Transplantation of Wnt4‐modified neural stem cells mediate M2 polarization to improve inflammatory micro‐environment of spinal cord injury

Abstract Neural stem cells (NSCs) transplantation has been considered as a potential strategy to reconnect the neural circuit after spinal cord injury (SCI) but the therapeutic effect was still unsatisfied because of the poor inflammatory micro‐environment of SCI. Previous study reported that neuroprotection and inflammatory immunomodulation were considered to be most important mechanism of NSCs transplantation. In addition, Wnt4 has been considered to be neurogenesis and anti‐inflammatory so that it would be an essential assistant agent for NSCs transplantation. Our single cells sequence indicates that macrophages are the most important contributor of inflammatory response after SCI and the interaction between macrophages and astrocytes may be the most crucial to inflammatory microenvironment of SCI. We further report the first piece of evidence to confirm the interaction between Wnt4‐modified NSCs and macrophages using NSCs‐macrophages co‐cultured system. Wnt4‐modified NSCs induce M2 polarization and inhibit M1 polarization of macrophages through suppression of TLR4/NF‐κB signal pathway; furthermore, M2 cells promote neuronal differentiation of NSCs through MAPK/JNK signal pathway. In vivo, transplantation of Wnt4‐modified NSCs improves inflammatory micro‐environment through induce M2 polarization and inhibits M1 polarization of macrophages to promote axonal regeneration and tissue repair. The current study indicated that transplantation of Wnt4‐modified NSCs mediates M2 polarization of macrophages to promote spinal cord injury repair. Our novel findings would provide more insight of SCI and help with identification of novel treatment strategy.

rehabilitation. 4,5 Reconstruction of the damaged neural circuits was once considered to be hopeless as the adult mammalian central nervous system has very poor ability to regenerate. 2,6 Cell transplantation has been considered as a potential strategy to reconnect the neural circuit after SCI. 1,2,7 Several studies have reported that neural stem cells (NSCs) transplantation results in partial repairing due to the neuronal differentiation of NSCs. 7 However, several studies reported that most exogenous NSCs at the lesion site differentiated into astrocytes, which are to the disadvantage of spinal cord repairing, rather than Neuron. 6,8 Thus, the outcome of NSCs transplantation was not satisfied.
Following transplantation of NSCs, the transplanted cells were considered to mediate functional improvements following SCI through a variety of mechanisms, including axonal regeneration, neuroprotection and inflammatory immunomodulation. 2 Many repair mechanisms rely on beneficial aspects of inflammation. However, the excessive inflammation led to lots of cell death and generation of astrocyte which are harmful to tissue repair and axonal regeneration [9][10][11][12][13] Macrophages/microglia lineages play crucial roles in the pathophysiology of many repair including SCI. 14,15 Macrophages/microglia after injury are organized on a continuum of phenotypes and these phenotypes have been defined as two major categories of proinflammatory M1 cells and anti-inflammatory M2 cells according to their primary functions. 16,17 Both these cells represent the extreme activation states of monocyte and macrophages lineages. 17 M1 cells initiate the inflammation by releasing proinflammatory cytokines, nitric oxide and reactive oxygen species that lead to glial scar formation, neuronal cell apoptosis and causing activation of astrocytes. M2 response to promote tissue repair by secreting anti-inflammatory cytokines to enhance axonal growth and promote the proliferation and differentiation of oligodendrocyte progenitor cells. [17][18][19] In fact, both M1 and M2 are important in the whole inflammatory response respectively. However, in SCI, it has been demonstrated that the lesion site of spinal cord is continuously filled with M1 cells and these cells cannot switch to M2 cells that leading to irreversible tissue impairing. 19 Thus, it is important to elevate the ratio of M2 in the micro-environment to promote tissue repair in SCI treatment. Previous studies reported that cell transplantation can improve the immune response by increasing antiinflammatory cytokines and reducing proinflammatory cytokines after SCI. 14 However, the involving mechanism is still unclear. Still, few studies investigated the direct interactions between transplanted cells and immune cells; instead, they measure global changes in cytokine release or the abundance of immune cells after SCI. 20,21 Recently, lot of wingless-type MMTV integration site family (Wnt) proteins such as Wnt4, Wnt5a, Wnt7b, has gain growing attention as an attractive factor for neural differentiation and anti-inflammation. 22 Wnt4 has been considered to have dual role of neuronal differentiation and anti-inflammation and has been proven to be an optimal assistant agent in cell transplantation after SCI. 23,24 Wnt4-modified NSCs have been considered to promote neuronal differentiation and functional recovery in SCI model according to our previous studies. 24 However, the effect and relative mechanism of Wnt4-modified NSCs on monocyte/macrophages niche and immune response in inflammatory micro-environment of SCI are still elusive.
In the current study, we investigated the effect and underlying mechanism of Wnt4-modified NSCs on macrophages in inflammatory micro-environment, the potential application of Wnt4-modified NSCs transplantation in SCI, that may provide useful information for translational application of NSCs transplantation.

