Transplantation of mesenchymal stem cells ameliorates secondary osteoporosis through interleukin-17-impaired functions of recipient bone marrow mesenchymal stem cells in MRL/lpr mice

Introduction Secondary osteoporosis is common in systemic lupus erythematosus and leads to a reduction in quality of life due to fragility fractures, even in patients with improvement of the primary disorder. Systemic transplantation of mesenchymal stem cells could ameliorate bone loss and autoimmune disorders in a MRL/lpr mouse systemic lupus erythematosus model, but the detailed therapeutic mechanism of bone regeneration is not fully understood. In this study, we transplanted human bone marrow mesenchymal stem cells (BMMSCs) and stem cells from exfoliated deciduous teeth (SHED) into MRL/lpr mice and explored their therapeutic mechanisms in secondary osteoporotic disorders of the systemic lupus erythematosus model mice. Methods The effects of systemic human mesenchymal stem cell transplantation on bone loss of MRL/lpr mice were analyzed in vivo and ex vivo. After systemic human mesenchymal stem cell transplantation, recipient BMMSC functions of MRL/lpr mice were assessed for aspects of stemness, osteogenesis and osteoclastogenesis, and a series of co-culture experiments under osteogenic or osteoclastogenic inductions were performed to examine the efficacy of interleukin (IL)-17-impaired recipient BMMSCs in the bone marrow of MRL/lpr mice. Results Systemic transplantation of human BMMSCs and SHED recovered the reduction in bone density and structure in MRL/lpr mice. To explore the mechanism, we found that impaired recipient BMMSCs mediated the negative bone metabolic turnover by enhanced osteoclastogenesis and suppressed osteoblastogenesis in secondary osteoporosis of MRL/lpr mice. Moreover, IL-17-dependent hyperimmune conditions in the recipient bone marrow of MRL/lpr mice damaged recipient BMMSCs to suppress osteoblast capacity and accelerate osteoclast induction. To overcome the abnormal bone metabolism, systemic transplantation of human BMMSCs and SHED into MRL/lpr mice improved the functionally impaired recipient BMMSCs through IL-17 suppression in the recipient bone marrow and then maintained a regular positive bone metabolism via the balance of osteoblasts and osteoclasts. Conclusions These findings indicate that IL-17 and recipient BMMSCs might be a therapeutic target for secondary osteoporosis in systemic lupus erythematosus. Electronic supplementary material The online version of this article (doi:10.1186/s13287-015-0091-4) contains supplementary material, which is available to authorized users.


Introduction
Osteoporosis is defined as a reduction in bone strength and is the most common bone disease [1]. The bone loss is primarily related to age and/or menopause and secondarily affected by underlying risk factors such as nutritional deficiencies, diseases, or drugs [2]. Systemic lupus erythematosus (SLE) is a refractory and chronic multiorgan autoimmune disease. Because recent medical advances have successfully increased the lifespan of patients with SLE, many clinical researchers have focused on the organ damage associated with the systemic chronic inflammation and/or long-term medications relating to quality of life [3]. Secondary osteoporosis frequently occurs in SLE patients, which causes fragility fractures [4]. Currently, there are no safe or efficient treatments for SLE-associated osteoporosis.
Mesenchymal stem cells (MSCs) are a typical type of adult stem cell with the capabilities of self-renewal and multilineage differentiation [5]. Recent studies show that MSCs have immunomodulatory effects on immune cells [6,7], and MSC-based cell therapy has been greatly focused on the treatment of various immune diseases such as acute graft-versus-host disease [8] and inflammatory bowel disease [9]. Previous allogeneic transplantation of human bone marrow MSCs (hBMMSCs) and human umbilical cord-derived MSCs (hUCMSCs) governs successful therapeutic efficacy in refractory SLE patients [10][11][12]. However, it is unclear whether MSC transplantation is an effective treatment for skeletal disorders in SLE patients.
MRL/lpr mice are a well-known model of human SLElike disorders with clinical manifestations including a short lifespan, abundant autoantibodies, glomerulonephritis, and a breakdown of self-tolerance [13]. Furthermore, MRL/lpr mice exhibit a severe reduction of the trabecular bone, which is associated with excessive osteoclastic bone resorption and limited osteoblastic bone formation [10]. Recent studies show that systemic transplantation of human MSCs, including hBMMSCs, hUCMSCs, stem cells from human exfoliated deciduous teeth (SHED), and human supernumerary tooth-derived stem cells, improves primary autoimmune disorders in MRL/lpr mice, such as elevated autoimmune antibodies, renal dysfunction, and abnormal immunity [14][15][16][17][18]. In addition, hBMMSC and SHED transplantation markedly recovers the bone loss in MRL/lpr mice [16,17]. These results indicate that MSC transplantation might be a therapeutic approach for SLE patients who suffer from secondary osteoporosis. However, little is known about the human MSC-mediated therapeutic mechanism in the skeletal disorder of MRL/lpr mice.
Osteoporosis is characterized by a disruption of the balance between the formation and resorption of bone, which is associated with abnormal development of osteoclasts and osteoblasts. Increasing evidence has shown that BMMSCs from SLE patients and SLE model MRL/lpr mice exhibit a reduction in their bone-forming capacity both in vitro and in vivo [10,19]. Therefore, the osteogenic deficiency of recipient BMMSCs might explain the origin of osteoporosis in SLE. Accordingly, the impaired BMMSCs might be a therapeutic target for osteoporosis. However, little is known about the processes through which recipient BMMSCs are damaged functionally or the underlying mechanism of human MSC transplantation in restoration of the reduced bone formation via recipient BMMSCs in the bone marrow under SLE conditions.
In this study, we used MRL/lpr mice to examine the therapeutic efficacy and mechanisms of systemically transplanted hBMMSCs and SHED in the secondary osteoporotic disorders of SLE. Moreover, we focused on the pathological and clinical contributions of recipient BMMSCs to the dysregulation of bone metabolism through osteoblasts and osteoclasts in the inflammatory bone disorder of SLE.

