Adipose‐derived mesenchymal stem cells overexpressing prion improve outcomes via the NLRP3 inflammasome/DAMP signalling after spinal cord injury in rat

Abstract Traumatic spinal cord injury (SCI) is a highly destructive disease in human neurological functions. Adipose‐derived mesenchymal stem cells (ADMSCs) have tissue regenerations and anti‐inflammations, especially with prion protein overexpression (PrPcOE). Therefore, this study tested whether PrPcOE‐ADMSCs therapy offered benefits in improving outcomes via regulating nod‐like‐receptor‐protein‐3 (NLRP3) inflammasome/DAMP signalling after acute SCI in rats. Compared with ADMSCs only, the capabilities of PrPcOE‐ADMSCs were significantly enhanced in cellular viability, anti‐oxidative stress and migration against H2O2 and lipopolysaccharide damages. Similarly, PrPcOE‐ADMSCs significantly inhibited the inflammatory patterns of Raw264.7 cells. The SD rats (n = 32) were categorized into group 1 (Sham‐operated‐control), group 2 (SCI), group 3 (SCI + ADMSCs) and group 4 (SCI + PrPcOE‐ADMSCs). Compared with SCI group 2, both ADMSCs and PrPcOE‐ADMSCs significantly improved neurological functions. Additionally, the circulatory inflammatory cytokines levels (TNF‐α/IL‐6) and inflammatory cells (CD11b/c+/MPO+/Ly6G+) were highest in group 2, lowest in group 1, and significantly higher in group 3 than in group 4. By Day 3 after SCI induction, the protein expressions of inflammasome signalling (HGMB1/TLR4/MyD88/TRIF/c‐caspase8/FADD/p‐NF‐κB/NEK7/NRLP3/ASC/c‐caspase1/IL‐ß) and by Day 42 the protein expressions of DAMP‐inflammatory signalling (HGMB1/TLR‐4/MyD88/TRIF/TRAF6/p‐NF‐κB/TNF‐α/IL‐1ß) in spinal cord tissues displayed an identical pattern as the inflammatory patterns. In conclusion, PrPcOE‐ADMSCs significantly attenuated SCI in rodents that could be through suppressing the inflammatory signalling.


| INTRODUC TI ON
Traumatic spinal cord injury (SCI) is a highly destructive disease that results in deficits in human neurological functions. 1,2 The global incidence of acute SCI is approximately 10 cases per 100,000 persons, resulting in over 700,000-800,000 new patients diagnosed annually worldwide. [2][3][4] Regrettably, despite advances in medical and surgical cares, the current clinical therapies for this devastating disease are primarily ineffective. [5][6][7][8][9] When acute SCI develops, it is generally considered to progress in two stages. The primary injury is the mechanical damage caused by a direct external force. It is followed by secondary injury, which refers to the delayed spread of damage brought about by factors such as inflammatory cytokines, tissue acidosis, glutamate, and dysregulation of the electrolyte homeostasis, leading to further functional deterioration. 10,11 Additionally, studies have further demonstrated that the secondary damage of SCI is orchestrated by the various pathophysiologic mechanisms, including inflammatory reaction, 12 mitochondrial dysfunction 13 and oxidative stress. 14 However, the detailed molecular mechanisms underlying SCI are currently not completely understood.
An inflammasome is now clearly defined by its sensor protein [i.e. a pseudo-response regulator (PRR)], which oligomerizes to form a pro-caspase-1 activating platform in response to DAMPs. There are four members of PRRs that have been identified to form inflammasomes, including nucleotide-binding oligomerization domain (NOD), leucine-rich repeat (NLR)-containing proteins (NLRP) family members NLRP1, NLRP3 and NLRC4. [15][16][17] The NLRP3 inflammasome pathway has been the most extensively investigated, and the results showed that dysregulation of this signalling is closely related to the development of many human diseases, such as neuroinflammation, metabolic inflammation and immune inflammation. 18 Some studies have previously demonstrated that acute SCI triggers NLRP3 inflammasome activation in spinal cord tissue [19][20][21] and spinal cord microglia. Additionally, NLRP3 was identified in neurons, microglia and astroglia, and microglia was the major contributors to the spinal cord damage in the setting of acute SCI. 21 These findings [15][16][17][18][19][20][21] raise the hypothesis that to develop a new strategic management that specifically inhibits the generation of NLRP3 inflammasome in the setting of acute SCI may be a fundamental issue for the treatment of SCI patients, especially in those who are refractory to conventional therapy.

