Osteoclasts (OCLs) are multinucleated phagocytes derived from monocytic progenitors and specialized in bone resorption (Madel et al., 2019). Similar to other cells of monocytic origin, they are also innate immunocompetent cells and heterogeneous in their phenotype, function, and origin (Ibáñez et al., 2016; Jacome-Galarza et al., 2019; Madel et al., 2020; Madel et al., 2019; Yahara et al., 2020). OCLs derived from steady-state bone marrow (BM) cells or from BM CD11b⁺ monocytic cells (MN-OCLs) promote tolerance by inducing CD4⁺ and CD8⁺ regulatory T cells (tolerogenic OCLs [t-OCLs]) (Ibáñez et al., 2016; Kiesel et al., 2009). In contrast, in the context of bone destruction linked to inflammatory bowel disease (IBD) or when derived from dendritic cells (DC-OCLs), OCLs induce Tnf-α-producing CD4⁺ T cells (inflammatory OCLs [i-OCLs]) (Ibáñez et al., 2016; Madel et al., 2020). OCLs associated with inflammation can be identified by expression of Cx3cr1 (fractalkine receptor) and the proportion of Cx3cr1⁺ OCLs increases in osteoporosis, IBD, and after Rank-L treatment (Ibáñez et al., 2016; Madel et al., 2020). However, Cx3cr1 is only expressed in approximately 20% of i-OCLs (Ibáñez et al., 2016; Madel et al., 2020), which highlights their heterogeneity while limiting the possibility to analyze them in the context of pathological bone loss and urges the identification of novel markers.

Current anti-resorptive therapies aim to globally inhibit OCLs, without considering their recently established diversity. In the long term, they result in poor bone remodeling which may increase the risk of atypical fractures (Reyes et al., 2016). Therefore, an in-depth characterization of OCLs associated with healthy versus inflammatory bone resorption would allow the identification of distinct characteristics that could help to specifically target i-OCL.

i-OCLs arise under the control of persistent high levels of Rank-L, IL-17, and TNFα mainly produced by CD4⁺ T cells that play a major role in pathological osteoclastogenesis observed in osteoporosis and IBD (Cenci et al., 2000; Ciucci et al., 2015; Ibáñez et al., 2016; Li et al., 2011). Interestingly, the emergence of such osteoclastogenic CD4⁺ T cells is associated with gut dysbiosis and increased intestinal permeability (Jones et al., 2018; Li et al., 2016). In line with this, bacterial probiotics such as Lactobacillus and Bifidobacteria have been shown to effectively reduce osteoporotic bone loss (Li et al., 2016; Britton et al., 2014; Ohlsson et al., 2014; Ibáñez et al., 2019b), but their specific effect on i-OCLs remains unknown.

Here, using a comparative RNAseq approach performed on sorted pure mature MN-OCLs and DC-OCLs as models of t-OCLs and i-OCLs, respectively, as already established (Ibáñez et al., 2016; Madel et al., 2020), we showed that the two OCL populations are distinctly equipped to respond to different signals that can modulate their differentiation. In particular, the pattern recognition receptors (PRRs) Dectin-1, Tlr2, and Mincle involved in the response to fungi (Li et al., 2019; Sancho and Reis e Sousa, 2012) are overexpressed in i-OCLs. Administration of the probiotic yeast Saccharomyces boulardii CNCM I-745 (Sb), which is used in the treatment of gastrointestinal disorders for its anti-inflammatory properties and its capacity to restore the gut microbiota (Czerucka and Rampal, 2019; Terciolo et al., 2019), significantly reduces bone loss and inflammatory parameters in vivo in ovariectomized (OVX) mice. In vitro, Sb derivates as well as low doses of agonists of the PRRs overexpressed in i-OCLs specifically inhibit the differentiation of these cells without affecting t-OCLs. These data open perspectives on targeting specific OCL populations and provide evidence for the protective effect of a probiotic yeast on inflammatory bone resorption. Our study unveils very new insights into the regulation and modulation of i-OCLs and enables a better understanding of the molecular mechanisms involved in inflammation-induced bone erosions.