Human subjects
Human exfoliated deciduous teeth were obtained as clinically discarded biological samples from five patients (5)(6)(7) years old) at the Department of Pediatric Dentistry of Kyushu University Hospital under the approval of the Kyushu University Institutional Review Board for Human Genome/Gene Research (protocol number: 393-01). Written informed consent was obtained from all parents on behalf of the participants.

Mice
C57BL/6J-lpr/lpr mice (female, 8 weeks old), and pregnant and young adult C57BL/6J mice (female, 8 weeks old) were purchased from Japan SLC (Shizuoka, Japan) and CLEA Japan (Tokyo, Japan), respectively. All animal experiments were approved by the Institutional Animal Care and Use Committee of Kyushu University (protocol number: A21-044-1).
To identify the isolated cells as MSCs, passage 3 (P3) cells (1×10 5 /100 μl) were stained with specific antibodies against stem cell markers including CD11b, CD14, CD35, CD45, CD73, CD90, CD105 and CD146 (1 μg/ml each; eBioscience, San Diego, CA, USA) and then analyzed using a flow cytometer (FACSVerse, BD Biosciences). The percentages of positive cells were determined by comparison with the corresponding control cells stained with an isotype-matched antibody, in which a false-positive rate of less than 1 % was acceptable. The isolated cells were positive for CD73, CD90, CD105, and CD146, and negative for CD11b, CD14, CD35, and CD45 (data not shown). Furthermore, P3 cells were cultured under osteogenic, chondrogenic, or adipogenic conditions [17,18], and showed differentiation capacities for osteoblasts (odontoblasts in the case of SHED), chondrocytes, and adipocytes (data not shown). These findings showed that our isolated cells were MSCs based on the standard MSC criteria [22].
Systemic MSC transplantation into MRL/lpr mice P3 hBMMSCs and SHED were collected and washed with PBS three times. The donor cells were diluted in PBS, and intravenously infused at 1×10 5 per 10 g body weight into 16-week-old MRL/lpr mice via the right cervical vein according to a previously published method [16]. The mice were analyzed at 20 weeks of age. Age-matched MRL/lpr mice that received PBS were used as controls.

Histological bone analysis
Tibias were fixed with 4 % paraformaldehyde in PBS and decalcified with 10 % ethylenediaminetetraacetic acid. Paraffin sections were prepared at a thickness of 6 μm and stained with tartrate-resistant acid phosphate (TRAP) [21]. The number of TRAP-positive cells per total bone area in the bone metaphysis was analyzed in five representative images by Image J software (National Institutes of Health, Bethesda, MA, USA).

Isolation and culture of mouse BMMSCs
Mouse BMMSCs were isolated based on the CFU-F method [23]. BMCs were seeded at 1-2×10 7 cells per 100-mm culture dish. After 3 h, the cells were washed with PBS twice to eliminate non-adherent cells, and the attached cells were cultured for 14-16 days. Attached colonies consisting of spindle-shaped cells were observed under a microscope. The colony-forming attached cells were passaged once. The cells were cultured in αMEM supplemented with 20 % FBS, 2 mM L-glutamine, 55 μM 2-mercaptoethanol, 100 U/ml penicillin, and 100 μg/ml streptomycin. Based on the MSC criteria [22], the colonyforming cells were characterized as described previously [24]: 1) flow cytometry demonstrated immunophenotypes of CD73, CD105, CD146, Sca-1, and SSEA-4, and negativity for CD14, CD34, and CD45; 2) mouse BMMSCs were evaluated for differentiation into osteoblasts, chondrocytes, and adipocytes under the corresponding specific culture conditions.

