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Bone remodeling is a tightly controlled mechanism in which osteoblasts (OB), the cells responsible for bone formation, osteoclasts (OC), the cells specialized for bone resorption, and osteocytes, the multifunctional mechanosensing cells embedded in the bone matrix, are the main actors. Increased oxidative stress in OB, the cells producing and mineralizing bone matrix, has been associated with osteoporosis development but the role of autophagy in OB has not yet been addressed. This is the goal of the present study. We first show that the autophagic process is induced in OB during mineralization. Then, using knockdown of autophagy-essential genes and OB-specific autophagy-deficient mice, we demonstrate that autophagy deficiency reduces mineralization capacity. Moreover, our data suggest that autophagic vacuoles could be used as vehicles in OB to secrete apatite crystals. In addition, autophagy-deficient OB exhibit increased oxidative stress and secretion of the receptor activator of NFKB1 (TNFSF11/RANKL), favoring generation of OC, the cells specialized in bone resorption. In vivo, we observed a 50% reduction in trabecular bone mass in OB-specific autophagy-deficient mice. Taken together, our results show for the first time that autophagy in OB is involved both in the mineralization process and in bone homeostasis. These findings are of importance for mineralized tissues which extend from corals to vertebrates and uncover new therapeutic targets for calcified tissue-related metabolic pathologies.

Osteoclasts are the cells responsible for physiological bone resorption. A specific organization of their most prominent cytoskeletal structures, podosomes, is crucial for the degradation of mineralized bone matrix. Each podosome is constituted of an F-actin-enriched central core surrounded by a loose F-actin network, called the podosome cloud. In addition to intrinsic actin dynamics, podosomes are defined by their adhesion to the extracellular matrix, mainly via core-linking CD44 and cloud-linking integrins. These properties allow podosomes to collectively evolve into different patterns implicated in migration and bone resorption. Indeed, to resorb bone, osteoclasts polarize, actively secrete protons, and proteases into the resorption pit where these molecules are confined by a podosome-containing sealing zone. Here, we review recent advancements on podosome structure and regulatory pathways in osteoclasts. We also discuss the distinct functions of different podosome patterns during the lifespan of a single osteoclast.

Two-step transcriptomic profiling of bone-resorbing OCs versus nonresorbing MGCs generated a list of 115 genes potentially involved in bone resorption. Of these, RhoE was investigated. Its role in podosome dynamics is central for OC migration, SZ formation, and, ultimately, bone resorption.

Podosomes are dynamic, actin-containing adhesion structures that self-organize as rings in osteoclasts. We observe that podosome rings generate tension forces and that osteoclasts migrate by periodically moving toward the actin-containing structures. Actin dynamics thus not only correlate with the cell migration, but drive it.

Podosomes are dynamic, actin-containing adhesion structures that collectively self-organize as rings. In this study, we first show by observing osteoclasts plated on bead-seeded soft substrates that podosome assemblies, such as rings, are involved in tension forces. During the expansion of a podosome ring, substrate displacement is oriented outward, suggesting that podosomal structures push the substrate away. To further elucidate the function of forces generated by podosomes, we analyze osteoclast migration. Determining the centers of mass of the whole cell (G) and of actin (P), we demonstrate that osteoclasts migrate by "jumps" and that the trajectories of G and P are strongly correlated. The velocity of the center of mass as a function of time reveals that osteoclasts rapidly catch up with podosomal structures in a periodic pattern. We conclude that actin dynamics inside the cell are not only correlated with cell migration, but drive it.

Survival and capability of cancer cells to form metastases fundamentally depend on interactions with their microenvironment. Secondary tumors originating from prostate carcinomas affect remodeling of bone tissue and can induce both osteolytic and osteocondensing lesions. However, particular molecular mechanisms responsible for selective homing and activity of cancer cells in bone microenvironment have not been clarified yet. Growth/differentiation factor-15 (GDF-15), a distant member of the TGF-β protein family, has recently been associated with many human cancers, including prostate.

