Maria-Bernadette Madel

He Fu

Dominique D Pierroz

Mariano Schiffrin

Carine Winkler

Anne Wilson

Cécile Pochon

Barbara Toffoli

Mahdia Taïeb

Jean- Yves Jouzeau

Federica Gilardi

Serge Ferrari

Nicolas Bonnet

Claudine Blin- Wakkach

Béatrice Desvergne

David Moulin

Lack of adiponectin drives hyperosteoclastogenesis in lipoatrophic mice

Keywords: Adiponectin, Osteoclast, Bone Marrow Adiposity, Cortical Bone porosity

Long bones from mammals host blood cell formation and contain multiple cell types, including adipocytes. Physiological functions of bone marrow adipocytes are poorly documented. Herein, we used adipocyte-deficient PPARγ-whole body null mice to investigate the consequence of total adipocyte deficiency on bone homeostasis in mice. We first highlighted the dual bone phenotype of PPARγ null mice: on the one hand the increase bone formation and subsequent trabecularization extending in the long bone diaphysis, due to the well-known impact of PPARγ deficiency on osteoblasts formation and activity; on the other hand, an increased osteoclastogenesis in the cortical bone. We then further explored the cause of this unexpected increased osteoclastogenesis using two independent models of lipoatrophy, which recapitulated this phenotype. This demonstrates that hyperosteoclastogenesis is not intrinsically linked to PPARγ deficiency, but is a consequence of the total lipodystrophy. We further showed that adiponectin, a cytokine produced by adipocytes and mesenchymal stromal cells is a potent inhibitor of osteoclastogenesis in vitro and in vivo. Moreover, pharmacological activation of adiponectin receptors by the synthetic agonist AdipoRon inhibited mature osteoclast activity both in mouse and human cells by blocking podosome formation through AMPK activation. Finally, we demonstrated that AdipoRon treatment blocks bone erosion in vivo in a murine model of inflammatory bone loss, providing potential new approaches to treat osteoporosis.

Introduction

Bone homeostasis is a result of constant remodelling activities, with a balance of bone resorption and bone formation. The most prevalent disorder of bone homeostasis is osteoporosis, affecting men and women, with a particular high prevalence in post-menopausal women. The low efficacy of actual anti-osteoporotic therapeutic options highlights the need of better understanding the pathophysiological processes at work.

The role of adipocyte in bone homeostasis is one of the factors raising great interest, but it faces quite some complexity. An illustration of this complexity, is the fact that obesity is traditionally considered to be protective against osteoporosis, consequently to an increased proliferation and differentiation of osteoblasts and osteocytes stimulated by mechanical load [START_REF] Felson | Effects of weight and body mass index on bone mineral density in men and women: the Framingham study[END_REF]. However, obesity in postmenopausal women and in men correlates with increased marrow adiposity, alteration of the bone microarchitecture, and increased risk of fracture [START_REF] Sheu | The role of bone marrow and visceral fat on bone metabolism[END_REF].Thus, the respective roles of ageing, increased BMI, and visceral vs bone marrow fat on bone homeostasis remain difficult to disentangle [START_REF] Cornish | Role of Marrow Adipocytes in Regulation of Energy Metabolism and Bone Homeostasis[END_REF][START_REF] Horowitz | Bone marrow adipocytes[END_REF].

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily. PPARγ is a key factor and master regulator in adipocytes development and functions [START_REF] Tontonoz | Fat and beyond: the diverse biology of PPARgamma[END_REF]. Indeed, loss-of-function studies both in vitro and in vivo have clearly demonstrated that PPARγ is essential for the formation of adipose tissue [START_REF] Barak | PPAR gamma is required for placental, cardiac, and adipose tissue development[END_REF][START_REF] Rosen | PPAR gamma is required for the differentiation of adipose tissue in vivo and in vitro[END_REF][START_REF] Wang | Lipoatrophy and severe metabolic disturbance in mice with fat-specific deletion of PPAR[END_REF] and mature adipocyte homeostasis [START_REF] Imai | Peroxisome proliferator-activated receptor gamma is required in mature white and brown adipocytes for their survival in the mouse[END_REF]. Recently, we demonstrated that mice carrying a full body deletion of PPARγ (Pparg Δ/Δ ) are totally devoid of adipocytes [START_REF] Sardella | Delayed Hair Follicle Morphogenesis and Hair Follicle Dystrophy in a Lipoatrophy Mouse Model of Pparg Total Deletion[END_REF]. The activity of PPARγ is also central in bone homeostasis by modulating both bone formation by osteoblasts and bone resorption due to osteoclast activity.

First, adipocytes and osteoblasts share same mesenchymal progenitors, and the cell fate decision between adipocyte and osteoblast lineages depends on various signals [START_REF] Calo | Rb regulates fate choice and lineage commitment in vivo[END_REF][START_REF] Idris | Cannabinoid receptor type 1 protects against age-related osteoporosis by regulating osteoblast and adipocyte differentiation in marrow stromal cells[END_REF][START_REF] Kawai | PPARγ: a circadian transcription factor in adipogenesis and osteogenesis[END_REF][START_REF] Wan | PPARγ in bone homeostasis[END_REF]. Activation of PPARγ inhibits osteoblast formation, and thus bone formation, by favouring adipocyte differentiation [START_REF] Ali | Rosiglitazone Causes Bone Loss in Mice by Suppressing Osteoblast Differentiation and Bone Formation[END_REF][START_REF] Rzonca | Bone is a target for the antidiabetic compound rosiglitazone[END_REF]. Along this line, in vivo evidence indicates that mice heterozygous for a null allele of the gene encoding PPARγ (Pparg) exhibit increased osteoblastogenesis, which results in increased bone mass with a doubling of the rate of new bone formation, when compared to control mice. Moreover, PPARγ also directly inhibits osteoblast activity, independently of adipogenesis, as demonstrated by the consequence of osteoblast-specific Pparg deletion in osteoblast differentiation and trabecular bone formation [START_REF] Sun | Osteoblast-Targeted suppression of PPARγ increases osteogenesis through activation of mTOR signaling[END_REF]. Second, in bone resorption, PPARγ promotes osteoclastogenesis, as illustrated by increased osteoclast numbers in aged mice treated with thiazolidinediones, which are high-affinity synthetic PPARg agonists, [START_REF] Lazarenko | Rosiglitazone Induces Decreases in Bone Mass and Strength that Are Reminiscent of Aged Bone[END_REF]. Conversely, mice in which Pparg is specifically deleted in endothelial and hematopoietic cells, (i.e., affecting osteoclast progenitors but not osteoblasts), exhibit decreased osteoclastogenesis compared to wild-type mice, provoking osteopetrosis with impaired bone resorption and reduced medullary cavities [START_REF] Wan | PPAR-gamma regulates osteoclastogenesis in mice[END_REF]. However, a recent controversy on the role of PPARγ in osteoclast was raised by Zou et al. who found that only the pharmacological, and not the physiological activation of PPARγ affects osteoclastogenesis [START_REF] Zou | PPAR-γ regulates pharmacological but not physiological or pathological osteoclast formation[END_REF]. Finally, hyperactivation of PPARγ via agonist treatment causes accumulation of adipocytes in the bone marrow in thiazolidinedione-treated rodents, whereas increased bone loss and bone fractures have been correlated with thiazolidinedione therapy in humans [START_REF] Grey | Skeletal consequences of thiazolidinedione therapy[END_REF][START_REF] Kahn | Rosiglitazone-Associated Fractures in Type 2 Diabetes: An analysis from A Diabetes Outcome Progression Trial (ADOPT)[END_REF][START_REF] Lazarenko | Rosiglitazone Induces Decreases in Bone Mass and Strength that Are Reminiscent of Aged Bone[END_REF].

Herein, we used mice carrying a PPARγ full body deletion to explore the consequences of the lack of adipocyte on bone homeostasis, and demonstrate that as expected, there is a strong increased bone density of the trabecular bone. However, this phenotype was associated with an exacerbated osteoclastogenesis. Using two other complementary models of geneticallyinduced lipoatrophic mice, i.e. mice carrying an adipose-tissue specific deletion of PPARγ and AZIP mice, we then further explore the cross-talk between adipocytes and osteoclasts, whose alteration leads to hyperosteoclastogenesis and osteoporosis.

Results

In order to evaluate the impact of adipose tissue on bone homeostasis, we studied lipoatrophic mice that we recently generated. These mice, hereby called Pparg Δ/Δ carry a whole body deletion of PPARγ and were obtained through epiblastic (Sox2 promoter) cre deletion of the Pparγ allele [START_REF] Sardella | Delayed Hair Follicle Morphogenesis and Hair Follicle Dystrophy in a Lipoatrophy Mouse Model of Pparg Total Deletion[END_REF]. Bone analyses of female one-year-old mice first showed that these mice displayed dramatic alterations of both trabecular and cortical bones (Fig 1).