Plentiful data from clinical trials and experimental studies have
shown that mesenchymal stem cell (MSC) therapy, especially those of adipose-derived mesenchymal stem cells (ADMSCs) have the capability of ameliorating inflammatory reaction [22][23][24] and suppressing the innate and adaptive immunity 23,25,26 through downregulating the immunogenicity. 23,[25][26][27][28][29][30] Our preclinical studies have shown that ADMSCs have a strong capacity for immunomodulation that significantly reduced post-heart transplanted acute rejection, 31 and effectively suppressed both overwhelming inflammatory and inflammatory-immune reactions, 23,25,[30][31][32][33] resulting in an improvement of prognostic outcomes. Recently, our study has demonstrated that valsartan therapy preserved residual renal function in chronic kidney disease (CKD) rats mainly through upregulating the cellular prion protein (PrPc) expression. 34 Intriguingly, our more recent study has demonstrated that overexpression of PrPc (PrPc OE ) in ADMSCs has a strong capacity for downregulating the inflammation and oxidative stress and augmentation of myocardial regeneration. 35 Surprisingly, there has been yet no data to address the impact of PrPc OE in ADMSCs on protecting the spinal cord in the setting of acute SCI. Accordingly, we tested the hypothesis that PrPc OE in ADMSCs (i.e. called PrPc OE -ADMSCs) could offer neuroprotective and neurogenerative effects by suppressing NLRP3 inflammasome and signalling in SCI rats.

| Ethics
All animal procedures were approved by the Institute of Animal Care and Use Committee at Kaohsiung Chang Gung Memorial Hospital (Affidavit of Approval of Animal Use Protocol No. 2020061706), followed the Guide for the Care and Use of Laboratory Animals, and housed in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-approved animal and inflammatory cells (CD11b/c+/MPO+/Ly6G+) were highest in group 2, lowest in group 1, and significantly higher in group 3 than in group 4. By Day 3 after SCI induction, the protein expressions of inflammasome signalling (HGMB1/TLR4/MyD88/ TRIF/c-caspase8/FADD/p-NF-κB/NEK7/NRLP3/ASC/c-caspase1/IL-ß) and by Day 42 the protein expressions of DAMP-inflammatory signalling (HGMB1/TLR-4/MyD88/ TRIF/TRAF6/p-NF-κB/TNF-α/IL-1ß) in spinal cord tissues displayed an identical pattern as the inflammatory patterns. In conclusion, PrPc OE -ADMSCs significantly attenuated SCI in rodents that could be through suppressing the inflammatory signalling.

K E Y W O R D S
adipose-derived mesenchymal stem cells, inflammasome, inflammatory signalling, prion protein, spinal cord injury facility in our institutes with controlled temperature and light cycles (24°C and 12/12 light cycle).

| Isolation of adipose tissue for culturing ADMSCs
For the preparation of allogenic ADMSCs, additional 12 rats were utilized in the present study. The procedure and protocol for ADMSCs isolation and culturing have been described in our previous reports. 22,[24][25][26] In detail, allogenic ADMSC donated rats were anaesthetised and then cut into <1 mm 3 size pieces using a pair of sharp, sterile surgical scissors. After serial incubation and centrifugation, the cells were obtained and cultured in a 100 mm diameter dish with 10 ml DMEM culture medium containing 10% FBS for 14 days.

| Transfection of ADMSCs with plasmids for cellular prion protein (PrPc) overexpression
The procedure and protocol have been reported in our recent study. 35 We purchased the pCS6-PRNP plasmid from Transomic Technologies . The plasmid transfection process was carried out with Lipofectamine 3000 according to the steps specified in the manual. The steps were briefly described as follows: 10 μg PRNP expression vector and 20 μl Lipofectamine 3000 were first incubated at room temperature for 15 minutes, followed by overnight incubation of cells at 37°C in a humidified atmosphere of 5% CO 2 and Lipofectamine (i.e. mixed them together), and relevant experiments were carried out.

