Macrophage colony-stimulating factor (M-CSF), encoded by the Csf1 gene, is a growth factor that plays a crucial role in the proliferation, differentiation, survival and function of myeloid lineage cells, including monocytes, macrophages, and osteoclasts (Stanley et al., 1997; Ross, 2006). The global absence of Csf1 in the Csf1^op/ Csf1^op mice, which carry an inactivating mutation in the coding region of Csf1, leads to tissue macrophage deficiency, growth retardation and skeletal abnormality. The severe osteopetrosis, shortened long bones and failure of dental eruption in Csf1^op/ Csf1^op mice attest to the essential role of M-CSF for osteoclast generation (Cecchini et al., 1994; Dai et al., 2002; Felix et al., 1990; Wiktor-Jedrzejczak et al., 1982; Wiktor-Jedrzejczak et al., 1990; Yoshida et al., 1990).

Osteoclasts are giant multinucleated cells responsible for bone resorption. They are derived from the monocyte/macrophage lineage and are a specialized terminally differentiated macrophage. Osteoclasts play an important role not only in physiological bone development and remodeling, but also actively contribute to musculoskeletal tissue damage and accelerating the progression of postmenopausal osteoporosis and inflammatory arthritis. Osteoclasts and their progenitors express the M-CSF receptor c-Fms. M-CSF is essential for the entire process of osteoclast differentiation, from the generation of osteoclast precursors to the formation and survival of mature osteoclasts (Ross, 2006; Tanaka et al., 1993; Feng and Teitelbaum, 2013). M-CSF also acts together with β3 integrin to regulate actin remodeling in osteoclasts to enable osteoclasts to spread, migrate, fuse, and form actin rings to facilitate bone resorption (Ross, 2006; Ross and Teitelbaum, 2005; Insogna et al., 1997). These findings underscore the indispensable role of Csf1 in not only monocyte to macrophage development, but also osteoclast differentiation, function and survival, which directly influences bone mass.

M-CSF is expressed by a variety of cells, such as endothelial cells, myoblasts, epithelial cells, and fibroblasts (Cecchini et al., 1994; Schrader et al., 1991; Clinton et al., 1992; Fibbe et al., 1988). This diversity of cellular sources of M-CSF is likely due to the need to support a widely distributed network of tissue-resident macrophages, which require M-CSF for their development, function, and homeostasis. Under physiologic conditions, circulating M-CSF is mainly produced by vascular endothelial cells (Clinton et al., 1992; Roth and Stanley, 1992; Ryan et al., 2001). The role of circulating M-CSF in tissue macrophage support appears to be highly context dependent. For example, macrophages in kidney and liver are highly dependent on circulating M-CSF. Bone marrow resident macrophages, however, appear to not require circulating M-CSF, indicating the importance of local M-CSF produced in bone marrow (Cecchini et al., 1994). Osteocytes embedded in bone express M-CSF that contributes to bone mass maintenance (Werner et al., 2020). Although in vitro studies show that M-CSF is expressed by an osteoblast cell line, calvarial osteoblastic cells and a cloned bone marrow stromal cell line (Tanaka et al., 1993; Elford et al., 1987; Zhao et al., 2002; Naparstek et al., 1986), there is no clear evidence supporting whether osteoblasts express M-CSF in vivo and the contributions of osteoblast-derived M-CSF to macrophage and bone homeostasis in vivo is unclear. Therefore, it is largely unknown what specific bone marrow cellular population mainly produces this critical cytokine.

The rapid evolution of scRNAseq technology provides an opportunity to investigate transcriptomics at the individual cell level. In this study, we utilized bone marrow scRNAseq datasets (Zhong et al., 2020; Wolock et al., 2019; Tikhonova et al., 2020; Baryawno et al., 2019; Baccin et al., 2020; Dolgalev and Tikhonova, 2021), and identified a group of unique bone marrow cells featuring Adipoq expression that highly express Csf1. The level of Csf1 expressed by these bone marrow Adipoq-lineage progenitor cells is much higher than that produced by osteoblast lineage cells. We further demonstrated the functional importance of the Csf1 expressed by bone marrow Adipoq-lineage progenitors in macrophage development, osteoclastogenesis and bone mass maintenance.

M-CSF is indispensable for myeloid lineage development and the differentiation and function of osteoclasts, thus playing a crucial role in the physiology and pathology of the immune and skeletal systems. In this study, we identified bone marrow Adipoq-lineage progenitors as a new cellular source of M-CSF expression, which contributes substantially to the bone marrow macrophage development, physiological bone mass maintenance and pathological bone destruction via controlling osteoclast formation. Bone marrow Adipoq-lineage progenitors also produce RANKL (Yu et al., 2021; Hu et al., 2021), an essential cytokine to induce osteoclast differentiation. These findings collectively highlight the significance of bone marrow Adipoq-lineage progenitors in the regulation of osteoclastogenesis and bone metabolism, as well as the potential translational implications of appropriately targeting this cell population in treating pathologic bone loss.

