an increased expression of FIZZ1 and Ym1 compared to M2 Mφs (Additional file 2: Fig. S1a, b). No significant upregulation of CD206 expression by IL-13 Mφs was detected (Additional file 2: Fig. S1c). In addition, the gene expression of the conserved anti-inflammatory markers interferon regulatory factor 4 (IRF4) and Kruppel-like factor 4 (KLF4) was analyzed [27]. Both IL-13 Mφs and M2 Mφs showed an upregulation of these genes compared to M0 Mφs (Fig. 2b, c). In contrast to M1 Mφs, IL-13 Mφs showed no upregulation iNOS expression or other M1-related genes (tumor necrosis factor-alpha [TNFα], CD38, and CD86) compared to M0 Mφs ( Fig. 2d and Additional file 2: Fig. S1d-f ). In line with these gene expression data, IL-13 Mφs showed a significant upregulation of Arg1 protein expression but no upregulation of iNOS (Fig. 2e-g). To verify that the IL-13 secreted by IL-13 Mφs was functional, we investigated whether CM of these cells could induce Arg1 expression in M1 Mφs, as the majority of perilesional Mφs tend to display a proinflammatory phenotype in vivo [28]. Incubation with CM of IL-13 Mφs or M2 Mφs was able to induce Arg1 expression by M1 Mφs (Fig. 2h, i).

IL-13 Mφs maintain their IL-13 secretion and anti-inflammatory markers upon a pro-inflammatory stimulus in vitro
Given the presence of a pro-inflammatory environment at the lesion site after SCI, we determined whether IL-13 Mφs could maintain their anti-inflammatory features when exposed to a pro-inflammatory stimulus in vitro. Interestingly, IL-13 secretion from IL-13 Mφs remained unaltered following LPS stimulation (Fig. 3a). Gene expression analyses showed that Arg1 expression significantly increased in IL-13 Mφs after LPS stimulation compared to their untreated control (Fig. 3b). While FIZZ1 level decreased considerably after LPS stimulation, Ym1 expression by IL-13 Mφs remained unaffected (Additional file 3: Fig. S2a, b). Notably, the expression of these anti-inflammatory markers by IL-13 Mφs was still significantly upregulated compared to LPS-stimulated M0 and M2 Mφs (Additional file 3: Fig. S2a, b). CD206 gene expression was significantly reduced in the LPS-stimulated IL-13 Mφs compared to the untreated control, although no upregulation  S2c). Concerning the pro-inflammatory markers, iNOS expression was induced in IL-13 Mφs after LPS stimulation compared to their unstimulated condition (Fig. 3c). This was similar to M0 and M2 Mφs, which also displayed increased iNOS expression compared to their controls (Fig. 3c). Similar results were obtained for TNF-α, CD38, and CD86 (Additional file 3: Fig. S2d-f ).
In contrast to this gene expression data, protein expression analysis showed that Arg1 and iNOS expression remained unaltered in IL-13 Mφs following LPS stimulation, whereas M0 and M2 Mφs displayed an increased iNOS protein level after LPS incubation compared to their untreated controls (Fig. 3d, e). In addition, nitric oxide (NO) secretion was measured to analyze iNOS activity. Upon LPS stimulation, IL-13 Mφs produced more NO compared to their untreated controls (Fig. 3f ). Notably, this was significantly decreased compared to the NO secreted by M2 Mφs stimulated with LPS (Fig. 3f ).

Transplantation of IL-13 Mφs improves functional and histopathological outcome after spinal cord injury
To investigate the therapeutic potential of IL-13 Mφs following SCI, mice received an intraspinal injection with either vehicle or IL-13 Mφs immediately after injury induction in the perilesional area. As the in vitro results showed that M2 Mφs adopt a mixed phenotype upon a pro-inflammatory stimulus, these cells were included to test whether stable IL-13 secretion and anti-inflammatory gene expression are prerequisites for beneficial effects. Functional recovery was measured for 4 weeks using the BMS score. Mice treated with IL-13 Mφs showed significantly improved functional recovery compared to vehicle and M2 Mφ groups (Fig. 4a). Accordingly, histological analyses indicated significantly reduced lesion size and a trend towards decreased demyelination in IL-13 Mφ-treated mice compared to vehicle control ( Fig. 4b-i). M2 Mφ treatment did not affect functional outcome, although a significant decrease in demyelinated area compared to vehicle was shown (Fig. 4a, c, g-i). No differences in astrogliosis were observed between the  Fig. S4a-d).

