Mouse model of uncoupled bone remodeling upon discontinuation of anti-RANKL antibody therapy

doi:10.21203/rs.3.rs-4720958/v1

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Mouse model of uncoupled bone remodeling upon discontinuation of anti-RANKL antibody therapy

https://doi.org/10.21203/rs.3.rs-4720958/v1

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The discontinuation of denosumab (anti-RANKL antibody) therapy may increase the risk of multiple vertebral fractures, however, the underlying pathophysiology is largely unknown. Eight patients who underwent discontinuation after multiple injections of denosumab had higher levels of TRAP-5b, the so-called “overshoot” phenomenon, compared to their pre-treatment levels. The rate of decrease in bone mineral density (BMD) during the withdrawal period was higher than in age-matched non-treated patients, suggesting a decrease in BMD independent of age-related physiological bone metabolism. Overshoot and significant bone loss were also observed in mice receiving continuous anti-RANKL antibody administration after treatment was interrupted, resembling the original pathology. In mice long out of overshoot, bone resorption recovered, but osteoblast numbers and bone formation remained markedly reduced. The bone marrow exhibited a significant reduction in Sca-1- and PDGFRα-expressing osteoblast progenitors (PαS cells) and ALP-positive early osteoblasts. Just before the overshoot phase, the osteoclast precursor cell population expands and RANKL-bearing extracellular vesicles (EVs) became abundant in the serum, leading to robust osteoclastogenesis after cessation of anti-RANKL treatment. Thus, accelerated bone resorption due to the accumulation of RANKL-bearing EVs and long-term suppression of bone formation uncoupled from bone resorption leads to the severe bone loss that is characteristic of denosumab discontinuation.

Health sciences/Diseases/Endocrine system and metabolic diseases/Metabolic bone disease/Osteoporosis

Health sciences/Endocrinology/Endocrine system and metabolic diseases/Metabolic bone disease/Osteoporosis

Osteoporosis, which is characterized by progressive bone loss and deterioration of the bone microarchitecture, is an enormous and growing public health problem. Bone homeostasis is maintained throughout life by a continuous remodeling that depends upon a tightly controlled balance between bone resorption by osteoclasts and bone formation by osteoblasts. Shifting this balance toward excessive bone resorption causes osteoporosis (1, 2). Several of the effective therapeutic agents for osteoporosis, such as denosumab and bisphosphonates, have been developed with a focus on their anti-catabolic effects, while teriparatide and romosozumab have been developed with a focus on their pro-anabolic effects (3). Yet despite the remarkable achievement of the pro-anabolic agents, the effective therapeutic window for these agents is limited to only a few years (3). Thus, long-term treatment of osteoporosis requires the sequential switching between different agents, including anti-resorptive drugs, so a detailed understanding of the characteristics and conditions of use of the drug of choice is essential.

Denosumab is a fully human monoclonal antibody against the receptor activator of NF-κB ligand (RANKL) (4, 5) that prevents RANKL from binding to its receptor RANK which is expressed on osteoclast precursor cells, thus preventing osteoclastogenesis (1). Denosumab is currently widely used for the treatment of osteoporosis and rheumatoid arthritis as well as the prevention of skeletal-related events in patients with solid tumors. The long-term efficacy and safety of denosumab in the treatment of osteoporosis have been established by demonstrating a progressive increase in bone mineral density (BMD) and a low incidence of fracture for up to 10 years (6). However, a post hoc analysis and numerous case reports have raised the concern that discontinuation of denosumab may cause an increased risk of fracture (7–10). Upon discontinuation of denosumab, certain bone turnover markers such as the serum C-terminal telopeptide of type I collagen and serum procollagen type 1 N-terminal propeptide were observed to be robustly increased above the pretreatment levels (11, 12). Consistent with these observations, the histology of patients who underwent denosumab discontinuation revealed increased bone turnover with an increase in both osteoclast number and eroded surface as well as an increase in osteoblast number (13, 14). The management of denosumab treatment and when and how to resume osteoporosis therapy after discontinuation are important issues, especially in the context of declining adherence (15).

The discontinuation of other anti-osteoporosis agents, including bisphosphonates and teriparatide, has also been shown to reduce bone mass to pretreatment levels, but it has not been reported to increase the risk of bone fractures, unlike denosumab (15, 16). Furthermore, a burst of bone resorption (referred to as “overshoot”) (12, 17) does not typically occur after the stopping of anti-catabolic agents, but so far at least only after the discontinuation of denosumab. The denosumab-specific overshoot seems to be explained by differences in the distinguishing features of denosumab and the bisphosphonates; the former has a rapid reversibility of its anti-resorptive effect once removed from the circulation, the latter has a long-lasting effect that persists for months or even years after the stoppage of treatment since they are incorporated into the bone matrix due to their high affinity for binding hydroxyapatite. However, there has been little experimental investigation of denosumab discontinuation-caused bone loss, and the molecular basis for the pathology remains to be elucidated.

