OPG:Fc treatment potently increases BMD and ablates osteoclasts from bone surfaces in mice, while treatment withdrawal leads to rebound resorption

To identify the cellular mechanisms that activate osteoclastogenesis and to address whether blocking RANKL may induce an increased abundance of local activation sites, we injected healthy 6–8-week-old female mice with OPG:Fc or vehicle 3 times a week for 2 weeks followed by treatment withdrawal, as illustrated in the study design (Fig. 1a). Longitudinal dual-energy X-ray absorptiometry (DEXA) revealed a significant interaction between time and treatment, with an initial increase in BMD, peaking at week 8 due to a prolonged half-life of OPG:Fc, followed by a decrease in BMD to vehicle levels by week 13 (Fig. 1b). Tibiae and femora were harvested following the 2 weeks of treatment, and at 8, 11, and 13 weeks after baseline (6, 9, and 11 weeks after withdrawal, respectively) (Fig. 1a). Micro-computed tomography (µCT) analysis of the femora revealed a notable increase in trabecular bone volume and trabecular thickness, accompanied by a decrease in trabecular separation at week 8, 11, and 13, in OPG:Fc-treated mice compared to vehicle, while trabecular thickness was increased at all time points compared to vehicle (Fig. 1c, d and Fig. S1a, b). Of note, at week 13, the distinction in trabecular architecture was less pronounced, suggesting a relative decrease in trabecular volume during the rebound bone loss. µCT-analysis further showed an increase in the diaphyseal marrow cross-sectional areas at week 8, 11, and 13, while total diaphyseal cross-sectional areas were increased at all timepoints compared to vehicle. This suggests that OPG:Fc treatment induced periosteal apposition and increased endosteal resorption during withdrawal (Fig. 1e and Fig. S1c). To confirm the absence of osteoclasts after OPG:Fc treatment and their reoccurrence after treatment withdrawal, we examined the presence of Acp5+ (encodes the osteoclast marker enzyme tartrate-resistant acid phosphatase [TRAcP]) osteoclasts on trabecular and endocortical bone surfaces using ISH. After 2 weeks, OPG:Fc treatment, like denosumab, successfully ablated Acp5+ osteoclasts from bone surfaces, while treatment withdrawal resulted in recruitment of Acp5+ osteoclasts to the bone surfaces at 11 and 13 weeks. Importantly, histological assessment revealed that Acp5+ osteoclasts predominantly occupied trabecular bone surfaces at 13 weeks during the rebound period, while endocortical bone surfaces were occupied by less osteoclasts in the OPG:Fc-treated mice (Fig. 1f, g and Fig. S2).

Fig. 1

OPG:Fc treatment Increases BMD and ablates osteoclasts, while treatment withdrawal results in rebound resorption. a Study design illustrating 6–8-week-old, female mice that were injected with vehicle or OPG:Fc (10 mg/kg) 3 times a week for 2 weeks followed by a withdrawal period. b Longitudinal DEXA scans of the left hind limb were carried out every second or third week, and at the end of the study. c–e Ex vivo µCT femoral parameters at week 2, 8 11 or 13. f ISH of Acp5 using tissue sections of the tibia shows Acp5+ osteoclasts (green) present on trabecular and endocortical bone surfaces in vehicle-treated but not in OPG:Fc-treated mice, with return of Acp5+ cells following treatment withdrawal. g Oc.Pm/B.Pm determined using Acp5 ISH-stained sections after 2 weeks of treatment and at week 11 and 13 following withdrawal. f White dashed lines highlight trabecular, endocortical, and periosteal bone surfaces. c–g Data is expressed as mean ± SD. P values are calculated using a mixed-effects analysis in (b), and using an unpaired t test in (c–e), and (g). ****P values < 0.000 1, ***P values < 0.001, **P values < 0.01, *P values < 0.05. n = 3–8/group. Scale bars: (f) overview = 1 mm and high magnifications = 100 µm. Ct cortical, Tb trabecular, Bm bone marrow, OPG:W OPG:Fc withdrawal, Oc.Pm/B.Pm osteoclast perimeter/Bone perimeter, CS cross-sectional