Transplantation of IL-13 Mφs induces an anti-inflammatory shift in phagocytes at the lesion site
As IL-13 is one of the main inducers of an alternatively activated Mφ/microglia phenotype, we assessed the phenotype of perilesional Mφs/microglia to investigate Kruskal-Wallis test with Dunn's correction. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001  S5a). Second, the number of alternatively (Arg1 + ) and classically (MHCII + ) activated phagocytes at the lesion site was determined (Fig. 5a, b, d-i). IL-13 Mφ treatment increased the amount of Arg1 + cells at the lesion site while reducing the number of MHCII + cells compared to vehicle (Fig. 5a, b, d-i). Besides MHCII expression, foamy Mɸs are considered to be detrimental inflammatory cells in SCI [29]. Therefore, we determined the perilesional lipid load as an indirect measurement for the presence of foamy cells. Mice treated with the IL-13 Mφs had a significantly reduced number of lipid droplets versus vehicle (Fig. 5c, j-l).

Transplantation of IL-13 Mφs skews the lesion site towards a neuroprotective environment
The abovementioned results indicate that IL-13 Mφs transplantation enforced an anti-inflammatory perilesional milieu. Next, we investigated whether this change in environment also provided neuroprotection. First, the neuroprotective effect of IL-13 Mφs was determined through double staining for cleaved caspase 3 and NeuN to detect apoptotic neurons. Mice treated with IL-13 Mφs had reduced neuronal cell death compared to vehicle (Fig. 6a, c, e). Furthermore, the M2 Mφs group only displayed a non-significant trend towards reduced neuronal cell death relative to control (Fig. 6a, c, d). Second, the impact of IL-13 Mφ transplantation on the detrimental phagocyte-axon contacts was investigated. This was determined via quantification of the contacts between Mφs/microglia (Iba-1 + cells) and dystrophic axons (NF + cells showing end bulbs) at the lesion site [4]. IL-13 Mφs significantly reduced the number of detrimental contacts compared to vehicle control (Fig. 6b, f, h). A similar effect was observed in the mice treated with the M2 Mφs (Fig. 6b, f, g).

IL-13 is critical for the beneficial effects of IL-13 Mφs after spinal cord injury
We have shown that IL-13 Mφs have anti-inflammatory and neuroprotective effects in vivo, accompanied by improved functional recovery after SCI. This can be attributed to either IL-13 secretion or the anti-inflammatory phenotype of the IL-13 Mφs (e.g., Arg1 expression).
To investigate this, we used two different approaches. First, IL-13 Mφs were administered to IL-4Rα KO mice. These mice lack not only IL-4 signaling but also IL-13 signaling as IL-13Rα1 activation requires heterodimerization with the IL-4Rα, which is prevented in the IL-4Rα KO model [30]. After SCI induction and cell transplantation, functional recovery was analyzed for 4 weeks using the BMS score. IL-13 secretion by IL-13 Mφs was confirmed by ELISA (Additional file 7: Fig. S6a). WT control and vehicle administration were included as a control for KO-induced effects and to ensure positive outcome after IL-13 Mφ transplantation, respectively. The IL4Rα KO did not affect functional recovery (Fig. 7a, IL-4Rα KO mice + vehicle versus WT controls + vehicle). Transplantation of IL-13 Mφs into the spinal cord of IL-4Rα KO mice did not affect functional recovery (Fig. 7a, IL-4Rα KO mice + IL-13 Mφs versus IL-4Rα KO + vehicle), whereas transplantation of IL-13 Mφs improved outcome in WT controls compared to vehicle treatment (Fig. 7a, WT + IL-13 Mφs versus WT + vehicle). Second, Arg1 Mφs were transplanted, which represent Mφs that do not secrete IL-13 but overexpress the typical anti-inflammatory Mφ/microglia marker Arg1 (Additional file 7: Fig. S6b, c). Again, functional recovery was analyzed. Transplantation of Arg1 Mφs did not improve functional recovery compared to vehicle control mice (Fig. 7b).
