Bone marrow Adipoq-lineage progenitors are a major cellular source of M-CSF that dominates bone marrow macrophage development, osteoclastogenesis, and bone mass

  1. Kazuki Inoue
  2. Yongli Qin
  3. Yuhan Xia
  4. Jie Han
  5. Ruoxi Yuan
  6. Jun Sun
  7. Ren Xu
  8. Jean X Jiang
  9. Matthew B Greenblatt
  10. Baohong Zhao  Is a corresponding author
  1. Arthritis and Tissue Degeneration Program and David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, United States
  2. Department of Medicine, Weill Cornell Medical College, United States
  3. 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, China
  4. Pathology and Laboratory Medicine, Weill Cornell Medical College, United States
  5. Department of Biochemistry & Structural Biology, University of Texas Health Science Center at San Antonio, United States
  6. Research Institute, Hospital for Special Surgery, United States
  7. Graduate Program in Cell and Development Biology, Weill Cornell Graduate School of Medical Sciences, United States
9 figures, 1 table and 1 additional file

Figures

Integrated analysis of the bone marrow niche datasets of scRNAseq shows that Adipoq-lineage progenitors (Adipoq+ MSPCs) express high level of Csf1.

(A) UMAP plot of the integrated analysis of the bone marrow niche datasets of scRNAseq based on Dolgalev and Tikhonova, 2021. EC, endothelial cell; MSPC: mesenchymal progenitor cell. (B) Dot plot of several typical marker gene expression for bone marrow stromal cells, adipocyte lineage, osteoblast lineage and endothelial cells across the listed scRNAseq clusters. Cell clusters are listed on the y-axis. Features are listed along the x-axis. Dot size reflects the percentage of cells in a cluster expressing each gene. Dot color reflects scaled average gene expression level as indicated by the legend. (C) Flowcytometry images and quantification of the bone marrow Adipoq-lineage progenitors (Adipoq+) incorporating BrdU in 10-week-old female Adipoq Cre-mTmG mice. n=5. (D) UMAP plots of the expression of Adipoq (upper left panel), Csf1 (upper right panel) and the co-expression of these two genes (lower left panel) in bone marrow cells. The lower right panel shows a relative expression scale for each gene. (E) Dot plot of Adipoq and Csf1 expression across the listed scRNA-seq clusters. Cell clusters are listed on y-axis. Features are listed along the x-axis. Dot size reflects the percentage of cells in a cluster expressing each gene. Dot color reflects the scaled average gene expression level as indicated by the legend. (F) Violin plots of the expression of Adipoq and Csf1.

Figure 2 with 1 supplement
scRNAseq analysis of human bone marrow unveils the existence of ADIPOQ-lineage progenitors highly expressing CSF1.

(A) UMAP plot analysis of the human bone marrow datasets of scRNAseq based on Wang et al., 2021. (B) Dot plot of several typical marker gene expression for bone marrow stromal cells, adipocyte lineage, osteoblast lineage and endothelial cells across the listed scRNA-seq clusters. Cell clusters are listed on the y-axis. Features are listed along the x-axis. Dot size reflects the percentage of cells in a cluster expressing each gene. Dot color reflects scaled average gene expression level as indicated by the legend. (C) UMAP plots of the expression of ADIPOQ (upper left panel), CSF1 (upper right panel) and the co-expression of these two genes (lower left panel) in bone marrow cells. The lower right panel shows a relative expression scale for each gene. (D) Dot plot of ADIPOQ and CSF1 expression across the listed scRNAseq clusters. Cell clusters are listed on y-axis. Features are listed along the x-axis. Dot size reflects the percentage of cells in a cluster expressing each gene. Dot color reflects the scaled average gene expression level as indicated by the legend. (E) Violin plots of the expression of ADIPOQ and CSF1.

Figure 2—figure supplement 1
Heatmap of ADIPOQ and CSF1 gene expression by LogFC (fold changes) values across the cell clusters based on human bone marrow scRNAseq dataset (Li et al., 2022).
Figure 3 with 1 supplement
M-CSF is mainly produced by the bone marrow Adipoq-lineage progenitors, but not by mature adipocytes in peripheral adipose or in bone marrow.