Tnfsf11 and Tnfrsf11b are expressed by distinct cell populations in mice

Next, we investigated the expression of Tnfsf11 and Tnfrsf11b to localize the cells that activate osteoclastogenesis. We found that Tnfsf11 and Tnfrsf11b expression is differently distributed when considering the zones beneath, above, and at the bone surfaces. The expression of Tnfsf11 was most prominent in bone surface cells located on periosteal, endocortical, and trabecular bone surfaces, which to our knowledge has not previously been directly illustrated (Fig. 2a). To determine the location of Tnfsf11+ cells we quantified the percentage of Tnfsf11+ cells and measured the distance of these cells to trabecular and endocortical bone surfaces. In vehicle-treated mice, we found that the percentage and staining intensities of Tnfsf11+ cells remained relatively stable across different distances from the bone surfaces, with a slight increase near the bone surface and deeper osteocytes (Fig. 2b and Fig. S3a). Likewise, we examined the spatial expression of Tnfrsf11b. Tnfrsf11b ISH revealed intense expression mainly amongst osteocytes, and to a lesser extent in the bone surface cells. Moreover, Tnfrsf11b+ cell populations residing in the marrow seemed less abundant (Fig. 2c). We further quantified the percentage of Tnfrsf11b+ cells and measured the distance of these cells from trabecular and endocortical bone surfaces. In vehicle-treated mice, osteocytes and cells near the bone surface were the most abundant population of Tnfrsf11b+ cells in both cortical and trabecular bone, while a lower proportion of Tnfrsf11b+ cells resided in the marrow (Fig. 2d). A similar pattern was seen in Tnfrsf11b staining intensities (Fig. S3b).

Fig. 2

OPG:Fc treatment induces a changed Tnfsf11 and Tnfrsf11b expression pattern. Tibiae from 8-week-old female mice were harvested after being injected thrice weekly for two weeks with either vehicle or OPG:Fc (10 mg/kg). Following the final dose of OPG:Fc at the end of the two-week period, Tnfsf11 and Tnfrsf11b ISH was performed on tissue sections. a Tnfsf11 (yellow) illustrated in bone sections from vehicle and OPG:Fc-treated mice. b Percentage of Tnfsf11+ cells against the distance from the trabecular or endocortical bone surface. c Tnfrsf11b (red) illustrated in bone sections from vehicle and OPG:Fc-treated mice. d Percentage of Tnfrsf11b+ cells against the distance from the trabecular or endocortical bone surface. a, c White dashed lines highlight trabecular, endocortical, and periosteal bone surfaces. Data is expressed as mean ± SEM. P values are calculated using a two-way ANOVA. ****P values < 0.000 1, ***P values < 0.001, **P values < 0.01, *P values < 0.05. n = 8/group. Scale bars: (a, b) overview = 1 mm and high magnifications (a) = 100 µm, (b) = 50 µm. Ct cortical, Tb trabecular, Bm bone marrow, OPG:W OPG:Fc withdrawal

OPG:Fc treatment induces a change in the Tnfsf11 and Tnfrsf11b expression pattern in mice

Since denosumab treatment is associated with rebound bone loss upon treatment discontinuation, we hypothesized that denosumab treatment may alter the expression of Tnfsf11 and Tnfrsf11b. Therefore, we examined the expression of Tnfrsf11 and Tnfrsf11b at the end of 2 weeks OPG:Fc treatment. ISH revealed a noticeable increase in the expression of Tnfsf11 especially on trabecular bone surfaces and the periosteum of the cortex, while endocortical surfaces seemed less affected (Fig. 2a). We further quantified the percentage of Tnfsf11+ cells and the Tnfsf11 staining intensity and found a significant interaction between regions and treatment (Fig. 2b and Fig. S3a). Moreover, the percentage of Tnfsf11+ cells and the Tnfsf11 staining intensity increased significantly on the trabecular bone surface in OPG:Fc-treated mice compared to vehicle-treated mice (Fig. 2b and Fig. S3a), suggesting that Tnfsf11 expression is mainly upregulated in trabecular bone surface cells upon OPG:Fc treatment. Examining the abundance of Tnfsf11 expressing cells in the cortical compartment, no interaction between region and treatment was detected. Moreover, the abundance of Tnfsf11+ cells was not increased in OPG:Fc-treated mice (Fig. 2b). However, interaction between treatment and region was detected when comparing staining intensities between vehicle- and OPG:Fc-treated mice across the regions (Fig. S3a). In contrast to the expression of Tnfsf11, the expression of Tnfrsf11b was strongly reduced upon OPG:Fc treatment in both the cortical and trabecular compartment (Fig. 2c). We further quantified the percentage of Tnfrsf11b+ cells and the Tnrfsf11b staining intensity and found a significant interaction between regions and treatment (Fig. 2d and Fig. S3b). Finally, the abundance of Tnfrsf11b+ cells was persistently lower on the trabecular and endocortical bone surfaces and among osteocytes residing close to the surface in OPG:Fc-treated mice compared to vehicle (Fig. 2d), while Tnrfsf11b staining intensity was lower in almost all cortical and trabecular osteocytes in OPG:Fc-treated mice compared to vehicle (Fig. S3b).

