Transplantation of tauroursodeoxycholic acid–inducing M2‐phenotype macrophages promotes an anti‐neuroinflammatory effect and functional recovery after spinal cord injury in rats

Abstract Objectives In this study, we study the transplantation of tauroursodeoxycholic acid (TUDCA)‐induced M2‐phenotype (M2) macrophages and their ability to promote anti‐neuroinflammatory effects and functional recovery in a spinal cord injury (SCI) model. Methods To this end, compared to the granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), we evaluated whether TUDCA effectively differentiates bone marrow–derived macrophages (BMDMs) into M2 macrophages. Results The M2 expression markers in the TUDCA‐treated BMDM group were increased more than those in the GM‐CSF‐treated BMDM group. After the SCI and transplantation steps, pro‐inflammatory cytokine levels and the mitogen‐activated protein kinase (MAPK) pathway were significantly decreased in the TUDCA‐induced M2 group more than they were in the GM‐CSF‐induced M1 group and in the TUDCA group. Moreover, the TUDCA‐induced M2 group showed significantly enhanced tissue volumes and improved motor functions compared to the GM‐CSF‐induced M1 group and the TUDCA group. In addition, biotinylated dextran amine (BDA)–labelled corticospinal tract (CST) axons and neuronal nuclei marker (NeuN) levels were increased in the TUDCA‐induced M2 group more than those in the GM‐CSF‐induced M1 group and the TUDCA group. Conclusions This study demonstrates that the transplantation of TUDCA‐induced M2 macrophages promotes an anti‐neuroinflammatory effect and motor function recovery in SCI. Therefore, we suggest that the transplantation of TUDCA‐induced M2 macrophages represents a possible alternative cell therapy for SCI.


| INTRODUC TI ON
Spinal cord injuries (SCIs) occur as a result of fractures, dislocations and compressed vertebra. The annual incidence of SCI in the United States alone is estimated to be 12,000 patients. 1 SCI patients suffer from dysfunctions of the central nervous system (CNS) and the peripheral nervous system (PNS). 2 Methylprednisolone (MP) has been used for the treatment of SCI patients. 3 However, MP has side effects such as wound infections, pneumonia and myopathy. 4 Therefore, the use of MP as a SCI treatment remains controversial. 5 Tauroursodeoxycholic acid (TUDCA) is a natural molecule containing taurine conjugated with ursodeoxycholic acid. It has been in use since ancient times as a component of traditional Asian medicine. 6 TUDCA is a hydrophilic nontoxic bile acid that is produced endogenously at low levels in humans and bears. 7 Food and Drug Administration (FDA) approved TUDCA for the treatment of liver diseases such as cirrhosis and hepatitis. 8 Moreover, TUDCA has protective effects in those with CNS diseases such as Huntington's disease and Alzheimer's disease. [9][10][11] In previous studies by the authors, we suggested that TUDCA can be an alternative drug based on its antineuroinflammatory effects in macrophages and in SCI rats. 6,12 Secondary injury reactions occur in SCI as a result of activated macrophages. 13 During the inflammatory reaction, M1-phenotype (M1) macrophages release inflammatory cytokines, including tumour necrosis factorα (TNFα), interleukin (IL)-1β and IL-6. 14, 15 On the other hand, M2-phenotype (M2) macrophages express macrophage mannose receptors (cluster of differentiation 206, CD206) and arginase-1 (Arg-1) and secrete anti-inflammatory cytokine (IL-4). 13,16 Therefore, M2 macrophages facilitate cellular processes such as tissue repair. 17 Hence, we hypothesized that TUDCA-induced M2 macrophages can promote spinal cord repair because they limit secondary injuries.