(A) qPCR analysis of Csf1 and Lpl expression in bone marrow Adipoq-linage progenitors that were sorted from the bone marrow of Adipoq Cre-mTmG reporter mice, mature bone marrow adipocytes (BMAd), mature peripheral white and brown adipocytes and white stromal vascular fraction (SVF). E-adipocyte: mature adipocytes isolated from the epididymal white adipose tissue. I-adipocyte: mature adipocytes isolated from inguinal white adipose tissue. B-adipocyte: Brown adipocytes. n=5 for E-, I- and B-adipocytes from 12-week-old male mice. Five replicates, each with a pooled sample from 12-week-old male mice for BMAd (6–7 mice) and bone marrow Adipoq-lineage progenitors (3–4 mice). Error bars: Data are mean ± SD. ****p<0.0001 by one-way ANOVA analysis followed by post hoc Bonferroni’s correction for multiple comparisons. (B) Immunofluorescence staining of M-CSF (purple) on femur bone slices from 12-week-old male Adipoq Cre-mTmG reporter mice. DAPI: blue. Arrows: co-localization of M-CSF and Adipoq-GFP in GFP +Adipoq-lineage progenitors. n=3 mice. (C) Immunofluorescence staining of M-CSF (purple) and Perilipin1 (green, mature adipocyte marker) on femur bone slices from 12-week-old male mice. n=3. (D) Immunoblot analysis of M-CSF and Adiponectin expression in mature adipocytes and stromal vascular fraction (SVF) in peripheral adipose. (E) epididymal white adipose tissue. (I) the inguinal white adipose tissue. p38 was used as a loading control.

Figure 3—source data 1

M-CSF is mainly produced by the bone marrow Adipoq-lineage progenitors, but not by mature adipocytes in peripheral adipose or in bone marrow.

https://cdn.elifesciences.org/articles/82118/elife-82118-fig3-data1-v2.zip
Figure 3—figure supplement 1
qPCR analysis of Csf1 expression relative to the geometric mean (Vandesompele et al., 2002) of Gapdh and Actb in bone marrow Adipoq-linage progenitors that were sorted from the bone marrow of Adipoq Cre-mTmG reporter mice, mature bone marrow adipocytes (BMAd), mature peripheral white and brown adipocytes.

E-adipocyte: mature adipocytes isolated from the epididymal white adipose tissue. I-adipocyte: mature adipocytes isolated from inguinal white adipose tissue. B-adipocyte: Brown adipocytes. n=5 for E-, I- and B-adipocytes from 12-week-old male mice. Five replicates, each with a pooled sample from 12-week-old male mice for BMAd (6–7 mice) and bone marrow Adipoq-lineage progenitors (3–4 mice). Error bars: Data are mean ± SD. ****p<0.0001.

Figure 3—figure supplement 1—source data 1

qPCR analysis of Csf1 expression relative to the geometric mean (Vandesompele et al., 2002) of Gapdh and Actb in bone marrow Adipoq-linage progenitors that were sorted from the bone marrow of Adipoq Cre-mTmG reporter mice, mature bone marrow adipocytes (BMAd), mature peripheral white and brown adipocytes.

https://cdn.elifesciences.org/articles/82118/elife-82118-fig3-figsupp1-data1-v2.zip
Figure 4 with 6 supplements
Csf1 deficiency in Csf1ΔAdipoq mice increases bone mass.

(A) Csf1 expression in BMSCs derived from Csf f/f and Csf1ΔAdipoq (n = 4/group). (B) Representative images of immunostaining of Adiponectin (red) and M-CSF (white) on femur bone slices from 12-week-old male Csf f/f and Csf1ΔAdipoq mice. DAPI: blue. n=3/group. (C) Gross appearance of the incisors from Csf f/f and Csf1ΔAdipoq mice. (D) Gross appearance of the femur (left panel), and the lengths of femur and tibia from Csf f/f and Csf1ΔAdipoq mice (right panels) (n = 6/group). (E) μCT images and (F) bone morphometric analysis of trabecular bone of the distal femurs isolated from 12-week-old male Csf f/f and Csf1ΔAdipoq mice (n = 5/group). (G) μCT images and bone morphometric analysis of cortical bone of the mid-shaft femurs isolated from 12-week-old male Csf f/f and Csf1ΔAdipoq mice (n = 5/group). BM, bone marrow; BMSC, bone marrow stromal cell; BV/TV, bone volume per tissue volume; BMD, bone mineral density; Conn-Dens, connectivity density; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation; Tb.N, trabecular number. Ct.Th, cortical bone thickness; BA/TA: Bone area/Tissue area. A, D, F, G *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns: not statistically significant by two tailed unpaired Student’s t test. Error bars: Data are mean ± SD. Scale bars: B, 50 µm; E, 500 µm; G, 500 μm.

