Specific targeting of inflammatory osteoclastogenesis by the probiotic yeast S. boulardii CNCM I-745 reduces bone loss in osteoporosis.

Bone destruction is a hallmark of chronic inflammation, and bone-resorbing osteoclasts arising under such a condition differ from steady-state ones. However, osteoclast diversity remains poorly explored. Here, we combined transcriptomic profiling, differentiation assays and in vivo analysis in mouse to decipher specific traits for inflammatory and steady-state osteoclasts. We identified and validated the pattern-recognition receptors (PRR) Tlr2, Dectin-1, and Mincle, all involved in yeast recognition as major regulators of inflammatory osteoclasts. We showed that administration of the yeast probiotic Saccharomyces boulardii CNCM I-745 (Sb) in vivo reduced bone loss in ovariectomized but not sham mice by reducing inflammatory osteoclastogenesis. This beneficial impact of Sb is mediated by the regulation of the inflammatory environment required for the generation of inflammatory osteoclasts. We also showed that Sb derivatives as well as agonists of Tlr2, Dectin-1, and Mincle specifically inhibited directly the differentiation of inflammatory but not steady-state osteoclasts in vitro. These findings demonstrate a preferential use of the PRR-associated costimulatory differentiation pathway by inflammatory osteoclasts, thus enabling their specific inhibition, which opens new therapeutic perspectives for inflammatory bone loss.

Differentiation and Phenotyping of Murine Osteoclasts from Bone Marrow Progenitors, Monocytes, and Dendritic Cells.

Bone physiology is dictated by various players, including osteoclasts (OCLs) as bone resorbing cells, osteoblasts (capable of bone formation), osteocytes, or mesenchymal stem cells, to mention the most important players. All these cells are in tight communication with each other and influence the constantly occurring process of bone remodeling to meet changing requirements on the skeletal system. In order to understand these interplays, one must investigate isolated functions of the various cell types. However, OCL research displays a special drawback: due to their giant size, low abundance, and tight attachment on the bone surface, ex vivo isolation of sufficient amounts of mature OCLs is limited or not conceivable in most species including mice. Moreover, OCLs can be obtained from different progenitors in vivo as well as in vitro. Thus, in vitro differentiation of OCLs from various progenitor cells remains essential in the analysis of OCL biology, underlining the importance of reliable gold standard protocols to be applied throughout OCL research. This chapter will deal with in vitro differentiation of OCLs from murine bone marrow cells, as well as isolated monocytes and dendritic cells that have already been validated in numerous studies.

Dissecting the phenotypic and functional heterogeneity of mouse inflammatory osteoclasts by the expression of Cx3cr1.

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.

The haemochromatosis gene Hfe and Kupffer cells control LDL cholesterol homeostasis and impact on atherosclerosis development.

AIMS: Imbalances of iron metabolism have been linked to the development of atherosclerosis. However, subjects with hereditary haemochromatosis have a lower prevalence of cardiovascular disease. The aim of our study was to understand the underlying mechanisms by combining data from genome-wide association study analyses in humans, CRISPR/Cas9 genome editing, and loss-of-function studies in mice. METHODS AND RESULTS: Our analysis of the Global Lipids Genetics Consortium (GLGC) dataset revealed that single nucleotide polymorphisms (SNPs) in the haemochromatosis gene HFE associate with reduced low-density lipoprotein cholesterol (LDL-C) in human plasma. The LDL-C lowering effect could be phenocopied in dyslipidaemic ApoE-/- mice lacking Hfe, which translated into reduced atherosclerosis burden. Mechanistically, we identified HFE as a negative regulator of LDL receptor expression in hepatocytes. Moreover, we uncovered liver-resident Kupffer cells (KCs) as central players in cholesterol homeostasis as they were found to acquire and transfer LDL-derived cholesterol to hepatocytes in an Abca1-dependent fashion, which is controlled by iron availability. CONCLUSION: Our results disentangle novel regulatory interactions between iron metabolism, KC biology and cholesterol homeostasis which are promising targets for treating dyslipidaemia but also provide a mechanistic explanation for reduced cardiovascular morbidity in subjects with haemochromatosis.