| Isolation and primary culture of bone marrowderived macrophages (BMDMs)
To obtain rat BMDMs, bone marrow (BM) cells derived from femurs and tibias were harvested and cultured. 18 The bone inner cavity was rinsed with 10 mL of a medium containing DMEM, 10% FBS, 2% glutamate and 1% PS to harvest BM cells. Collected BM cells were centrifuged (10 minutes, 450 × g). Erythrocytes were lysed in a red blood lysis buffer (Cat. 37757; Sigma). To exclude resident macrophages, cells were incubated for 4 hours at 37℃ in culture-treated petri dish (Cat. 430591; Corning). Afterwards, the supernatant was collected and centrifugated (10 minutes, 450 × g). The pellet was dissociated in 150 mL complete DMEM (cDMEM) containing 10% FBS, 2% glutamate, 1% PS and L929-conditioned medium. 19 10 mL of suspended cells was distributed to a petri dish. The dish was cultivated at 37℃ in 5% CO 2 .
After 3 days, we added 10 mL of cDMEM to each petri dish and the cells were incubated for another 4 days. Finally, the BMDMs were harvested and seeded for the following experiments ( Figure 1).

| Immunocytochemistry (ICC) staining
We conducted ICC staining to quantify differentiated M1 and M2

| Fluorescence-activated cell sorting (FACS) analysis
A FACS analysis of the GM-CSF or TUDCA-treated BMDMs was used for macrophage differentiation. The cells were detached with ice-cold phosphate-buffered saline (PBS) containing 0.02% ethylenediaminetetraacetic acid (EDTA). 1 × 10 6 BMDMs were incubated with primary antibodies, including anti-liver Arg-1 antibodies All rats were housed in a pathogen-free facility with controlled temperature and humidity. Rats were allowed free access to food.
On each day at 8 am and 8 pm, urination was manually performed.
All surgeries were operated by the same spine neurosurgeon (S. Sohn).

| Enzyme-linked immunosorbent assay (ELISA)
Ten mm epicentre segment of the spinal cord lesion was obtained 7 days after the SCI. The collected segments were then homogenized in a 1X RIPA buffer. The segments were then centrifugated at 15 000 RPM for 10 minutes at 4℃. The protein concentration was calculated using a BCA protein analysis kit (Thermo Scientific). 22 Protein levels were measured using ELISA kits (Koma Biotech). Subsequently, equal membranes were stripped and reprobed with the total forms of ERK (t-ERK; 1:1000; Cat. 9102S), JNK (t-JNK; 1:100; Cat. 9258S) and p38 (t-p38; 1:1000; Cat. 9212S). All primary antibodies were purchased from Cell Signaling Technology except for β-actin (1:5000; ABM). As an internal control, β-actin was also probed into the membranes. All of the primary antibodies were then incubated with secondary antibodies (1:5000, Gene Tex). The visualized signal bands were detected using an ECL solution (Amersham) through a G: Box Chemi-XX6 gel doc system (Syngene). The p/t form volumes for the predetermined days were calculated and quantified using ImageJ software (NIH).

| Haematoxylin & eosin (H&E) staining
At 7 days after SCI, the rats were anesthetized. Rapid perfusion was performed using ice-cold saline after cannulation of the left ventricular-ascending aorta. When the efflux became clear, 4% PFA/ F I G U R E 1 Schematic procedures of the transplantation therapy of tauroursodeoxycholic acid (TUDCA)-induced M2-phenotype macrophages in the contusion spinal cord injury (SCI) rat model in this study PBS was perfused for 5 minutes. 22 The 10 mm spinal cord segments of the lesion epicentres were collected and fixed overnight in 4% PFA/PBS. They were dehydrated, and paraffin embedding was followed. 23 Sagittal sections were cut to a thickness of 5 μm. Sections were stained using haematoxylin and eosin. The changes in morphology were observed under a light microscope (IX71: Olympus).

| Behavioural tests
Basso, Beattie and Bresnahan (BBB) scores for hindlimb function were measured using open-field locomotion. 24 The rats were evaluated on 1, 3, 5, 7, 9, 12, 14, 16, 18 and 21 via BBB tests. Two trained investigators who were blind to the experimental conditions performed the behavioural analyses.