Western blotting
All the samples were lyzed in M-PER mammalian protein extraction reagent (Thermo, Rockford, IL, USA) containing proteinase inhibitor cocktail (Nacalai Tesque). They were separated by sodium dodecyl sulfatepolyacrylamide gel and transferred to Immobilon-P membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5 % skimmed milk in Tris-buffered saline (150 mM NaCl and 20 mM Tris-HCl, pH 7.2) for 1 h at room temperature and then incubated with anti-mouse IL-17 antibody overnight at 4°C. They were then treated with horseradish peroxidase-conjugated donkey antirabbit or anti-mouse IgG antibody (1:1000; Santa Cruz Biotechnology) for 1 h at room temperature. The bound antibodies were visualized using SuperSignal West Pico (Thermo).

Statistical analysis
Data were analyzed by the one-way analysis of variance F-test. P-values of less than 0.05 were considered to be significant.

Supplementary materials and methods
Supplementary materials and methods are provided as Additional file 1.

Systemic human MSC transplantation ameliorates SLE disorders in MRL/lpr mice
According to a previous method [16], hBMMSCs and SHED (1×10 5 per 10 g body weight) were systemically transplanted through the right cervical vein into MRL/ lpr mice at 16 weeks of age (Fig. S1a in Additional file 2), following which they exhibited severe autoimmune disorders including abnormal autoantibody increments and severe renal nephritis (Fig. S1b and c in Additional file 2) [10]. The systemic transplantation of human MSCs was followed by evaluation of their immune therapeutic efficacy in the SLE-like disorders, such as hyper-autoimmune antibody production and renal dysfunction in MRL/lpr mice at 4 weeks post-transplantation (Fig. S1b and c in Additional file 2) as described previously [16,17].

Systemic human MSC transplantation improves secondary bone loss in MRL/lpr mice
Severe osteoporosis with progressive trabecular bone breakdown occurs secondarily in SLE model MRL/lpr mice at 16 weeks of age ( Fig. 1) [10] and SLE patients [4]. To investigate effects of transplanted human MSC . These findings suggested that human MSC transplantation recovered the bone reduction in MRL/lpr mice via accelerated bone formation and suppressed bone resorption, but did not elucidate the detailed therapeutic mechanisms at cellular and molecular levels.