Here, we have developed a mouse model exhibiting overshoot of bone resorption, robust bone loss, and bone fragility, which resemble the human phenotypes after denosumab discontinuation. Mice after discontinuation of the anti-RANKL antibody exhibited not only excessive bone resorption, but also long-lasting suppression of bone formation due to a marked reduction of osteoblastic lineage cells even after bone resorption recovered, resulting in a significant decrease in bone mass. Osteoclast precursors as well as RANKL-bearing extracellular vesicles (EVs) accumulated during the anti-RANKL antibody-treatment period, and caused an osteoclastic burst after discontinuation. The anti-RANKL antibody suppressed phagocytic clearance of EVs, possibly through binding to RANKL on the EV surface. This study provides new mechanistic insights into how discontinuation of the anti-RANKL antibody causes an osteoclastic burst and impaired bone metabolism, and has important implications for the clinical management of patients with osteoporosis in need of long-term treatment and diverse therapeutic options.

Bone fragility after discontinuation of the anti-RANKL antibody treatment

In order to identify aberrant bone metabolism after denosumab discontinuation, we first analyzed 8 patients (79.9 ± 5.4 years old) who met the inclusion criteria for this study as described in S1 Fig (e.g., multiple doses of denosumab and an appropriate period after discontinuation). After discontinuation of denosumab treatment (for more than 10 months), the serum TRAP-5b levels in patients were markedly higher than before treatment in each (Fig. 1A, left), indicating an overshoot. Bone mineral density (BMD) in both the lumbar spine and femoral neck was also decreased to pre-treatment levels in these patients (Fig. 1A, middle and right). Compared to the rate of decrease in BMD over a similar time period in the age- and sex-matched non-treated controls, those who had discontinued treatment showed significantly higher rate of decrease (Fig. 1B), despite there being no differences in blood parameters indicative of altered parathyroid or kidney function (S1 Table). This suggested that BMD was decreased independently of the physiological bone loss due to age-related osteoporosis, consistent with previous reports (7–14).

Mice were injected with anti-mouse RANKL antibody (OYC1) three times at 2-week intervals (Fig. 1C) to investigate the effect of discontinuation on bone metabolism in detail, and analyzed the bone phenotype over time, including before and after the last injection (0 week). Once injected with the anti-RANKL antibody, the serum TRAP levels immediately declined, but increased rapidly at 6 weeks after the last injection, reaching levels significantly higher than in control mice over the next several weeks (the overshoot period) (Fig. 1D). The mRNA of osteoclastic marker genes (Acp5 and Ctsk) in the long bones also increased when the serum TRAP levels peaked (at 10 weeks) (Fig. 1E). The in vivo longitudinal CT analysis revealed rapid bone loss and a significantly higher rate of bone loss at the end of the overshoot period (10–12 weeks) (Fig. 1F, left). Notably, the reduction in bone mass was still evident long after discontinuation (Fig. 1F, right, 10–16 weeks), indicating that bone loss continues for an unexpectedly long time after TRAP overshoot.

These model mice exhibited the altered bone features after discontinuation as shown by three-dimensional microcomputed tomography (µCT), in which the BMD of the trabecular and cortical bone of the femur as well as the spine was significantly decreased, and trabecular bone volume, trabecular number, and cortical bone thickness were all markedly reduced (Fig. 2A and B). Notably, mice that received risedronate as bisphosphonate for 4 weeks and then discontinued the treatment did not have less bone mass than control mice even after a similar period of time after discontinuation as mice that discontinued anti-RANKL antibody treatment (Fig. 3A and B).

When mice received a single injection, moderate TRAP overshoot was detected, although there was no significant difference at 8 weeks after the injection (S2A, B Fig). While bone mass peaked at 4 weeks post-injection, then declined rapidly and continued to decline over a long period (S2C Fig, 4–12 weeks), it was not less than the bone mass of the control mice (S2D Fig). The BMD in cortical bone was significantly reduced even in the single-dose model mice compared to pre-treatment, but no significant difference was observed in trabecular bone (S2D Fig). Thus, our multiple-injected mouse model effectively captures the characteristics of patients with denosumab discontinuation.

On the other hand, in ovariectomized mice, a postmenopausal osteoporosis model, serum TRAP overshoot was observed not only in the multiple injection model but also in the single-dose model of anti-RANKL antibody (S3A, B Fig), suggesting that postmenopausal conditions accelerated the responsiveness to discontinuation. In addition, discontinuation after multiple injection of the anti-RANKL antibody led reduction of BMD both in the cortical bone and spine, but not trabecular BMD (S3C Fig).