The present study demonstrates that i-OCLs and t-OCLs are two distinct OCL populations with specific molecular signatures that differ in their preferential use of differentiation pathways and their capacity to sense the environment through the PRRs Dectin-1, Tlr2, and Mincle. Based on these specificities, we could show that targeting these receptors reduces bone loss in OVX mice and inhibits specifically the differentiation of i-OCLs while sparing t-OCLs.

OCL heterogeneity has been neglected for a long time, and the mechanisms regulating the emergence and function of i-OCLs are still largely unknown. Our transcriptomic profiling shows that, while being comparable in their expression of bone resorption-associated genes, t-OCLs and i-OCLs differ in their expression of genes associated with immune responses, which provides additional evidence for their previously reported divergent immune properties (Ibáñez et al., 2016). It also highlights differences in genes related to OCL differentiation. Osteoclastogenesis is regulated by two main pathways, the classical Rank-associated and the co-stimulatory Ig-like receptor-associated pathways, both of which are required to promote OCL differentiation (Humphrey and Nakamura, 2016; Koga et al., 2004; Seeling et al., 2013). Increased Rank-L levels as well as stimulation of FcγR and ITAM signaling are involved in pathological bone resorption in rheumatic diseases such as rheumatoid arthritis (Herman et al., 2008; Ochi et al., 2007). However, to date these pathways have not been described to be preferentially used by specific populations of OCLs. Here, we show that the implication of the co-stimulatory pathway in diseases associated with high bone resorption could be linked to its upregulation during i-OCL differentiation, which suggests that specific targeting of this pathway and its related molecules could limit inflammatory bone loss with a minimal impact on OCLs involved in physiological bone remodeling.

Remarkably, Tlr2, Dectin-1, and Mincle are increased in i-OCLs and OCLs from OVX mice compared to t-OCLs and to those from SHAM mice. These receptors are expressed in myeloid cells and sense microbial structures from bacteria, fungi, or parasites, including lipoproteins, glycoproteins, peptidoglycan for Tlr2, β-glucans for Dectin-1 and Tlr2, and α-mannose for Mincle (Sancho and Reis e Sousa, 2012; Savva and Roger, 2013). Moreover, they interact with each other and with Fcγ receptors for the induction of inflammatory responses (Gantner et al., 2003; Sato et al., 2003). Very few studies have investigated the effect of microbial β-glucans and α-mannose on osteoclastogenesis. Tlr2 stimulation was shown to reduce osteoclastogenesis from non-committed OCL progenitors but to increase OCL survival and differentiation of Rank-L-primed pre-OCLs (Souza and Lerner, 2019; Takami et al., 2002). High concentrations of curdlan (in the range of µg/mL) have been previously reported to decrease the overall differentiation of OCLs from Dectin-1-transfected RAW cells or from BM cells by upregulating Mafb and reducing Nfatc1 expression (Zhu et al., 2017). Our results extend these data by revealing that with much lower doses of curdlan (range of ng/mL), only i-OCL formation is inhibited whereas other OCLs are not affected, in line with the huge difference in Dectin-1 and Tlr2 expression by their respective progenitors. Interestingly, here we show that the yeast probiotic Sb significantly reduces bone loss in OVX mice. Sb has proven its beneficial probiotic effects in several gastrointestinal disorders. It favors regeneration of the integrity of the epithelial barrier in colitis and restores the gut microbiota after antibiotic treatment or diarrhea by increasing SCFA-producing bacteria such as Lachnospiraceae, Bacteroides, Ruminococcus, and Prevotellaceae (Moré and Swidsinski, 2015; Terciolo et al., 2019; Yu et al., 2017). In line with this, our results demonstrate that Sb improves the gut barrier function in the context of OVX-induced osteoporosis. It also normalizes the concentration of SCFAs and lactate in OVX mice, which strongly suggests that Sb favors bacteria species producing these metabolites. Indeed previous studies reported that the diversity of intestinal microbiota is lower in OVX mice than in SHAM mice, and its composition is altered (Li et al., 2021; Zaiss et al., 2019). LAB as well as SCFA-producing bacteria have a beneficial effect on bone (Zaiss et al., 2019) which probably participates in the reduced bone loss observed in Sb-treated OVX mice. Moreover, gut dysbiosis and increased intestinal permeability reported in osteoporosis (He et al., 2020; Xu et al., 2020) induce activation of osteoclastogenic CD4⁺ T cells over-producing Rank-L and Tnf-α (D’Amelio et al., 2008; Li et al., 2016). Thus, the reduction in BM Tnf-α-producing CD4⁺ T cells observed after Sb treatment in OVX mice is likely due to its beneficial effect on the gut, as described for bacterial probiotics (Li et al., 2016). As Tnf-α⁺ CD4⁺ T cells are major inducers of i-OCLs (Ciucci et al., 2015; Ibáñez et al., 2016; Madel et al., 2019), this effect probably also participates in the decreased proportion of i-OCLs observed in Sb-treated OVX mice. In addition, in various models of gut disorders, Sb has been described to increase the production of anti-inflammatory cytokines such as IL-10 and IL-4 (Czerucka and Rampal, 2019; Pais et al., 2020) that are potent inhibitors of OCL formation (Fujii et al., 2012; Park-Min et al., 2009). Despite we did not detect significant difference in serum IL-10 concentration after Sb treatment in OVX mice (data not shown), we could not rule out local cytokine modification in the BM.