Systemic human MSC transplantation recovers dysregulation of osteoblast and osteoclast development via recipient BMMSCs in MRL/lpr mice
Dysregulation of osteoblast and osteoclast development in bone marrow leads to skeletal dysfunction. BMMSCs play a crucial role in the development of osteoblasts and osteoclasts in bone marrow [25][26][27]. Recent studies showed that recipient BMMSCs in SLE mice and patients impaired the stemness and osteogenic capacity [10,19]. Therefore, we hypothesized that recipient BMMSCs might participate in the osteoporosis of SLE and may be a therapeutic target. We isolated recipient BMMSCs from non-, hBMMSC-, and SHED-transplanted MRL/lpr mice, and designated them as MSC-MRL/lpr, MSC-hBMMSC, and MSC-SHED, respectively. We also isolated recipient BMMSCs from pre-transplant MRL/lpr mice at 16  to wild-type C57/BL6 mice (data not shown), as shown previously [10]. We examined the immunoregulatory effect of hBMMSCs and SHED on IL-17 production using a co-culture system of human MSCs with human T cells in the presence of transforming growth factor-β 1 and IL-6. This co-culture system demonstrated that hBMMSCs and SHED suppressed both Th17 cell differentiation and IL-17 secretion by flow cytometric analysis and ELISA, respectively ( Fig. S5a and b in Additional file 2). To confirm in vivo immunological effects of hBMMSCs and SHED on IL-17 secretion, we analyzed peripheral levels of Th17 cells and IL-17 in MRL/lpr mice. Immunological analyses showed that hBMMSCs and SHED suppressed systemic Th17 cells and IL-17 expression in MRL/lpr mice, as well as pre-transplant MRL/lpr mice ( Fig. S5c and d in Additional file 2). These findings indicated that hBMMSCs and SHED expressed a suppressive regulation of Th17 cell differentiation in vitro and in vivo, as reported previously [10,16].
Carboxyfluorescein diacetate succinimidyl ester (CFSE)labeled hBMMSCs and SHED were infused intravenously into MRL/lpr mice. Subsequently, these cells were found in the recipient bone marrow space at days 1 and 7 after infusion, but the positive cell number was lower at day 7 than that at day 1 (Fig. S6a in Additional file 2). IL-17 is significantly increased in the bone marrow of MRL/lpr mice [10]. We analyzed IL-17 levels in bone marrow tissues of hBMMSC-and SHED-transplanted MRL/lpr mice in comparison with non-transplanted MRL/lpr mice. In immunofluorescence analysis, hBMMSC and SHED transplantation resulted in a marked decrease in IL-17-positive cells in the recipient bone marrow (  (Fig. 2e). BMC-hBMMSC and BMC-SHED exhibited lower production of IL-17 than BMC-MRL/lpr, as well as BMC-Pre-MRL/lpr (Fig. 2f ), reflecting the recipient bone marrow IL-17 conditions. Taken together, these findings indicated that transplanted hBMMSCs and SHED might suppress the abnormal IL-17 production in the recipient bone marrow of MRL/lpr mice, but did not evaluate whether the recipient IL-17 conditions effected on the dysregulation by osteoclastic bone resorption and osteoblastic bone formation in MRL/lpr mice.
To understand the mechanistic transplant studies, we transplanted hBMMSCs and SHED (1×10 5 per 10 g body weight) into wild-type C57BL/6 mice at 16 weeks of age, and analyzed the effects of the transplants 4 weeks after the infusion (Fig. S1a in Additional file 2). There was no effect of hMSC transplantation on bone metabolism and IL-17 levels (Fig. S7 in Additional file 2). These findings suggested that recipient inflammatory conditions might influence the transplants' ability to treat recipient bone metabolism. capacity of MSC-WT as demonstrated by Alizarin Red staining ( Fig. S8a and b in Additional file 2). Anti-IL-17 antibody treatment reversed the suppressed osteogenic capacity in IL-17-and CM-MRL/lpr-treated MSC-WT when co-treated with the control antibody treatment ( Fig. S8a and b in Additional file 2). These preliminary data suggested that IL-17 conditions in the recipient bone marrow might modulate the osteoblastic differentiation of recipient BMMSCs in MRL/lpr mice. We then examined whether IL-17-dependent immune conditions in recipient bone marrow affected recipient BMMSC-mediated bone formation. Mouse BMMSCs were cultured under osteogenic conditions with or without CM collected from BMC-hBMMSC and BMC-SHED cultures (CM-hBMMSC, and CM-SHED, respectively), as well as CM-MRL/lpr, in the presence or absence of antimouse IL-17 antibody (Fig. 3a). The control antibody for anti-mouse IL-17 antibody was used as the control treatment. Alizarin Red staining results showed that CM-MRL/lpr significantly suppressed the osteogenic capacity of MSC-WT (Fig. 3b, Fig. S8b in Additional file 2), whereas CM-hBMMSC and CM-SHED showed little suppressive effects on the osteogenic capability of MSC-WT (Fig. 3b). Anti-IL-17 antibody treatment neutralized the inhibited osteogenic capability by the individual CMs, especially in the CM-MRL/lpr-treated group (Fig. 3b,  Fig. S8b in Additional file 2). These results suggested that the hyperactivity of IL-17 in recipient bone marrow of MRL/lpr mice might cause the severe defective bone formation mediated by recipient BMMSCs.
Next, we investigated the effect of abnormal IL-17 in recipient bone marrow of MRL/lpr mice on recipient BMMSC-mediated bone formation. Recipient BMMSCs, including MSC-MRL/lpr, MSC-hBMMSC and MSC-SHED, were treated with CM-MRL/lpr under osteogenic conditions. CM-MRL/lpr treatment significantly suppressed the mineralized deposition induced by MSC-hBMMSC and MSC-SHED (Fig. 3c). The suppressed MSC-hBMMSCand MSC-SHED-induced mineralization showed similar rates to MSC-MRL/lpr-induced bone formation (Fig. 3c). The CM-MRL/lpr treated suppression was recovered by the treatment with anti-IL-17 antibody (Fig. 3c). We then cultured individual recipient BMMSCs under osteogenic conditions in the presence of IL-17. IL-17 significantly suppressed the mineralized deposition induced by MSC-hBMMSC and MSC-SHED, which showed similar deposition rates to that of MSC-MRL/lpr (Fig. 3d). Anti-IL-17 antibody treatment completely neutralized the IL-17suppressed osteogenic capacities of MSC-hBMMSC and MSC-SHED (Fig. 3d). These findings indicated that abnormal IL-17 in the bone marrow of MRL/lpr mice impaired the osteogenic capacity of recipient BMMSCs, and suggested that hBMMSC and SHED transplantation recovered the osteogenic dysfunction of recipient BMMSCs through inhibiting hyperactivated IL-17 in the recipient bone marrow of MRL/lpr.