Bone fracture is attributed to multiple factors in combination, including the biochemical properties of bone tissue and mechanical loading as well as bone structure. Chemical mapping by Fourier transform infrared spectroscopy (FTIR) revealed a reduced ratio of the mineral to organic matrix and carbonate to phosphate in the cortical area, indicating an alteration of the tissue composition in bone (Fig. 2C). As the bone biomechanical function is reflected in the bone microstructure and quality, a three-point bending test was performed. The mice after discontinuation exhibited lower maximum load, breaking force and stiffness, along with increasing displacement, suggesting severe bone fragility (Fig. 2D). Taken together, discontinuation of the anti-RANKL treatment resulted in significant bone fragility caused by bone loss and a deterioration of bone quality.

Accumulation of osteoclast precursor cells in an expanded cell population

During TRAP overshoot, the mice that underwent discontinuation (8 weeks after the last injection) exhibited a significant increase in the osteoclast number, osteoclast surface and eroded surface (Fig. 4A), while bone formation was significantly suppressed (S4 Fig). The osteoclastogenesis that occurred after discontinuation was investigated using bone marrow monocyte/macrophage lineage cells (BMMs) to gain insights into the mechanism underlying the bone resorption. After an overshoot, there was no difference between the control and anti-RANKL injected mice in terms of the number of TRAP-positive multinucleated osteoclasts differentiated from the same number of BMMs, suggesting there was no cell-intrinsic alteration related to the osteoclastogenic activity (Fig. 4B). Interestingly, before the overshoot, the BMMs derived from the anti-RANKL antibody-treated mice formed much more osteoclasts compared to the BMMs from control mice (Fig. 4C), which led us to hypothesize an accumulation of osteoclast precursors in vivo.

The cell population characterized as c-kit⁺CD11b^dull/−c-fms⁺ has been described to contain classical precursor cells with high osteoclastogenic potential (18), and now c-kit⁻CD11b^dull/−c-fms⁺ cells have also been shown to have a potential for osteoclastogenesis in vitro (19). In fact, before the overshoot period (2 weeks after the last injection), mice treated with anti-RANKL antibody had more CD11b^dull/−c-fms⁺ cells in any subpopulation distinguished by c-kit expression, although not in bisphosphonate-treated mice (Fig. 3C, Fig. 4D-F, and S5 Fig).

On the other hand, high expression of Ly6C in myeloid cells is also reported to be a cell surface marker of osteoclast precursors (20). Anti-RANKL antibody-treated mice had higher expression of c-fms in the CD11b^dull/−Ly6C^high population than control mice, suggesting an expansion of osteoclast precursor cells (Fig. 4G and S5 Fig). Although CD11b^dull/−Ly6C^high monocytes are known to increase in response to pathological conditions, including rheumatoid arthritis (20), mice treated with anti-RANKL antibody had a comparable number of these cells as the control mice (S5 Fig).

Interestingly, we additionally found that cell populations were altered not only in the osteoclast lineage but also in the broader lineage of bone marrow cells. The frequency of a lineage negative hematopoietic stem cell population (LSK: Lin⁻Sca-1⁺c-kit⁺) as well as Lin⁻Sca-1^loc-kit^loIL-7Rα⁺Flk2^high common lymphoid progenitors (CLPs) and Lin⁻Sca-1⁻c-kit⁺IL-7R⁻αCD34⁺FcγR⁻ common myeloid progenitors (CMPs) were increased in the anti-RANKL antibody-treated mice (Fig. 4H-J). No significant differences were observed for the several inflammatory cytokines tested in this study, except for a slight increase in IL-6 and G-CSF (S2 Table). It is unlikely that the accumulation of osteoclast precursor cells and alteration of the hematopoietic cell populations were caused by an inflammatory condition due to anti-RANKL antibody injection.

Accumulation of RANKL-bearing EVs during anti-RANKL treatment

The question remained as to what had triggered the osteoclast differentiation of the accumulated precursors. A recent clinical study showed increased production of RANKL in the serum of patients who had undergone denosumab discontinuation (21). Consistent with this, the model mice in the present study had an increased ratio of RANKL versus OPG levels in serum (Fig. 5A), although, interestingly, mRNA induction of Rankl was rather on the decline in bone (Fig. 5B). In addition, we found that serum derived from both normal mice and healthy humans had a stimulatory effect on the osteoclastogenesis of normal mouse BMMs and human PBMCs, respectively (S6A and B Fig). These findings suggested the presence of osteoclastogenic factors in serum.