OPG:Fc treatment in mice induces an increase in Tnfsf11 and a decrease in Tnfrsf11b expression in the trabecular bone

Since denosumab discontinuation is associated with multiple spontaneous vertebral fractures, we hypothesized that the rebound mainly affects trabecular and not cortical bone.9 Therefore, we aimed to assess the expression of Tnfsf11 and Tnfrsf11b across cell types and in the two bone compartments. In each compartment, we defined three distinct cell types based on their spatial position. Cells residing on the bone surface, including bone lining cells, osteoblasts, and reversal cells were collectively defined as bone surface cells. Moreover, bone matrix embedded cells were defined as osteocytes, while cells residing in the marrow were defined as marrow cells. Then, we examined the impact of 2 weeks of OPG:Fc treatment on these cell types.

In the trabecular compartment, we found that the vehicle-treated mice had a significantly higher proportion of trabecular bone surface cells expressing Tnfsf11+ compared to osteocytes and proximate marrow cells. Next, we compared the percentage of Tnfsf11+ in vehicle and OPG:Fc-treated mice and found that Tnfsf11+ was strongly upregulated among bone surface cells compared to vehicle. Notably, the percentage of Tnfsf11+ bone surface cells were significantly higher compared to osteocytes, and proximate marrow cells in OPG:Fc treated mice (Fig. 3a). Although less prominent, the percentage of Tnfsf11+ osteocytes were also significantly increased in OPG:Fc-treated mice compared to vehicle-treated mice (Fig. 3a). No difference was detected in proximate marrow cells between vehicle and OPG:Fc treated mice (Fig. 3a). We further quantified the percentage of Tnfrsf11b+ cells and found a higher proportion of osteocytes expressing Tnfrsf11b+ compared to bone surface cells and proximate marrow cells in vehicle-treated mice. In addition, surface cells also presented with a higher percentage of Tnfrsf11b+ compared to proximate marrow cells (Fig. 3a). Similarly, we examined the effect of OPG:Fc treatment on Tnfrsf11b expression. Here, we found that OPG:Fc treatment resulted in a significant decrease in the percentage of Tnfrsf11b+ bone surface cells and osteocytes (Fig. 3a). No difference was detected between proximate marrow cells upon OPG:Fc treatment (Fig. 3a). The increase in Tnfsf11+ and decrease in Tnfrsf11b+ cells resulted in a significantly increased Tnfsf11+/Tnfrsf11b+ cell ratio among all cell types upon OPG:Fc treatment, with the most prominent increase observed in bone surface cells (Fig. S3c). Examining the changes in Tnfsf11 and Tnfrsf11b on histological sections, the Tnfsf11 and Tnfrsf11b staining intensity appeared highly affected by OPG:Fc treatment (Fig. 2a, c). Therefore, we quantified the mean staining intensity and found that the Tnfsf11 staining intensity was increased almost 3-fold in bone surface cells, and by 2-fold in osteocytes upon OPG:Fc treatment (Fig. 3b). Notably, bone surface cells exhibited a significantly higher staining intensity compared to osteocytes and proximate marrow cells in the OPG:Fc-treated mice (Fig. 3b). Tnfrsf11b staining intensity was decreased almost 3-fold in bone surface cells, more than 3-fold in osteocytes, and were unaltered in marrow cells (Fig. 3b). The changes in Tnfsf11 and Tnfrsf11b staining intensities, resulted in an 11-fold increase in the Tnfsf11/Tnfrsf11b staining intensity ratios amongst bone surface cells and approximately a 3-fold increase in the Tnfsf11/Tnfrsf11b staining intensity ratios in marrow cells proximate to the bone surface (Fig. S3c).