| Basso, Beattie, Bresnahan (BBB) functional scale for the assessment of the locomotor capacity of rats after acute SCI
The animals were followed for 6 weeks (i.e. 42 days) after the acute SCI procedure. The procedure and protocol of the BBB scale were according to the previous reports. 36,37 Briefly, each rat was placed in an 80 × 80 × 30 cm 3 clear box lined with a blue non-slippery material and stimulated to move freely. All movements were recorded by video. Identical copies of the edited videos were monitored by two independent evaluators blinded to the degree of injury severity.
Each evaluator accessed the locomotor capacity of rats using the BBB functional scale. At the end of the experiments, the parameters in each rat were calculated and averaging scores from these two evaluators.

| Western blot analysis
Western blot analysis was performed as described previously. [22][23][24] Equal amounts (50 μg) of protein extracts were loaded and separated by SDS-PAGE and transferred to PVDF membranes. Next, the membranes were incubated with the indicated primary antibodies as Table S1 for 1 h at room temperature. Horseradish peroxidaseconjugated anti-rabbit IgG (1:2000, Cell Signalling) was used as a secondary antibody. Immuno-reactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences) and digitized using Labwork software (UVP).

| Immunofluorescent (IF) staining
IF staining proceeded as we previously reported. 22

| Statistical analysis
Quantitative data are expressed as mean ± SD. The statistical analysis was performed by anova followed by Bonferroni multiple comparison post-hoc test. We utilized SAS statistical software for Windows

| Impact of PrPc OE -ADMSCs on time courses of cell viability and anti-oxidative stress underwent oxidative stress and inflammatory stimulation in vitro
First, to verify the protective capacity of PrPc OE in ADMSCs (i.e. PrPc OE -ADMSCs) on cell viability against oxidative stress or inflammatory stimulation, the in vitro study was categorized into G1 (ADMSCs only), The result of the MTT assay demonstrated that as compared with G1, the cell viability at the time intervals of 24, 48 and 72 h was significantly lower in G2 and was significantly reversed in G3 ( Figure 1A). To verify these issues of oxidative stress and inflammation, the in vitro study was categorized into six groups as shown in Figure 1A,B, that is G1 to G6 and the flow cytometric analysis was utilized in the present study. The result showed that the fluorescent intensity of total intracellular and mitochondrial ROS under H 2 O 2 treatment were significantly increased in G2 than in G1 and G3 and significantly increased in G3 than in G1 ( Figure 1C). Additionally, under LPS treatment these parameters were significantly increased in G5 than in G4 and G6, and significantly increased in G6 than in G4 ( Figure 1D). Our findings supported that PrPc gene overexpression in ADMSCs enhanced the resistant capacity against the generation of ROS resulted from oxidative stress and inflammatory stimulation.

| Impact of PrPc OE -ADMSCs on cell migration under oxidative stress and inflammatory stimulations
To elucidate whether PrPc OE -ADMSC treatment would preserve the ADMSCs migratory ability with respect to oxidative stress and inflammatory stimulations, the in vitro study was categorized into six groups as shown in Figure 1.
Under the oxidative stress, that is treated by H 2 O 2 , the ADMSCs migratory ability was significantly lower in G2 than in G1 and G3 and significantly lower in G3 than in G1 (Figure 2A-G). Additionally, under the inflammatory stimulation, that is treated by LPS, the ADMSCs migratory ability also exhibited an identical pattern of oxidative stress among groups G4 to G6 ( Figure 2H-N). These findings implied that PrPc gene overexpression in ADMSCs offered much more excellent benefits than the ADMSC-only counterpart in maintaining the migratory cell ability against oxidative stress or inflammatory stimulation. Next, we collected the circulatory blood sample from post SCI rats and utilized the ELISA to determine the circulating levels of TNFα and IL-6. The results showed that these two parameters were highest in group 2 (SCI), lowest in group 1 (SC), and significantly higher in group 3 (SCI + ADMSCs) than in group 4 (SCI + PrPc OE -ADMSCs) at time points of 6 h and 72 h after SCI induction in rodent ( Figure 3E,F). Our in vitro and in vivo studies indicated that PrPc OE -ADMSCs were better than merely ADMSCs alone at attenuating the inflammatory reaction.