Systemic human MSC transplantation rescues impaired recipient BMMSC-mediated osteoclast induction through IL-17-dependent immune conditions in the recipient bone marrow of MRL/lpr mice
We also preliminarily examined the effects of IL-17 and CM-MRL/lpr on osteoclast differentiation in the coculture system with BMC-WT and wild-type mousederived calvarial cells (Calvaria-WT). BMC-WT were co-cultured with Calvaria-WT under the stimulation of vitamin D 3 and prostaglandin E 2 in the presence or absence of IL-17 and CM-MRL/lpr (Fig. 4a). The control antibody for anti-mouse IL-17 antibody was also used as the control treatment. TRAP staining showed that both IL-17 and CM-MRL/lpr treatments induced TRAPpositive MNCs in the co-culture system (  (Fig. 4b). Anti-IL-17 antibody treatment reduced the number of TRAP-positive MNCs under the stimulation of CM-hBMMSC and CM-SHED (Fig. 4b). Subsequently, we co-cultured Calvaria-WT with recipient BMCs. Induction of TRAP-positive MNCs in the co-culture systems with BMC-hBMMSC and BMC-SHED showed a fewer number of TRAP-positive MNCs than that with BMC-MRL/lpr ( Fig. 4c and d). Treatment with CM-MRL/lpr and IL-17 stimulated the reduced osteoclast induction in the co-culture systems with BMC-hBMMSC and BMC-SHED ( Fig. 4c and d). Anti-IL-17 antibody treatment neutralized the CM-MRL/lpr-and IL-17enhanced TRAP-positive MNC-induction in the coculture systems with BMC-hBMMSC and BMC-SHED ( Fig. 4c and d). These results suggested that IL-17dependent immune conditions in the recipient bone marrow might modulate osteoclastic differentiation and bone resorption in MRL/lpr mice.
To examine whether IL-17-dependent immune conditions in recipient bone marrow affect recipient BMMSCmediated osteoclast induction, we pretreated recipient BMMSCs with CM of recipient BMC culture or IL-17 in the presence or absence of anti-IL-17 antibody. In the co-culture system of MSC-MRL/lpr and BMC-MRL/lpr, TRAP staining demonstrated that CM-MRL/lpr-enhanced TRAP-positive MNC induction was reduced when pretreated with CM-hBMMSC and CM-SHED (Fig. 4e). BMC-MRL/lpr were then co-cultured with individual recipient MSCs. MSC-hBMMSC and MSC-SHED showed less TRAP-positive MNC-inductive functions in comparison with MSC-MRL/lpr ( Fig. 4f and g). CM-MRL/lpr-pretreated recipient BMMSCs significantly enhanced the TRAP-positive MNC induction from BMC-MRL/lpr in the respective co-culture system, but the pretreatment effects on MSC-hBMMSC and MSC-SHED were less than those on MSC-MRL/lpr (Fig. 4f ). Anti-IL-17 antibody co-pretreatment blocked the CM-MRL/lpr-enhanced TRAP-positive MNCs in the individual co-culture system (Fig. 4f ). We then examined whether IL-17 treatment in recipient BMMSCs directly affected the osteoclast inductivity in the co-culture system with BMC-MRL/lpr. IL-17-pretreated MSC-hBMMSC and MSC-SHED, as well as MSC-MRL/lpr, significantly induced TRAP-positive MNCs, but the inductivity by IL-17-pretreated MSC-hBMMSC and MSC-SHED was less than that by IL-17-pretreated MSC-MRL/lpr (Fig. 4g). The increased TRAP-positive MNC inductivity was inhibited when the recipient BMMSCs were copretreated with anti-IL-17 antibody (Fig. 4g). These findings suggested that IL-17-dependent hyperimmune conditions in the recipient bone marrow of MRL/lpr mice might impair recipient BMMSCs to accelerate abnormal osteoclast induction while human MSC transplantation might target the impaired recipient BMMSCs to suppress the enhanced osteoclast inductivity.