These results indicate that IL-13 is critical for functional recovery and neuroprotective effects induced by IL-13 Mφs, which are absent in mice deficient for IL-4Rα signaling. Next, we investigated in vitro whether IL-13 was directly responsible for neuroprotection. We used murine and human neuroblastoma cell lines and induced cell death via NO, which plays a vital role in secondary injury neurotoxicity [31]. Of note, we confirmed the presence of the IL-13Rα1 and IL-4Rα on these cells (Additional file 8: Fig. S7a, b). Overall, treatment with different concentrations of IL-13 did not affect cell viability (Additional file 8: Fig. S7c, d). Incubation (72 h) of these cells with varying concentrations of SNAP (100-800 µM), an NO donor, significantly induced cell death at high doses (75% at 800 µM, Fig. 8a, b). No rescue effect was observed by incubation with IL-13 (5-500 ng/ml, Fig. 8a, b). Likewise, 48 h co-treatment or 24 h pretreatment with IL-13 did not affect cell viability (Additional file 8: Fig. S7e-h). In addition, we used a neurospheroid culture obtained from hiPSC-NSCs to analyze the direct effects of IL-13 in a more complex set-up which approaches the in vivo structural organization of cells. Four-week old spheroid cultures strongly expressed the neuronal marker Tuj1 (Fig. 8c, d). Using an OGD assay, we mimicked a state of hypoxia, which is also induced after SCI [31]. Following these experimental conditions, neuronal viability was reduced by 50% (Fig. 8e). However, IL-13 treatment did not influence cell viability (Fig. 8e).
The abovementioned results suggest that IL-13 does not have direct neuroprotective actions. Therefore, we used a murine brain slice model, which is a more complex in vitro trauma model to maintain the interaction between CNS neural and immune cells to include analyses of indirect effects induced by IL-13 on neurons. In this case, we used a model of NMDA-induced toxicity as NMDA contributes to secondary cell death via glutamate excitotoxicity [31]. In this model, NMDA treatment resulted in 18% cell death (Fig. 8f ). A low concentration of IL-13 (5 ng/ml) protected against NMDA-induced cell death (Fig. 8f, g). Surprisingly, a high dose of IL-13 (500 ng/ml) was cytotoxic even in the absence of NMDA treatment (Fig. 8f ).

Discussion
It is generally accepted that neuroinflammation is a significant driver of secondary injury-related damage in SCI. During the last years, the potential of IL-13 as a treatment for neuroinflammation has been shown by us and others in different animal models of neurodegeneration [9, 12-15, 19, 32]. Here, we show for the first time that IL-13 delivery by genetically modified Mφs improves functional repair after SCI and that these beneficial effects are mediated by IL-13 and not the M2a phenotype of the IL-13 Mφs. Mφs were selected as carriers above MSCs due to their strong homing ability towards inflammatory sites and because MSC-mediated actions after SCI were attributed to the induction of M2a Mφs [15,33]. In this study, we demonstrate that transplantation of IL-13 Mφs after SCI is an efficient approach for eliciting anti-inflammatory effects and thereby create a neuroprotective environment less prone to neuronal death and axonal retraction. Our study supports IL-13 administration as an effective immunomodulatory therapy and underlines the importance of reprogramming local Mφs/ microglia to provide neuroprotection and improve functional outcome after SCI. IL-13 secretion by Mφs has not been previously reported under physiological conditions, only in the context of pulmonary fibrosis [34]. Through genetic transfer, we induced IL-13 secretion by Mφs. Phenotyping of these IL-13 Mφs prior to in vivo administration showed that IL-13 Mφs secreted IL-13 at similar concentrations to that used for ex vivo polarization of M2a Mφs [12,13]. In addition, CCR2 and CCR5 upregulation by IL-13 Mφs suggests an increased homing potential towards the lesion site, as their ligands, CCL2, and CCL3 are upregulated during the 1st days after SCI [16,26]. Whether the upregulation of these factors in the IL-13 Mφ group also contributes to neuroprotection remains to be elucidated. The effects of the CCL2-CCR2 pathway after SCI are controversial. While CCR2 deficiency improves outcome, CCL2 contributes to M2 Mφ polarization [26,35]. Interestingly, CCL2 treatment counteracted NMDA-induced cell death of motor neurons [36,37]. The CCR5 axis has been described to play a pro-inflammatory role after SCI, as in the absence of CCR5 or its ligand, CCL3, recovery was improved [38,39]. Therefore, it was rather surprising that CCR5 was upregulated by IL-13 Mφs. However, the role of CCR5 in SCI seems to be more complex as recombinant CCL5 treatment caused upregulation of M2 Mφ and axonal regeneration markers [26]. Overall, CCL2 and CCL5 have been described to be able to provide anti-inflammatory actions [26,35]. Yet, since recombinant IL-13 alone has a similar neuroprotective effect in the brain slice culture, it is feasible that the CCR2 and CCR5 upregulation in IL-13 Mφs plays a minor role in protecting the spinal cord after lesion. The polarization of the IL-13 Mφs was indicated by the upregulation of the primary M2a markers Arg1, FIZZ1, and Ym1. Furthermore, their anti-inflammatory phenotype was more pronounced than M2 Mφs, potentially due to continuous autocrine signaling provided by IL-13.