Figure 4—source data 1

Csf1 deficiency in Csf1ΔAdipoq mice increases bone mass.

https://cdn.elifesciences.org/articles/82118/elife-82118-fig4-data1-v2.zip
Figure 4—figure supplement 1
The flowcytometry images (left) and the percentage (right) of Adipoq-lineage progenitors (GFP+) in bone marrow (without red blood cells) from 12-week-old male mice.

n=3. Data are mean ± SD.

Figure 4—figure supplement 1—source data 1

The percentage of Adipoq-lineage progenitors (GFP+) in bone marrow (without red blood cells).

https://cdn.elifesciences.org/articles/82118/elife-82118-fig4-figsupp1-data1-v2.xlsx
Figure 4—figure supplement 2
qPCR analysis of Csf1 expression in SVF from 12-week-old male Csf f/f and Csf1ΔAdipoq mice (n = 3 for Csf f/f and n=2 for Csf1ΔAdipoq mice).
Figure 4—figure supplement 2—source data 1

qPCR analysis of Csf1 expression in SVF.

https://cdn.elifesciences.org/articles/82118/elife-82118-fig4-figsupp2-data1-v2.xlsx
Figure 4—figure supplement 3
qPCR analysis of cytokines expressed in bone barrow from 12-week-old male Csf f/f and Csf1ΔAdipoq mice (n = 3/group; For Il34, Csf f/f n=4, Csf1ΔAdipoq n=6).
Figure 4—figure supplement 3—source data 1

qPCR analysis of cytokines expressed in bone barrow.

https://cdn.elifesciences.org/articles/82118/elife-82118-fig4-figsupp3-data1-v2.xlsx
Figure 4—figure supplement 4
Gross appearance and body weight of 12-week-old male Csf f/f and Csf1ΔAdipoq mice (n = 6/group).

ns: not statistically significant by two tailed unpaired Student’s t test. Data are mean ± SD.

Figure 4—figure supplement 4—source data 1

Body weight of 12-week-old male Csf f/f and Csf1ΔAdipoq mice.

https://cdn.elifesciences.org/articles/82118/elife-82118-fig4-figsupp4-data1-v2.xlsx
Figure 4—figure supplement 5
Bone morphometric analysis of trabecular bone of the distal femurs isolated from 12-week-old male Csf f/f and Csf1f/+ΔAdipoq mice (n = 5/group).

BV/TV, bone volume per tissue volume; BMD, bone mineral density; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation; Conn-Dens., connectivity density. Data are mean ± SD. n.s., not statistically significant by two tailed unpaired Student’s t test analysis.

Figure 4—figure supplement 5—source data 1

Bone morphometric analysis of trabecular bone of distal femurs.

https://cdn.elifesciences.org/articles/82118/elife-82118-fig4-figsupp5-data1-v2.xlsx
Figure 4—figure supplement 6
μCT images (A) and bone morphometric analysis (B) of lumbar vertebrae (L5) isolated from 12-week-old male Csf f/f and Csf1ΔAdipoq mice (n = 5/group).

BV/TV, bone volume per tissue volume; BMD, bone mineral density; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation; Conn-Dens., connectivity density. Data are mean ± SD. n.s., not statistically significant by two tailed unpaired Student’s t test analysis.

Figure 4—figure supplement 6—source data 1

Bone morphometric analysis of lumbar vertebrae (L5).

https://cdn.elifesciences.org/articles/82118/elife-82118-fig4-figsupp6-data1-v2.xlsx
Figure 5 with 2 supplements
Csf1 deficiency in Csf1ΔAdipoq mice suppresses the populations of bone marrow macrophages and osteoclasts.