The three-dimensional images generated by trabecular bone microcomputed tomography (3D micro-CT) reconstruction of the femurs of one-year old female mice highlighted a remarkably high trabecularization of the bone architecture in Pparg Δ/Δ mice, which extended along the shaft and almost reached the mid-diaphysis (VIDEO 1, Fig. 1a, Table 1a). In contrast, the cortical bone was found to be extremely porous at the epiphysis and the diaphysis. Owing to hypertrabecularization and high cortical porosity, the frontier between these two types of bone along the diaphysis remained difficult to establish. The phenotype was also observed in Pparg Δ/Δ male mice. However, the difficulties to obtain an appropriate number of old Pparg Δ/Δ males (due to premature death, still being investigated), and the fact that old female mice had a marked phenotype lead us to pursue all further explorations in females.

We first further evaluated in female whether the bone alterations appear during development or appear progressively with age, by performing micro CT measurements at 5 -6 months. A quantification of the diverse parameters is shown in Table 1b. More particularly, we found that the increased trabecular number and connectivity density in both vertebral bodies and distal femoral metaphysic bones appeared at 5-6 months of age, whereas the alteration of cortical parameters were only seen in one year-old mice.

Molecular analyses confirmed an increased trabecular bone formation, as exemplified by increased levels of alkaline phosphatase mRNA (Alp1) in the bone, evaluated by RT-qPCR and increased levels of alkaline phosphatase (ALP) and osteocalcin (OCN) in the serum measured by ELISA (Fig. 1b andc). To evaluate whether osteocytes, which come from osteoblasts and are mature permanent bone cells, versus osteoblast populations were altered in these mice, we measured the mRNA levels of sclerostin (Sost) and keratocan (Kera), which are expressed by osteocytes and osteoblasts, respectively [START_REF] Jilka | Dysapoptosis of osteoblasts and osteocytes increases cancellous bone formation but exaggerates cortical porosity with age[END_REF]. Both markers were unchanged (supplementary Fig. S1). Three-D micro-CT of the fourth lumbar vertebrae clearly demonstrated increased trabecular bone volume fraction and trabecular connectivity density, indicating increased osteoblastogenesis in Pparg Δ/Δ (supplementary Fig. S1), in agreement with previous report [START_REF] Akune | PPARgamma insufficiency enhances osteogenesis through osteoblast formation from bone marrow progenitors[END_REF]. These results are consistent with the pivotal role of PPARg on the fate of bone marrow mesenchymal stromal cells, with previous reports showing increased osteoblastogenesis when PPARg signalling is pharmacologically or genetically blocked [START_REF] Takada | Wnt and PPARγ signaling in osteoblastogenesis and adipogenesis[END_REF]. As mentioned above, the role of PPARg in osteoblastogenesis has been well documented and was therefore not further investigated in the present report.

Remarkably, the expression of bone resorbing markers such as the Tartrate-Resistant Acid Phosphatase Type 5 (Acp5), cathepsin K (Ctsk) was increased in the long bones of Pparg Δ/Δ mice compared to control mice. Mmp9, which is a key proteinase involved in the recruitment and activity of osteoclasts and endothelial cells for the development of long bones, particularly in the diaphysis core [START_REF] Engsig | Matrix metalloproteinase 9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bones[END_REF][START_REF] Jemtland | Heterogeneity among cells that express osteoclast-associated genes in developing bone[END_REF], was also increased (Fig. 1d) suggesting an increase in osteoclastogenesis. This is further supported by an augmentation of serum levels of carboxy-terminal collagen I telopeptide (CTX) (Fig. 1e).

Indeed, TRAcP staining on femoral sections (Fig. 1f) confirmed the increased presence of bone-resorbing osteoclasts explaining the observed cortical porosity.

PPARg is known to play a role in activating osteoclastogenesis. [START_REF] Wan | PPAR-gamma regulates osteoclastogenesis in mice[END_REF] used a Tie2Cre-directed PPARg deletion, which specifically target endothelial cells among which the myeloid-lineage cells -from which the osteoclast is derived-. These authors show that this cell-specific PPARg deletion severely impaired osteoclastogenesis, whereas PPARg synthetic agonists exacerbated it [START_REF] Wan | PPAR-gamma regulates osteoclastogenesis in mice[END_REF]. This role of PPARg in osteoclast has been recently questioned by the study of Zou et al., showing that the in vivo effect of PPARγ on osteoclast formation is only seen upon pharmacological activation of PPARγ but neither on physiological nor on pathological osteoclast formation [START_REF] Zou | PPAR-γ regulates pharmacological but not physiological or pathological osteoclast formation[END_REF]. This raised the question whether the elevated osteoclast activity observed in cortical bone is attributable to lipoatrophy, or to unforeseen intrinsic effects of PPARg deficiency in the osteoclast lineage.

To disentangle these two possibilities, we used another PPARγ-independent lipodystrophic mouse model. AZIP tg/+ mouse is a hemizygote transgenic mouse strain in which a dominant negative protein of key B-ZIP transcription factors (A-ZIP) blocks adipocyte development.

AZIP tg/+ mice are born with no white adipose tissue and markedly decreased brown adipose tissue [START_REF] Moitra | Life without white fat: a transgenic mouse[END_REF]. In support of the lipoatrophy hypothesis, micro-CT analyses of the femur of one year old female AZIP tg/+ mice revealed an increased cortical porosity (Fig 2a).

As observed in Pparg Δ/Δ mice, TRAcP staining of longitudinal bone sections revealed increased osteoclast numbers (Fig 2b), further confirmed by the increased expression levels of the osteoclastogensis markers (Fig 2c). Finally, we generated a third mouse model by invalidating PPARg in adipose tissue (Adipoq cre PPARg fl/fl, γ FatKO [START_REF] Wang | Lipoatrophy and severe metabolic disturbance in mice with fat-specific deletion of PPAR[END_REF], called herein PPARγ adipoKO ). These mice are lipoatrophic, but in contrast to Pparg Δ/Δ , they have an unaltered hematopoietic lineage. Here again, micro-CT analysis and osteoclastogenesis markers in one-year old females revealed increased osteoclast numbers and activity with an enhanced cortical porosity, as observed in Pparg Δ/Δ mice (Fig. 2d and2e).

These two independent mouse models confirmed that adipocyte deficiency is pivotal to the cortical bone phenotype and the increased osteoclast number observed here.

One consequence of lipodystrophy observed in all three models is a severe type 2 diabetes with signs of diabetic nephropathy [START_REF] Moitra | Life without white fat: a transgenic mouse[END_REF][START_REF] Toffoli | Nephropathy in Pparg-null mice highlights PPARγ systemic activities in metabolism and in the immune system[END_REF][START_REF] Wang | Lipoatrophy and severe metabolic disturbance in mice with fat-specific deletion of PPAR[END_REF]. Given that chronic kidney diseases are often accompanied by hyperparathyroidism and result in complex, combined catabolic and anabolic activities in the bones [START_REF] Levin | Prevalence of abnormal serum vitamin D, PTH, calcium, and phosphorus in patients with chronic kidney disease: results of the study to evaluate early kidney disease[END_REF], we evaluated parathyroid hormone (PTH) levels. As shown in supplementary Figure S2, no statistically significant differences were observed between Pparg Δ/Δ mice and their control littermates in either the circulating levels of PTH or the mRNA levels of the PTH receptor Pth1r in the bones or kidneys. Notably, serum PTH levels were also unchanged in AZIP tg/+ mice (supplementary Fig S2 ), suggesting that the PTH pathway is unlikely to support the increased bone remodelling observed in lipodystrophic mice.

To investigate how adipocytes regulate osteoclastogenesis, and to evaluate whether factors produced by adipocytes within the bone marrow can influence osteoclastogenesis, we performed an osteoclastogenesis assay using bone marrow cells from wild-type mice cultured in the presence of either control medium (CTL in Fig. 3) or adipocyte-conditioned medium (ACM) (see Fig. 3a for a graphic summary of the experiment). Adipocyte-conditioned medium was isolated from culture of adipocyte-differentiated 3T3-L1 cells whereas control medium was obtained from cultures of undifferentiated pre-adipocytes 3T3-L1 cells. The presence in bone marrow cells culture of adipocyte-conditioned medium, compared to preadipocyte-conditioned medium, provoked a substantial decrease in the number of mature osteoclasts (Fig. 3b). Consistently, adipocyte-conditioned medium significantly inhibited bone resorption as assessed by the decrease number of resorption pits in the calvaria resorption assay (Fig. 3c). These data demonstrate that adipocyte-secreted mediators are responsible for the inhibition of RANKL-induced osteoclast differentiation in vitro.