The osteopetrotic phenotype in long bones is similar between Adipoq Cre-driven Csf1 (our results) and RANKL conditional KO mice (Yu et al., 2021). Interestingly, Adipoq Cre-driven RANKL conditional KO mice also exhibit osteopetrosis in lumbar bone (Yu et al., 2021), while Csf1^∆Adipoq mice do not. The mechanisms underlying this difference are unclear. A possibility is that cells other than Adipoq-expressing cells in lumbar presumably produce sufficient M-CSF or IL34 that could compensate the loss of M-CSF by Adipoq-lineage progenitors. This difference also implicates the presence of distinct cellular compartments and microenvironments between long bones and vertebral bones.

Adipoq is not only highly expressed in the bone marrow Adipoq-lineage progenitors but also in lipid-laden adipocytes in fat tissues (Berry and Rodeheffer, 2013), such as peripheral inguinal and epididymal adipose tissue. However, unlike the wide expression of Adipoq in lipid-laden adipocytes to Adipoq-lineage progenitors in bone marrow, Adipoq is expressed in mature lipid-laden adipocytes but not their progenitors in peripheral adipose tissue (Berry and Rodeheffer, 2013). Additionally, unlike bone marrow Adipoq+ progenitors, these peripheral Adipoq+ cells nearly do not produce M-CSF. The cells producing RANKL usually appear to simultaneously express M-CSF, such as osteocytes and bone marrow Adipoq-lineage progenitors (our findings and Werner et al., 2020; Zhong et al., 2020; Yu et al., 2021; Hu et al., 2021). This is not the case for peripheral Adipoq+ adipocytes, which express neither RANKL (Fan et al., 2017) nor M-CSF (our findings). The unique expression of M-CSF and RANKL by bone marrow Adipoq-lineage progenitors is a feature that distinguishes this cell population from other adipose depots.

Bone marrow macrophages are reduced by approximately half in the Csf1^∆Adipoq mice, but macrophages in other tissues/organs are not significantly affected. These results indicate a strong local regulation of macrophage development by bone marrow Adipoq-lineage progenitors via M-CSF. It was thought that M-CSF secreted by cells in certain tissues could affect M-CSF levels in other tissues/organs via circulation. However, research using Csf1^op/ Csf1^op mice with the restoration of circulating M-CSF shows that some tissues/organs are dependent much more on local than circulating M-CSF, such as bone marrow (Cecchini et al., 1994). Our results support this finding and further demonstrate that Adipoq-lineage progenitors are a crucial cellular source of M-CSF that controls macrophage and osteoclast populations in bone marrow. It is unclear but interesting why macrophage development and osteoclastogenesis in bone marrow is more dependent on local M-CSF. This is presumably a unique mechanism by which bone marrow microenvironment decouples osteoclastogenesis and marrow monocytic development from systemic regulation of tissue-resident macrophages, allowing for marrow homeostasis.

The bone marrow Adipoq-lineage progenitors only constitute approximately 0.08% of bone marrow cells. However, the deficiency of M-CSF produced by this small Adipoq+ cell population leads to around a 50% increase in trabecular bone mass and an over 50% reduction of bone marrow macrophages. These results together with the scRNAseq data in Figure 1 demonstrate that Adipoq-lineage progenitor cells in bone marrow produce substantially more M-CSF than osteoblast lineage cells. Thus, Adipoq-lineage progenitors are a major cellular source of M-CSF for bone marrow. We also observed that the osteopetrotic phenotype in Csf1^∆Adipoq mice is not as strong as Csf1^op/ Csf1^op mice. Literature show that M-CSF expression was found in cultures of osteoblastic cells, an osteocyte cell line and osteocytes (Tanaka et al., 1993; Werner et al., 2020; Elford et al., 1987; Zhao et al., 2002). Despite scRNAseq analysis showing that osteoblasts do not express Csf1, the MSPC-Osteo population does (Figure 1). The possibility of M-CSF expression by osteoblasts also exists, which needs further assessment in vivo in addition to scRNAseq approach. Collectively, the M-CSF produced by MSPC-Osteo cells, osteocytes and potentially osteoblasts could partially compensate for the loss of M-CSF from bone marrow Adipoq-lineage progenitor cells.