| NSCs isolation and culturing
NSCs cultures were obtained from the foetal brains of embryonic day 14 rats, which were extracted from pregnant Sprague-Dawley (SD) rats (Laboratory Animal Center of Sun Yat-sen University, Guangzhou, China) and identified as previously described. 24,26 Briefly, the brain tissue was mechanically dissected and dissociated in Hanks Balanced Salt Solution to prepare cell suspensions which were centrifuged at 1000 rpm for 5 min. The cell pellet was diluted to a singlecell suspension after the supernatant was discarded. NSCs were plated on a T25 culture flask (Corning, Acton, MA) containing Dulbecco modified eagle medium (DMEM)/F-12 nutrient mixture, 2% B27, 1% penicillin/streptomycin, 1% L-glutamine (Gibco, Grand Island, NY), 20 ng/mL fibroblast growth factor-2 (FGF-2) and 20 ng/mL epidermal growth factor (EGF) (Peprotech, Rocky Hill, NJ). NSCs were cultured at 37 C in 5% CO 2 and were passaged via weekly digestion with accutase (Millipore, Bedford, MA) in the medium described above. All NSCs used in this research were between passages 2 and 4.
To induce neural differentiation, cells were plated at a density of 2 Â 10 5 cells/well in 6-or 12-well tissue-culture plates and allowed to adhere for 24 h at 37 C, at which time cells were switched to neural differentiated medium consisting of basic medium supplemented with 2% B27, 1% penicillin/streptomycin, 1% L-glutamine. The medium was changed every 2-3 days.
Monocytes were isolated from the femurs of adult SD rats (Laboratory Animal Center of Sun Yat-sen University, Guangzhou, China) as previously described. 27 To induce M1 and M2 polarization of macrophages, cells were plated at a density of 2 Â 10 5 cells/well in 6-or 12-well tissue-culture plates and allowed to adhere for 24 h at 37 C, at which time cells were switched to polarization medium consisting of basic medium supplemented with LPS (10 μg/mL) for M1 and IL-4 (10 ng/mL) for M2 polarization ( Figure S1A-C). 28,29

| Lentiviral vector construction
The lentiviral vectors carrying green fluorescent protein (GFP) and a sequence that specifically overexpression the Wnt4. Lentiviruses carrying GFP and Wnt4 gene overexpression were constructed as previously described 24

| Surgical procedures and cell transplantation
Adult female Sprague-Dawley (SD) rats (weighing 200-220 g, supplied by the Experimental Animal Center of Sun Yat-sen University, Guangzhou, China) were divided into four groups for this study: Sham group (n = 10), SCI group (n = 10), NSC vector group (n = 10), and NSC Wnt4 group (n = 10). After 72 h of lentiviral transfection, NSCs were collected for transplantation. Briefly, animals were anaesthetized with 1% pentobarbital sodium (40-45 mg/kg), and the spinal cord was exposed at the 10th thoracic vertebral level (T10) via laminectomy.
The animals underwent spinal cord exposure with no injury in the sham group. In the other three groups, the exposed spinal cord was contused with a weight-drop device by dropping a 10 g rod from a precalibrated height of 12.5 mm. 30 Once the bleeding had stopped, 5 μL NSCs were implanted at a density of 1 Â 10 5 cells/μL to the rostral and caudal of injured site using microsyringe. 31,32 After SCI and NSCs transplantation, the T8-T11 spinal cord segments were dissected at 8 weeks. including joint movements, stepping ability, coordination, and trunk stability. A score of 21 indicates unimpaired locomotion as observed in uninjured rats. All animals underwent behavioural testing, and the duration of each session was 5 min per rat. Finally, we calculated the overall score. The evaluation was performed by three independent observers who were blinded to the treatment group of the tested animals. 33