Next, we searched for component(s) of ACM responsible for this anti-osteoclastogenic activity. Interestingly, the ACM inhibitory activity was maintained after filtering out all components larger than 35 kDa from the conditioned medium. The molecular weights of the major adipokines, adiponectin and leptin, are below 35 kDa and their circulating levels are not or barely detectable in Pparg Δ/Δ mice, as we previously demonstrated [START_REF] Gilardi | Systemic PPARγ deletion in mice provokes lipoatrophy, organomegaly, severe type 2 diabetes and metabolic inflexibility[END_REF]. As expected, the gene expression levels of Leptin and Adiponectin in long bones of Pparg Δ/Δ mice as well as AZIP tg/+ mice were extremely low (supplementary Fig S3). Unlike Leptin, Adiponectin is preferentially expressed by adipocyte and by bone-marrow MSC, and is a wellcharacterized target gene of PPARg [START_REF] Iwaki | Induction of adiponectin, a fat-derived antidiabetic and antiatherogenic factor, by nuclear receptors[END_REF]. Western-blot analyses confirmed that adiponectin was present in large amounts in ACM but not in CTL medium (Fig 3d). We therefore tested whether blocking adiponectin would affect ACM-mediated inhibition of osteoclastogenesis in vitro. As shown in Figure 3b, the addition of a blocking adiponectin antibody reversed the reduction in osteoclast number in a dose-dependent manner. These results support that the soluble factors secreted by adipocytes, in particular adiponectin, are inhibitors of osteoclastogenesis. Consistently, the total lack of adipocytes, including in the bone marrow, results in a complete absence of this adipokine, leading to the loss of its repressive activity in osteoclast differentiation.

AdipoRon is a non-peptidic agonist of adiponectin receptors (Okada- [START_REF] Okada-Iwabu | A small-molecule AdipoR agonist for type 2 diabetes and short life in obesity[END_REF]. We thus perform the reverse experiment of the antibody blocking assay, adding AdipoRon to culture of bone marrow cells. Consistently, AdipoRon recapitulated the blocking effect of ACM on osteoclast differentiation irrespective of the origin of the osteoclast progenitors (bone marrow, spleen, or monocytic-enriched fraction) with IC50 in the micromolar range (Fig. 4a,b, supplementary Fig. S4). Furthermore, AdipoRon blocked osteoclast differentiation in Pparg deficient cells, confirming that intrinsic PPARg alterations do not play a role in the observed phenotype (Fig. 4c).

Previous studies demonstrated in vivo that mice treated with adiponectin expressing adenovirus have reduced osteoclast numbers and bone-resorption markers [START_REF] Oshima | Adiponectin increases bone mass by suppressing osteoclast and activating osteoblast[END_REF]. Using the macrophage cell line RAW264, prone to differentiate in osteoclasts, these authors further showed that adiponectin blocked in vitro osteoclastogenesis by inhibiting NFATc1. Blockade of NFATc1 was suggested to occur through activation of the AMPK signalling pathway, a key integrator of metabolic signals and target of adiponectin signalling [START_REF] Yamaguchi | Adiponectin inhibits induction of TNF-alpha/RANKL-stimulated NFATc1 via the AMPK signaling[END_REF]. We thus tested whether this mechanism would apply in vivo and to primary cells. Flow cytometry analysis combining nuclei staining [START_REF] Madel | A Novel Reliable and Efficient Procedure for Purification of Mature Osteoclasts Allowing Functional Assays in Mouse Cells[END_REF] and anti-AdipoR1 antibody, confirmed that the adiponectin receptor AdipoR1 is expressed also by mature osteoclasts generated from murine bone marrow cells (Fig. 4d). Furthermore, RT-PCR performed on osteoclasts from different origins (Bone marrow, bone-marrow derived dendritic cells, bone-marrow monocytic cells) showed that osteoclasts express both adiponectin receptors AdipoR1 andAdipoR2 (Fig 4e). In agreement, western-blot analysis of AMPK phosphorylation confirmed that the Adiponectin signalling pathway is active in both precursors and mature osteoclasts sorted on the basis of their nuclei number (≥ 3) [START_REF] Madel | A Novel Reliable and Efficient Procedure for Purification of Mature Osteoclasts Allowing Functional Assays in Mouse Cells[END_REF](Fig. 4f-h).

AdipoRon was thus used to investigate the consequences of adiponectin receptor activation on murine and human osteoclasts. AICAR was used as a synthetic activator of AMPK. As seen in Figure 5a,b each of these molecules inhibited the formation of podosomes that are essential for bone resorption. Moreover, dorsomorphin, a reversible AMP-kinase inhibitor, could reverse the AdipoRon inhibitory effect (Fig. 5b). This is in line with the recently published effect of recombinant adiponectin on RAW264.7 cells, confirming the impact of adiponectin on both osteoclasts differentiation and podosome formation [START_REF] Chen | Adiponectin inhibits osteoclastogenesis by suppressing NF-κB and p38 signaling pathways[END_REF] and endorsing the use of AdipoRon as a surrogate activator for adiponectin receptors. Finally, adiponectin signalling regulates numerous cellular metabolic pathways including mitochondrial functions [START_REF] Iwabu | Adiponectin and AdipoR1 regulate PGC-1alpha and mitochondria by Ca(2+) and AMPK/SIRT1[END_REF]. AdipoRon treatment provoked disruption of the perinuclear mitochondrial network when added to culture of mature osteoclasts (Fig. 5c). Altogether, these results indicate that, beside its inhibiting action in osteoclast differentiation, adiponectin signalling exerts a pivotal role in mature osteoclast energy homeostasis regulation.

The inhibitory effect of AdipoRon was further tested in vivo on the well-described model of osteoclast bone resorption induced by lipopolysaccharide (LPS) injection into the mouse calvaria. Both micro-CT and TRAcP staining (Fig. 5d,e) showed that AdipoRon administration resulted in a strong decrease in osteoclast numbers and bone resorption pits on calvaria.

We thus aimed to validate our findings on human cells and therefore investigated the impact of AdipoRon on hPBMC-derived osteoclasts. Consistent with the results obtained with mouse cells, low dose of AdipoRon significantly inhibited RANKL-induced human osteoclast differentiation (supplementary Fig. S5a,b). AdipoRon also activated AMPK phosphorylation in human PBMC-derived osteoclasts within minutes (supplementary Fig. S5c). At later timepoint, i.e. within hours after the challenge with AdipoRon, the number of actin-containing podosome was drastically reduced in human osteoclasts (supplementary Fig. S5d). These original findings offer new therapeutic perspectives in inflammatory bone loss management by targeting adiponectin/AdipoR.

Discussion

In the present report, using lipodystrophic mouse models, we evaluated how the total lack of adipose tissue may affect bone homeostasis. Taken together our results demonstrate that mature osteoclasts are sensitive to extrinsic adipose-derived metabolic signals such as adipokine and that bone marrow adiposity must be considered as a physiological regulator of bone homeostasis. Consistently with the literature, we identified adiponectin as one main inhibitory signal for osteoclastogenesis. Herein, we further showed that AdipoRon, a pharmacological agonist of adiponectin receptors, inhibits osteoclast differentiation of precursors from different origins (bone-marrow, dendritic and monocytic cells). We also demonstrated that adiponectin signaling inhibits human osteoclast differentiation as well as activity of human mature osteoclast and explored how AMPK signaling act on mature osteoclast activity via podosome formation blockade. These original findings reveal a conserved adiponectin-mediated metabolic control of osteoclast activity and are clinically promising, in particular with regard to the treatment and prevention of excessive pathological bone-resorption conditions, such as in osteoporotic patients.

The first model used in the present study, the PPARg null mice, is bringing up some decisive arguments with respect to the controversy on the role of PPARg in osteoclastogenesis.

Whereas initial studies reported that PPARg is a key factor for osteoclastogenesis [START_REF] Wan | PPAR-gamma regulates osteoclastogenesis in mice[END_REF], they could not confirm such activity and proposed that only pharmacological activation of PPARg had impact on osteoclastogenesis [12,23]. Herein, we definitely demonstrate that osteoclastogenesis can occur in the absence of PPARg signal, and that other adipose tissue-derived signals, such as adipokines, affect osteoclast differentiation and activity. In contrast, the increase trabecular bone formation seen in vertebrae, in femoral epiphyses and distal diaphysis of Pparg Δ/Δ mouse is consistent with the known role of PPARg in inhibiting osteogenesis.