L’interface entre le système immunitaire et la physiologie osseuse a conduit à l’émergence de l’ostéo-immunologie, nouveau champ de recherche qui ouvre de nouvelles perspectives dans la compréhension et le traitement des pathologies inflammatoires ostéo-articulaires. De nombreuses molécules et voies de signalisation, décrites initialement dans les cellules du système immunitaire, agissent également dans les ostéoclastes, cellules ayant pour rôle le maintien de l’homéostasie osseuse en assurant le remodelage du tissu osseux. Ainsi, la différenciation ostéoclastique requiert deux voies de signalisation « immunologiques » : la voie RANK/RANKL (receptor activator of NF-κB ligand) et celle des protéines adaptatrices à domaine ITAM (immunoreceptor tyrosine based activation motif), DAP12 (DNAX-activating protein) et FcRγ (récepteur du fragment Fc des immunoglobulines). Mais la façon dont les deux voies s’associent pour induire le facteur de transcription clé de l’ostéoclastogenèse NFATc1 (nuclear factor of activated T-cells), restait à élucider. Une étude réalisée par le groupe de H. Takayanagi vient de permettre d’identifier les protéines tyrosine kinases Bruton et Tec, qui forment un complexe protéique capable d’intégrer ces deux voies de signalisation et, ainsi, permettent l’expression des gènes nécessaires à la différenciation ostéoclastique.

Finding that activated T cells control osteoclast (OCL) differentiation has revealed the importance of the interactions between immune and bone cells. Dendritic cells (DCs) are responsible for T-cell activation and share common precursors with OCLs. Here we show that DCs participate in bone resorption more directly than simply through T-cell activation. We show that, among the splenic DC subsets, the conventional DCs have the higher osteoclastogenic potential in vitro. We demonstrate that conventional DCs differentiate into functional OCLs in vivo when injected into osteopetrotic oc/oc mice defective in OCL resorptive function. Moreover, this differentiation involves the presence of activated CD4+ T cells controlling a high RANK-L expression by bone marrow stromal cells. Our results open new insights in the differentiation of OCLs and DCs and offer new basis for analyzing the relations between bone and immune systems.

Finding that activated T cells control osteoclast (OCL) differentiation has revealed the importance of the interactions between immune and bone cells. Dendritic cells (DCs) are responsible for T-cell activation and share common precursors with OCLs. Here we show that DCs participate in bone resorption more directly than simply through T-cell activation. We show that, among the splenic DC subsets, the conventional DCs have the higher osteoclastogenic potential in vitro. We demonstrate that conventional DCs differentiate into functional OCLs in vivo when injected into osteopetrotic oc/ocmice defective in OCL resorptive function. Moreover, this differentiation involves the presence of activated CD4+T cells controlling a high RANK-L expression by bone marrow stromal cells. Our results open new insights in the differentiation of OCLs and DCs and offer new basis for analyzing the relations between bone and immune systems.

In Rous sarcoma virus (RSV)-transformed baby hamster kidney (BHK) cells, invadopodia can self-organize into rings and belts, similarly to podosome distribution during osteoclast differentiation. The composition of individual invadopodia is spatiotemporally regulated and depends on invadopodia localization along the ring section: the actin core assembly precedes the recruitment of surrounding integrins and integrin-linked proteins, whereas the loss of the actin core was a prerequisite to invadopodia disassembly. We have shown that invadopodia ring expansion is controlled by paxillin phosphorylations on tyrosine 31 and 118, which allows invadopodia disassembly. In BHK-RSV cells, ectopic expression of the paxillin mutant Y31F-Y118F induces a delay in invadopodia disassembly and impairs their self-organization. A similar mechanism is unraveled in osteoclasts by using paxillin knockdown. Lack of paxillin phosphorylation, calpain or extracellular signal-regulated kinase inhibition, resulted in similar phenotype, suggesting that these proteins belong to the same regulatory pathways. Indeed, we have shown that paxillin phosphorylation promotes Erk activation that in turn activates calpain. Finally, we observed that invadopodia/podosomes ring expansion is required for efficient extracellular matrix degradation both in BHK-RSV cells and primary osteoclasts, and for transmigration through a cell monolayer.