| Footprint analysis
Hindlimb and locomotor behaviour were assessed at 8 weeks post-injury. The fore and hind limbs were coated with different colours dyes, and the rats of different groups were placed on a 10 cm Â 100 cm runway which was covered by a paper. The rats were encouraged to run in a straight line in order to obtain and evaluate the gait of the rats in the different groups. Then the digital footprints were shown as representative pictures to assess the coordination variability. 34

| Spinal cord-evoked potential (SCEP) recording
At 8 weeks post-injury, total 20 rats (n = 5 in the sham group and n = 5 in each cell-transplanted group) were anaesthetized with 1% pentobarbital sodium (40-45 mg/kg) and fixed stereotaxically. The T5-L1 vertebrae were exposed completely. Briefly, the stimulation electrode was inserted into the T5-T6 interspinous ligaments, and a pair of needle electrodes was inserted into the interspinous ligaments of T12-L1 for SCEP recording. Then, the electrodes were connected to a BL-420 Biological Function Experiment System (Taimeng, Chengdu, China). The variables of the SCEP signals were set according to previous reports as follows: gain of 2000 time constant of 0.01 s, and filtering at 300 Hz. A single pulse stimulation (50 ms in duration at a frequency of 5.1 Hz and a voltage increase of 1 mV) was transmitted to elicit a SCEP through the electrodes until a mild twitch of the vertebral body of the animal was observed. One hundred SCEP responses were averaged for each rat to obtain high-quality waveforms for the SCEP signals. 35  overnight, and soaked in 10% sucrose followed by 30% sucrose at 4 C overnight. Samples were embedded in optimal cutting temperature compound, frozen at À20 C and sliced in the longitudinal or transverse plane at a thickness of 20 μm. Animals (n = 5 per group) were killed for haematoxylin-eosin (HE) staining to visualize the cavity area. The T8-T11 longitudinal spinal cord sections from each group were stained with HE according to standard protocols and examined under bright field microscope. 24,35 For neuron counting, animals (n = 5 per group) were killed, and transverse sections of the injured spinal cord were used to stain neurons with Nissl. Sections at 2 mm rostral and caudal to the lesion epicentre were counted for each rat. The numbers of positively stained cells were counted and averaged per section in a blinded manner. 24,35 For axonal tract tracing, dorsal laminectomy was performed at T12 and Fluorogold (FG; Biotium, Fremont, CA) was injected into the spinal cord at 7 weeks after operation. One week after injection, the animals were perfused and T8 segment of the spinal cord was removed, cryopreserved, embedded in OCT compound, and sliced into 10 μm frozen sections. A fluorescence microscope (Olympus, Tokyo, Japan) was used to detect FG-labelled neurons. 31  3.2 | Macrophages but not microglia were the major contributor in inflammatory response of SCI Microglia are the main cell type in SCI. However, the scRNA-seq results indicated that activated microglia were increased after SCI, these cells may not involve in the inflammatory response (detailed results were in Figure S3). Thus, we focused on macrophages subtypes and its function in SCI. Clustering analysis was performed on macrophages and visualized on a separate UMAP (Figure 2A). Macrophages were divided into five subtypes and M1-like macrophages were identified by expression of IL1-b, whereas M2-like macrophages were identified by Arg1 ( Figure 2B,C). Although the proportion of M1-like macrophages was not changed significantly among uninjured and 1, 3 and 7 dpi group, the numbers of M1-like macrophages were significantly increased ( Figure 2D). The activation of inflammatory pathways was increased in macrophages after SCI. Taken together, our analysis identified M1-like macrophages were increased in the injury group, indicating that macrophages were the major contributor in the inflammatory environment of SCI.