Using three complementary genetic models (2 distinct conditional null mice of PPARg and the AZIP tg/+ mice, a PPARg-independent lipoatrophy model), we showed that the dramatic cortical bone alteration due to an excessive osteoclastogenesis in mice is linked to the lack of adipocyte and adipocyte-derived signals. The systemic and local activities of adipokines such as leptin and adiponectin have been recently reviewed [START_REF] Cornish | Role of Marrow Adipocytes in Regulation of Energy Metabolism and Bone Homeostasis[END_REF]. Although leptin can act as a positive regulator of osteogenesis in in vitro systems, other studies in mice show that systemic leptin acting via the central nervous system is a potent inhibitor of bone formation [START_REF] Ducy | Leptin inhibits bone formation through a hypothalamic relay: a central control of bone mass[END_REF]. Systemic adiponectin was reported to promote bone formation through its depressive action on the sympathetic tone in mice in age-dependent manner [START_REF] Kajimura | Adiponectin regulates bone mass via opposite central and peripheral mechanisms through FoxO1[END_REF]. However, in vitro differentiation assay showed that adiponectin inhibits osteoclast formation [START_REF] Oshima | Adiponectin increases bone mass by suppressing osteoclast and activating osteoblast[END_REF][START_REF] Yamaguchi | Adiponectin inhibits induction of TNF-alpha/RANKL-stimulated NFATc1 via the AMPK signaling[END_REF]. The observations in vivo are more controversial. Adiponectin-null mice exhibit an increased trabecular volume and fewer osteoclasts according to Wang et al. [START_REF] Wang | Adiponectin exerts its negative effect on bone metabolism via OPG/RANKL pathway: an in vivo study[END_REF], whereas Shinoda et al. [START_REF] Shinoda | Regulation of bone formation by adiponectin through autocrine/paracrine and endocrine pathways[END_REF] found normal bone development in these mice. In contrast, Naot et al. [START_REF] Naot | Reduced Bone Density and Cortical Bone Indices in Female Adiponectin-Knockout Mice[END_REF] described a decreased cortical thickness in adiponectin-null female mice and Yang et al. [START_REF] Yang | Adiponectin Deficiency Triggers Bone Loss by Up-Regulation of Osteoclastogenesis and Down-Regulation of Osteoblastogenesis[END_REF] a decreased bone mass. Interestingly, the female bias of the phenotype observed herein was also found in adiponectin-null mice. Thus, adiponectin may have different effects on bone remodelling, depending on the activated autocrine, paracrine or systemic pathways [START_REF] Tu | Adiponectin inhibits osteoclastogenesis and bone resorption via APPL1-mediated suppression of Akt1[END_REF]. Our results from the osteoclastogenesis assay using bone marrow-derived cells and adiponectin-specific blocking antibodies support the important paracrine role of adiponectin in the inhibition of osteoclastogenesis in the normal BM environment. Direct paracrine activity is also consistent with the presence of the adiponectin receptors, AdipoR1 and AdipoR2, on the surface of differentiating osteoclasts [START_REF] Pacheco-Pantoja | Adiponectin receptors are present in RANK-L-induced multinucleated osteoclastlike cells[END_REF]. Nonetheless, we cannot exclude that other adipokines may also contribute to the paracrine effects of adipocytes on osteoclasts.

Identifying the source of adiponectin in the vicinity of osteoclast precursors and/or mature osteoclasts in bone marrow and bone compartment is challenging. Indeed, levels of adiponectin production by BM adipocytes remain controversial. Cawthorn et al. [START_REF] Cawthorn | Bone Marrow Adipose Tissue Is an Endocrine Organ that Contributes to Increased Circulating Adiponectin during Caloric Restriction[END_REF] reported that BM adipocytes serve as a major source of circulating adiponectin exerting systemic effects during caloric restriction whereas others groups reported that bone marrow adipocytes express lower levels of adiponectin than adipocytes in adipose tissue [START_REF] Li | The Unique Metabolic Characteristics of Bone Marrow Adipose Tissue[END_REF][START_REF] Liu | Characterization of age-related gene expression profiling in bone marrow and epididymal adipocytes[END_REF][START_REF] Poloni | Molecular and functional characterization of human bone marrow adipocytes[END_REF]. Others sources of bone marrow adiponectin have been recently described. Bone marrow PDGFRβ + VCAM-1 + stromal cells have been identified as major producers of adiponectin in the bone marrow [START_REF] Mukohira | Mesenchymal stromal cells in bone marrow express adiponectin and are efficiently targeted by an adiponectin promoter-driven Cre transgene[END_REF].

Single cell transcriptomic profiling of the mouse bone marrow stromal compartment indicated that LepR + bone marrow mesenchymal stromal cells, which form a functional continuum with osteolineage cells, are expressing adiponectin [START_REF] Baryawno | A Cellular Taxonomy of the Bone Marrow Stroma in Homeostasis and Leukemia[END_REF]. Beside their proliferative action on HSC and antiproliferative effects on myelomonocytic and early B lymphoid cells, these adiponectin-producing cells appears to be essential to the hematopoietic niche. In this context, our finding that adiponectin regulates osteoclast activity and differentiation in bone further strengthens the argument that a metabolic pathway driven by adiponectin controls many osteoimmunology processes, including remodeling of bone marrow hematopoietic stem cell niche, with potential implications for multiple human disease states including cancer.

The distinct behaviour and response of trabecular bone versus cortical bone is particularly highlighted in Pparg Δ/Δ mice. Such contrasting phenotype has been previously observed. For example, increased or decreased mechanical strain [START_REF] Ausk | Metaphyseal and diaphyseal bone loss in the tibia following transient muscle paralysis are spatiotemporally distinct resorption events[END_REF] or fluoride treatment increase the density of trabecular bone but decrease the density of cortical bone (Connett, Fluoride Action network, April 2012, http://fluoridealert.org/studies/bone03/). This bivalent phenotype is reminiscent of that observed when osteoblast and osteocyte apoptosis is prevented via targeted deletion of the proapoptotic proteins Bak/Bax [START_REF] Jilka | Dysapoptosis of osteoblasts and osteocytes increases cancellous bone formation but exaggerates cortical porosity with age[END_REF]. In this context, a severe increase in cortical porosity was accompanied by increased osteoclast formation. The prolonged lifespan of osteoblasts was proposed by the authors to trigger diametrically opposed biological effects on bone homeostasis with an exaggeration of the adverse effects of ageing due to long-lived osteocytes [START_REF] Jilka | Dysapoptosis of osteoblasts and osteocytes increases cancellous bone formation but exaggerates cortical porosity with age[END_REF]. In particular, osteocytes may increase intracortical remodelling via their secretion of RANKL and the subsequent activation of osteoclasts [START_REF] Fuller | TRANCE is necessary and sufficient for osteoblast-mediated activation of bone resorption in osteoclasts[END_REF][START_REF] Jilka | Dysapoptosis of osteoblasts and osteocytes increases cancellous bone formation but exaggerates cortical porosity with age[END_REF][START_REF] Rinotas | Novel genetic models of osteoporosis by overexpression of human RANKL in transgenic mice[END_REF]. In Pparg Δ/Δ mice, the levels of Sost and Keratocan gene expression as markers of osteocytes and osteoblasts, respectively, are unchanged with respect to those found in their control littermates, thus refuting the possibility of an increased number of both cell types. In parallel, the increased expression of Ctsk and Mmp9 likely contributes to the loss of cortical bone owing to high osteoclast activity, whereas the well-established role of PPARγ deficiency in bone formation via osteoblast activation is particularly highlighted by the increased trabecular bone density. Finally, it has been recently shown that osteoclasts have some sitedependent specificities and heterogeneity [START_REF] Madel | Immune Function and Diversity of Osteoclasts in Normal and Pathological Conditions[END_REF], that could contribute to the distinct behaviour of the cortical bone vs trabecular bone.

The difference in the intensity of the cortical porosity observed between the three lipodystrophic models we used may stem from different causes. On the one hand, the fact that the phenotype is more marked in the Pparg Δ/Δ mice indicates that the expression of PPARγ in other cell types including osteoblasts and osteoclasts may dampen the phenotype in the AZIP and in the PPARγ adipoKO models, irrespectively of adiponectin signals. However, a clear contribution of adipocytes remains highlighted by the results in AZIP mice which expressed functional PPARγ in all cell types. On the other hand, adiponectin gene expression, barely detectable in Pparg Δ/Δ mice, is still present in the AZIP model and could contribute to the difference of phenotype penetrance between these two mouse models.

One important finding of the present report is the mechanism by which AdipoR signaling affects osteoclast activity. Indeed, here we demonstrated that AMPK was activated following AdipoRon challenge, which resulted in alteration of osteoclast metabolism, as exemplified by the alteration of the perinuclear mitochondrial network. We cannot exclude that direct or indirect signaling events may occur following adipoRon challenge that could interfere with osteoclast activity genetic program, such as Akt or NFATc1 signaling pathway as previously demonstrated with recombinant adiponectin [START_REF] Tu | Adiponectin inhibits osteoclastogenesis and bone resorption via APPL1-mediated suppression of Akt1[END_REF][START_REF] Yamaguchi | Adiponectin inhibits induction of TNF-alpha/RANKL-stimulated NFATc1 via the AMPK signaling[END_REF].

However, AMPK is central in this process, since its activation induced mitochondrial network alteration and podosome formation blockade, whereas AMPK blockade reverted these effects.

Similarly, we also demonstrated that adiponectin signaling inhibits human osteoclast differentiation as well as activity of human mature osteoclast.