Fig. 3

OPG:Fc treatment has differential effects on Tnfsf11 and Tnfrsf11b expression in cortical and trabecular bone. Tnfsf11+ and Tnfrsf11b+ cell percentage and cell staining intensities were quantified in bone surface cells, osteocytes, and marrow cells and compared across cell types and between vehicle and OPG:Fc-treated mice. All data is collected from ISH stained sections of the tibia. a Percentage of Tnfsf11+ and Tnfrsf11b+ trabecular surface cells, trabecular osteocytes, and proximate marrow cells. b Mean Tnfsf11 and Tnfrsf11b cell staining intensities in trabecular surface cells, trabecular osteocytes and proximate marrow cells c Percentage of Tnfsf11+ and Tnfrsf11b+ endocortical surface cells, cortical osteocytes and marrow cells. d Mean Tnfsf11 and Tnfrsf11b cell staining intensities in endocortical surface cells, cortical osteocytes, and marrow cells. Data are shown as mean ± SD. P values are calculated using a two-way ANOVA. ****P values < 0.000 1, ***P values < 0.001, **P values < 0.01, *P values < 0.05. n = 8/group

OPG:Fc treatment does not result in an increased expression of Tnfsf11 in the cortical compartment

In the cortical compartment, the percentage of Tnfsf11+ cells did not differ between the vehicle and the OPG:Fc-treated mice. However, a significantly higher proportion of endocortical surface cells and cortical osteocytes were Tnfsf11+ compared to marrow cells. Interestingly, we found a 40% reduction in Tnfrsf11b+ bone surface cells, while the abundance of osteocytes and marrow cells were unaffected by OPG:Fc treatment (Fig. 3c). Accordingly, Tnfsf11+/Tnfrsf11b+ cell ratios were significantly higher among bone surface cells in the OPG:Fc-treated mice compared to vehicle, but were unchanged in osteocytes and marrow cells (Fig. S3d). Like in the trabecular bone, we examined the effect of OPG:Fc treatment on Tnfsf11 and Tnfrsf11b staining intensity on cortical osteocytes, endocortical surface cells, and marrow cells (Fig. 3d). In the OPG:Fc-treated mice, Tnfsf11 staining intensity in bone surface cells was increased by approximately 1.5-fold compared to vehicle. Moreover, Tnfsf11 staining intensity was higher in bone surface cells than in osteocytes and marrow cells, in the OPG:Fc-treated mice (Fig. 3d). Tnfrsf11b staining intensity was higher in cortical osteocytes than in endocortical surface cells and marrow cells, and was significantly reduced in cortical osteocytes and endocortical surface cells upon OPG:Fc treatment (Fig. 3d). Tnfsf11/Tnfrsf11b staining intensity ratios seemed unaffected by OPG:Fc treatment, but were generally higher in marrow cells (Fig. S3d).

Tnfrsf11b expression by chondrocytes in the growth plate and articular cartilage is high compared to the primary spongiosa, where it becomes even lower upon OPG:Fc treatment