In Rous sarcoma virus (RSV)-transformed baby hamster kidney (BHK) cells, invadopodia can self-organize into rings and belts, similarly to podosome distribution during osteoclast differentiation. The composition of individual invadopodia is spatiotemporally regulated and depends on invadopodia localization along the ring section: the actin core assembly precedes the recruitment of surrounding integrins and integrin-linked proteins, whereas the loss of the actin core was a prerequisite to invadopodia disassembly. We have shown that invadopodia ring expansion is controlled by paxillin phosphorylations on tyrosine 31 and 118, which allows invadopodia disassembly. In BHK-RSV cells, ectopic expression of the paxillin mutant Y31F-Y118F induces a delay in invadopodia disassembly and impairs their self-organization. A similar mechanism is unraveled in osteoclasts by using paxillin knockdown. Lack of paxillin phosphorylation, calpain or extracellular signal-regulated kinase inhibition, resulted in similar phenotype, suggesting that these proteins belong to the same regulatory pathways. Indeed, we have shown that paxillin phosphorylation promotes Erk activation that in turn activates calpain. Finally, we observed that invadopodia/podosomes ring expansion is required for efficient extracellular matrix degradation both in BHK-RSV cells and primary osteoclasts, and for transmigration through a cell monolayer.

The actin cytoskeleton of mature osteoclasts (OCs) adhering to nonmineralized substrates is organized in a belt of podosomes reminiscent of the sealing zone (SZ) found in bone resorbing OCs. In this study, we demonstrate that the belt is composed of two functionally different actin-based domains: podosome cores linked with CD44, which are involved in cell adhesion, and a diffuse cloud associated with β3 integrin, which is involved in cell adhesion and contraction. Wiskott Aldrich Syndrome Protein (WASp) Interacting Protein (WIP)–/– OCs were devoid of podosomes, but they still exhibited actin clouds. Indeed, WIP–/– OCs show diminished expression of WASp, which is required for podosome formation. CD44 is a novel marker of OC podosome cores and the first nonintegrin receptor detected in these structures. The importance of CD44 is revealed by showing that its clustering restores podosome cores and WASp expression in WIP–/– OCs. However, although CD44 signals are sufficient to form a SZ, the presence of WIP is indispensable for the formation of a fully functional SZ.

The actin cytoskeleton of mature osteoclasts (OCs) adhering to nonmineralized substrates is organized in a belt of podosomes reminiscent of the sealing zone (SZ) found in bone resorbing OCs. In this study, we demonstrate that the belt is composed of two functionally different actin-based domains: podosome cores linked with CD44, which are involved in cell adhesion, and a diffuse cloud associated with β3 integrin, which is involved in cell adhesion and contraction. Wiskott Aldrich Syndrome Protein (WASp) Interacting Protein (WIP)−/− OCs were devoid of podosomes, but they still exhibited actin clouds. Indeed, WIP−/− OCs show diminished expression of WASp, which is required for podosome formation. CD44 is a novel marker of OC podosome cores and the first nonintegrin receptor detected in these structures. The importance of CD44 is revealed by showing that its clustering restores podosome cores and WASp expression in WIP−/− OCs. However, although CD44 signals are sufficient to form a SZ, the presence of WIP is indispensable for the formation of a fully functional SZ.

Human polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy, also known as Nasu-Hakola disease, has been described to be associated with mutations affecting the immunoreceptor tyrosine-based activation motif-bearing KARAP/DAP12 immunoreceptor gene. Patients present bone fragilities and severe neurological alterations leading to presenile dementia. Here we investigated whether the absence of KARAP/DAP12-mediated signals in loss-of-function (KΔ75) mice also leads to bone and central nervous system pathological features. Histological analysis of adult KΔ75 mice brains revealed a diffuse hypomyelination predominating in anterior brain regions. As this was not accompanied by oligodendrocyte degeneration or microglial cell activation it suggests a developmental defect of myelin formation. Interestingly, in postnatal KΔ75 mice, we observed a dramatic reduction in microglial cell numbers similar to in vitromicroglial cell differentiation impairment. Our results raise the intriguing possibility that defective microglial cell differentiation might be responsible for abnormal myelin development. Histomorphometry revealed that bone remodeling is also altered, because of a resorption defect, associated with a severe block of in vitroosteoclast differentiation. In addition, we show that, among monocytic lineages, KARAP/DAP12 specifically controls microglial and osteoclast differentiation. Our results confirm that KARAP/DAP12-mediated signals play an important role in the regulation of both brain and bone homeostasis. Yet, important differences exist between the symptoms observed in Nasu-Hakola patients and KΔ75 mice.