| The time courses of neurological function after acute SCI procedure
The BBB functional scale for assessing the locomotor capacity of rats was performed for each rat on Days 1, 3, 7, 14, 21, 35 and 42 after acute SCI induction ( Figure 4A). By Days 1 and 3, the BBB score of the left or right lower limb was significantly higher in group 1 than in groups 2, 3 and 4, but it did not differ among groups 2-4 at these time inter-

| Flow cytometric analysis of circulatory inflammatory cells by Days 3 and 7 after acute SCI procedure
To elucidate the circulatory levels of inflammatory cells at the acute phase of SCI, the flow cytometric analysis was utilized in the present study. Consistent with in vitro study, the circulatory levels of

| Role of DAMP-signalling inflammatory pathway in chronic phase (i.e. by Day 42) of SCI
To deeply uncover the crucial role of the DAMP signalling pathway on neurological damage in the setting of SCI, six animals in each group were euthanized and the Western blot analysis was utilized again by Day 42 after the SCI procedure. The result showed that F I G U R E 4 Time courses of BBB scores (i.e. an indicator of neurological function) and circulatory inflammatory cells after acute SCI procedure. (A) By Days 1 and 3: * vs. †, p < 0.0001; symbol † indicated the p value >0.5 among the groups of SC, SCI only, SCI + ADMSCs and SCI + PrPc-ADMSCs at these time points with respect to both right and left lower limbs. By Days 7, 14, 21 and 35, ‡ vs. other groups with different symbols ( §, ¶, α), p < 0.0001 at these time points with respect to both right and left lower limbs. By day 42, ß vs. other groups with different symbols (γ, κ), p < 0.0001 at these time points with respect to both right and left lower limbs; γ indicated the p value >0.5 between the groups of SCI + ADMSCs and SCI + PrPc-ADMSCs at this time point (i.e. by Day 42). All statistical analyses were performed by one-way anova, followed by Bonferroni multiple comparison post hoc test (n = 8 for each group). Symbols ( ‡, §, ¶, α) or (ß, γ, κ) indicate significance for each other (at 0.05 level). (B-C) Flow cytometric analysis of circulatory inflammatory cells (CD11b/c+, Ly6G+, myeloperoxidase (MPO)+) by Days 3 and 7 after acute SCI procedure. * vs. other groups with different symbols ( †, ‡, §), p < 0.0001. All statistical analyses were performed by one-way anova, followed by Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (*, †, ‡, §) indicate significance for each other (at 0.05 level). ADMSCs, adipose-derived mesenchymal stem cells; PrPc OE -ADMSCs, overexpression of cellular prion protein in ADMSCs; BBB score, Basso, Beattie, Bresnahan score; SC, sham-operated control; SCI, spinal cord injury the protein expressions of HGMB1, TLR4, MyD88, TRIF, TRAF6 and p-NF-κB, six indicators of upstream DAMP-inflammatory signalling, and the protein expressions of TNFα, MMP-9 and IL-1ß, three indicators of downstream inflammatory DAMP signalling, were highest in group 2, lowest in group 1 and significantly higher in group 3 than in group 4 ( Figure S1). Additionally, the protein expression of PrPc was highest in group 1, lowest in group 2 and significantly higher in group 4 than in group 3. These finding implicated that the PrPc OE -ADMSCs was better than ADMSCs merely for ameliorating the activation of DAMP-inflammatory signalling. Based on these

| Gene and protein expressions of PrP C in ADMSC and the expression of MSCs surface markers
The relative gene expression and protein expression of PrP C in PrPc OE -ADMSCs was significantly increased than in ADMSCs, suggesting that PrP C overexpression in ADMSCs was successful conducted ( Figure S2A,B). Additionally, the flow cytometric analysis showed that the ADMSCs surface markers, including CD29, CD73 and CD99 were highly populated by day-14 cell culture ( Figure S2C-E). In this way, our findings strengthened the findings of our previous studies. [22][23][24][25][26] Originally, PrPc was identified as a glycosylphosphatidylinositolanchored glycoprotein that was enriched in the brain and nerve cells and also displayed in other tissues. 38,39 Subsequently, the PrPc, acting as a neuroprotective or survival protein for cell proliferation and growth, has been discovered because this cellular protein prevents Bcl-2-associated protein X (Bax)-mediated cell apoptosis and death. 40 To further prove these doctrines, [38][39][40] we performed another in vitro study. Our result demonstrated that as compared with ADMSCs only, the cell viability/proliferation rate and migratory ability were much better in PrPc OE in ADMSCs. Our above-mentioned in vitro studies and the findings of previous studies 38-40 encouraged us to perform an acute SCI animal model by using the PrPc OE in ADMSCs therapy.