M-CSF functions through its receptor c-Fms to exert various biological activities. IL34 was identified later as an additional ligand for c-Fms. Although it also binds to other receptors aside from c-Fms, IL34 has, at least partially, functional redundancy with M-CSF (Lin et al., 2019; Lelios et al., 2020). Unlike M-CSF that is broadly and highly expressed by almost every tissue, IL34 is found highly expressed in brain and skin and plays an important role in the differentiation and maintenance of microglia and Langerhans cells (Lin et al., 2019; Lelios et al., 2020). The vascular endothelial cells in spleen also produce IL34 (Nakamichi et al., 2012). The results from scRNAseq datasets show that IL34 is expressed in bone marrow, largely by Adipoq-lineage progenitors but also moderately by pericytes and slightly by MSPC-Osteo cells (Dolgalev and Tikhonova, 2021). This indicates that Adipoq-lineage progenitors are highly likely a major cellular source of IL34 in bone marrow. Spleen has a reservoir of RANK+/CSF-1R+osteoclast precursors, which can home to bone marrow to differentiate to mature osteoclasts in response to RANKL, IL34 and/or M-CSF (Nakamichi et al., 2012). Il34 expression in bone marrow was not affected in Csf1^∆Adipoq mice. These mechanisms collectively may partially compensate for M-CSF function and alleviate the osteopetrotic and macrophage deficiency phenotype in Csf1^∆Adipoq mice.

Recently, increasing attention has been paid to the question on whether Adipoq-lineage progenitors consist exclusively of adipogenic cells. In addition to Adipoq-lineage progenitors, Adipoq Cre also labels other clusters. However, the expression levels of Adipoq and frequency of Adipoq+ cells in other cell populations are relatively low. For example, the integrated scRNAseq dataset we analyzed shows that Adipoq is expressed at a low level (with scaled mean expression at 0.68, Dolgalev and Tikhonova, 2021) in a small proportion of MSPC-osteo cells (Figure 1), and small amounts (Yu et al., 2021; Jeffery et al., 2022; about 4%) of osteoblasts in 8 or 12-week-old mice are Adipoq-lineage. A recent report found that in 24-week-old mice, about 15–40% of osteoblasts are marked with Adipoq Cre (Jeffery et al., 2022). This raises a few important possibilities that will need to be distinguished in future work. One possibility is that the Adipoq-lineage cells (adipo-CAR cells/MALPs) have minor or latent osteogenic potential that may become more evident under specific conditions, such as in older animals. However, balanced against this is the alternative that Adipoq-cre could primarily target a population of solely adipogenic adipo-CAR cells but that its specificity is imperfect, leading to progressive low levels of deletion in a separate population expressing very low levels of Adipoq, such as osteo-CAR cells. An additional possibility is that the Adipoq-lineage cells may themselves actually be further subdivided into multiple component cell types, including a major adipogenic and a separate minor osteogenic subpopulation. Ultimately, at the root of these issues is that Adipoq cre primarily defines one or possibly more lineages of cells rather than a cell type within those lineages. Therefore, application of further markers to fractionate the adipoq-lineage into its component cell types will be needed to resolve these possibilities, focusing on whether any potential osteogenic activity present can be fractionated away from the primary adipogenic activity present.

Of note, the Adipoq expression level and positive cell proportion are much higher in bone marrow Adipoq lineage progenitors than the levels seen in osteoblast lineage (Figure 1, Figure 2, Zhong et al., 2020; Dolgalev and Tikhonova, 2021; Yu et al., 2021) or endothelial cells in bone marrow (Zhang et al., 2021; Emoto et al., 2022). For example, the MSPC-Adipo cluster (Adipoq-lineage progenitors) has 6441 cells with the highest level (scaled mean expression level at 3.01 per Dolgalev and Tikhonova, 2021 at Single Cell Portal) of Adipoq seen among bone marrow cells analyzed. In contrast, the MSPC-osteo cluster consists of 2247 cells with a very low Adipoq expression level (scaled mean expression level at 0.68 per Dolgalev and Tikhonova, 2021 at Single Cell Portal). Taken together with both average expression level and cell numbers in each cluster, the relative overall contribution to Adipoq expression by MSPC-osteo vs the Adipoq-lineage progenitors is 7.8% ([2247x0.68]/[6441x3.01]). Therefore, the expression of Adipoq in MSPC-osteo cluster is marginal compared to that in the Adipoq-lineage progenitors. These data make Adipoq as an important marker to identify bone marrow Adipoq lineage progenitors. Overall, our work not only validates prior research identifying adipoq-lineage cells, identified as MALPs (Zhong et al., 2020; Yu et al., 2021), as a key osteoclast regulatory population, but also further extends the scope of their functions to encompass M-CSF production and regulation of macrophages.