In addition to its systemic effects on inflammation and microbiota, Sb is also likely to directly affect i-OCL differentiation. The yeast cell wall components, ß-glucans, are well known to cross the gut barrier and translocate to the blood from where they can disseminate to organs (Isnard et al., 2021; Rice et al., 2005) and can directly modulate cells from the monocytic family (Ibáñez et al., 2019a), which includes circulating OCL progenitors, through stimulation of CLRs. Consequently, in vivo they can possibly exert the same direct inhibitory effect on the differentiation of i-OCLs as observed in vitro. This is confirmed by our in vitro analysis showing a specific inhibition of i-OCL differentiation compared to t-OCLs by Sb, as well as low doses of agonists of Tlr2, Dectin-1, and Mincle, which is in line with the much higher expression of these receptors in BM-derived DCs than in BM MNs.

The inhibitory effect of Mincle stimulation observed here is contrasting with a recent publication reporting that sensing of necrotic cells through Mincle by OCL progenitors stimulates OCL formation (Andreev et al., 2020). Clec4e KO mice have increased bone mass (Andreev et al., 2020), a result confirmed by our study. However, we also observed a similar or even higher OCL formation from progenitors from Clec4e KO compared to control mice. Mincle is not expressed by osteoblasts (Andreev et al., 2020), but the increased bone mass observed in Clec4e KO mice could be related to indirect effects of Mincle on osteoblasts and OCLs through myeloid cells that are well-known regulators of bone cell differentiation and activity (Yahara et al., 2021). Moreover, Mincle KO mice display reduced CD4⁺IL-17⁺ T cells (Martínez-López et al., 2019), which may also contribute to a reduced OCL differentiation and increased bone mass. It is therefore likely that depending on whether Mincle is stimulated by exogenous microbial signals (as in our study) or endogenous necrotic signals (as in Andreev’s study), this receptor has divergent effects on osteoclastogenesis. These observations revealed the complex effects of Mincle on osteoclastogenesis, as already reported for other monocytic cells (Patin et al., 2017).

The kinase Syk plays a major role in Dectin-1 and Mincle signaling pathways, and indeed, Syk is rapidly phosphorylated upon curdlan stimulation in BM-DCs. On the other hand, Syk is required for efficient OCL differentiation (Zou et al., 2007), including from DCs as shown here. However, in DCs stimulated with curdlan in osteoclastogenic medium, the expression of Syk decreases with time together with Nfatc1, a master gene of OCL differentiation, and Ctsk, a main marker of OCLs required for their activity, which participates in reducing i-OCL formation. These results are in agreement with the literature showing in the same conditions a degradation of Syk after treatment with high doses of curdlan (Yamasaki et al., 2014). In DCs, the interaction between Tlr2 and Dectin-1 is involved in the stimulation of NF-κB and in the production of inflammatory cytokines such as Tnf-α and IL-12 (Gantner et al., 2003; Sancho and Reis e Sousa, 2012). This strongly induces DC activation and therefore potent phagocytic and anti-microbial responses (Gantner et al., 2003; Sancho and Reis e Sousa, 2012). Accordingly, we show that treatment with curdlan or with Sb-CM induces DC maturation while it decreases the proportion of Rank⁺ cells expressing Csf1r and FcgrII/III, all of which are required for OCL formation. Thus, stimulation of DCs with the PRR agonists has a dual effect, i.e., increasing their maturation and simultaneously reducing their ability to give rise to i-OCLs.