We explored other local sources of RANKL and OPG. ISH for Tnfsf11 and Tnfrsf11b revealed a prominent expression of Tnfrsf11b in chondrocytes of the epiphyseal growth plate and the articular cartilage, while Tnfsf11 expression seemed higher in the primary spongiosa below the growth plate (Fig. 4a). Moreover, Tnfsf11 expression seemed to be upregulated upon OPG:Fc treatment in the primary spongiosa (Fig. 4a). Quantification of the Tnfsf11 and Tnfrsf11b expression in the chondrocytes of the growth plate and primary spongiosa revealed that approximately 40% of chondrocytes expressed Tnfsf11, while the proportion of Tnfsf11+ cells increased to almost 60% in the primary spongiosa immediately below the growth plate (Fig. 4b). The proportion of Tnfsf11+ cells increased in the primary spongiosa, but not in chondrocytes, upon OPG:Fc treatment. In contrast, the proportion of Tnfrsf11b+ cells were higher in chondrocytes than in the primary spongiosa (Fig. 4b). OPG:Fc treatment significantly reduced the proportion of Tnfrsf11b+ cells in the primary spongiosa (Fig. 4b). Moreover, OPG:Fc treatment increased the Tnfsf11+/Tnfrsf11b+ cell ratio significantly in the cells in the primary spongiosa, but not in the chondrocytes (Fig. S4a). To further examine the expression of Tnfsf11 and Tnfrsf11b, we quantified staining intensities and found a higher Tnfsf11 staining intensity in the primary spongiosa than in chondrocytes, while Tnfrsf11b staining intensity was higher in chondrocytes (Fig. 4c). Moreover, Tnfsf11 staining intensity was enhanced by OPG:Fc treatment, while Tnfrsf11b staining intensities were generally not affected (Fig. 4c). The Tnfsf11/Tnfrsf11b staining intensity ratio increased more than 4-fold in the primary spongiosa upon OPG:Fc treatment (Fig. S4a). To visualize the expression pattern of Tnfsf11 and Tnfrsf11b, we quantified the percentage of positive cells, and related their distance to an interface line separating the chondrocytes from the primary spongiosa (Fig. 4d, e and Fig. S4b, c). For both Tnfsf11 and Tnfrsf11b, a significant interaction between regions and treatment was detected (Fig. 4d, e and Fig. S4b, c). Moreover, The percentage of Tnfsf11+ cells and Tnfsf11 staining intensities remained low in the cartilage, and increased as the tissue transitioned to primary spongiosa, with OPG:Fc treatment increasing the Tnfsf11+ cells and Tnfsf11 staining intensity in the primary spongiosa (Fig. 4d and Fig. S4b). Conversely, Tnfrsf11b+ cells and Tnfrsf11b staining intensity increased gradually in chondrocytes of the growth plate, while it decreased again in the primary spongiosa (Fig. 4e and Fig. S4c). OPG:Fc treatment induced a decrease in Tnfrsf11b staining intensity in the primary spongiosa (Fig. S4c).

Fig. 4

Chondrocytes of the epiphysial growth plate of the tibia express high levels of Tnfrsf11b, while cells residing in the primary spongiosa express high levels of Tnfsf11. All results on this figure are female mice treated with vehicle or OPG:Fc for two weeks. a ISH of Tnfrsf11b (red) present at high levels in the growth plate of the tibia and articular cartilage and Tnfsf11 (yellow), present at the primary spongiosa. b Percentage of Tnfsf11+ and Tnfrsf11b+ cells, and Tnfsf11+/Tnfrsf11b+ cell ratios in chondrocytes and cells within the primary spongiosa. c Mean staining intensities of Tnfsf11 and Tnfrsf11b, and Tnfsf11/Tnfrsf11b staining intensity ratios in chondrocytes and cells within the primary spongiosa. d, e Percentage of Tnfsf11+ and Tnfrsf11b+ cells against the distance from the border between the primary spongiosa and chondrocytes. b, c Data are shown as mean ± SD. d, e Data are shown as mean ± SEM. P values are calculated using a two-way ANOVA or a mixed-effects analysis. b–e ****P values < 0.000 1, ***P values < 0.001, **P values < 0.01, *P values < 0.05. n = 8/group. Scale bars: Overview = 1 mm, high magnification images = 100 µm. GP growth plate, AC articular cartilage, PS primary spongiosa

Bone surface osteoprogenitors expressing the gene encoding matrix metallopeptidase 13 (Mmp13) also express Tnfsf11