In addition to its function in development and postnatal homeostasis, M-CSF is a key factor contributing to pathological bone destruction. In OVX induced osteoporosis model, we found that the extent of bone loss in Csf1^∆Adipoq mice was less than that of the control mice, and the bone mass was significantly higher in Csf1^∆Adipoq mice than control mice after OVX. These results indicate a significant role for the M-CSF produced by Adipoq + cells in estrogen-deficiency induced bone loss. Interestingly, however, no genotype by OVX interaction term was seen in two-way ANOVA, even though there is a significant difference in the post-OVX bone mass between genotypes. The difference in baseline bone mass pre-OVX between genotypes could presumably complicate and compromise the significance analysis of genotype by OVX interaction. Further study, for example, using inducible Adipoq-cre mice, is needed to compare whether Adipoq-lineage progenitors-derived M-CSF specifically regulates OVX responses or pathological bone loss induced by other stimuli, or is instead more involved in basal homeostasis. Broad blockade of M-CSF, however, is not clinically feasible because this may suppress macrophage survival and function in a variety of important tissues and organs. Our results show that lack of M-CSF in bone marrow Adipoq-lineage progenitors does not affect macrophage populations outside of the bone marrow. These findings suggest that targeting bone marrow Adipoq-lineage progenitors themselves or the mechanisms regulating their production of M-CSF could be a potential therapeutic strategy to prevent pathological bone destruction.

The current manuscript does not contain sequencing data. The Source Data files for figures have been submitted.

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Author details

1. Kazuki Inoue

   1. Arthritis and Tissue Degeneration Program and David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, United States
   2. Department of Medicine, Weill Cornell Medical College, New York, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Writing – review and editing, Performed most experiments for the first submission

   Contributed equally with

   Yongli Qin and Yuhan Xia

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6305-9374

2. Yongli Qin

   1. Arthritis and Tissue Degeneration Program and David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, United States
   2. Department of Medicine, Weill Cornell Medical College, New York, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Writing – review and editing, Performed flowcytometry

   Contributed equally with

   Kazuki Inoue and Yuhan Xia

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6133-0511

3. Yuhan Xia

   1. Arthritis and Tissue Degeneration Program and David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, United States
   2. Department of Medicine, Weill Cornell Medical College, New York, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Validation, Writing – review and editing, Performed most experiments for the first submission

   Contributed equally with

   Kazuki Inoue and Yongli Qin

   Competing interests

   No competing interests declared

4. Jie Han

   The first Affiliated Hospital of Xiamen University-ICMRS Collaborating Center for Skeletal Stem Cells, State Key Laboratory of Cellular Stress Biology, Faculty of Medicine and Life Sciences, Fujian Provincial Key Laboratory of Organ and Tissue Regeneration, School of Medicine, Xiamen University, Xiamen, China

   Contribution

   Software, Writing – review and editing, Assisted YQ for bioinformatics analysis

   Competing interests

   No competing interests declared

5. Ruoxi Yuan

   1. Arthritis and Tissue Degeneration Program and David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, United States
   2. Department of Medicine, Weill Cornell Medical College, New York, United States

   Contribution

   Software, Writing – review and editing, Assisted YQ for bioinformatics analysis

   Competing interests

   No competing interests declared

6. Jun Sun

   Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, United States

   Contribution

   Methodology, Writing – review and editing, Assisted flowcytometry and cell sorting

   Competing interests

   No competing interests declared

7. Ren Xu

   The first Affiliated Hospital of Xiamen University-ICMRS Collaborating Center for Skeletal Stem Cells, State Key Laboratory of Cellular Stress Biology, Faculty of Medicine and Life Sciences, Fujian Provincial Key Laboratory of Organ and Tissue Regeneration, School of Medicine, Xiamen University, Xiamen, China

   Contribution

   Software, Writing – review and editing, Assisted YQ for bioinformatics analysis

   Competing interests

   No competing interests declared

8. Jean X Jiang

   Department of Biochemistry & Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, United States

   Contribution

   Resources, Funding acquisition, Writing – review and editing

   Competing interests

   Reviewing editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0002-2185-5716

9. Matthew B Greenblatt

   1. Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, United States
   2. Research Institute, Hospital for Special Surgery, New York, United States

   Contribution

   Resources, Funding acquisition, Writing – review and editing

   Competing interests

   No competing interests declared

10. Baohong Zhao

    1. Arthritis and Tissue Degeneration Program and David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, United States
    2. Department of Medicine, Weill Cornell Medical College, New York, United States
    3. Graduate Program in Cell and Development Biology, Weill Cornell Graduate School of Medical Sciences, New York, United States

    Contribution

    Conceptualization, Resources, Supervision, Funding acquisition, Writing - original draft, Writing – review and editing

    For correspondence

    zhaob@hss.edu

    Competing interests

    Reviewing editor, eLife

    "This ORCID iD identifies the author of this article:" 0000-0002-1286-0919

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