| Immunofluorescence staining
According to standard procedures for immunofluorescence staining, sections were incubated in a blocking solution to prevent any non-specific binding reaction for 1 hour. Afterwards, primary anti-

| Statistical analyses
All values were presented as the mean ± standard deviation (SD).
A one-way analysis of variance (ANOVA) followed by a post hoc test was used to verify statistical differences among the groups.
Behavioural scores were analysed by Student's t-tests. Differences with P-values for which *P < .05, **P < .01 and ***P < .001 were considered as statistically significant.

| TUDCA treatment increases the number of M2 macrophages on BMDMs
To observe whether TUDCA upregulates M2 differentiation, we stained BMDMs in the GM-CSF-treated group and the TUDCAtreated group by ICC staining (Figure 2A Moreover, the CD206 expression levels in the TUDCA-treated group were significantly increased relative to those in the GM-CSF-treated group ( Figure 2K; GM-CSF-treated group vs TUDCA-treated group: 38.08 ± 0.73 vs 48.08 ± 1.55; *** P < .001). On the other hand, the IL-4 secretion level in the TUDCA-induced M2 group was significantly increased compared to those in the GM-CSF-induced M1 group and the TUDCA group ( Figure 3D; **P < .01 and ***P < .001).

| Transplantation of TUDCA-induced M2 macrophages inhibits the phosphorylation of ERK, JNK and p38 in the mitogen-activated protein kinase (MAPK) pathway
The phosphorylation activities of ERK, JNK and the p38 signals in the MAPK pathway are key processes during the inflammatory response after SCI ( Figure 4A-G). As shown in Figure 4A, the phos-  This tendency was also found at 18 days ( Figure 6C; TUDCA group vs TUDCA-induced M2 group: 7.6 ± 0.35 vs 10.5 ± 0.71; # P < .05).

| Transplantation of TUDCA-induced M2 macrophages increases CST axons past the lesion
CST axons were traced by injecting BDA into the grey matter 7 days post-SCI. We undertook staining using an axonal tract marker and a neuronal marker (NeuN) ( Figure 7A,B and Figure S3A,B). BDA is routinely used to trace axonal tracts. NeuN has not been detected in tissues other than nervous tissues. Therefore, NeuN was considered as a specific neuronal marker. 28 The BDA fluorescence
The TUDCA-induced M2 macrophages improved histopathological damage and promoted functional recovery to spinal cords in SCI rats ( Figure 5). Moreover, the TUDCA-induced M2 macrophages decreased GFAP and increased CD206 levels ( Figure 6). In addition, BDA fluorescence intensity was detected at significant levels in the caudal site of the TUDCA-induced M2 group (Figure 7).
Macrophages are key players in immunity. Macrophages express two differential phenotypes, specifically classically activated inflammatory M1 and alternatively activated anti-inflammatory M2. 29 After the BMDMs underwent culturing for 2 days, the M2 marker (CD206 and Arg-1) levels in the TUDCA-treated cells were increased to a greater extent than those in GM-CSF-treated cells. SCI activates the expression of astrocytes. Activated astrocytes increase the production of GFAP. GFAP produces numerous pro-inflammatory cytokines. 42,43 In this study, TUDCA-induced M2 macrophages were transplanted in the injured spinal cord. The TUDCA-induced M2 macrophages were demonstrated to be able to inhibit astrocytes and enhance the anti-inflammatory effect.
The transplantation of TUDCA-induced M2 macrophages shows an anti-neuroinflammatory effect and axon regeneration in the injured spinal cord. Therefore, we suggest that the transplantation of TUDCA-induced M2 macrophages can be a useful cell therapy after SCI.

CO N FLI C T O F I NTE R E S T
The authors declare no competing financial interest.

AUTH O R CO NTR I B UTI O N S
GHH, SJK and SIS conceived the idea and conceptualized the study.
GHH, SJK, WKK and DYL conducted the bioinformatics analysis and interpreted results. IBH, SHS and JBH generated the figures. GHH and SJK wrote the paper. SIS supervised the whole study process and revised the manuscript. All authors have read and approved the version of the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
Research data are all within the present manuscript and the additional files.