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

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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 boulardi CNCM I-745 ( Sb ) in vivo reduced bone loss in OVX but not sham mice by reducing inflammatory osteoclasts. 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.

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  1. eLife assessment

    In this work, the authors provide convincing evidence about the existence of two distinct osteoclast populations with specific expression profiles and properties and show that the probiotic yeast S. boulardii may be useful in managing inflammation-mediated bone loss, including estrogen deprivation-mediated osteoporosis. The reported study aims to bring the concept of heterogeneous osteoclasts into a proof-of-principle therapeutic application, which may mean that the use of probiotics might combat osteoporosis towards a better bone quality than current therapies. The molecular mechanism of how the probiotic yeast S. boulardii treatment acts via the receptors remains obscure since it might act via changes in the gut permeability or by components directly released by the fungus.

  2. Reviewer #1 (Public Review):

    Osteoclasts, giant multinucleated bone-resorbing cells, are crucial regulators of bone homeostasis and pathology. An underestimated aspect of their biology is that they are very heterogeneous, with at least 2 sub-populations (inflammatory osteoclasts and tolerogenic osteoclasts) existing, and exerting different actions, especially in the context of inflammatory bone loss. In this report, Madel, Halper (co-first authors), and colleagues present an interesting report investigating this heterogeneity, and showing that the probiotic yeast S. boulardii (probably through β-glucans) may be useful in managing inflammation-mediated bone loss, including oestrogen deprivation-mediated osteoporosis, as the authors show in vivo using an OVX mouse model.

    The authors first evaluate the differences in the transcriptional landscape of tolerogenic vs inflammatory osteoclasts with RNAseq, and then they evaluate the differences in miRNA expression between the two. Finding that some of the pathways/genes that vary are related to pattern recognition receptors (PRRs), specialized in recognizing non-self antigens including those arising from bacteria and yeasts, they wonder if the probiotic yeast S. boulardii could influence the balance between tolerogenic and inflammatory osteoclasts. Indeed, when the authors treated OVX mice, characterised by an increase in inflammatory osteoclasts and estrogen deprivation/inflammation-induced bone loss, with the probiotic, the bone loss is avoided and inflammatory osteoclasts are reduced. This challenges the classical way in which osteoclast-mediated bone loss is treated, since targeting specifically the inflammatory osteoclasts could allow the good osteoclasts to keep working and improving bone health and immunity, while only the bad osteoclasts are targeted. Current treatments are not able to distinguish between the two, which can cause a paradoxical degradation in bone health and atypical fractures. The report is therefore potentially very important for the field, and although quite focused on a specific strain, it can pave the way to treating bone diseases with probiotics, or specific molecules derived from them including beta-glucans.

  3. Reviewer #2 (Public Review):

    The authors apply their previously developed concept that osteoclasts exist in at least two flavors, tolerogenic and inflammatory osteoclasts towards the treatment of osteoporosis. They suggest that selectively targeting inflammatory osteoclasts attenuates ovariectomy-induced bone loss by agonists of pattern recognition receptors (PRR) that are higher expressed on inflammatory osteoclasts. The vision would be that the tolerogenic osteoclasts are still functioning, allowing bone remodeling with high bone quality, while the strong resorbing inflammatory osteoclasts are resorbed. By expression profiling, they detected PPR differentially expressed and confirmed these by flow cytometry and RT-QPCR. The activation of the Tlr2, Dectin-1, and Mincle reduced inflammatory osteoclast generation in vitro and affected their resorptive activity. Dendritic syk cell-specific deletion abrogated the differentiation of this osteoclast subset as well. The application of yeast Saccharomyces boulardii (Sbb) into mice attenuated trabecular bone loss (but not cortical) and seemed to inhibit in vitro the generation of inflammatory osteoclasts.

    - The expression profiling between very defined in vitro generated osteoclasts, which are somehow extreme phenotypes, provides a good tool to discern gene signatures on the osteoclast level.
    - The candidate of PPR were evaluated in their expression at the protein level by flow cytometry and their function was evaluated by loss of function studies.
    - The effect of S.b. treatment is striking and exploiting such probiotic fungi could be an elegant way to treat osteoporosis.

    - The osteoclasts are generated in vitro in the presence of M-CSF to induce tolerogenic osteoclasts or GM-CSF / Il-4 to generate inflammatory osteoclasts. The demonstration of these cell populations in the S.b. treated mice in vivo is not present, despite the challenge to do this. The author tried to tackle this, by analyzing the differentiation potential of bone marrow progenitor cells of S.b. treated animals, which provides some information.
    - The effect on tolerogenic osteoclasts could have been further evaluated, whether they are not affected at all, or whether there are also effects.

    The authors strikingly show that agonists for PPR are affecting strongly GM-CSF/IL-4 progenitor-derived osteoclasts. They show that t-Ocl and i-Ocl differ in their gene signature and convincingly show the differential expression of the PPR, with exception of mincle which is clearly acknowledged. The molecular mechanism of how Sb treatment acts via the receptors remains obscure since it might act via changes in the gut permeability or by components directly released by the fungus. The kinase syk could play a role, at least some data in vitro suggest this.

    Conceptionally the authors tried to utilize the previously generated knowledge by the group published 2016 and 2020 into an approach. If the use of a probiotic fungus would be beneficial indeed this could be a suitable drug with few side effects much superior to current treatments of osteoporosis.
    For me, an intriguing question arises from this study, in case these i-Ocl express these receptors and are thus so "vulnerable" to the agonists to decrease their activity, evt. a negative feedback to prevent overshooting reactions?

  4. Reviewer #3 (Public Review):

    The general objective of this work is the dissection of osteoclast diversity; in particular, the authors intend to identify the specific features and properties that distinguish inflammatory and steady-state (tolerogenic) osteoclasts. To this end, the authors perform a transcriptional analysis of inflammatory and tolerogenic osteoclasts and identify the pattern recognition receptors TLR2, Dectin-1, and Mincle as differentially expressed genes. Agonists of these receptors or yeast probiotics regulating the elicited mechanisms in vitro and in vivo caused a specific inhibition of the differentiation of inflammatory rather than tolerogenic osteoclasts, thus highlighting the preferential use of different differentiation pathways by the two distinct osteoclast populations.

    The project is based on the previous knowledge and know-how of the authors on this peculiar skeletal cell population. The work is well conceived; the experiments are clearly designed and exploit state-of-the-art technologies. The results confirm the heterogeneity of osteoclasts and provide new insights in this respect. The in vitro and in vivo studies suggest that osteoclast heterogeneity can be purposedly modulated; which might be useful and advisable for therapeutic purposes. Overall, the work provides hints for further implementation and future broad applications to diseases featuring pathological bone loss.