Altogether, these original findings reveal a conserved adiponectin-mediated metabolic control of osteoclast activity and are clinically promising, in particular with regard to the treatment and prevention of excessive pathological bone-resorption conditions, such as in osteoporotic patients. The present study implies that preserving functional adiponectin sensitivity in patients suffering from metabolic diseases (e.g. diabetes, obesity, metabolic syndrome) might be also beneficial for preventing bone fragility. Experimental design of the in vitro assay used to test anti-osteoclastogenic activity from mature adipocyte secreted factors. b. One day after plating, wild type BM cells were cultured for 6 more days in the presence of M-CSF and RANKL (osteoclast differentiation media) in addition to conditioned medium prepared from differentiated (ACM: adipocyte-conditioned medium) or undifferentiated (CTL: control-conditioned medium) 3T3-L1 preadipocytes. Different doses of a specific blocking antibody against adiponectin (1.5-12.0 µg/ml) were added to the conditioned medium (ACM50) during osteoclast differentiation (ACM 100 means pure ACM; ACM50 means 1:1 diluted ACM with osteoclast differentiation medium). Data are presented as mean ± SEM of biological triplicates.. Statistical significance was determined by two-tailed unpaired t-test. c. Calvaria resorption pit assay. Representative sections of skulls from 14-day-old wild-type pups cultured for 2 weeks in complete aMEM in the presence of conditioned medium from either undifferentiated 3T3-L1 preadipocytes (CTL, n=3) or mature 3T3-L1 adipocytes (ACM, n=3), after TRAP staining. Top panel: the white arrow indicates one example of resorption pit seen in many places on the skull with CTL medium, while absent with the ACM culture medium. d. Western-blot analysis of conditioned medium from undifferentiated (CTL) and differentiated 3T3-L1 adipocyte (A-CM) against adiponectin. Recombinant mouse adiponectin (200ng rmAdiponectin) was added to demonstrate specificity of the signal. RT-qPCR analysis of Adiponectin Receptor 1 and 2 (Adipor1 and Adipor2) on osteoclasts differentiated from bone marrow (BM) cells (BM-OCL), from BM-derived dendritic cells (DC-OCL) and BM monocytic cells (MN-OCLs). f. Western-blot analysis of sorted mature bone marrow-derived osteoclasts treated with AdipoRon (5µM). AdipoRon treatment activates AMPK phosphorylation (p-AMPK) in murine BM-derived osteoclasts g. Ratio of P-AMPK over total AMPK measured by densitometry h. Kinetic of AMPK phosphorylation (P-AMPK) after AdipoRon challenge in RAW264.7-derived osteoclasts. Data are presented as mean ± S.E.M. Statistical significance was determined by two-tailed unpaired t-test.

Material & Methods

Key resources table

Mouse models

Genotype denomination follows the rules recommended by the Mouse Genome Database (MGD) Nomenclature Committee. Animal procedures were authorized by the Canton of Vaud veterinary service. Sox2CRE transgenic mice (SOX2CRE tg/+ ; Tg(Sox2-cre)1Amc/J) and AdipoQCRE (Adipoq-Cre tg/+ ) from The Jackson Laboratory, were maintained in the University of Lausanne Animal Facility. The series of matings allowing the generation of the SOX2CRE tg/+ Pparg emΔ/Δ mouse (hereafter called Pparg Δ/Δ ), which expressed the SOX2CRE transgene and have no Pparg functional alleles, as well as their control littermates (CTL) that have no SOX2CRE transgene and two functional Pparg alleles (Pparg fl/+ ) have been described in [START_REF] Gilardi | Systemic PPARγ deletion in mice provokes lipoatrophy, organomegaly, severe type 2 diabetes and metabolic inflexibility[END_REF]. The resulting genetic background of these mice are a mix of C57Bl6 and SV129L. PPARg adipoKO and their control littermates were generated as described in [START_REF] Wang | Lipoatrophy and severe metabolic disturbance in mice with fat-specific deletion of PPAR[END_REF], in a mixed genetic background C57Bl6 and SV129L.

AZIP/F1 mice on FVB background (Tg(AZIP/F)1Vsn in MGD, herein called AZIP tg/+ ) and wild type FVB controls were a kind gift from Dr. Charles Vinson and were generated as previously described [START_REF] Moitra | Life without white fat: a transgenic mouse[END_REF].

Primary cell culture

Mouse bone marrow cells were isolated by crushing entire long bones into DMEM/3% FCS (GIBCO). Bone fragments were removed by filtration through a 70 µm filter mesh.

Splenocyte suspensions were obtained by mashing the spleen through sieves into DMEM/3% FCS, washing by centrifugation then filtering through 70 µm filter caps. Primary osteoblasts from neonatal calvaria of Pparg fl/+ and Pparg Δ/Δ were harvested by sequential collagenase type II (3 mg/mL) digestions of individual calvarium from 2 to 3-day-old mice. After genotyping, calvaria cells of the same genotype were pooled and plated accordingly. The cells were incubated at 37° C with 5% CO2 and media was changed every two days until they reached 80% confluency. To assess proliferation, primary osteoblasts were plated in 24-well plates at a concentration of 10,000 cells/well in EMEM media (GIBCO) containing 10% FBS, 100 U/ml penicillin and 100 ug/ml streptomycin (GIBCO).

Osteoclastogenesis Assays and TRAcP staining of murine and human primary cells

Erythrocytes were eliminated from murine and BM preparation or human PBMC using ACK lysis buffer. When indicated, osteoclasts were also derived from CD11b + monocytic cells (MN-OCLs) and bone marrow-derived CD11c + dendritic cells (DC-OCLs) as described previously (Ibanez et al., 2016). Cells were cultured in complete αMEM with 40 ng/ml M-CSF (ProSpec-Tany TechnoGene, Rehovat, Israel) and l0 ng/ml RANKL (R&D Systems, MN). Tartrate-resistant acid phosphatase (TRAcP) staining was performed after 4-6 days, using the Acid Phosphatase, Leukocyte (TRAP) Kit according to the manufacturer's instructions (Sigma). Mature osteoclasts were identified as multi-nucleate TRAcP + cells using light microscopy. 3T3-L1 cells (ATCC) were grown to sub-confluence in DMEM/10% FBS.

Cells were then cultured in adipogenic conditions (DMEM/10%FBS, 0.5 mM 3-isobutyl-1methylxanthine, 1 µM dexamethasone (Sigma), and 10 µg/ml bovine insulin (Sigma)) for 2 weeks, after which the medium was changed back to DMEM/10% FBS. Adipocyte Conditioned Medium (A-CM) was collected after 24 hours exposure to differentiated cells.

Similarly, conditioned control medium (CTL) was obtained from undifferentiated 3T3-L1 cells. A-CM and CTL were concentrated 5X using Centriprep TM (Millipore). Conditioned medium from either differentiated or undifferentiated 3T3-L1 cells was added during osteoclastogenesis from multipotent progenitor cells. For neutralisation experiments, 50%

CM was incubated with different amounts of anti-adiponectin antibody (Enzo Life Sciences)

for 30 min at room temperature then added during osteoclastogenesis. TRAcP staining to evaluate osteoclast numbers was performed as mentioned above.

Osteoclast differentiation and TRAcP staining of RAW 264.7 cells

For osteoclastogenesis, a total of 1x10 4 RAW 264.7 (ATCC) cells were seeded per well on 24-well plates in 500 µl αMEM containing 5% FCS (Hyclone, GE Healthcare), 1% penicillinstreptomycin (Gibco) as well as 50 μM β-mercaptoethanol (Gibco) and 30 ng/ml murine RANKL (R&D Systems). To study the impact of adiporon on osteoclast differentiation, 10 µM AdipoRon (Sigma-Aldrich) were added to the culture medium. Tartrate-resistant acid phosphatase (TRAcP) activity was examined using the leucocyte acid phosphatase kit (Sigma-Aldrich) according to manufacturer's specifications. TRAcP + cells with ≥3 nuclei were considered as osteoclasts. In addition, 5x10 3 RAW 264.7 cells were seeded per well in a Nunc Lab-Tek II 8-well Chamber Slide system (ThermoFisher Scientific) in a total of 300 µl osteoclast differentiation medium containing 30 ng/ml RANKL. Fully differentiated osteoclasts were treated with 10 µM AdipoRon for the indicated time points. FACS analysis as well as sorting of mature OCLs for subsequent Western-blot analyses was performed as described previously [START_REF] Madel | A Novel Reliable and Efficient Procedure for Purification of Mature Osteoclasts Allowing Functional Assays in Mouse Cells[END_REF].

Measurement of bone morphology and microarchitecture

Unless otherwise indicated, one-year old females of each genotype were used for bone analyses.

High-resolution micro-computed tomography (UCT40, ScancoMedical AG, Bassdorf, Switzerland) was used to scan the femur and the 5 th lumber vertebral body. Data were calibrated against a phantom, which has a known hydroxyapatite value.For the vertebral trabecular region, we evaluated ∼300 transverse CT slices between the cranial and caudal end plates, excluding 100 μm near each endplate. Femoral cortical geometry was assessed in a 1mm-long region centered at the femoral midshaft. CT images were reconstructed in 1024 × 1024 pixel matrices using a standard convolution-backprojection procedure, and the resulting gray-scale images were segmented using a constrained 3D Gaussian filter (σ = 0.8, support = 1.0) to remove noise, and a fixed threshold (22% and 30% of maximal gray scale value for trabecular bone and cortical bone, respectively) was used to extract the structure of mineralized tissue.