In conclusion, we here demonstrated that i-OCLs differ from t-OCLs in the control of their differentiation and in their capacity to sense their environment and in particular to respond to stimuli through CLR and TLR activation. Based on these properties, we demonstrated that the probiotic yeast Sb has a beneficial effect on bone loss in osteoporotic mice by restoring gut barrier integrity and reducing osteoclastogenic CD4⁺ T cells thereby reducing indirectly i-OCLs. Moreover, it also directly targets i-OCL progenitors to interfere with their differentiation.

These insights open new interesting perspectives for the treatment of pathological bone resorption by demonstrating that the Ig-like receptor costimulatory pathway and the associated PRR pathway, rather than the Rank pathway, could represent efficient therapeutic targets to specifically impact on inflammatory osteoclastogenesis while maintaining physiological OCL resorption. The dose of Sb used in mice is much higher than that recommended for humans. However, this is also true in studies evaluating probiotic lactobacillus strains in osteoporosis that have shown protective effects at much lower doses in humans than in mice (Britton et al., 2014; Nilsson et al., 2018; Jansson et al., 2019). Therefore, Sb administration in osteoporotic patients would represent a feasible possibility. These novel insights and regulatory mechanisms mediated by yeast probiotics could provide new therapeutic options to overcome the global inhibition of OCLs and the resulting impaired bone quality associated with current anti-resorptive therapies.

RNAseq data have been deposited in ENA (European Nucleotide Archive) accession number PRJEB42043.

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Author details

1. Maria-Bernadette Madel

   1. Université Côte d’Azur, CNRS, LP2M, Nice, France
   2. LIA ROPSE, Laboratoire International Associé Université Côte d’Azur - Centre Scientifique de Monaco, Nice and Monaco, France

   Contribution

   Formal analysis, Investigation, Methodology, Validation, Writing – original draft, Writing – review and editing

   Contributed equally with

   Julia Halper

   Competing interests

   No competing interests declared

2. Julia Halper

   1. Université Côte d’Azur, CNRS, LP2M, Nice, France
   2. LIA ROPSE, Laboratoire International Associé Université Côte d’Azur - Centre Scientifique de Monaco, Nice and Monaco, France

   Contribution

   Formal analysis, Investigation, Methodology, Validation, Writing – review and editing

   Contributed equally with

   Maria-Bernadette Madel

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3967-6633

3. Lidia Ibáñez

   Department of Pharmacy, Cardenal Herrera-CEU University, Valencia, Spain

   Contribution

   Formal analysis, Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2217-8817

4. Lozano Claire

   IRMB, Université Montpellier, Montpellier, France

   Contribution

   Formal analysis, Investigation, Supervision

   Competing interests

   No competing interests declared

5. Matthieu Rouleau

   1. Université Côte d’Azur, CNRS, LP2M, Nice, France
   2. LIA ROPSE, Laboratoire International Associé Université Côte d’Azur - Centre Scientifique de Monaco, Nice and Monaco, France

   Contribution

   Formal analysis, Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0075-9880

6. Antoine Boutin

   1. Université Côte d’Azur, CNRS, LP2M, Nice, France
   2. LIA ROPSE, Laboratoire International Associé Université Côte d’Azur - Centre Scientifique de Monaco, Nice and Monaco, France

   Contribution

   Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

7. Adrien Mahler

   1. Université Côte d’Azur, CNRS, LP2M, Nice, France
   2. LIA ROPSE, Laboratoire International Associé Université Côte d’Azur - Centre Scientifique de Monaco, Nice and Monaco, France

   Contribution

   Formal analysis, Funding acquisition

   Competing interests

   No competing interests declared

8. Rodolphe Pontier-Bres

   1. LIA ROPSE, Laboratoire International Associé Université Côte d’Azur - Centre Scientifique de Monaco, Nice and Monaco, France
   2. Centre Scientifique, de Monaco, Monaco