Dendritic cells (DCs), the mononuclear cells that initiate immune response, and osteoclasts, the multinucleated bone-resorbing cells, are derived from monocyte/macrophage precursor cells. Granulocyte-macrophage colony-stimulating factor and macrophage colony-stimulating factor (M-CSF) reciprocally regulate the differentiation of both lineages in mice. Using human monocyte-derived DCs generated in vitro, we show that immature DCs transdifferentiate into functional osteoclasts (OCs) in the presence of M-CSF and receptor activator of nuclear factor-κB ligand (RANKL). Transdifferentiation operates through fusion of intermediate adherent bipolar fusiform mononuclear cells expressing CD14, CD1a, and RANKL and able to induce RANKL+ T-cell proliferation. Surprisingly, DC fusion in vitro is faster and more efficient than monocyte fusion to form multinucleated giant cells. The transdifferentiation process reported here supports the existence of a high cellular plasticity within differentiated myeloid phagocytes. Importantly, this process is greatly enhanced by rheumatoid arthritis synovial fluid and involves proinflammatory cytokines such as interleukin 1 or tumor necrosis factor α, as well as components of the extracellular matrix such as hyaluronic acid. Our data therefore suggest that DC-derived OCs may be directly involved in the osteolytic lesions observed in human inflammatory bone diseases such as rheumatoid arthritis or in particular forms of Langerhans cell histiocytosis, characterized by accumulation of immature skin DCs and chronic lytic bone lesions. (Blood. 2004;104:4029-4037)

Dendritic cells (DCs), the mononuclear cells that initiate immune response, and osteoclasts, the multinucleated bone-resorbing cells, are derived from monocyte/macrophage precursor cells. Granulocyte-macrophage colony-stimulating factor and macrophage colony-stimulating factor (M-CSF) reciprocally regulate the differentiation of both lineages in mice. Using human monocyte-derived DCs generated in vitro, we show that immature DCs transdifferentiate into functional osteoclasts (OCs) in the presence of M-CSF and receptor activator of nuclear factor-κB ligand (RANKL). Transdifferentiation operates through fusion of intermediate adherent bipolar fusiform mononuclear cells expressing CD14, CD1a, and RANKL and able to induce RANKL+T-cell proliferation. Surprisingly, DC fusion in vitro is faster and more efficient than monocyte fusion to form multinucleated giant cells. The transdifferentiation process reported here supports the existence of a high cellular plasticity within differentiated myeloid phagocytes. Importantly, this process is greatly enhanced by rheumatoid arthritis synovial fluid and involves proinflammatory cytokines such as interleukin 1 or tumor necrosis factor α, as well as components of the extracellular matrix such as hyaluronic acid. Our data therefore suggest that DC-derived OCs may be directly involved in the osteolytic lesions observed in human inflammatory bone diseases such as rheumatoid arthritis or in particular forms of Langerhans cell histiocytosis, characterized by accumulation of immature skin DCs and chronic lytic bone lesions. (Blood. 2004;104:4029-4037)

The actin cytoskeleton is essential for osteoclasts main function, bone resorption. Two different organizations of actin have been described in osteoclasts, the podosomes belt corresponding to numerous F-actin columns arranged at the cell periphery, and the sealing zone defined as a unique large band of actin. To compare the role of these two different actin organizations, we imaged osteoclasts on various substrata: glass, dentin, and apatite. Using primary osteoclasts expressing GFP-actin, we found that podosome belts and sealing zones, both very dynamic actin structures, were present in mature osteoclasts; podosome belts were observed only in spread osteoclasts adhering onto glass, whereas sealing zone were seen in apico-basal polarized osteoclasts adherent on mineralized matrix. Dynamic observations of several resorption cycles of osteoclasts seeded on apatite revealed that 1) podosomes do not fuse together to form the sealing zone; 2) osteoclasts alternate successive stationary polarized resorption phases with a sealing zone and migration, nonresorption phases without any specific actin structure; and 3) apatite itself promotes sealing zone formation though c-src and Rho signaling. Finally, our work suggests that apatite-mediated sealing zone formation is dependent on both c-src and Rho whereas apico-basal polarization requires only Rho.