Extracellular vesicles (EVs) are emerging as important mediators of a range of pathological as well as physiological processes, including the communication of bone cells (22), and thus are of interest as a potential causative factor for TRAP overshoot after anti-RANKL discontinuation. Immunoblot and immuno-transmission electron microscopy revealed that RANKL was present in all three types of EVs, including exosomes, microvesicles (MVs), and apoptotic bodies (ABs) isolated from human and mouse serum (Fig. 5C, and S6C and D Fig). Compared to the same amount of serum, a higher amount of ABs and MVs were collected from the anti-RANKL-treated mice, but not bisphosphonate-treated mice, during the overshoot period than untreated mice (Fig. 3D, Fig. 5D). The isolated EVs were tested to determine the contribution of EVs to osteoclastogenesis. The ABs and MVs from untreated mice, and all of the EV types from healthy humans effectively promoted osteoclastogenesis of BMMs and PBMC, respectively (S6E and F Fig). Conversely, the osteoclastogenesis of both mouse BMMs and human PBMCs was decreased in the presence of sera after depletion of EVs (Fig. 5E and F). All of the EVs, even the exosomes derived from anti-RANKL antibody-treated mice exhibited greater promotion of osteoclastogenesis than that observed in untreated mice (Fig. 5G). Collectively, the accumulation of RANKL in serum, a part of which is present on EVs, could trigger the burst of osteoclastogenesis from the accumulated osteoclast precursor cells. Notably, the EVs collected from the serum of patients during denosumab treatment lost the promotive effect on osteoclastogenesis compared with those obtained from treatment-naïve osteoporotic patients (Fig. 5H), suggesting that the RANKL expressed on EVs did not have a stimulatory role due to a masking effect of denosumab.

EVs are released in various tissues and organs, and cleared through the phagocytic system (23) in response to diverse circumstances, but the details of such release and clearance depending on cell type have not been fully elucidated. When the circulating EVs collected from EGFP-tg mice were incubated with BMMs from wild-type mice, the EGFP signals were found to have been incorporated into the BMMs, and the EGFP intensity was reduced in the presence of anti-RANKL antibody (Fig. 5I). A masking of RANKL on EVs by the anti-RANKL antibody might induce resistance to phagocytosis by macrophages and cause the accumulation of the RANKL-carrying EVs, and then the disappearance of the anti-RANKL antibody might result in the osteoclastic burst mediated by RANKL-bearing EVs.

Sustained inhibition of osteoblastic bone formation after discontinuation

Anti-RANKL treatment has been reported to simultaneously reduce levels of bone turnover markers for bone formation as well as resorption (6, 24), and suppression of bone formation was generally thought to be coupled with inhibition of bone resorption. However, our bone histomorphometric analysis revealed that after discontinuation in mice, even after a long period without any exposure to the anti-RANKL antibody (16 weeks after the last injection) the animals exhibited osteoblastic defects such as a decreased osteoblast number and bone formation rate despite there being no osteoclastic defects (Fig. 6A and S7 Fig). Consistent with this, serum ALP activity remained suppressed in mice after discontinuation for long period (Fig. 6B). Considering our data showing transient TRAP overshoot and long-lasting bone reduction (Fig. 1D and F), the bone formation appeared to be impaired independently of a coupling with bone resorption during this period.

In order to address this in greater detail, the osteogenic potential of the bone marrow-derived mesenchymal stem/stromal cells (BMSCs) was examined by colony formation unit (CFU) assay. The BMSCs derived from mice 16 weeks after the last injection formed a markedly lower number of CFUs positive for ALP (CFU-ALP) and Alizarin Red S (CFU-Ob) (Fig. 6C). In addition, even BMSCs from mice that received a single injection displayed reduced CFUs (S8A and B Fig), suggesting that even a single exposure to the anti-RANKL antibody resulted in a sustained reduction in the number of osteoblast progenitor cells, rather than inhibition of the differentiation into mature osteoblasts. Flow cytometric analysis strengthened these results by showing a significant reduction of ALP-positive immature osteoblasts (25) in the bone marrow of mice after discontinuation (16 weeks after the last injection) (Fig. 6D and S8C Fig). Moreover, BMSCs expressing Sca-1 and PDGFRα (PαS cells: Ter119⁻CD45⁻Sca-1⁺PDGFRα⁺) (26), which give rise to osteoblastic lineage cells, were also significantly reduced following discontinuation (Fig. 6E and S8D Fig). There was no direct effect of anti-RANKL treatment on either osteoblast differentiation or bone nodule formation in calvarial cell-derived osteoblasts (S8E Fig). Collectively, the anti-RANKL treatment caused suppression of bone formation not only through a coupling effect associated with decreased bone resorption but also by decreasing the number of osteoblastic lineage cells, especially in that the latter may be involved in long-lasting bone reduction.