Functional downregulation of IL-17 in BMCs of MRL/lpr mice inhibits recipient BMMSC-mediated osteoclast differentiation
We examined whether IL-17 levels in recipient BMCs of MRL/lpr mice play a significant role in osteoclast differentiation through recipient BMMSCs. BMC-MRL/lpr were treated with mouse IL-17 siRNA or the control siRNA, and were co-cultured with recipient BMMSCs under stimulation with 1α, 25-(OH) 2 vitamin D 3 and prostaglandin E 2 (Fig. 5a). Some co-cultures were incubated in the presence of anti-mouse IL-17 antibody or the control antibody (Fig. 5a). Western blotting confirmed that siRNA for mouse IL-17 significantly downregulated the expression of IL-17 in BMC-MRL/lpr (Fig. 5b). IL-17 siRNA-treated BMC-MRL/lpr inhibited individual recipient BMMSCmediated osteoclast induction in the co-culture system, but control siRNA-treated BMC-MRL/lpr did not (Fig. 5c). IL-17 antibody treatment also suppressed osteoclast differentiation in a co-culture system of intact BMC-MRL/lpr and individual recipient BMMSCs (Fig. 5d). Furthermore, to understand whether IL-17 levels in recipient BMCs affect direct capabilities of osteoclast differentiation of the BMCs, recipient BMCs were cultured in a RANKL-M-CSF culture system (Fig. 5e). Although IL-17 siRNA treatment in individual BMCs efficiently induced TRAPpositive MNCs, the efficiency was similar to the control cultures treated with control siRNA (Fig. 5f). Anti-IL-17 antibody treatment showed no effect on TRAP-positive MNC formation from individual recipient BMCs in a RANKL-M-CSF culture system when compared to the control antibody treatment (Fig. 5g). In addition, osteoclast formation in a RANKL-M-CSF system expressed a similar efficacy among individual recipient BMCs ( Fig. 5f and g). These finding suggested that IL-17 levels in recipient BMCs might affect recipient BMMSC-mediated osteoclast differentiation of recipient BMCs, but not direct osteoclastogenesis of recipient BMCs.

Systemic treatment of anti-IL-17 antibody improves secondary bone loss in MRL/lpr mice
To investigate a role of IL-17 on skeletal metabolism in MRL/lpr mice, we systemically infused anti-IL-17 antibody to MRL/lpr mice at 16 weeks of age for 4 weeks (