To identify the main cell populations expressing Tnfsf11 and Tnfrsf11b we utilized the previously published and publicly available scRNAseq datasets.32 Enriched bone marrow stromal cells were classified into 13 different cell clusters (Fig. 5a). In these 13 identified cell clusters, Tnfsf11+ and Tnfrsf11b+ cells were abundant in cluster 0, 3, and 7 (Fig. 5b). Key gene markers defining the identity of bone marrow cell clusters included Lepr, Cxcl12, Adipoq, and Bglap for cluster 0, lepr, Cxcl12, Adipoq, and Bmp4 for cluster 3, and Col1a1, Alpl, Bglap, Bglap2, and Bglap3 for cluster 7. Moreover, Mmp13 was expressed in all three cell clusters (Fig. S5a). Since our ISH assessment showed that Tnfrsf11b was mainly expressed by osteocytes, and the scRNAseq data only includes bone marrow stromal cells, we decided to continue with Tnfrsf11 for further characterization of the cell populations that express this gene. We identified 58 differentially expressed genes in Tnfsf11+ cells compared to Tnfsf11− cells in cluster 0, 3, and 7 (Fig. 5c). Of these genes, a high proportion of Tnfsf11+ cells had a high expression of genes such as Mmp13, Wisp2, Limch1, and Wif1 and a low expression of genes such as Col1a1, Lepr, and Lpl (Fig. 5c). Conversely, a high proportion of Tnfsf11- cells had a high expression of Col1a1, Lepr. and Lpl, and a low expression of Mmp13, Wisp2, Limch1 (Fig. 5c). Since we have previously established that osteoprogenitor cells including reversal cells, canopy cells and bone lining cells express MMP13 in humans,33,34 we hypothesized that these cells also express Tnfsf11. Multiplex ISH of Tnfsf11 or Tnfrsf11b together with Mmp13, and Col1a1, revealed that the majority of Tnfsf11+ bone surface cells were Mmp13+, and Col1a1-, and that these Tnfsf11/Mmp13+ cells remained on the bone surfaces after OPG:Fc treatment (Fig. 5d). Tnfrsf11b/Mmp13/Coll1a1 multiplex ISH confirmed that Tnfrsf11b was mainly expressed by Mmp13-/Col1a1- osteocytes, and showed that only a few bone surface cells were Tnfrsf11b+/Mmp13+/Col1a1+(Fig. 5e) and found some Tnfrsf11b+/ Col1a1high osteoblasts (data not shown).

Fig. 5

Single cell RNA-seq data of enriched bone marrow stroma of male mice and multiplex ISH of tissue sections from OPG:Fc-treated female mice. a Bone marrow cells were classified into 13 cell clusters by UMAP: (0) Lepr+ Bglap+ mesenchymal stem cell (MSC), (1) bone marrow endothelial cell (BMEC), (2) megakaryocyte (Mk), (3) Lepr+ Bmp4hi MSC, (4) B cell, (5) myeloerythroid progenitor (MEP), (6) neutrophil (NEUT), (7) osteolineage cell (Osteo), (8) myofibroblast (Myofibro), (9) fibroblast (Fibro), (10) lymphocyte (LYM), (11) B cell, (12) neutrophil (NEUT), (13) basophil (Baso). b Expression of Tnfrsf11b and Tnfsf11 within each bone marrow cell cluster. c All differentially expressed genes identified in Tnfsf11+ cell compared to Tnfsf11− cells within clusters 0, 3, 7. d Tnfsf11/Mmp13/Col1a1, or (e) Tnfrsf11b/Mmp13/Col1a1 multiplex ISH conducted on adjacent sections of tibiae collected from mice treated with either vehicle or OPG:Fc for two weeks. d, e White dashed lines outline trabecular bone surfaces. Tb Trabecular, Bm bone marrow

Bone surface cells and osteocytes near the bone surface respond to systemic PTH stimulus

Previous studies suggest that Tnfsf11 and Tnfrsf11b expression is regulated upon PTH treatment with the most notable effect observed one hour after treatment.30,31 Therefore, we utilized a single PTH treatment dose as a biological probe to induce Tnfsf11 expression in mice, in order to (1) identify the spatial location of cells that respond to systemic PTH stimuli by regulating the expression of Tnfsf11 and Tnfrsf11b, and (2) to validate that the main activator of osteoclastogenesis is cells residing near or on the bone surfaces. Histological assessment of Tnfsf11 mRNA expression in vehicle-treated mice revealed subtle expression of Tnfsf11 in bone surface cells, with limited expression in osteocytes. Upon PTH treatment, mRNA expression was highly increased in cells near the trabecular and endocortical bone surfaces (Fig. 6a). Next, we quantified cells and related their distances to the trabecular and cortical bone surfaces. A significant interaction between treatment and region was detected in both the trabecular and cortical compartment. Specifically, we found that PTH treatment highly increased Tnfsf11 expression in bone surface cells, and osteocytes and marrow cells near the bone surface compared to vehicle—a finding similar to what we observed in the mice treated with OPG:Fc (Fig. 6b and Fig. S6a). Similarly, we assessed the expression of Tnfrsf11b using ISH and found that mainly osteocytes near the bone surfaces expressed Tnfrsf11b compared to marrow cells and surface cells—this osteocytic Tnfrsf11b expression was reduced by PTH(1–34) treatment (Fig. 6c). Quantification of Tnfrsf11b expressing cells, and their distances to the trabecular or endocortical bone surfaces revealed a significant interaction between treatment and region in all cases, but not when relating the abundance of Tnfrsf11b+ cells to the distance to the trabecular bone surfaces (Fig. 6d and Fig. S6b). PTH treatment reduced Tnfrsf11b expression in bone surface cells, and osteocytes near the bone surface compared to vehicle—this was reflected by a decreased staining intensity in trabecular osteocytes and a decrease in both the abundance and staining intensity of Tnfrsf11b+ cells in cortical osteocytes near the bone surfaces (Fig. 6d and Fig. S6b).