| DISCUSS ION
The most important finding of the present study was that as compared with the SCI animals, the BBB score (i.e. an index of recovery of neurological function) was substantially preserved in that of SCI animals after receiving ADMSCs treatment. Interestingly, our previous studies have clearly identified that ADMSCs therapy significantly reduced brain infarct size and preserved neurological function in rodents after acute ischemic stroke. 41,42 In this way, our finding was comparable with that of our previous studies. 41,42 Of distinctive finding was that PrPc OE -ADMSCs therapy was found to be better than ADMSCs therapy for the improvement of BBB score at the early and convalescent stages of SCI. However, this advantage was lost at the chronic phase of SCI, suggesting that it could be attributed to the admirably inherent capacity of rodents for self-regeneration. The other cause may also be because one dosage of PrPc OE -ADMSCs therapy is not enough when further improvement of neurological function is considered in the chronic phase of SCI.

F I G U R E 7
Schematically illustrated the proposed mechanism of NLRP3 inflammasome signalling participated in the acute and chronic phases of SCI. ADMSCs, adipose-derived mesenchymal stem cells; PrPc OE -ADMSCs, overexpression of cellular prion protein in ADMSCs; SCI, spinal cord injury In response to the distinctive outcome of neurological functional recovery was identified, the next step had to perform the bench works to delineate the possible underlying mechanism of these promising treatments. Intriguingly, when carefully reviewing the previous studies, we could find that acute tissue/organ injury always elicits vigorous inflammatory reaction. [31][32][33]35,41,42 By acute phase after acute SCI procedure, we found the circulatory levels of inflammatory cells (i.e. by flow cytometry) and proinflammatory cytokines (i.e. by ELISA) were markedly increased in animals with SCI injury, suggesting that systemically inflammatory reaction was elicited in the setting of SCI. Additionally, when looking at the molecular level (i.e. Western blot analysis), we found the NLRP3 inflammasome was substantially upregulated in SCI animals. In addition to corroborating the findings of previous studies, [31][32][33]35,41,42 our findings indicated that this acute inflammatory signalling pathway might play a crucial role in the damage of the spinal cord after SCI induction. An important finding was that PrPc OE -ADMSCs were superior to ADMSCs only in suppressing upstream and downstream inflammatory signalling.
Further, we were curious about the inflammatory reaction and what the crucial inflammatory signalling in the chronic phase (i.e., by day 42 after SCI induction) of SCI was, the Western blot analysis was again utilized. As we expected, not only one inflammatory signalling (i.e. NLRP3 inflammasome) but also the DAMP inflammatory signalling biomarkers were found remaining to be notably increased, suggesting that the chronic inflammatory reaction could be persistently present after SCI for the destruction of the neurons and tissues, resulting in impeding the neurological functional recovery in a distant future. Of promising findings was that these two inflammatory signalling were markedly suppressed by ADMSCs and further markedly suppressed by PrPc OE -ADMSCs, highlighting that this strategic management, particularly in the utilization of PrPc, which is the critical protein for neuron growth and proliferation, could be considered for the SCI patients, especially when they are refractory to conventional treatment.

| Study limitation
This study has limitations. First, although the study period was 42 days, we remain concerned about whether such a time interval was long enough to define a true 'chronic phase' of SCI. Second, we did not test whether two doses of cell therapy would be better than a single dose for improving the outcome in the setting of SCI. Third, we remain uncertain whether two doses of PrPc OE in ADMSCs would be superior to two doses of ADMSCs for augmenting the recovery of neurological function. Finally, we did not utilize agonists or antagonists (i.e. such as drugs, specific inhibitors, or gene manipulations) to prove or disprove the proposed signalling pathways of NLRP3 inflammasome and DAMPinflammation involved in SCI. Thus, we cannot rule out there may also have other signalling pathways to participate in the damage of the injured spinal cord.

| CON CLUS ION
Overall, inflammatory signalling might play a fundamental role in spinal cord damage and retardation of neurological functional recovery. PrPc OE -ADMSCs might be superior to AMDSCs in improving the outcomes after acute SCI.

ACK N OWLED G EM ENT
This study was supported by a program grant from Chang Gung Memorial Hospital, Chang Gung University (CMRPG8K1511).

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data available on request from the authors.