| Macrophages were as major contributor to have various interaction with astrocytes
Previous studies demonstrated that the activation of astrocytes was associated with inflammatory response modulated by M1-like macrophages. 12 Ascc1, Nes, Bcan were used to identify naive, reactive and scar-forming astrocytes subtypes according to previous study. 36 The scRNA-seq results (detailed results were in Supporting Information) showed the differentiation paths from naive to scarforming astrocytes and indicated naive astrocytes would be differentiated into scar-forming astrocytes after SCI ( Figure S4). This analysis indicated that astrocytes become activated in SCI which contributes to scar formation and is to the disadvantage of tissue repair.
We further investigated the interaction between macrophages and astrocytes. We compared the outgoing and incoming interaction strength in 2D space allows ready identification of the macrophages and astrocytes subtypes with significant changes in receiving signals. M1 and M2-like macrophages play an important role in sending signals ( Figure 3A). There were various communications between macrophages and astrocytes subtypes ( Figure 3B,C,E). Particularly, M1 and M2 macrophages had the strong interaction with scar forming astrocytes ( Figure 3D,F). These results confirmed the obvious interaction between macrophages and astrocytes subtypes.    Figure S5). According to our scRNA-seq results, M1-like macrophages maybe the most important inflammatory contributor after SCI. Thus, we further investigated the interaction of NSCs and macrophages. Macrophages were co-cultured with wild type NSCs or Wnt4-modified NSCs to establish the co-culture system ( Figure S6B).
PCR and WB results showed that the M2 relative makers including CD163 and CD206 were increased in macrophages co-cultured with wild type NSCs and further increased in macrophages co-cultured with Wnt4-modified NSCs at mRNA and protein levels. In contrast, M1 makers CD68 were decreased in macrophages co-cultured with wild type NSCs or Wnt4-modified NSCs at protein levels ( Figure 4A-C). The results of FACS demonstrated that macrophages tended to polarize into M2 cells rather than M1 cells when cocultured with Wnt4-modified NSCs ( Figure 4D-F). These results suggested that Wnt4-modified NSCs promote M2 polarization of macrophages.
Toll like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signalling was considered to a critical signalling pathway in neuroinflammation 37,38 and M1 polarization of macrophages. 38 We further confirm whether the TLR4/NF-κB signalling was involved in anti-inflammatory effect of Wnt4-modified NSCs. We first examined the mRNA and protein levels of TLR4. The results showed that the expression of TLR4 was significantly decreased in macrophages co-cultured with wild type NSCs and further decreased in macrophages co-cultured with Wnt4-modified NSCs ( Figure 5A-C). The immunofluorescence staining showed that the expression and nucleus translocation of p65 (NF-κB) was significantly decreased in macrophages co-cultured with Wnt4-modified NSCs ( Figure 5D,E). Immunoblots of p65 in cytosolic extract (CE) and nuclear extract (NE) revealed that p65 nuclear These results indicated that transplantation of Wnt4-modified NSCs improved the inflammatory micro-environment to promote axonal regeneration and suppress tissue destruction after SCI.

| Wnt4-modified NSCs tended to differentiate into neuron to promote tissue repair and locomotor recovery
The appropriated micro-environment is to the benefit of neuronal differentiation of NSCs. We further investigated the differentiated status of the drafted cells. Immunofluorescence results showed that MAP2 + positive NSCs (GFP + ) were significantly increased and GFAP + positive NSCs (GFP + ) were significantly decreased at the injured site of spinal cord after transplantation of Wnt4-modified NSCs ( Figure S9A-D).
Furthermore, HE, Nissl and Flurogold staining indicated that the cavity was significantly smaller and there was more survival neuron at the injured site of spinal cord after transplantation of Wnt4-modified NSCs ( Figure 8A,B). Furthermore, behavioural test including BBB motor function scores, footprint analysis and SCEP result demonstrated that transplantation of Wnt4-modified NSCs improved the motor function of hindlimb in SCI rats ( Figure 8C-H). These results indicated that Wnt4-modified NSCs tended to differentiate into neuron to promote tissue repair and locomotor functional recovery. (The data are presented as the means ± SD from one representative experiment of three independent experiments performed in triplicate. **p < 0.01 compared between groups; *p < 0.05 compared between groups.) damage to the spinal cord. 19 Thus, it is significant to simultaneously achieve valid M2 ratio to promote tissue repair in the microenvironment for the treatment of SCI.