Discussion
The incidence of secondary osteoporosis in SLE patients ranges from 6.3 % to 28 % worldwide [30,31]. Osteoporosis is generally caused by disruption of bone remodeling, which is related to menopause and aging, whereas secondary osteoporosis in SLE is multifactorial and involves excessive systemic inflammation and long-term anti-inflammatory/immunosuppressive medications such as glucocorticoids [25]. These risk factors lead to fragility fractures, which are mainly vertebral fractures, and can proceed to new fractures, increase the mortality rate, and reduce quality of life [32,33]. Among the numerous antiosteoporosis drugs to prevent and treat bone loss in SLE patients, bisphosphonates are a first-line, anti-resorptive agent but show little effect on bone reconstruction and exhibit side effects such as negative fetus development, osteonecrosis of the jaw, and musculoskeletal pain. Other anti-osteoporotic agents, such as estrogen, calcitonin, and raloxifene, exhibit various limitations in terms of potency, population effects, and side effects [34]. From the viewpoint of a quality of life-based medicine, novel therapeutics have been strongly desired to recover the bone loss in SLE patients. In this study, according to recent findings of human MSC-based therapy [14][15][16][17][18], we systemically transplanted hBMMSCs and SHED into SLE model MRL/lpr mice with a severe osteoporotic phenotype and assessed their therapeutic effects on bone loss. Our results demonstrated that human MSC-based therapy ameliorated bone reduction in MRL/lpr mice. Upon dysregulation of the bone remodeling balance by osteoblasts and osteoclasts, the skeletal system enters a pathological condition. In bone marrow, BMMSCs not only serve as a source of osteoblasts, but also support osteoclast differentiation [25][26][27]. Increasing evidence has shown impaired functions in BMMSCs derived from SLE patients [10,19,35,36] and SLE model mice [10], suggesting that a recipient BMMSC deficiency might participate in the pathology of secondary osteoporosis in SLE. However, recent studies demonstrate that systemic MSC transplantation into MRL/lpr mice improves the bone loss, although no study has focused on recipient BMMSCs in the bone regeneration process by systemic transplantation of MSCs [10,16,17]. Therefore, we hypothesized that the therapeutic effects of systemically transplanted MSCs on bone regeneration in SLE were mediated by recovery of deficient host BMMSCs. Interestingly, in the present study, the exogenous hBMMSCs and SHED recovered the impaired bone-forming capability of recipient BMMSCs and reduced abnormal osteoclast induction via recipient BMMSCs in the bone marrow of MRL/lpr mice. These findings suggest that recovery of the impaired recipient BMMSCs might be a critical process to regenerate the skeletal loss in MRL/ lpr mice after human MSC transplantation. However, further studies should explore the cellular and molecular mechanisms through which exogenous MSCs improve the deficiency of recipient BMMSCs.
Inflammation shifts skeletal homeostasis to a bone resorptive condition. Secondary osteoporosis in SLE involves complex interplay of hyperactivated immune reactions and abnormal bone metabolism. The proinflammatory cytokine IL-17, which is produced by Th17 cells [37], has been investigated as a participant in various autoimmune diseases [38,39]. These findings suggest a novel role for IL-17 in bone diseases such as rheumatoid arthritis and osteoporosis [40,41]. In this study, we demonstrated that IL-17dependent hyperimmune conditions in the recipient bone marrow in MRL/lpr mice impaired recipient BMMSCs to suppress the osteogenic function and accelerate the osteoclast induction. Our IL-17 neutralization experiments also provided strong evidence that abnormal IL-17 expression in the bone marrow of MRL/lpr mice impaired recipient BMMSC-mediated osteogenesis and recipient BMMSCmediated osteoclast induction. However, our siRNA and neutralizing antibody of IL-17 experiments also demonstrated that IL-17 levels in recipient BMCs might affect recipient BMMSC-mediated osteoclast differentiation of recipient BMCs, but not influence osteoclastogenesis of recipient BMCs directly. In bone metabolism, IL-17 (See figure on previous page.) Fig. 5 Functional downregulation of IL-17 in bone marrow cells of MRL/lpr mice suppressed recipient BMMSC-mediated osteoclastogenesis. a-d Osteoclast induction assay in bone marrow cell MRL/lpr (BMC-MRL/lpr) and recipient bone marrow mesenchymal stem cell (BMMSC) co-culture. a A schema of osteoclast induction in a co-culture system. BMC-MRL/lpr were pretreated with or without mouse interleukin-17 siRNA (IL-17 siRNA) and the control siRNA (Cont siRNA). Some BMCs were co-cultured with recipient BMMSCs under the stimulation with 1α, 25-(OH) 2 vitamin D 3 (VD3) and prostaglandin E 2 (PGE2) in the presence of anti-mouse IL-17 antibody (Anti-IL-17 Ab) or control antibody for anti-mouse IL-17 antibody (Cont Ab). b Expression of IL-17 in BMC-MRL/lpr pretreated with IL-17 siRNA and Cont siRNA by Western blotting. c, d Osteoclast induction assay by tartrate-resistant acid phosphate (TRAP) staining. c Effects of IL-17 siRNA. d Effects of Anti-IL-17 Ab. e-g Direct osteoclast induction assay of recipient BMCs. e A schema of a direct osteoclast induction in a receptor activator for nuclear factor κB ligand and macrophage-colony stimulating factor (RANKL + M-CSF) system. Recipient BMCs were pretreated with or without IL-17 siRNA and Cont siRNA and cultured under a stimulation of M-CSF and RANKL. Some BMCs were cultured in the presence of Anti-IL-17 Ab or Cont Ab. f, g Osteoclast induction assay by TRAP staining. f Effects of IL-17 siRNA. g Effects of Anti-IL-17 Ab. n = 5 for all groups. Values are shown as means ± SD. *P < 0.05. c ¶ P < 0.05, ¶ ¶ ¶ P < 0.005, versus co-culture of Cont siRNA-treated BMC-MRL/lpr and MSC-MRL/lpr. # P < 0.05, versus co-culture of IL-17 siRNA-treated BMC-MRL/lpr and MSC-MRL/lpr co-culture. d ¶ P < 0.05, ¶ ¶ P < 0.01, ¶ ¶ ¶ P < 0.005, versus co-culture of BMC-MRL/lpr and MSC-WT in the presence of Cont Ab. # P < 0.05, versus co-culture of BMC-MRL/lpr and MSC-MRL/lpr in the presence of Anti-IL-17 Ab. MNC multinucleated cell; MSC mesenchymal stem cell; SHED stem cells from exfoliated deciduous teeth induces osteoclastic differentiation through osteoclastogenesis-supporting cells, such as mesenchymal cells and osteoblasts, but not in direct osteoclast induction stimulated by M-CSF and RANKL [42,43]. IL-17 also directly inhibits osteoblastic differentiation of MSCs [44]. Therefore, these findings suggest that hyperactivated IL-17 in recipient bone marrow may be responsible for secondary osteoporosis in MRL/lpr mice by impairing recipient BMMSCs. Furthermore, the present systemic transplantation of hBMMSCs and SHED suppressed the increased expression of bone marrow IL-17 in MRL/lpr mice, and restored the impaired functions of recipient BMMSCs in bone metabolism. NF-κB activation by proinflammatory cytokines including IL-17 is known to reduce the bone formation capacity of host BMMSCs [19,44]. Further efforts will be required to elucidate the crucial mechanism of IL-17 in the recipient BMMSCbased pathology of secondary osteoporosis in SLE, which may lead to novel recipient BMMSC-targeting therapeutics for skeletal disorders.
IL-17 antibody treatments have emerged as a novel therapeutic approach for immune-mediated diseases such as psoriasis, rheumatoid arthritis, psoriatic arthritis and ankylosing spondylitis [45,46]. Experimental evidence demonstrated anti-IL-17 therapy could protect bone destruction in rheumatoid arthritis by reducing the number of osteoclasts in joints as well as Th17 cells [47]. Anti-IL-17 antibody also preserves skeletal loss in osteoporosis by enhancing osteoblastic bone formation and suppressing osteoclastic bone resorption, as well as protecting against IL-17-mediated immune damages [48]. Several direct IL-17 inhibitors (for example, secukinumab and ixekizumab, anti-IL-17A monoclonal antibody) have shown exciting advances in proof-of-concept and phase II clinical trials, but also expected are further evaluations from phase III clinical trials in multiple autoimmune and immunerelated inflammatory diseases [49]. In this study, we demonstrated that systemic treatment of anti-IL-17 antibody recovered bone reduction in MRL/lpr mice. The usage of IL-17 antibody in treating secondary osteoporosis will not only provide a therapeutic method but also improve the understanding of disease pathogenesis.
In this study, we found that pathological immune conditions affected recipient MSCs, and indicated that hBMMSCs and SHED transplantation improved the bone reduction by restoring IL-17-impaired recipient BMMSCs after migration into the damaged bone marrow. These results suggest that abnormal recipient BMMSCs may undergo correction of their primary functions by the post-transplantation actions of human MSCs. Although the therapeutic mechanism of engrafted human MSCs in the target bone site is not fully understood, several possibilities may be involved in the posttransplantation behaviors of human MSCs in impaired bone marrow. Migrated human MSCs have the potential to participate directly in bone regeneration by differentiating into osteoblasts and suppressing osteoclast induction [25][26][27]. However, bone reconstruction is affected by proinflammatory cytokines at bone defect sites [50]. In addition, MSCs act as cellular modulators through immunomodulatory and trophic effects [51]. Immunomodulatory functions are inducted by cell-cell contact and include FasL-mediated T cell apoptosis [52], CCR6mediated Th17 cell inhibition [53], and MSC-secreted molecule (e.g., IL-10)-mediated Th17 cell suppression [54]. Trophic molecules released from MSCs can inhibit apoptosis and scar formation, such as macrophage inflammatory protein-1, stromal cell-derived factor-1, transforming growth factor-β1, and vascular endothelial growth factor [55]. Therefore, exogenous MSCs may exert their immunomodulatory and trophic functions by secreting bioactive molecules to recover impaired recipient BMMSCs. Further studies will be necessary to explore the deeper mechanisms of transplanted MSCmediated recovery of recipient BMMSCs.