Cortical and trabecular bones were evaluated using isotropic 12µm voxels. For trabecular bones, we assessed bone volume fraction (BV/TV, %), trabecular thickness (Tb.Th, μm), trabecular number (Tb.N, mm -1 ), and trabecular separation (Tb.Sp, μm). For cortical midshaft femur, we measured the average total volume (TV, mm 3 ), bone volume (BV, mm 3 ) and average cortical thickness (μm). SkyScan software (CTAn and CTVol) was employed to calculate cortical bone porosity of femur using a dedicate method after acquisition using a Skyscan 1272 (SkyScan 1272, Bruker, Brussels, Belgium) at a resolution of 10 μm/pix, and a threshold set up at 100-255, finally visualized in 3D.

Serum protein assays

Blood was obtained by cardiac puncture immediately after euthanasia. After clotting and centrifugation, serum was collected and stored at -80°C until use. Serum CTX (carboxyterminal collagen crosslinks), osteocalcin (OCN), alkaline phosphatase (ALP) and parathyroid hormone (PTH) were measured by ELISA assays according to the manufacturer's protocol (LifeSpan Biosciences, Inc., Seattle, WA).

Calvaria injection

Nine 6-week old C57/BL6 male mice were purchased from Envigo and housed in the local animal facility under a 12h light/12h dark cycle. The animals were randomly divided into three experimental groups, each group comprising three mice. Mice received daily s.c.

injections on the calvaria before analysis on the third day. Group 1 was injected with 100 µg lipopolysaccharide from Salmonella abortus equi (LPS, Sigma-Aldrich), group 2 with 100 µg LPS + 500 ng AdipoRon (Sigma-Aldrich) and experimental group 3 underwent vehicle (PBS) injection. Calvariae were collected and fixed overnight in 4% formaldehyde at 4°C and then stored in 70% EtOH. Subsequent micro-CT analysis were used to evaluate the resorbed area.

Calvariae were decalcified in 10% EDTA, embedded in paraffin and 5-micron sections were cut and stained for tartrate-resistant acid phosphatase (TRAcP, Sigma-Aldrich).

Quantitative RT-PCR

Total RNA was isolated from total long bones, including the crude BM, or primary cell cultures, using TRIzol LS (Invitrogen) and purified with the RNeasy kit (Qiagen). RNA quality was verified by microfluidic (Agilent 2100 Bioanalyzer) and concentration determined with a NanoDrop spectrophotometer (Wilmington). Total RNA (500 ng/µg) was reverse transcribed using Script TM cDNA synthesis kit (Bio-Rad) according to the manufacturer's instructions. Real time qPCR was performed with SYBR® Green PCR mastermix using an ABI PRISM® 7900 PCR machine (Applied Biosystems). Results were normalized to glyceraldehyde-3-phosphate dehydrogenase (Gapdh) or beta-actin beta (Actb). The primer sets are indicated in the supplementary table 1.

Western blot analysis

Mature OCLs were sorted based on their nuclei number (≥ 3) following the protocol described in Madel et al. [START_REF] Madel | A Novel Reliable and Efficient Procedure for Purification of Mature Osteoclasts Allowing Functional Assays in Mouse Cells[END_REF]. This protocol ensures that further analyses (FACS and western-blot analysis) are made with a pure mature cell population, avoiding contaminant effects due to the important number of non-osteoclastic cells always present in classical culture, as reported [START_REF] Madel | A Novel Reliable and Efficient Procedure for Purification of Mature Osteoclasts Allowing Functional Assays in Mouse Cells[END_REF]. The sorted mature OCLs were washed twice with ice-cold PBS and scraped in 1X Laemmli buffer. Cells were disrupted by sonication and centrifuged at 3000 rpm for 10 min. Protein samples were analyzed by SDS/PAGE (gradient 4-15% acrylamide, Criterion, BioRad) and electroblotted onto 0.2% PVDF membrane using Transblot Turbo (BioRad). After 1 h in blocking buffer (Tris-buffered saline (TBS)-Tween 20 with 5% fat free dry milk), membranes were blotted overnight at 4°C with Rabbit mAb against AMPKα (D63G4) (dilution 1:1 000, Cell Signaling Technology) or Phospho-AMPKα (Thr172) (40H9) (dilution 1 :1000, Cell Signaling Technology), diluted in TBS-Tween with 5% BSA. After three washing steps with TBS-Tween 20, blot was incubated for 1 h at room temperature with anti-rabbit IgG conjugated with horseradish peroxidase diluted 1:2000 in blocking buffer. After four additional washing steps with TBS-Tween 20, protein bands were detected by chemiluminescence using Chemi Doc XRS+ (BioRad).

Imaging

Podosomes were visualized by phalloidin staining (Sigma-Aldrich) following manufacturer's instructions. After removing unbound phalloidin conjugates, cells were labelled with 1 µg/ml DAPI (Sigma-Aldrich). Microscopic imaging was performed using a fluorescence microscope (Zeiss Axio Observer D1). For mitochondrial analysis, live cells were labeled with 200 nM MitoTracker Orange (ThermoFisher) according to manufacturer's recommendations. After staining, cells were fixed in 4% formaldehyde, labelled with 1 µg/ml DAPI and subjected to z-stack confocal microscopy analyses (Zeiss LSM 710 on an inverted Axio Observer.Z1 stand). Images were processed using Fiji/Image J software [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF].

Statistical analysis

The data were analyzed using one-or two-factor ANOVA for multiple comparisons as appropriate. Two-group comparisons were performed using the Student's t-test. ns: nonsignificant, * p<0.05, ** p<0.01, *** p< 0.001, **** p<0.0001.

Figure 2 :

 2 Figure2: Lipoatrophy is responsible for unregulated osteoclastogenesis a. Representative 3D sections of midshaft femur from one year-old female A-ZIP tg/+ and CTL littermates (left panels) and femoral cortical porosity measured by micro-CT analysis of femur from A-ZIP tg/+ and control (CTL) littermates (right panel). b. Representative images of TRAcP staining of longitudinal sections of decalcified femurs, at mid-shaft levels, from control (CTL) A-ZIP tg/+ (left panels; cortical bone; BM: bone marrow). The right panel is a quantification of TRAcP positive osteoclasts. c. RT-qPCR analysis of Acid Phosphatase 5-Tartrate Resistant (Acp5) Cathepsin K (Ctsk) and Matrix Metallopeptidase 9 (Mmp9) expression in femur from A-ZIP tg/+ mice and their control littermates. d. Representative 3D sections of midshaft femur from one year-old females PPARγ adipoKO and CTL littermates (left panels) and femoral cortical porosity measured by micro-CT analysis of femur from PPARγ adipoKO and control (CTL) littermates (right panel). e. RT-qPCR analysis of Acid Phosphatase 5-Tartrate Resistant (Acp5) Cathepsin K (Ctsk) and Matrix Metallopeptidase 9 (Mmp9) expression in femur from PPARγ adipoKO mice and their control littermates. Each dot represents an individual animal. Statistical significance was determined by two-tailed unpaired t-test.

Figure 3 :

 3 Figure3: Adipocyte conditioned medium inhibits osteoclastogenesis a. Experimental design of the in vitro assay used to test anti-osteoclastogenic activity from mature adipocyte secreted factors. b. One day after plating, wild type BM cells were cultured for 6 more days in the presence of M-CSF and RANKL (osteoclast differentiation media) in addition to conditioned medium prepared from differentiated (ACM: adipocyte-conditioned medium) or undifferentiated (CTL: control-conditioned medium) 3T3-L1 preadipocytes. Different doses of a specific blocking antibody against adiponectin (1.5-12.0 µg/ml) were added to the conditioned medium (ACM50) during osteoclast differentiation (ACM 100 means pure ACM; ACM50 means 1:1 diluted ACM with osteoclast differentiation medium). Data are presented as mean ± SEM of biological triplicates.. Statistical significance was determined by two-tailed unpaired t-test. c. Calvaria resorption pit assay. Representative sections of skulls from 14-day-old wild-type pups cultured for 2 weeks in complete aMEM in

Figure 4 :

 4 Figure 4: Adiponectin signalling activation blocks osteoclast differentiation a. Representative images of TRAcP stained BM-derived osteoclasts differentiated in the presence or absence of AdipoRon (5µM). Scale bars=100µm. b. Dose response effect of AdipoRon on osteoclast differentiation using RAW264.7 cells (n=3). c. Effect of AdipoRon on osteoclast differentiation assay using bone marrow cells from Pparg Δ/Δ (γ Δ/Δ , grey bars) mice and control littermates (CTL, black bars). d. Expression of Type 1 Adiponectin Receptor (AdipoR1) at the surface of BM-derived osteoclasts demonstrated by flow cytometry. Grey curve represents unstained negative control; blue curve represents BM-derived osteoclasts. e.RT-qPCR analysis of Adiponectin Receptor 1 and 2 (Adipor1 and Adipor2) on osteoclasts differentiated from bone marrow (BM) cells (BM-OCL), from BM-derived dendritic cells (DC-OCL) and BM monocytic cells (MN-OCLs). f. Western-blot analysis of sorted mature bone marrow-derived osteoclasts treated with AdipoRon (5µM). AdipoRon treatment activates AMPK phosphorylation (p-AMPK) in murine BM-derived osteoclasts g. Ratio of P-AMPK over total AMPK measured by densitometry h. Kinetic of AMPK phosphorylation (P-AMPK) after AdipoRon challenge in RAW264.7-derived osteoclasts. Data are presented as mean ± S.E.M. Statistical significance was determined by two-tailed unpaired t-test.