| DISCUSSION
The micro-environment of the injured spinal cord is characterized by complex and imbalance. The imbalance micro-environment is considered to be the principal reason of the poor regeneration and recovery of SCI. 15 The cellular level imbalance in the microenvironment mainly involves the differentiation of stem cells and transformation phenotypes of microglia and macrophages. 15 After SCI, astrocytes and other neural cells can release lot of inflammatory cytokines such as IL-1β, TNF-α, to induce the M1 activation of macrophages that cause a sustained imbalance in the M1/M2 ratio. We reached the conclusion that macrophages and astrocytes were increased after SCI by scRNA-seq analysis. Macrophages were the most important contributor and modulator in this inflammatory environment. In addition, astrocytes were activated and differentiated into scar-forming status due to an increase in macrophages ( Figures 1-3, S3 and S4). It is worth noting that there was increasing of activated microglia after SCI, however, the inflammatory signal pathways in microglia were suppressed which indicated that microglia may not be involved in the modulation of inflammatory response after SCI ( Figure S3).
Cell transplantation has emerged as a potential strategy to promote tissue repair after SCI. 2,6 The primary mechanism of cell transplantation mediate functional improvement and tissue repair is axonal regeneration, neuroprotection and immunomodulation. 2 Previous studies mainly focus on expression and secretion in various cytokines from different immune cells after SCI. Previous study also considered that NSPC transplantation improves hindlimb movement and it is able to increase T cells and decrease B cells. 44 MSCs also were considered to modify the immune response by elevating of anti-inflammatory and reducing of proinflammatory cytokines after SCI. 45,46 However, whether transplanted cells directly alter the inflammatory micro-environment and the direct interactions between transplanted cells and immune cells remain unclear. In the current study, we provided first evidence to investigate the interactions between NSCs and macrophages using our co-cultured system.
In one hand, Wnt4-modified NSCs could secret various antiinflammatory cytokines and induces M2 polarization of macrophages (Figures 4 and S5). On the other hand, M2 cells promote neuronal differentiation of NSCs through MAPK/JNK signal pathway ( Figures S7 and S8).
Previous studies have indicated that M1 polarization of macrophages resulted in the activation of the NF-κB/p65 signalling pathway. 47,48 This activation was also regulated by many other signal pathways, such as TLRs, AMPK. 49  inducing the expression of multiple inflammatory cytokines. 50,51 Thus, we explored that whether TLR4 and NF-κB/p65 signal pathways were involved in the interaction between macrophages and NSCs. Our results showed that the expression of TLR4 was decreased and NF-κB/p65 signal pathway was suppressed in macrophages co-cultured with Wnt4-modified NSCs. These results suggested that the suppression of M1 macrophages polarization was associated with TLR4/NF-κB signalling pathways ( Figure 5).
NSCs transplantation is considered a potential treatment for SCI because NSCs can differentiate into neurons and oligodendrocytes for neuronal rewiring and recruitment in the lesion. 1,2 However, previous studies have reported that most NSCs transplanted into lesion have differentiated into astrocytes rather than neuron due to the inflammatory micro-environment of SCI. 6,8 Therefore, assisting agents that maintain a good rate of neuronal differentiation and improve the inflammatory micro-environment are the research direction for the treatment of SCI. Wnt4 has been considered to have neurogenesis and anti-inflammatory capacities as an important ligand of non-canonical Wnt signal pathway. [22][23][24]52 Previous studies reported that Wnt4 suppressed p65 translocation and NF-κB activation through transforming TAK1 in macrophages. 52 Our previous studies reported that Wnt4 promotes neuronal differentiation by activating both Wnt/β-catenin and MAPK/JNK signal pathways.
Wnt4-modified NSCs transplanted into the lesion efficiently differentiated into neurons and promoted functional recovery after SCI. 24 In the current study, we also conducted a series of in vivo experiments to prove the beneficial therapeutic effect of Wnt4-modified NSC. Our results showed that transplantation of Wnt4-modified NSCs promoted M2 polarization and suppressed M1 polarization, which resulted in the propensity of macrophages to polarize into M2 cells at the injured site of spinal cord ( Figure 6). The increased M2 cells provided an appropriate micro-environment, which was beneficial for axonal regeneration and suppressing of cell apoptosis and tissue destruction at the injured site ( Figure 7). Thus, the drafted NSCs tended to differentiate into neuron rather than astrocytes in this appropriated micro-environment after SCI ( Figure S9) and these effects may facilitate tissue repair and functional recovery after SCI ( Figure 8, Video S1) due to promotion of neurite growth and synaptic plasticity.

| CONCLUSION
In summary, our finding first provides evidence to confirm the interaction between Wnt4-modified NSCs and macrophages and the involved downstream mechanism by using co-cultured system. Transplantation of Wnt4-modified NSCs effectively improves the inflammatory micro-environment through inducing M2 polarization and suppressing M1 polarization of macrophages after SCI ( Figure 8I).
Considering these positive therapeutic effects, Wnt4 may also have remarkable potential assistant agent in NSCs transplantation for SCI.

CONFLICT OF INTEREST STATEMENT
None of the authors have conflicts of interest to disclose.

DATA AVAILABILITY STATEMENT
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.