Osteoclasts (OCLs) are key players in controlling bone remodeling. Modifications in their differentiation or bone resorbing activity are associated with a number of pathologies ranging from osteopetrosis to osteoporosis, chronic inflammation and cancer, that are all characterized by immunological alterations. Therefore, the 2000s were marked by the emergence of osteoimmunology and by a growing number of studies focused on the control of OCL differentiation and function by the immune system. At the same time, it was discovered that OCLs are much more than bone resorbing cells. As monocytic lineage-derived cells, they belong to a family of cells that displays a wide heterogeneity and plasticity and that is involved in phagocytosis and innate immune responses. However, while OCLs have been extensively studied for their bone resorption capacity, their implication as immune cells was neglected for a long time. In recent years, new evidence pointed out that OCLs play important roles in the modulation of immune responses toward immune suppression or inflammation. They unlocked their capacity to modulate T cell activation, to efficiently process and present antigens as well as their ability to activate T cell responses in an antigen-dependent manner. Moreover, similar to other monocytic lineage cells such as macrophages, monocytes and dendritic cells, OCLs display a phenotypic and functional plasticity participating to their anti-inflammatory or pro-inflammatory effect depending on their cell origin and environment. This review will address this novel vision of the OCL, not only as a phagocyte specialized in bone resorption, but also as innate immune cell participating in the control of immune responses.

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Bone destruction relies on interactions between bone and immune cells. Bone-resorbing osteoclasts (OCLs) were recently identified as innate immune cells activating T cells toward tolerance or inflammation. Thus, pathological bone destruction not only relies on increased osteoclast differentiation, but also on the presence of inflammatory OCLs (i-OCLs), part of which express Cx3cr1. Here, we investigated the contribution of mouse Cx3cr1+ and Cx3cr1neg i-OCLs to bone loss. We showed that Cx3cr1+ and Cx3cr1neg i-OCLs differ considerably in transcriptional and functional aspects. Cx3cr1neg i-OCLs have a high ability to resorb bone and activate inflammatory CD4+ T cells. Although Cx3cr1+ i-OCLs are associated with inflammation, they resorb less and have in vitro an immune-suppressive effect on Cx3cr1neg i-OCLs, mediated by PD-L1. Our results provide new insights into i-OCL heterogeneity. They also reveal that different i-OCL subsets may interact to regulate inflammation. This contributes to a better understanding and prevention of inflammatory bone destruction.

Osteoclasts (OCLs) are multinucleated phagocytes of monocytic origin responsible for physiological and pathological bone resorption including aging processes, chronic inflammation and cancer. Besides bone resorption, they are also involved in the modulation of immune responses and the regulation of hematopoietic niches. Accordingly, OCLs are the subject of an increasing number of studies. Due to their rarity and the difficulty to isolate them directly ex vivo, analyses on OCLs are usually performed on in vitro differentiated cells. In this state, however, OCLs represent a minority of differentiated cells. Since up to date a reliable purification procedure is still lacking for mature OCLs, all cells present in the culture are analyzed collectively to answer OCL-specific questions. With the development of in-depth transcriptomic and proteomic analyses, such global analyses on unsorted cells can induce severe bias effects in further results. In addition, for instance, analysis on OCL immune function requires working on purified OCLs to avoid contamination effects of monocytic precursors that may persist during the culture. This clearly highlights the need for a reliable OCL purification procedure. Here, we describe a novel and reliable method to sort OCLs based on cell multinucleation while preserving cell viability. Using this method, we successfully purified multinucleated murine cells. We showed that they expressed high levels of OCL markers and retained a high capacity of bone resorption, demonstrating that these are mature OCLs. The same approach was equally applied for the purification of human mature OCLs. Comparison of purified OCLs with mononucleated cells or unsorted cells revealed significant differences in the expression of OCL-specific markers at RNA and/or protein level. This exemplifies that substantially better outcomes for OCLs are achieved after the exclusion of mononucleated cells. Our results clearly demonstrate that the in here presented procedure for the analysis and sorting of pure OCLs represents a novel, robust and reliable method for the detailed examination of bona fide mature OCLs in a range that was previously impossible. Noteworthy, this procedure will open new perspectives into the biology of osteoclasts and osteoclast-related diseases.

The Cre/Lox system is a powerful tool in the biologist's toolbox, allowing loss-of-function and gain-of-function studies, as well as lineage tracing, through gene recombination in a tissue-specific and inducible manner. Evidence indicates, however, that Cre transgenic lines have a far more nuanced and broader pattern of Cre activity than initially thought, exhibiting "off-target" activity in tissues/cells other than the ones they were originally designed to target. With the goal of facilitating the comparison and selection of optimal Cre lines to be used for the study of gene function, we have summarized in a single manuscript the major sites and timing of Cre activity of the main Cre lines available to target bone mesenchymal stem cells, chondrocytes, osteoblasts, osteocytes, tenocytes, and osteoclasts, along with their reported sites of "off-target" Cre activity. We also discuss characteristics, advantages, and limitations of these Cre lines for users to avoid common risks related to overinterpretation or misinterpretation based on the assumption of strict cell-type specificity or unaccounted effect of the Cre transgene or Cre inducers.

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The skeleton is heavily innervated by sympathetic nerves and represents a common site for breast cancer metastases, the latter being the main cause of morbidity and mortality in breast cancer patients. Progression and recurrence of breast cancer, as well as decreased overall survival in breast cancer patients, are associated with chronic stress, a condition known to stimulate sympathetic nerve outflow. Preclinical studies have demonstrated that sympathetic stimulation of β-adrenergic receptors in osteoblasts increases bone vascular density, adhesion of metastatic cancer cells to blood vessels, and their colonization of the bone microenvironment, whereas β-blockade prevented these events in mice with high endogenous sympathetic activity. These findings in preclinical models, along with clinical data from breast cancer patients receiving β-blockers, support the pathophysiological role of excess sympathetic nervous system activity in the formation of bone metastases, and the potential of commonly used, safe, and low-cost β-blockers as adjuvant therapy to improve the prognosis of bone metastases.

Receptor activator of NF-κB (RANK), a member of the TNF-receptor superfamily, plays an important role in bone resorption and stimulates immune and epithelial cell activation. Denosumab, a human monoclonal antibody that blocks the RANK ligand (RANKL), is approved for the treatment of osteoporosis and bone metastasis. However, a small molecule that inhibits the RANK-RANKL interaction would be beneficial to decrease cost and to facilitate treatments with orally available therapeutic agents. Herein we report the discovery of the first nonpeptidic inhibitors of RANK-RANKL interactions. In screening a chemical library by competitive ELISA, the porphyrin verteporfin was identified as a hit. Derivatives were screened, and the chlorin-macrocycle-containing pheophorbide A and purpurin 18 were found to bind recombinant RANKL, to inhibit RANK-RANKL interactions in the ELISA, and to suppress the RANKL-dependent activation of model cells and the differentiation of RANK-expressing precursors into osteoclasts. This discovery of a family of small molecules that inhibit RANK activation presents an initial basis for further development of nonpeptidic therapeutic agents targeting the interaction between RANK and RANKL.