The present study reports the development of a mouse model of anti-RAKNL antibody discontinuation and demonstrated that discontinuation of anti-RANKL treatment caused uncoupled bone remodeling in which bone formation remained suppressed despite rapid acceleration of bone resorption, resulting in prolonged bone loss and bone fragility. Osteoclast precursor cells were expanded across not only the physiological cell population, but also across other cell populations, including c-kit-negative cells and Ly6C-positive cells during the treatment period. The accumulated precursor cells also contributed to the osteoclastic burst that occurred in response to the increased RANKL-bearing EVs once the anti-RANKL treatment had been discontinued. Furthermore, osteoblast progenitors were reduced and did not recover long after the discontinuation of treatment. Thus, even after the transient increase in bone resorption was restored, the coupled bone remodeling remained disrupted, leading to long-term bone loss.

Several population-based cohort studies have reported that a longer duration of treatment with denosumab and an increased time interval since the last denosumab injection are associated with greater bone loss and fracture risk. Consistent with these findings, the most severe bone loss was observed in mice that underwent discontinuation after multiple doses in the present study. In addition, consistent with previous reports (27), the ovariectomized mice showed a significant reduction in cortical and vertebral BMD after discontinuation compared to pre-treatment, but not in trabecular BMD. It is likely that trabecular bone volume and BMD were so reduced by ovariectomy that no further reduction was observed. Notably, however, ovariectomized mice exhibited TRAP overshoot even after a single dose of anti-RANKL antibody, suggesting that increased turnover in the postmenopausal background was associated with acceleration of bone loss after discontinuation.

Denosumab, like the anabolic agent teriparatide, has a reversible effect on BMD after treatment discontinuation, and is markedly different in nature from bisphosphonates, which lose their anti-resorptive effect very slowly due to their affinity for hydroxyapatite. Consistent with previous report (28), we have indeed found that bisphosphonate did not cause bone loss below pre-treatment levels, although it gradually decreased after treatment discontinuation. In addition, there was no accumulation of osteoclast precursor cells and EVs in bisphosphonate-treated mice, suggesting that the bone loss following discontinuation of anti-RANKL antibody therapy might be due to a breakdown of the RANKL specific biological system. However, the mechanism responsible for RANKL-specific rebound remained unclear.

In the present study, it was demonstrated that the anti-RANKL antibody suppressed the phagocytosis of EVs by macrophages, possibly resulting in a delayed clearance of RANKL-bearing EVs from the circulation and acceleration of osteoclastogenesis through the accumulated EVs. In recent years, intensive studies have revealed the role of EVs in the physiological and pathological bone microenvironment through their bioactive cargo (23, 29), and it is now known that the RANKL-bearing EVs derived from osteoblast lineages are involved in osteoclast-osteoblast communication in bone metabolism (30), which fully supports the present study. Future studies will clarify the extent to which quantitative changes in RANKL-bearing EVs contribute to bone metabolism, and may be one of the triggers of TRAP overshoot after discontinuation of anti-RANKL antibody therapy.

A recent study using intravital imaging reported that multinucleated osteoclasts on the bone surface into smaller daughter cells that are recycled and fused into larger osteoclasts in response to soluble RANKL administration. In addition, during suppression of RANKL signaling by OPG-Fc administration, these daughter cells are accumulated and re-fused after OPG administration is interrupted (31). It is possible that such recycling of osteoclast-derived cells may contribute to the overshoot of bone resorption observed in mice after the discontinuation of anti-RANKL antibody treatment in the present study. However, during anti-RANKL antibody treatment, we observed an expansion of hematopoietic stem and progenitor cells, including HSC, CLP and CMP as well as osteoclast progenitors, indicating that inhibition of the RANKL signaling not only suppresses bone resorption, but also broadly affects the homeostasis of the bone marrow environment.

The present study also demonstrates that inhibition of RANKL signaling with an anti-RANKL antibody decreases the number of osteoblast progenitor cells, resulting in decreased bone formation. In a recent clinical study, human biopsies revealed higher number of empty osteocyte lacunae, which indicates a lack of viable osteocytes during denosumab treatment, and number of empty osteocyte lacunae remained high after discontinuation (14). The primary pathogenesis of bone loss and high fracture risk after discontinuation of anti-RANKL antibody therapy would be characterized by a disruption of the coupling between bone resorption and formation, that would have occurred thereafter overshoot. The reverse signaling of RANKL into osteoblasts is reported to promote bone formation (32), but the anti-RANKL antibody did not act directly on osteoblasts in the present study (S8D Fig). On the other hand, RANK is also reported to be expressed on BMSCs, including PαS cells, and to inhibit commitment from BMSC to the osteoblast lineages (33). Although further studies are needed to clarify the mechanism by which RANKL antibodies decrease BMSCs and osteoblasts, the finding that G-CSF levels were elevated in mice treated with the anti-RANKL antibody may help to explain the disruption of bone metabolism in these mice.