(A) TRAP staining and histomorphometric analysis of histological sections obtained from the metaphysis region of distal femurs of 12-week-old male Csf f/f and Csf1ΔAdipoq mice (n = 5/group). (B) ELISA analysis of serum TRAP levels in 12-week-old male Csf f/f and Csf1ΔAdipoq mice (n = 6/group). (C) Flowcytometry image (left) and quantification (right) of monocytes and macrophages in bone marrow. n=6/group. (D, E) Osteoclast differentiation directly from the cultures of bone marrows harvested from Csf f/f and Csf1ΔAdipoq mice stimulated with RANKL (40 ng/ml) but without recombinant M-CSF for ten days (D) or with both RANKL and recombinant M-CSF (20 ng/ml) for five days (E). TRAP staining (left panel) was performed and the area of TRAP-positive MNCs (≥3 nuclei/cell) per well was calculated (right panel). TRAP-positive cells appear red in the photographs. (n = 3/group). Oc.S/BS, osteoclast surface per bone surface; N.Oc/B.Pm, number of osteoclasts per bone perimeter. (A, B), C, D, E **p<0.01; ***p<0.001; ****p<0.0001; ns: not statistically significant by two tailed unpaired Student’s t test. Error bars: Data are mean ± SD. Scale bars: A, 100 µm; D, E, 200 µm.

Figure 5—source data 1

Csf1 deficiency in Csf1ΔAdipoq mice suppresses the populations of bone marrow macrophages and osteoclasts.

https://cdn.elifesciences.org/articles/82118/elife-82118-fig5-data1-v2.zip
Figure 5—figure supplement 1
Osteoblastic function in Csf1ΔAdipoq mice is normal.

(A) Images of calcein double labelling of the femur of 12-week-old male Csf f/f and Csf1ΔAdipoq mice. Dynamic histomorphometric analysis of trabecular bones (B) and cortical bones (C) of femurs isolated from 12-week-old male Csf f/f and Csf1ΔAdipoq mice (n = 5/group). BFR/BS, bone formation rate per bone surface; MAR, mineral apposition rate. Data are mean ± SD. ns: not statistically significant by two-tailed unpaired Student’s t test. Scale bars: A, 100 µm.

Figure 5—figure supplement 1—source data 1

Dynamic histomorphometric analysis of trabecular bones (B) and cortical bones (C) of femurs.

https://cdn.elifesciences.org/articles/82118/elife-82118-fig5-figsupp1-data1-v2.xlsx
Figure 5—figure supplement 2
Inflammatory response to LPS is not altered in Csf1ΔAdipoq BMMs.

(A) qPCR analysis of inflammatory gene expression and (B) Immunoblot analysis of the activation of signaling pathways in response to LPS stimulation (10 ng/ml) in BMMs derived from the Csf f/f and Csf1ΔAdipoq mice. Data are mean ± SD. ns: not statistically significant by two-way ANOVA.

Figure 5—figure supplement 2—source data 1

(A) qPCR analysis of inflammatory gene expression and (B) Immunoblot analysis of the activation of signaling pathways in response to LPS stimulation (10 ng/ml) in BMMs.

https://cdn.elifesciences.org/articles/82118/elife-82118-fig5-figsupp2-data1-v2.xlsx
Csf1 deficiency in Csf1ΔAdipoq mice does not affect monocyte and macrophage populations in spleen and peripheral adiposes.

(A) Gross appearance (left panel) and weight (right panel) of the spleen from Csf f/f and Csf1ΔAdipoq mice (n = 6/group). (B) Gross appearance and weight of the inguinal (left panels) and the epididymal (right panels) adipose from Csf f/f and Csf1ΔAdipoq mice (n = 6/group). (C) Flowcytometry quantification of monocytes and macrophages (gated on CD45 +Ly6G- cells) in the indicated tissues. n=6/group. (A, B, C) ns: not statistically significant by two tailed unpaired Student’s t test. Error bars: Data are mean ± SD.

Figure 6—source data 1

Csf1 deficiency in Csf1ΔAdipoq mice does not affect monocyte and macrophage populations in spleen and peripheral adiposes.

https://cdn.elifesciences.org/articles/82118/elife-82118-fig6-data1-v2.zip
Figure 7 with 1 supplement
Csf1 deficiency in Csf1ΔAdipoq mice protects bone in OVX model.