Podosomes, small actin-based adhesion structures, differ from focal adhesions in two aspects: their core structure and their ability to organize into large patterns in osteoclasts. To address the mechanisms underlying these features, we imaged live preosteoclasts expressing green fluorescent protein-actin during their differentiation. We observe that podosomes always form inside or close to podosome groups, which are surrounded by an actin cloud. Fluorescence recovery after photobleaching shows that actin turns over in individual podosomes in contrast to cortactin, suggesting a continuous actin polymerization in the podosome core. The observation of podosome assemblies during osteoclast differentiation reveals that they evolve from simple clusters into rings that expand by the continuous formation of new podosomes at their outer ridge and inhibition of podosome formation inside the rings. This self-organization of podosomes into dynamic rings is the mechanism that drives podosomes at the periphery of the cell in large circular patterns. We also show that an additional step of differentiation, requiring microtubule integrity, stabilizes the podosome circles at the cell periphery to form the characteristic podosome belt pattern of mature osteoclasts. These results therefore provide a mechanism for the patterning of podosomes in osteoclasts and reveal a turnover of actin inside the podosome.

We have shown that chick macrophages express RANK at their surface and human RANKL (hRANKL) triggers the formation of osteoclasts able to degrade dentine. As described for mammalian osteoclasts, hRANKL also stimulates the resorbing activity of chick bone-derived osteoclasts. In other hands, in culture, chick macrophages spontaneously form polykaryons sharing most of the osteoclast markers but unable to resorb bone. Since both bone-resorbing osteoclasts and macrophage polykaryons found in inflammatory tissues are multinucleated cells deriving from the fusion of macrophages, we examined whether macrophage polykaryons could be induced toward bone-resorbing osteoclasts. Long-term exposure of macrophage polykaryons to hRANKL failed to activate any resorbing activity, indicating that although deriving from the same precursors macrophage polykaryons and osteoclasts are independent cell types and polykaryons are not immature osteoclasts.

Macrophages and osteoclasts develop unique contact sites with the extracellular matrix called podosomes. Podosomes have been associated with migratory and invasive cell characteristics, but a basic mechanism outlining their function is lacking. We have used chicken and human monocytes differentiating in vitro into osteoclast-like cells in the presence of RANKL-ODF to study these cytoskeletal structures. During the differentiation process, podosomes are redistributed from the cell body in early macrophages to the cell periphery in increasingly spread and multinucleated cells expressing high levels of integrin αVβ3. Immunofluorescence with anti-phosphotyrosine antibodies revealed increased tyrosine-phosphorylation at the basal tips of these podosomes. RANKL-ODF treatment reinforced the peripheral location of podosomes and initiated their partial fusion to larger F-actin-containing structures that displayed reduced levels of tyrosine phosphorylation. Paxillin and the FAK-related kinase Pyk2 colocalized with integrin αVβ3 in the juxtamembrane region surrounding individual podosomes. In lysates of macrophages and differentiated osteoclasts both paxillin and Pyk2 associated with synthetic and recombinant polypeptides containing the C-terminal region of the integrin β3 cytoplasmic domain. These in vitro interactions were direct and they were abolished by substitutions in the β3 integrin peptides known to disrupt integrin function in vivo. The marked adhesion-dependent tyrosine-phosphorylation of Pyk2 and paxillin however did not detectably alter their interaction with β3 tail peptides in cell lysates. Our results provide novel insight into the molecular architecture and the phosphorylation dynamics in podosomes. Moreover, they outline a novel potential mechanism for the recruitment of paxillin and Pyk2 to β3 integrin-dependent cell contacts.