Fig. 6

PTH treatment induces a changed Tnfsf11 and Tnfrsf11b expression pattern. Tnfsf11 and Tnfrsf11b ISH performed on sections of femora obtained from 12-week-old female mice, 1 h after they were injected with a single dose of PTH (80 µg/kg). a Tnfsf11 (yellow) ISH illustrated in sections of the femur from vehicle and PTH treated mice. b Percentage of Tnfsf11+ cells against the distance from the trabecular or endocortical bone surface. c Tnfrsf11b (red) ISH illustrated in bone sections from vehicle and PTH treated mice. d Percentage of Tnfrsf11b+ cells against the distance from the trabecular or endocortical bone surface. e Percentage of Tnfsf11+ and Tnfrsf11b+ trabecular surface cells, trabecular osteocytes, and proximate marrow cells. f Mean Tnfsf11 and Tnfrsf11b cell staining intensities in trabecular surface cells, trabecular osteocytes and proximate marrow cells. g Percentage of Tnfsf11+ and Tnfrsf11b+ endocortical surface cells, cortical osteocytes and marrow cells. h Mean Tnfsf11 and Tnfrsf11b cell staining intensities in endocortical surface cells, cortical osteocytes, and marrow cells. a–c White dashed lines highlight trabecular, endocortical, and periosteal bone surfaces. In (b, d) data is expressed as mean ± SEM. In (e–h) data is expressed as mean ± SD. P values are calculated using a two-way ANOVA or mixed-effects analysis. ****P values < 0.000 1, ***P values < 0.001, **P values < 0.01, *P values < 0.05. n = 3/group. Scale bars: (a, b) Overview = 1 mm and high magnifications (a) = 100 µm or (b) = 50 µm. Ct cortical, Tb trabecular, Bm bone marrow

We subdivided the cell populations into bone surface cells, osteocytes and marrow cells to compare Tnfsf11 and Tnfrsf11b expression between cell types and to determine the impact of PTH treatment on the mRNA expression. In the trabecular compartment PTH induced a 2–3-fold increase in the proportion of surface cells, osteocytes and marrow cells that expressed Tnfsf11 (Fig. 6e). Notably, the proportion of bone surface cells expressing Tnfsf11 was 2-fold higher compared to marrow cells and osteocytes in the PTH-treated mice (Fig. 6e). Moreover, the Tnfsf11 staining intensities in bone surface cells, osteocytes and marrow cells were 18–35-fold higher in the PTH-treated mice compared to vehicle. Notably, bone surface cells had approximately a 2-fold higher Tnfsf11 staining intensity than osteocytes and marrow cells after PTH-treatment (Fig. 6e). Assessing the expression of Tnfrsf11b across cell types in the vehicle group, we found nearly a 3-fold higher proportion of osteocytes expressing Tnfsf11b compared to bone surface cells, while a 20-fold higher proportion was observed in osteocytes compared to marrow cells (Fig. 6e). The proportion of osteocytes expressing Tnfrsf11b was significantly reduced by nearly 2-fold in osteocytes and bone surface cells after PTH treatment, while marrow cells were not affected (Fig. 6e). Similar findings in osteocytes were found for staining intensities, while the Tnfrsf11b staining intensities were not changed in surface cells and marrow cells in the PTH-treated mice compared to vehicle (Fig. 6f).