As the inflammatory imbalance following SCI impedes regenerative processes, increasing the fraction of the anti-inflammatory Mφ/microglia phenotype has become a major therapeutic strategy. Ma et al. demonstrated that intravenous injection of M2 Mφs improved functional recovery after SCI [40]. However, Kigerl et al. showed that 3 days after intraspinal injection of GFP + -M2 Mφs, only 20-40% maintained their polarization status [28]. This is not surprising given that lesion-associated factors favor the classically activated Mφ phenotype. Based on these results, it is tempting to speculate that preventing an M1 switch of grafted cells is a prerequisite for functional improvement. To overcome this pitfall, we used genetically manipulated Mφs to secrete IL-13. Even under a robust inflammatory stimulus, these cells maintained IL-13 secretion, and their anti-inflammatory marker expression (Arg1, FIZZ1, and Ym1 expression) was more pronounced during inflammation than rIL-13 stimulated M2 Mφs. A similar observation was made by Boehler et al., who showed that IL-10 transduction was more potent than rIL-10 stimulation to maintain an M2 polarization upon inflammation [41]. Importantly, in our study, only IL-13 Mφs improved functional recovery after SCI, whereas the M2 Mφs did not. It is important to note that this is only a partial and no full recovery. IL-13 Mφ-treated mice reached an average score of 3.4, which is more than 2 points higher than the vehicle group. We consider this difference as biologically and therapeutically relevant, because it means that highly impaired mice which show only slight ankle movement develop a stepping pattern after IL-13 Mφ transplantation [20]. However, these data have to be interpreted with care, because in this study, only female mice have been used, and there is evidence that sex differences may influence the BMS outcome or the inflammatory response after SCI [42][43][44]. For example, female mice have a reduced lesion size and a higher Mφ number compared to males [42,43]. Surprisingly, IL-13 Mφs did show an upregulation of iNOS mRNA but not of its protein expression after LPS stimulation. The link between IL-13 and iNOS gene expression was already observed in our previous studies [32,45]. For example, IL-13 MSC treatment induced upregulation of Arg1 and iNOS mRNA levels in a cuprizone model [32]. Still, IL-13 delivery was associated with anti-inflammatory effects and suppressed demyelination.