The Carbonic Anhydrase II (CAII) gene that encodes an enzyme involved in proton production is expressed in several cell types including monocyte/macrophage-derived osteoclasts. We have analyzed the regulation of the chicken CAII promoter/reporter construct by nuclear hormone receptors of the VDR subfamily in HD11 avian macrophages. The CAII expression is stimulated by 1,25-dihydroxyvitamin D3 but not by 9-cis retinoic acid and repressed by VDR overexpression due to RXR squelching. It is also stimulated by all-trans retinoic acid only when RARα is overexpressed, and is dependent on a RARE located in the distal part of the promoter and bound by RARα homodimer. Finally, in macrophages, unlike in erythrocytes, the CAII promoter is unresponsive to thyroid hormone. Our results demonstrate the first retinoic acid response element in the CAII promoter and show that according to cell type, different nuclear receptors of the VDR subfamily can regulate the CAII gene.

Osteoclasts are postmitotic, multinucleated giant cells generated by the fusion of hematopoietic mononuclear precursors from the monocyte–macrophage lineage. In culture, adherent macrophages from blood-derived monocytes grow, gather, and fuse together to form multinucleated osteoclast-like cells. These events are controlled by 1,25(OH)2D3. To sort out new 1,25(OH)2D3target genes involved in osteoclast differentiation, we have performed an RT-PCR differential display using mRNA from macrophages induced for 10 h by 1,25(OH)2D3compared to nontreated cells. We have identified a new target gene, a chick ATP-dependent Ca2+pump, ChkSERCA3. Although the level of the corresponding transcript increases during the differentiation process from macrophages to osteoclast-like cells, its steady-state level is downregulated by hormone treatment. The action of 1,25(OH)2D3on the Ca2+-ATPase gene expression is independent of de novoprotein synthesis and is hormone dose dependent. This expression in adult chick was restricted to the hematopoietic cell lineage, spleen, lung, intestine, and brain, whereas no expression was detected in embryos. The function of the protein can be predicted from its high homology with the other members of the SR ATP-dependent Ca2+pump family, i.e., storage and control of cytosolic Ca2+directly regulated by 1,25(OH)2D3, further supporting the critical role for intracellular calcium in highly specialized cells such as osteoclasts.

The carbonic anhydrase II gene, whose transcription is enhanced by 1,25-dihydroxyvitamin D3(1,25-(OH)2D3), encodes an important enzyme in bone-resorbing cells derived from the fusion of monocytic progenitors. We analyzed the 1,25-(OH)2D3-mediated activation of the avian gene by transient transfection assays with promoter/reporter constructs into HD11 chicken macrophages and by DNA mobility shift assays. Deletion and mobility shift analyses indicated that the −62/−29 region confers 1,25-(OH)2D3responsiveness and forms DNA-protein complexes. The addition of an anti-vitamin D receptor (VDR) antibody inhibited binding to this sequence, whereas anti-retinoid X receptor (RXR) antibody generated a lower mobility complex. Therefore, we concluded that this element binds a VDR·RXR heterodimer, but the addition of extra 1,25-(OH)2D3had no effect on the formation of this complex. Moreover, the use of nuclear extracts from 1,25-(OH)2D3-treated macrophages led to the formation of an additional high mobility complex also composed of VDR·RXR heterodimer. Mutations provided evidence that the 1,25-(OH)2D3-mediated activation of the carbonic anhydrase II gene is mediated by VDR·RXR heterodimers bound to a DR3-type vitamin D response element with sequence AGGGCAtggAGTTCG. This vitamin D response element is also functional in the ROS 17/2.8 osteoblasts.

We describe here the use of PCR-generated templates incorporating T3 polymerase sites in order to prepare digoxigenin (DIG)-labelled cRNA probes against any gene of known sequence. This method was applied to the preparation of probes specific for chicken glyceraldehyde-3-phosphate dehydrogenase messenger RNAs and we demonstrate that such probes can be used forin situhybridization (ISH). This technique therefore represents a rapid and convenient means to prepare DIG-labelled cRNA probes for use in a non-radioactive ISH. It adds speed and convenience of probe preparation to the previously described advantages of non-radioactive detection techniques.