   Contribution

   Formal analysis

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4139-5913

9. Thomas Ciucci

   Laboratory of Immune Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, United States

   Present address

   David H. Smith Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, United States

   Contribution

   Conceptualization, Writing – review and editing, Writing – original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5828-0207

10. Majlinda Topi

    1. Université Côte d’Azur, CNRS, LP2M, Nice, France
    2. LIA ROPSE, Laboratoire International Associé Université Côte d’Azur - Centre Scientifique de Monaco, Nice and Monaco, France

    Contribution

    Formal analysis

    Competing interests

    No competing interests declared

11. Christophe Hue

    Université Paris-Saclay, UVSQ, INSERM, Infection et inflammation, Montigny-Le-Bretonneux, France

    Contribution

    Writing – review and editing

    Competing interests

    No competing interests declared

12. Jerome Amiaud

    Inserm, Universite de Nantes, Nantes, France

    Contribution

    Formal analysis, Investigation

    Competing interests

    No competing interests declared

13. Salvador Iborra

    Department of Immunology, Ophthalmology and ENT. School of Medicine, Universidad Complutense de Madrid, Madrid, Spain

    Contribution

    Writing – review and editing, Validation

    Competing interests

    No competing interests declared

14. David Sancho

    Immunobiology Laboratory, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain

    Contribution

    Writing – review and editing, Investigation

    Competing interests

    No competing interests declared

15. Dominique Heymann

    Université de Nantes, Institut de Cancérologie de l’Ouest, Saint Herblain, France

    Contribution

    Conceptualization, Supervision, Writing – review and editing

    Competing interests

    No competing interests declared

16. Henri-Jean Garchon

    1. Université Paris-Saclay, UVSQ, INSERM, Infection et inflammation, Montigny-Le-Bretonneux, France
    2. Genetics Division, Ambroise Paré Hospital, AP-HP, Boulogne-Billancourt, France

    Contribution

    Formal analysis, Funding acquisition, Supervision, Writing – review and editing, Provided and participated in experiments on CD11c-Syk mutant mice

    Competing interests

    No competing interests declared

17. Dorota Czerucka

    1. LIA ROPSE, Laboratoire International Associé Université Côte d’Azur - Centre Scientifique de Monaco, Nice and Monaco, France
    2. Centre Scientifique, de Monaco, Monaco

    Contribution

    Funding acquisition, Writing – review and editing, Provided and participated in experiments on CD11c-Syk mutant mice

    Competing interests

    received a research grant from Biocodex. Biocodex had no role in the design of the study, in the analysis and interpretation of the data and in the preparation of the manuscript

18. Florence Apparailly

    IRMB, Université Montpellier, Montpellier, France

    Contribution

    Formal analysis, Funding acquisition, Supervision, Writing – review and editing

    Competing interests

    No competing interests declared

19. Isabelle Duroux-Richard

    IRMB, Université Montpellier, Montpellier, France

    Contribution

    Formal analysis, Investigation, Writing – review and editing

    Competing interests

    No competing interests declared

20. Abdelilah Wakkach

    1. Université Côte d’Azur, CNRS, LP2M, Nice, France
    2. LIA ROPSE, Laboratoire International Associé Université Côte d’Azur - Centre Scientifique de Monaco, Nice and Monaco, France

    Contribution

    Conceptualization, Supervision, Methodology, Writing – review and editing

    Contributed equally with

    Claudine Blin-Wakkach

    Competing interests

    No competing interests declared

21. Claudine Blin-Wakkach

    1. Université Côte d’Azur, CNRS, LP2M, Nice, France
    2. LIA ROPSE, Laboratoire International Associé Université Côte d’Azur - Centre Scientifique de Monaco, Nice and Monaco, France

    Contribution

    Conceptualization, Formal analysis, Funding acquisition, Methodology, Project administration, Supervision, Writing – original draft, Writing – review and editing

    Contributed equally with

    Abdelilah Wakkach

    For correspondence

    claudine.blin@univ-cotedazur.fr

    Competing interests

    received a research grant from Biocodex. Biocodex had no role in the design of the study, in the analysis and interpretation of the data and in the preparation of the manuscript

    "This ORCID iD identifies the author of this article:" 0000-0002-2621-3907

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