G-CSF is widely used clinically to mobilize HSCs from the bone marrow niche to the peripheral blood stream for transplantation. G-CSF directly regulates the proliferation and differentiation of HSCs, resulting in an expansion of phenotypic HSCs, including LSK. G-CSF also acts on mesenchymal stem cells to suppress their differentiation into osteoblasts, while simultaneously downregulating CXCL12 expression, which is essential for HSC maintenance in the niche (34). Furthermore, G-CSF strongly inhibits osteoblast differentiation and function and also causes osteoblast ablation (35, 36). Thus, G-CSF is associated with dynamic changes in the bone marrow microenvironment along with decreased bone volume.

Taken together, the unknown functions of RANKL in directly regulating the bone marrow environment, such as EV clearance and maintenance of hematopoietic and mesenchymal bone marrow cells, may be a clue to the specific bone loss that occurs after anti-RANKL antibody discontinuation.

Patients with osteoporosis require lifelong medical care, and therefore alternative treatments should be recommended if denosumab therapy is discontinued. However, in studies of drug switching after long-term denosumab use, bisphosphonate treatment did not completely prevent bone loss and fracture (37). Given the defects of bone formation after discontinuation in the present study, a catalytic agent such as romosozumab, an anti-sclerostin antibody, may be a good option after denosumab discontinuation, but clinical investigation is still ongoing (38). Thus, the present study may provide a molecular basis for future therapeutic approaches to the long-term management of osteoporosis.

Study design

The main objective of this study was to investigate whether and how an osteoclastogenic burst is induced after discontinuation of denosumab in patients with osteoporosis. To address this, we examined the bone architecture and metabolism in mice after discontinuation of anti-RANKL injection by focusing both osteoblasts and osteoclasts. We then analyzed the progenitors of osteoblasts and osteoclasts in mice after discontinuation. We also tested the effects of serum taken from mice after discontinuation on osteoclastogenesis. To identify what cues the osteoclastogenic burst, we determined the effect of EVs isolated from patients and mice after discontinuation of anti-RANKL treatment on osteoclastogenesis. Finally, we elucidate that the anti-RANKL antibody inhibits the clearance of extracellular vesicles by macrophages. All “replication” referred in this study means biological replicates. Data were acquired and analyzed in a blinded manner and are from at least three independently repeated experiments. The exact number and P value are given in the respective figure legends.

Ethics

This study was approved by the institutional research committee at the Showa University and Yamanashi red cross hospital and conducted in accordance with the Helsinki Declaration.

Mice and administration of anti-RANKL antibody

All animals were maintained under specific pathogen-free conditions, and all experiments were performed with the approval of the Institutional Animal Care and Use Committee of Showa University. C57BL/6J mice and C57BL/6-Tg (CAG-EGFP) mice were purchased from CLEA Japan and Japan SLC, respectively. For generation of the experimental model of denosumab discontinuation, a rat anti-mouse-RANKL antibody (clone OYC1, ORIENTAL YEAST) was used because there is no cross-reactivity of denosumab against rodents. Eight-week-old female mice were subcutaneously injected with 5 mg/kg anti-RANKL antibody or phosphate-buffered saline (PBS).

Bone analyses

The BMD of the lumber spine and femoral neck in patients was evaluated using a dual-energy x-ray absorptiometry (DXA) device. The changes in bone volume in the mice during the experimental periods were analyzed every 2 weeks using an in vivo 3D µCT system (R_mCT2, Rigaku Corp.). Changes in bone mass were assessed using images of the coronal plane of the proximal femur using the software ImageJ 1.44p (National Institute of Health, USA). For detailed analysis of bone structure, the femur was scanned using a ScanXmate-L090H (Comscantecno) and 3D images were reconstructed with a TRI/3D-Bon-FSC system (RATOC System Engineering) as previously described (27). Bone strength was analyzed with a 3-point bending test of the tibia. To distinguish chemical distribution in bone, FTIR images of the femur acquired using the Spectrum Spotight 400 Imaging System (PerkinElmer) as previously described (39, 40). For bone morphometric analysis, tibiae were dehydrated and embedded in glycol methacrylate, sectioned into 3-µm thick longitudinal sections with a Microtome, and stained with toluidine bule or TRAP. Static parameters of bone resorption and formation were measured using an OsteoMeasure bone histomorphometry system (OsteoMetrics) as previously described (27).