αvβ3 is a key integrin mediating adhesion of multinucleated osteoclasts during bone resorption. 1,25-dihydroxyvitamin D3 upregulates αvβ3 integrin expression in mononucleated osteoclast precursors and concomitantly stimulates their differentiation into osteoclasts. This suggests that this integrin could play a major role during osteoclast differentiation.We have developed an in vitro model, in which 1,25-dihydroxyvitamin D3 sequentially modifies the behavior of macrophages: Ìt first induces rounding up of these cells, then their subsequent aggregation and spreading, which finally leads to cell fusion and the formation of osteoclast-like multinucleated giant cells. We show that, while 1,25-dihydroxyvitamin D3 stimulates the de novo synthesis of αvβ3 in macrophages early in this process, its accumulation on the surface is triggered by cell aggregation. A high level of integrin αvβ3 cell surface expression correlates with macrophage spreading preceding fusion. This was confirmed by means of novel cell permeable peptides containing the C-terminal sequence of the integrin β3 tail to specifically block αvβ3 function. Although this peptide has no effect on the aggregation step, it disrupts the spreading of osteoclast precursors and consequently inhibits their fusion. These findings suggest a novel role of the integrin αvβ3 in a discrete step of osteoclast differentiation.

Les infections ostéo-articulaires (IOA), majoritairement causées par S. aureus(SA), sont associées à une destruction osseuse importante. Les interactions entre SA vivants et les ostéoclastes (OC) n’ont jamais été étudiées, pourtant seules cellules responsables de la résorption osseuse. Notre objectif a été d’étudier l’impact de l’internalisation de SA sur l’ostéoclastogenèse et l’ostéolyse induite par les ostéoclastes infectés. Les précurseurs (obtenus à partir de moelle osseuse de souris) et les OC matures (obtenus par différenciation des précurseurs en présence de RANK-L et M-CSF), ont été infectés pendant 2h avec une souche de SA (8325-4) vivante (MOI – 10bactéries cellule) puis incubées en présence de gentamicine pour tuer les bactéries extracellulaires. L’effet de l’infection sur l’ostéoclastogenèse a été évalué à j5 par (i) dénombrement des OC matures, (ii) caractérisation des cytokines sécrétées (Luminex) et des marqueurs cellulaires membranaires par cytométrie. L’effet de l’infection sur des OC matures a, lui, été évalué en mesurant la capacité de résorption des OC matures infectés sur une matrice d’hydroxyapatite (calcul de surface résorbée par analyse d’image). Après infection des précurseurs et en l’absence démontrée de cytotoxicité, le nombre d’OC matures par puits obtenus à j5 était significativement abaissé par rapport aux cellules non infectés (25A28 vs. 713A184, p<0,05). Les cellules mononuclées alors présentes dans les puits « infectés » présentaient un profil de marqueurs membranaires (F4 80+, CD11b+) et de cytokines pro-inflammatoires (MIP1, KC, RANTES, MCP-1) typique des macrophages. De leur coté, les OC matures après infection voyaient leurs capacités de résorption tripler par rapport aux OC non infectés (p=0,02). Nos travaux permettent pour la première fois de mettre en évidence que l’infection des précurseurs par des SA vivants les rend incapables de se différencier en OC mais provoque leur différenciation en macrophage avec sécrétion massive de cytokines pro-inflammatoires. D’un autre côté, l’infection des OC matures induit une augmentation de leurs capacités de résorption. Ces travaux suggèrent que l’interaction OC–SA pourrait jouer un rôle pivot dans l’inflammation locale ainsi que dans les pertes et remaniements osseux observés au cours des IOA à SA.

The mouse inbred line C57BL/6J is widely used in mouse genetics and its genome has been incorporated into many genetic reference populations. More recently large initiatives such as the International Knockout Mouse Consortium (IKMC) are using the C57BL/6N mouse strain to generate null alleles for all mouse genes. Hence both strains are now widely used in mouse genetics studies. Here we perform a comprehensive genomic and phenotypic analysis of the two strains to identify differences that may influence their underlying genetic mechanisms.