In the cortical compartment, a 4.5-fold higher proportion of bone surface cells expressed Tnfsf11 in the PTH-treated compared to vehicle-treated mice (Fig. 6g). Moreover, nearly a 3-fold higher proportion of osteocytes expressed Tnfsf11 in PTH-treated mice compared to vehicle-treated mice. No significant differences were detected in marrow cells between PTH- and vehicle-treated mice (Fig. 6g). Comparing the proportion of bone surface cells to osteocytes and marrow cells within the PTH-treated mice, the proportion of Tnfsf11+ bone surface cells was 2.5-fold higher compared to osteocytes and nearly 4-fold higher compared to marrow cells (Fig. 6g). Similar findings were found for Tnfsf11 staining intensities (Fig. 6h). Furthermore, we assessed the expression of Tnfrsf11b across cell types in the vehicle group and found approximately a 3-fold higher proportion of osteocytes expressing Tnfsf11b compared to bone surface cells, while the proportion of Tnfrsf11b+ osteocytes was nearly 58-fold than that of marrow cells (Fig. 6g). Moreover, the proportion of osteocytes expressing Tnfrsf11b, and the Tnfrsf11b staining intensities were 1–2-fold lower in the PTH-treated mice compared to vehicle-treated mice (Fig. 6g, h).

When comparing PTH- to vehicle-treated mice, the ratio of Tnfsf11/Tnfsf11b positive cells and ratio of staining intensity trended toward an increase in the PTH treated group but was only significantly different for marrow cells (Fig. S6c, d).

The inner ear of rats is protected from bone remodeling by high Tnfrsf11b and low Tnfsf11 expression

To better understand the cellular mechanisms that influence the activation of osteoclastogenesis, we utilized the inner ear as a unique anatomic location. Here, bone remodeling is highly inhibited in the bone immediately surrounding the inner ear, while the bulla of the middle ear undergo bone modeling with resorption on one side and formation on the other side.35,36,37,38 ISH of Tnfsf11 and Tnfrsf11b expression in the inner ear, revealed an extremely high local expression of Tnfrsf11b in the fibrocytes of the spiral ligament of the cochlea, while a minority of the osteocytes expressed Tnfrsf11b (Fig. 7a). In the bone immediately adjacent to the spiral ligament, bone remodeling was clearly inhibited, as bone remodeling took place at a distance from the Tnfrsf11b+ fibrocytes (Fig. 7a). We examined Tnfsf11 expression in adjacent to Tnfrsf11b-stained sections, and found little or no Tnfsf11+ cells in the spiral ligament and the bone immediately outside the spiral ligament, while Tnfsf11+ cells were increased in the bone remodeling spaces more distant from the spiral ligament, including in the bony labyrinth and in the tympanic bulla of the middle ear (Fig. 7b).

Fig. 7

Fibrocytes of the spiral ligament express high levels of Tnfrsf11b. All data are obtained from a 10-day-old Sprague-Dawley rat. ISH on sections through the inner and middle ear, with representative overview image and high magnification images of the spiral ligament lining the bony labyrinth of the inner ear and the tympanic bulla of the middle ear for (a)Tnfrsf11b and (b) Tnfsf11 ISH of an adjacent section. The green dashed line separates the fibrocytes of the spiral ligament from the bony labyrinth. The black dashed line separates bone protected from remodeling from bone undergoing remodeling. The red dashed line outlines a region of the tympanic bulla of the middle ear. Scale bars: overview = 1 mm, high magnification images = 50 µm. BL bony labyrinth, SL spiral ligament

Bone surface cells proximate to osteoclasts are the most abundant source of Tnfsf11 in mice

Previously, it has been illustrated that reversal cells form cell extensions that extend beneath the bone resorbing osteoclast, demonstrating direct cell-cell contact between reversal cells and bone resorbing osteoclasts.33,39 Therefore, we investigated Tnfsf11 and Tnfrsf11b expression in the local environment surrounding the osteoclasts in female and male mouse vertebral bone serial sections hybridized to Tnfsf11, Tnfrsf11b, or Acp5 probes in order to characterize the environment close to the osteoclasts and to address the possibility of sexual dimorphism in Tnfsf11 and Tnfrsf11b expression. We quantified the expression in cells that were up