Figure 5 :

 5 Figure 5: Adiponectin regulates OCL activity and podosome formation a. Confocal microscopy of phalloidin-marked podosomes in RAW264.7-OCL in the presence of increasing concentrations of AdipoRon (representative images of n=3 per concentration) Scale bars=100 µm. b. Confocal microscopy of phalloidin-marked podosomes in the presence of AMPK modulating drugs (AdipoRon, 10µM; AICAR, 50nM; Dorsomorphin, 1.2µM) (representative image of n=3 per conditions white asterisks marking the podosomes). Scale bars=50 µm. Blue arrows indicate podosome rings. c. Confocal microscopy of MitoTracker Orange in mature RAW264.7-OCL after AdipoRon (10µM) challenge at different time points. Scale bars=20 µm. d. Micro-CT of mouse skulls collected 3 days after mice received daily s.c. injections on the calvaria. Mice were injected with either 100 µg lipopolysaccharide from Salmonella abortus equi (LPS), or 100 µg LPS + 500 ng AdipoRon (LPS + AdipoRon) or vehicle (PBS) injection (control, CTL). (n=3 animals per group) e.TRAcP staining of mouse skull after LPS +/-AdipoRon challenge, (representative sections from 3 animals per group). Scale bars=100 µm.

Table 1 A-Micro CT analyses of 1 year-old female mice

 1

	1 year	Pparg fl/+	Pparg Δ/Δ
	No. of animals assessed	15	14
	Cortical TV (mm 3 )	1.498 ± 0.023	1.327 ± 0.031*
	Cortical BV (mm 3 )	0.723 ± 0.012	0.667 ± 0.028*
	Cortical thickness (µm)	241.8 ± 4.5	208.0 ± 10.4*
	Dist Femoral BV/TV (%)	3.41 ± 0.49	14.38 ± 2.26*
	Dist Femoral conn density (mm 3 )	9.18 ± 2.57	81.89 ± 13.62*
	Dist Femoral Tb N (/mm)	2.39 ± 0.09	3.61 ± 0.22*
	Dist Femoral Tb Th (µm)	54.37 ± 2.48	57.28 ± 3.52*
	Dist Femoral Tb Sp (µm)	431.34 ± 18.4	291.10 ± 9.70*
	Vertebral BV/TV (%)	17.33 ± 1.38	29.57 ± 3.30*
	Vertebral conn density (mm 3 )	127.02 ± 14.89	519.44 ± 73.33*
	Vertebral Tb N (/mm)	3.42 ± 0.17	6.29 ± 0.53*
	Vertebral Tb Th (µm)	51.43 ± 1.88	47.04 ± 2.30
	Vertebral Tb Sp (µm)	311.13 ± 16.23	177.30 ± 15.73*
	B-

Complete bone analysis by Micro CT of 5/6 month-old female mice

		Pparg fl/+	Pparg Δ/Δ
	5-6 months
	No. of animals assessed	8	10
	Cortical TV (mm 3 )	1.18 ± 0.05	1.18 ± 0.04
	Cortical BV (mm 3 )	0.0541 ± 0.023	0.0548 ± 0.014
	Cortical thickness (µm)	203.75 ± 4.80	202.80 ± 5.33
	Dist Femoral BV/TV (%)	7.78 ± 1.17	8.55 ± 1.07
	Dist Femoral conn density (mm 3 )	38.90 ± 8.70	84.30 ± 14.85*
	Dist Femoral Tb N (/mm)	3.13 ± 0.13	3.75 ± 0.22*
	Dist Femoral Tb Th (µm)	53.01 ± 1.62	45.93 ± 2.24*
	Dist Femoral Tb Sp (µm)	327.46 ± 15.03	283.59 ± 21.33
	Vertebral BV/TV (%)	15.49 ± 1.31	21.44 ± 1.79*
	Vertebral conn density (mm 3 )	112.73 ± 12.56	275.53 ± 33.10*
	Vertebral Tb N (/mm)	3.71 ± 0.13	4.76 ± 0.17*
	Vertebral Tb Th (µm)	49.03 ± 1.17	46.39 ± 1.14
	Vertebral Tb Sp (µm)	278.37 ± 10.66	215.1 ± 8.32*

Table 1 .

 1 A) Micro-CT data were obtained from various bones in Pparg fl/+ and Pparg Δ/Δ of 1 year-old female mice. B) Similar evaluation of bones from 5/6 month-old female mice. Values are given as mean ± standard deviation. TV, total volume; BV, bone volume; Tb N, trabecular number; Tb Th, trabecular thickness; Tb Sp, trabecular separation.

* : p <0.05 vs Pparg fl/+ .

Figures legends Figure 1: Epiblastic PPARγ-deletion induces trabecular and cortical bone alteration a.

  Micro-CT analysis of femur from Pparg Δ/Δ (γ Δ/Δ ) one-year old female mice or control female littermates (CTL). Dot plots on the right show for the different genotypes (empty square for CTL and black triangle for γ Δ/Δ ) the percentage bone volume/trabecular volume (BV/TV) in % and connectivity density (ConnDens) of distal femoral trabecular bone (upper panels), and the cortical bone volume (BV) in mm 3 and the percentage of cortical porosity of femur (lower panels). All mice were 1-year-old. b. analysis of Alkaline phosphatase (AlpI) gene expression in femur from CTL and γ Δ/Δ . c. ELISA measurements of serum Alkaline phosphatase (ALP) and osteocalcin (OCN). d. RT-qPCR analysis of Acid Phosphatase 5-Tartrate Resistant (Acp5), Cathepsin K (Ctsk) and Matrix Metallopeptidase 9 (Mmp9) expression in femur from CTL and γ Δ/Δ . e. ELISA measurements of serum Carboxyterminal Collagen I telopeptide (CTX);. f. Representative images of TRAcP staining of decalcified sections of femurs, at mid-shaft levels, from control (CTL) or Pparg Δ/Δ (γ Δ/Δ ) mice.

CB: cortical bone; BM: bone marrow. Data are presented as mean ± S.E.M. Each dot represents an individual animal. Statistical significance was determined by two-tailed unpaired t-test.

Acknowledgements

This work was supported by the Etat de Vaud (BD and AW) the FNRS (BD), the Région Grand Est (J-YJ), the Fondation Arthritis (DM), the french PIA project Lorraine Université d'Excellence, reference ANR-15-IDEX-04-LUE (J-YJ and DM) and the Fondation pour la Recherche Médicale (FRM, ECO20160736019) (MBM). The authors would like to acknowledge the Montpellier preclinical platform of ECELLFRANCE for the µCT analysis (IRMB, Montpellier, France) and the microCT platform SC3M from the RMeS Lab, INSERM UMRS 1229 ( Nantes, France).

Supplementary data

Video 1: Video of 3D micro-CT reconstruction of genetically induced lipoatrophic mice showing alterations of both trabecular and cortical femoral bone.

Supplementary Table 1: Primers used for q-RT-PCR.

Supplementary Figure S2: Parathyroid hormone (PTH) signalling

The serum levels of PTH were assessed by Elisa Assay in 1-year-old male and female mice (Pparg Δ/Δ , AZIP tg/+ , and their control littermates). Six males and six females were included per genotype. As there were no statistically significant differences between males and females, the analysis was performed using the total number of mice (mean ± SD). Lower panels: mRNA expression levels of Pth1r in the bones and in the kidneys of CTL vs Pparg Δ/Δ mice.

Supplementary Figure S3: Adiponectin expression levels in long bone and in adipocyteconditioned medium

RT-qPCR analysis of Leptin and AdipoQ mRNA levels in long bone from A-ZIP tg/+ (n=8) and their CTL littermates (n=8) and from γ Δ/Δ (n=7) and their CTL littermates (n=6). Data are presented as mean ± S.E.M. Statistical significance was determined by two-tailed unpaired ttest.

Supplementary Figure S4: AdipoRon inhibits osteoclast (OCL) differentiation of various origins.

Left panels: representative images of TRAcP stained (a.) BM-derived, (b.) spleen derived and (c.) macrophages enriched fraction osteoclasts differentiated in the presence or absence of AdipoRon (5µM). Right panels: the mean IC50 of AdipoRon inhibition of OCL differentiation was determined from the curve with the error of the fit (S.E.M.) Data are mean ± S.E.M. (n = 3 biological replicates).