Measurement of biomarkers and cytokines

The serum levels of TRAP, RANKL and OPG were determined using a Mouse TRAP™ ELISA Kit (Immunodiagnostic Systems), Mouse TRANCE/RANKL/TNFSF11 Quantikine ELISA Kit (R&D systems), and Mouse Osteoprotegerin/TNFRSF11B Quantikine ELISA Kit (R&D systems), respectively, according to the manufacturer’s instructions. The serum levels of ALP activity were determined using a LabAssay (p-nitrophenyl phosphatate (pNPP) assay, Wako) accoding to the manufacturers’ instructions. The concentrations of mouse chemokines were measured by Bio-Plex Pro cytokine assay using the following agents according to the manufacturer’s instructions; Eotaxin (171G5014M), G-CSF (171G5015M), INF-γ (171G5017M), IL-1α (171G5001M), IL-1β (171G5002M), IL-3 (171G5004M), IL-4 (171G5005M), IL-5 (171G5006M), IL-6 (171G5007M), IL-9 (171G5008M), IL-10 (171G5009M), IL-12 (p40) (171G5010M), IL-12 (p70) (171G5011M), IL-17A (171G5013M), KC(171G5018M), MCP-1 (171G5019M), MIP-1α (171G5020M), MIP-1β (171G5021M), RANTES (171G5022M), TNF-α (171G5023M, Bio-lad).

Quantitative RT-PCR

For the measurement of Acp5 and Ctsk mRNA expression, total mRNA was purified from bone marrow cells containing osteoclasts and osteoclast precursor cells obtained from the tibia and femur of the hindlimb. For Tnfsf11 mRNA expression, total mRNA was purified whole tibia and femur without removing bone marrow cells. Quantitative real-time reverse transcriptase (RT)-PCR was performed using a QuantStudio3 with PowerUp SYBR Green Master Mix (appliedbiosystems), according to the manufacturer’s protocol. The level of mRNA expression was normalized with that of Gapdh. The following primers were used: Acp5, 5′-ttgcgaccattgttagccac-3′ (sense) and 5′-cacaccgttctcgtcctgaa-3′ (antisense); Ctsk, 5′-tgtgaaccatgcagtgttgg-3′ (sense) and 5′-ggcgttgttcttattccgagc-3′ (antisense); Tnfsf11, 5′-gcagaaggaactgcaacaca-3′ (sense) and 5′-gatggtgaggtgtgcaaatg-3′ (antisense); and Gapdh, 5′-aactttggcattgtggaagg-3′ (sense) and 5′-ggatgcagggatgatgttct-3′ (antisense).

Osteoblast differentiation and colony forming unit assays

In vitro osteoblast differentiation was performed as previously described (41). Briefly, osteoblast progenitor cells derived from the mouse calvarial bone of newborn mice were cultured in osteogenic medium (50 µM ascorbic acid, 10 nM dexamethasone, 10 mM β-glycerophosphate), then ALP activity (after 7 days) and bone nodule formation (after 21 days) were analyzed. ALP activity was evaluated by ALP staining and bone nodule formation by Alizarin Red S staining. For the CFU assay, bone marrow cells were cultured for 3 days in α-MEM containing 10% FBS. CFU-ALP and CFU-Ob were detected as ALP-positive colonies and Alizarin Red S-positive colonies, respectively.

Osteoclast differentiation

In vitro osteoclast differentiation was performed as previously described (42). Briefly, nonadherent bone marrow cells from the mouse femur were cultured in α-MEM with 10% FBS containing 10 ng ml^− 1 of M-CSF (R&D systems) for 2 days to obtain BMMs. These cells were further cultured for 3 days with 50 ng ml^− 1 of RANKL (R&D systems) in the presence of 10 ng ml^− 1 of M-CSF with 10% FBS. Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient and cultured in the same manner as osteoclastogenesis from the mouse BMMs. Osteoclastogenesis was evaluated by counting TRAP positive multinucleated (more than 3 nuclei) cells.

Flow cytometric analysis

For the analysis of bone marrow-derived osteoclast precursor cells, a single cell suspension of mouse bone marrow cells was stained with the following antibodies; PerCP-Cy5.5 anti-CD3 (17A2, BioLegend), PerCP-Cyanine5.5 anti-CD45R (B220) (RA3-6B2), Super Bright 436 c-Kit (2B8), APC CD115 (c-fms) (AFS98), PE-Cyanine7 anti-CD11b (M1/70), and Alexa Fluor 488 anti-Ly6C (HK1.4, eBioscience). Dead cells were excluded as Zombie Aqua (BioLegend)-stained cells. For the analysis of osteoblast progenitor cells, a single cell suspension of mouse bone marrow cells and bone cells collected by collagenase and DNase treatment was stained with following antibodies; Zombie Aqua, FITC anti-TER-119/Erythroid Cells (TER-119, BioLegend), FITC anti-CD45 (30-F11), APC anti-Sca-1 (D7), Biotin anti-CD140a (PDGFRA) (APA5, eBioscience), Brilliant Violet 421 Streptavidin (BioLegend), goat anti-mouse ALPL polyclonal antibody, PE anti-goat IgG (Invitrogen). Flow cytometric analysis was performed using LSRFortessa (BD Biosciences) and analyzed with FlowJo software (BD Biosciences).