The M1:M2 ratio is a crucial determinant of functional recovery after SCI [1]. As IL-13 is well-known for inducing alternative activation of phagocytes, we analyzed whether IL-13 Mφ injection caused a polarization shift in vivo. The perilesional environment of IL-13 Mφ-treated mice was converted as shown by an increased anti-inflammatory cell population (Arg1 + cells) and a decreased pro-inflammatory cell number (MHCII + cells) without affecting the global Mφs/microglia cell presence. Notably, the increase in Arg1 + cells was absent Mφs were isolated from C57BL/6J mice. M0, M2, and IL-13 Mφs were left unstimulated (control) or were stimulated with LPS for 24 h. a IL-13 secretion, determined by ELISA, was not changed following LPS stimulation in IL-13 Mφs. Data represent mean ± SEM. n = 5-6. b, c Gene expression, determined by qPCR, showed that LPS exposure significantly increased Arg1 (b) and iNOS (c) expression by IL-13 Mφs compared to their untreated control. Data were normalized to M0 Mφs and represent mean ± SEM. n = 8-9. d, e Quantification of the western blot analysis showed that the protein expression of Arg1 (d) and iNOS (e) were not altered by LPS stimulation in IL-13 Mφs. Data were normalized to M0 Mφs and represent mean ± SEM. n = 4-6. f Using a Griess assay, NO was measured in the medium. Although NO secretion was significantly increased in the LPS-stimulated IL-13 Mφs compared to their untreated control, the secretion was significantly reduced compared to the M2 Mφs stimulated with LPS. Data represent mean ± SEM. n = 9. Kruskal-Wallis test with Dunn's correction. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 in the mice who received M2 Mφs. This immunomodulation of local Mφs/microglia by IL-13 is in line with previous stroke and TBI reports [12][13][14]. Consistently, we previously showed in a study using IL-13 MSC that the Arg1-expressing population consisted mainly of invading monocytes/Mφs [15]. Lesional lipid load was analyzed as an indirect measurement of foamy cells, which are detrimental in SCI [46]. Mφs phagocytose apoptotic cells and myelin debris to pave the way for regeneration [47]. However, in SCI, excessive uptake overwhelms the intracellular lipid homeostasis, subsequently transforming them into large foamy, pro-inflammatory cells [46]. Wang et al. showed that Arg1 expression was reduced 7 dpi, while foam cell formation was observed [47]. As our results indicate an increase in Arg1 + cells, it may be expected that foam cell formation is reduced. Indeed, the ORO staining results showed an overall reduction in lipid load at the lesion site following IL-13 Mφ transplantation. Even though the underlying mechanism remains to be resolved, several studies indicate a role of IL-13 in phagocytosis. The importance of IL-13 in stimulating efferocytosis was described in a TBI animal model where repeated intranasal rIL-13 treatment increased the NeuN + Iba-1 + area [13]. In stroke, the STAT6/Arg1 pathway was found to induce efferocytosis by Mφs/microglia [48]. Mechanistically, Proto et al. revealed that IL-13 secretion activated IL-10 production by Mφs, which enhanced efferocytosis via autocrine signaling [49]. In addition, in a mouse model of atherosclerosis, IL-13 acted anti-atherogenic by inducing M2 polarization. In vitro, Mφs stimulated with IL-13 increased the uptake of oxLDL by CD36 upregulation and induced the expression of the lipid exporter (ABCA1), preventing foam cell formation, which is a major driver of atherosclerosis [50]. Based on these data, it is plausible that enhanced phagocytosis and/or  processing may explain a reduction in foamy cells in our model. Notably, the beneficial immunomodulatory effects of IL-13 Mφs were accompanied by neuroprotection. In animal models of TBI and stroke, IL-13 treatment also facilitated anti-inflammatory effects and reduced neuronal cell loss [12,13]. Likewise, we observed a significant decrease in the number of cleaved caspase 3 + neurons in IL-13 Mφ-treated mice. Furthermore, damaged axons retract from the injury site after SCI, a process called axonal dieback [46,51]. The significant contribution of Mφs to this phenomenon has been shown by Horn et al., who demonstrated a substantial reduction in axonal dieback following peripheral Mφ depletion [4]. In addition, M1 Mφs, in particular, have been reported to induce axonal retraction via destructive physical contacts [4]. IL-13 Mφ treatment did not affect the number of lesional Iba-1 + cells compared to vehicle. However, it reduced the number of contacts between dystrophic axons and phagocytes. Since the number of Iba-1 + cells was not influenced, this suggests that axonal dieback, a limiting factor for regeneration, was reduced [52]. Therefore, it can be speculated that induction of an anti-inflammatory perilesional environment by IL-13 Mφs creates a permissive milieu for axon regeneration by reducing neuronal cell death and axonal dieback. It cannot be excluded that direct effects on axonal plasticity may contribute to the observed beneficial effects. However, Vogelaar et al. have shown that there were no direct effects of IL-13 on axonal outgrowth in vitro [53].