Supplementary

Authors contribution

HF designed the experimental plan, performed the experiments, analyzed the data, and contribute to the original draft preparation. MBM, DDP, MS, CW, AW, CP, MT and BT performed experiments, analyzed the data, and prepared the vizualisation. JYJ, FG, SF, NB and CBW contributed to the conceptualization, analyzed the data, and reviewed/edited the manuscript. BD and DM conceived the study, designed the experimental plan, supervised the study, analyzed the data, wrote the manuscript, and acquired financial support for the project.

All authors read and edited the manuscript. BD and DM are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analyses.


A Meffray

Y Ardagna

B Sillano

S Parmentier

B Pouget

M Signoli

P Biagini

Variola virus DNA in skeletal remains, 17 th -18 th centuries, southeastern France

J o u r n a l P r e -p r o o f

To the Editor, Smallpox is one of the most devastating diseases known to mankind. Its deadly hold, now vanished from the face of the earth thanks to a large-scale eradication campaign launched by the World Health Organization (WHO) in 1967, has since become a subject of medical, epidemiological and historical interest for the scientific community [START_REF] Hopkins | The greatest killer: smallpox in history[END_REF]. Ancient biological remains have been investigated for variola virus (VARV) DNA, broadening our knowledge on its evolutionary history in medieval / modern Northern Europe and modern Siberia [START_REF] Biagini | Variola virus in a 300-year-old Siberian mummy[END_REF][START_REF] Duggan | 17 th century variola virus reveals the recent history of smallpox[END_REF][START_REF] Muhlemann | Diverse variola virus (smallpox) strains were widespread in northern Europe in the Viking Age[END_REF]; however, we are still unable to estimate efficiently its presence in ancient urban or rural populations from Western Europe.

Our study, stemming from previous palaeomicrobiological investigations performed on contemporary foetus and infants possibly exposed to treponemal disease [START_REF] Meffray | Molecular detection of Treponema pallidum subspecies pallidum in 150-year-old foetal remains, southeastern France[END_REF], aimed to analyse 26 subjects excavated from the chapel of "Saint-Laurent de la Capelette" in Marseille city, southeastern France (see Supplementary material, Historical and archaeological context and Table S1). We carried out macroscopic and molecular analysis to evaluate the prevalence of smallpox infection among this osteoarchaeological corpus dating back to the mid-17 th -early-18 th centuries.

Bone samples (small cranial and costal elements) were collected and fully documented before proceeding to molecular investigations. VARV DNA presence was investigated using several PCR detection systems (<150 bp) targeting A38R, B7R, A30L, and E9L genomic regions; a 430 bp PCR amplification (E9L) to analyze the fragmented nature of the viral genome was also performed (see Supplementary material, Methods and Table S2). Extensive precautions were taken at each step of the study to meet with the 'authenticity criteria' expected for ancient DNA studies [START_REF] Meffray | Molecular detection of Treponema pallidum subspecies pallidum in 150-year-old foetal remains, southeastern France[END_REF]. No studies on modern smallpox were ever conducted in our facilities, and positive Sequences analysis confirmed the VARV origin of amplified material, and revealed strict identities with partial sequences obtained from Siberian [START_REF] Biagini | Variola virus in a 300-year-old Siberian mummy[END_REF] and Lithuanian [START_REF] Duggan | 17 th century variola virus reveals the recent history of smallpox[END_REF] mummies dating from similar periods (17 th -18 th centuries; PoxSib and VD21 isolates, respectively) (see Supplementary material, Figure S1). Phylogenetic and temporal analysis of concatenated sequences confirmed the divergent position of these 300-year-old VARV sequences, distinct from 20 th century P-I and P-II clades [START_REF] Biagini | Variola virus in a 300-year-old Siberian mummy[END_REF][START_REF] Duggan | 17 th century variola virus reveals the recent history of smallpox[END_REF] (Figure 1, and Supplementary material, Figure S2).

Our findings are consistent with smallpox being a Eurasian pandemic at this time, spreading through the Ancient Continent via trade, migratory, exploratory and invasion routes [START_REF] Hopkins | The greatest killer: smallpox in history[END_REF][START_REF] Biagini | Variola virus in a 300-year-old Siberian mummy[END_REF][START_REF] Duggan | 17 th century variola virus reveals the recent history of smallpox[END_REF][START_REF] Muhlemann | Diverse variola virus (smallpox) strains were widespread in northern Europe in the Viking Age[END_REF]. It echoes previous results, to the extent that the French SP2108 strain could either be an ancestor of the 20 th century VARV strains, or a member of an ancient lineage with no connections, however, to the recent times outbreaks strains [START_REF] Biagini | Variola virus in a 300-year-old Siberian mummy[END_REF].

Importantly, above results entail also the possibility of bone samples from archaeological context as a substrate for VARV molecular detection, the bone marrow being a known site of replication for the virus, thus freeing palaeomicrobiologists from the necessity of using mummified and/or frozen ancient remains to investigate this pathogen. Of note, the skeleton of this young child displayed no signs of pathological conditions such as periosteal remodelling or osteomyelitis variolosa (see Supplementary material, Figure S3).

J o u r n a l P r e -p r o o f

In the present case, there is no reason to assume that this specimen was contaminated by any other than the natural ways, as opposed to voluntary transmission by inoculation, since the advent of this practice in modest populations did not occur in southeastern France until the second half of the 18 th century [START_REF] Hopkins | The greatest killer: smallpox in history[END_REF][START_REF] Darmon | La longue traque de la variole: les pionniers de la médecine préventive[END_REF]. On the same note, the first immunization session in Marseille took place only on March 27, 1801 at the "Hospice de la Charité", involving cowpox virus [START_REF] Darmon | La longue traque de la variole: les pionniers de la médecine préventive[END_REF]. In the deteriorated sanitary context of the 17 th and 18 th centuries, smallpox was a classic companion of Western populations, whether they were poor or favoured. As an endemo-epidemic disease, it affected mainly children under 5 years of age and caused a high mortality among them [START_REF] Darmon | La longue traque de la variole: les pionniers de la médecine préventive[END_REF]. The parish archives of the "Saint-Laurent dite la Capelette" chapel show the recurrence of young children burials, especially in the early 18 th (see Supplementary material, Methods and Table S3), with the occasional occurrence of a greater number of losses among adults, that could result from more violent epidemic outbreaks. As pointed out by P. Darmon, contrary to plague, "smallpox, in a much more diffuse way, decimated consistently and piercingly by small touches, eliminating the generations at the base without really slashing into the mass" [START_REF] Darmon | La longue traque de la variole: les pionniers de la médecine préventive[END_REF]. Considered as a common and inevitable scourge, public health policies of the time struggled little against its omnipresence and did not classify it as part of quarantine diseases. The presence of smallpox throughout the year, with a mortality pattern that staggered over the four seasons, did not lead to the use of mass burials, which explains why this infected specimen was found in a normal burial context (parish church) [START_REF] Hopkins | The greatest killer: smallpox in history[END_REF][START_REF] Darmon | La longue traque de la variole: les pionniers de la médecine préventive[END_REF].

The uncovering of conserved, though fragmented, VARV DNA in 300-year-old bone samples of a child's skeleton identified in south France adds clues to the dispersion of smallpox in past populations; subsequent investigations are now expected.

J o u r n a l P r e -p r o o f

  r n a l P r e -p r o o f Letter to the Editor Variola virus DNA in skeletal remains, 17 th -18 th centuries, southeastern France A. Meffray 1 , Y. Ardagna 1 , B. Sillano 2 , S. Parmentier 2 , B. Pouget 1 , M. Signoli 1 , P. Biagini

  J o u r n a l P r e -p r o o f controls were never used; negative controls (sterile water) were included systematically in extraction and DNA amplification procedures.Remains of one individual only (SP2108, child 1-4 year-old at the time of death, fairly complete skeleton except for the skull; costal fragment tested) yielded positive PCR signals;the four detection systems used gave amplicons of expected size on agarose gel electrophoresis, whereas no signal was observed for the longer amplification assay highlighting an extensive degradation of the viral genome.

Figure 1 .

 1 Figure 1. Neighbor-joining (NJ) phylogenetic trees generated using 20 th and 17 th -18 th

Acknowledgments

The authors are grateful to ML. Deligny for excellent technical assistance.

J o u r n a l P r e -p r o o f

Funding information

The work was supported by the UMR 7268 ADES, the French Blood Agency PACA Corse and the French Ministry of Research (no specific grant).

Author contributions

All authors have made substantial contributions to the work. Study concept and design: AM, YA, PB. Data acquisition: AM, YA, BS, SP, BP, MS. Analysis and/or interpretation of data: AM, YA, PB. Drafting the manuscript: AM, PB.

Conflict of interest

The authors declare that there are no conflicts of interest.