Isolation of extracellular vesicles and phagocytosis assay

EVs were isolated from the pooled serum of mice or untreated patients by differential ultracentrifugation as described previously (43). In brief, serum was centrifugated at 2000 g for 10 min. at 4 °C followed by ultracentrifugation at 10000 g for 30 min. to separate the pellet containing apoptotic bodies from the supernatant. The supernatant was then ultracentrifugated at 30000 g for 70 min. at 4 °C and the pellet containing the microvesicles and supernatant was separated. The supernatant was further ultracentrifugated at 100000 g for 70 min. at 4 °C to collect the final supernatant lacking EVs and the exosome-containing pellet was collected after washing with further ultracentrifugation at the same speed. To examine the effects on osteoclastogenesis, the EVs isolated from 1 ml of mouse or human serum were added to mouse BMM or human PBMC cultures in the presence of M-CSF and RANKL, respectively. The effect of mouse or human EV-depleted serum on osteoclastogenesis was tested by dilution with medium to 5% or 10%, respectively. For the phagocytosis assay, fluorescent EVs were isolated from the serum of C57BL/6-Tg (CAG-EGFP) mice after incubation with the anti-RANKL antibody (OYC1, 100 µg ml⁻¹) or isotype control IgG for 2 hours and then added to BMMs.

Immunoblot analysis and immunogold labelling of EVs

Ten µg of total protein extracted from the pellet containing AB, MV or exosomes was subjected to immunoblot analysis using rabbit anti-CD63 (System Biosciences) and mouse anti-RANKL (G-1, Santa Cruz) as the primary antibodies, HRP-conjugated anti-rabbit IgG and HPR-conjugated anti-mouse IgG as the secondary antibodies, and the ECL Plus Western Blotting Detection system (GE Healthcare). For electron microscope scanning (S-4700, Hitachi), EVs were immunolabelled with an anti-RANKL antibody (C30535, Assay Biotechnology) or isotype control IgG (2729, Cell signaling) followed by staining with 6 nm colloidal gold-conjugated anti-rabbit secondary antibody (Jackson Immuno Research).

Statistical analysis

Statistical analysis was performed using the unpaired tow-tailed Student’s t-test or one-way ANOVA (*p < 0.05; **p < 0.01; ***p < 0.001, throughout the paper). When the ANOVA indicated a significant difference, the specific difference was calculated by Holm-Sidak post-hoc test. Data are expressed as the mean ± SEM. Results are presented as representative examples of more than three independent experiments.

Author Contributions:

K. I. designed and performed most of the experiments, interpreted the results and prepared the manuscript. N. S. and S. T. contributed to the experiments and data interpretation. H.H. and H. K-S performed FTIR analysis and data interpretation. Y. K., M. Takami, M. Tshuji, Y. K., K. I contributed to data interpretation and scientific discussion. T. N.-K. directed the project and wrote the manuscript.

Competing Interest Statement:

Authors have no conflicts of interest to disclose.

Acknowledgements

We would like to express our sincere gratitude to the staff at the Center of Pharmacology Laboratory of Showa University for their helpful discussion and assistance. We are also grateful to Tomoko Towatari, Tomoko Nagai, Takashi Takagi, and Fumiya Nakamura for their valuable technical assistance. Finally, we thank Ayano Oyamada, Kei Gonsho and Miho Mochizuki for their cooperation in collecting clinical data.

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Mouse model of uncoupled bone remodeling upon discontinuation of anti-RANKL antibody therapy

Status:

Version 1

Abstract

Figures

Introduction

Results

Bone fragility after discontinuation of the anti-RANKL antibody treatment

Accumulation of osteoclast precursor cells in an expanded cell population

Accumulation of RANKL-bearing EVs during anti-RANKL treatment

Sustained inhibition of osteoblastic bone formation after discontinuation

Discussion

Methods

Study design

Ethics

Mice and administration of anti-RANKL antibody

Bone analyses

Measurement of biomarkers and cytokines

Quantitative RT-PCR

Osteoblast differentiation and colony forming unit assays

Osteoclast differentiation

Flow cytometric analysis

Isolation of extracellular vesicles and phagocytosis assay

Immunoblot analysis and immunogold labelling of EVs

Statistical analysis

Declarations

Author Contributions:

Acknowledgements

References

Additional Declarations

Supplementary Files

Status:

Version 1