Conclusions
The present study demonstrates that systemic transplantation of human MSCs, including SHED and hBMMSCs, ameliorates severe bone reduction, as well as primary SLE disorders, in MRL/lpr mice. The therapeutic efficacy is mediated by recovery of the impaired functions of recipient BMMSCs to regulate osteogenesis and osteoclastogenesis via IL-17 suppression in the recipient bone marrow. These data indicate that IL-17, as a cause of secondary osteoporosis in SLE, might be a therapeutic target of transplanted human MSCs in the skeletal disorder. Further studies will be necessary to explore new cellular and molecular strategies to overcome the recipient BMMSCbased pathology of secondary osteoporosis in SLE and develop a novel recipient BMMSC-targeting approach in MSC-based therapy for skeletal regeneration. Abbreviations ALP: alkaline phosphatase; αMEM: alpha minimum essential medium; BMC: bone marrow cell; BMC-WT: wild-type mice-derived bone marrow cells; BMD: bone mineral density; BMMSC: bone marrow mesenchymal stem cell; BV/TV: bone volume/trabecular volume; Calvaria-WT: wild-type mouse-derived calvarial cells; CFSE: carboxyfluorescein diacetate succinimidyl ester; CFU-F: colony-forming unit fibroblasts; CM: conditioned medium; CTX: C-terminal telopeptides of type I collagen; ELISA: enzyme-linked immunosorbent assay; FBS: fetal bovine serum; hBMMSC: human bone marrow mesenchymal stem cell; hUCMSC: human umbilical cord-derived mesenchymal stem cell; IFN: interferon; IL: interleukin; M-CSF: macrophagecolony stimulating factor; microCT: micro-computed tomography; MNC: multinucleated cell; MSC: mesenchymal stem cell; MSC-WT: wild-type mice-derived mesenchymal stem cells; NFATc1: nuclear factor of activated T-cells; NF-κB: nuclear factor κB; PBS: phosphate-buffered saline; RT-PCR: reverse transcription polymerase chain reaction; Runx2: runt-related transcription factor 2; SHED: stem cells from exfoliated deciduous teeth; SLE: systemic lupus erythematosus; sRANKL: soluble receptor activator for nuclear factor κB ligand; Tb.N: trabecular number; Tb.Sp: trabecular separation; Tb.Spac: trabecular space; Tb.Th: trabecular thickness; Th17: IL-17 producing helper T; TRAP: tartrate-resistant acid phosphate.