To analyze whether the beneficial effects of IL-13 Mφs were either dependent on IL-13 itself or their altered M2a phenotype, we used an IL-4Rα KO mouse model. IL-13 binding to its receptor, IL-13Rα1, will evoke the dimerization with the IL-4Rα for downstream pathway activation [54]. Deletion of this part will prevent IL-13-induced signaling. Noteworthy, IL-4 also exerts its effects through this receptor complex and IL-4 signaling will be lost as well [54]. However, following SCI, IL-4 levels are undetectable, challenging a significant role in the pathology [55]. In addition, we demonstrate that IL-13 Mφs do not secrete detectable levels of IL-4 (below detection range, Additional file 9: Table S2). Vehicle-treated WT and IL-4Rα KO mice did not show differences in functional recovery after SCI. This is in line with Fenn et al., who did not observe reduced recovery after SCI in adult IL-4Rα KO mice [56]. The functional improvement induced by IL-13 Mφs was abrogated in IL-4Rα KO mice, highlighting the role of proper IL-4Rα signaling. Besides their IL-13 secretion, IL-13 Mφs represent anti-inflammatory Mφs which overexpress Arg1 compared to rIL-13-stimulated M2 Mφs. To investigate whether this polarization was involved in their beneficial effects, Arg1 overexpressing Mφs, which do not secrete IL-13, were also grafted following SCI. However, no significant impact on functional recovery was observed. These results point towards IL-13 signaling as the critical mechanism by which IL-13 Mφs induce immunomodulation, neuroprotection, and functional recovery after SCI. Consistently, IL-13 delivery by MSCs was able to modulate Mφ/microglia polarization during the active inflammation phase in a cuprizone model [14]. As we were unable to trace the IL-13 Mφs 8 days after SCI, initial IL-13 secretion seems to be crucial for the functional recovery, while persistence of the cells at the lesion site appears not to be required. Data were normalized to untreated control and are shown as mean ± SEM. n = 14-15. f Hippocampalentorhinal brain slices derived from BALB/c mice were incubated for 72 h with IL-13 (ng/ml) with or without NMDA (100 µM). A low concentration of IL-13 (5 ng/ml) significantly reduced cell death measured via a PI staining. However, even without NMDA administration, a high dose of IL-13 (500 ng/ml) induced cell death. Data were normalized to untreated control and are shown as mean ± SEM. n = 17-38 slices/condition. g Representative images of the PI assay, depicting the hippocampal cornu ammonis (CA) area 1 and 3, showing a decrease in fluorescence intensity when slices were treated with NMDA + IL-13 (5 ng/ml) compared to NMDA treatment alone. DG dentate gyrus. Scale bar = 250 µm. Kruskal-Wallis test with Dunn's correction (a, b) or one-way ANOVA with Bonferroni post hoc test (e, f). *P < 0.05, **P < 0.01, and ***P < 0.001 Finally, our results indicate that the neuroprotective effect by IL-13 Mφs was not mediated by direct action of IL-13 on neurons. In both murine and human neuroblastoma cell lines, neither pre-nor co-treatment with IL-13 protected against NO-induced cell death. Similarly, no protective effect of IL-13 on the cell viability of OGDsubjected neurospheroids was observed. However, in an murine organotypic brain slice model, IL-13 (5 ng/ml) rescued neurons from NMDA-induced cell death. This suggests that IL-13 indirectly affects the survival of neurons via IL-13R-expressing microglia, which are present in organotypic brain slice culture but not in the neuronal monocultures. Consistent with these findings, Shin et al. reported that the induction of IL-13 in microglia contributed to the survival of neurons upon inflammation [57]. Accordingly, Miao et al. showed that IL-13 reduced neuronal death in an Mɸ neuronal co-culture by suppressing NO secretion, again highlighting the indirect neuroprotective effect of IL-13 [13]. Aside from the impact of IL-13 on Mφs/microglia, astrocytes can also be influenced. Stimulation of astrocytes with IL-13 induced brain-derived neurotrophic factor secretion, which reduces neuronal death, stimulates axonal regeneration and improves functional recovery following SCI [58,59]. Regardless, we did not observe a significant difference in astrogliosis.

Conclusions
To summarize, this study shows for the first time that perilesional transplantation of genetically modified Mφs that secrete IL-13 is a promising approach to improve functional recovery after SCI. Our data suggest that IL-13 elicits indirectly neuroprotection via stimulating antiinflammatory responses in Mφs/microglia. In addition, IL-13 also possibly ameliorated axonal dieback facilitating regeneration. Our data strongly support the therapeutic use of IL-13 and consider it a prime candidate for effective immunomodulation after SCI to improve repair.