12-week-old female Csf f/f and Csf1ΔAdipoq mice were subjected to OVX or sham surgery and analyzed 6 weeks after surgery. (A) Gross appearance (left panel) and weight (right panel) of uterus, (B) μCT images, and (C) bone morphometric analysis of trabecular bone of the distal femurs isolated from the Csf f/f and Csf1ΔAdipoq mice with sham or OVX surgery (n = 7/group). (D) TRAP staining (left panels) and histomorphometric analysis (right panels) of histological sections obtained from the metaphysis region of distal femurs isolated from the indicated mice (n = 7/group). BV/TV, bone volume per tissue volume; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation; Conn-Dens., connectivity density; Ct.Th, cortical thickness; Oc.S/BS, osteoclast surface per bone surface; N.Oc/B.Pm, number of osteoclasts per bone perimeter. Data are mean ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; n.s., not statistically significant by two-way ANOVA analysis followed by post hoc Bonferroni’s correction for multiple comparisons. Scale bars: B, 500 µm; D, 100 µm.

Figure 7—source data 1

Csf1 deficiency in Csf1ΔAdipoq mice protects bone in OVX model.

https://cdn.elifesciences.org/articles/82118/elife-82118-fig7-data1-v2.zip
Figure 7—figure supplement 1
qPCR analysis of Csf1 expression in the bone marrow from the Sham and OVX mice.

Sham n=3, OVX n=4. Data are mean ± SD. ns: not statistically significant by two-tailed unpaired Student’s t test.

Figure 7—figure supplement 1—source data 1

qPCR analysis of Csf1 expression in the bone marrow from the Sham and OVX mice.

https://cdn.elifesciences.org/articles/82118/elife-82118-fig7-figsupp1-data1-v2.zip
Author response image 1
Author response image 2

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (M. musculus)Csf1flox/floxPMID:21958845RRID:MGI:5305712
Genetic reagent (M. musculus)Rosa26mT/mGPMID:17868096RRID:IMSR_JAX:007676
Genetic reagent (M. musculus)Adipoq-CrePMID:21356515RRID:IMSR_JAX:028020
AntibodyAnti-mouse CD45-PerCP/Cyanine5.5 (Rat monoclonal)BioLegendCat# 103132
RRID: AB_893340
FACS (1:200)
AntibodyAnti-mouse Ly-6G- Brilliant Violet 711 (Rat monoclonal)BioLegendCat# 127643
RRID:AB_2565971
FACS (1:200)
AntibodyAnti-mouse/human CD11b- Alexa Fluor 700 (Rat monoclonal)BioLegendCat# 101222
RRID:AB_493705
FACS (1:200)
AntibodyAnti-mouse F4/80- APC/Cyanine7 (Rat monoclonal)BioLegendCat# 123118
RRID:AB_893477
FACS (1:200)
AntibodyAnti-mouse Ly-6C- Brilliant Violet 510 (Rat monoclonal)BioLegendCat# 128033
RRID:AB_2562351
FACS (1:200)
AntibodyAnti-mouse CD45- APC (Rat monoclonal)BioLegendCat# 103112
RRID:AB_312977
FACS (1:200)
AntibodyAnti-mouse CD31-APC (Rat monoclonal)BioLegendCat# 102510
RRID:AB_312917
FACS (1:200)
AntibodyAnti-mouse TER-119 /Erythroid Cells- APC (Rat monoclonal)BioLegendCat# 116212
RRID:AB_313713
FACS (1:200)
AntibodyAnti-BrdU-APC (Mouse monoclonal)BioLegendCat# 364114
RRID:AB_2814315
FACS (5 ug per test)
AntibodyMouse IgG1, κ Isotype-APC (Mouse monoclonal)BioLegendCat# 400119
RRID:AB_2888687
FACS (5 ug per test)
AntibodyAnti-Adiponectin (Rabbit polyclonal)Thermo Fisher ScientificCat# PA1-054
RRID:AB_325789
IF(1:200)
WB(1:1000)
AntibodyAnti-Mouse IgG-
Alexa Fluor 647 (Goat polyclonal)
Thermo Fisher ScientificCat# A-21235
RRID:AB_2535804
IF(1:2000)
AntibodyAnti-Rabbit IgG-
Alexa Fluor 594 (Goat polyclonal)
Thermo Fisher ScientificCat# A-11012
RRID:AB_2534079
IF(1:2000)
AntibodyAnti-M-CSF (Mouse monoclonal)Santa Cruz BiotechnologyCat# sc-365779
RRID:AB_10846852
IF(1:200)
WB(1:1000)
AntibodyAnti-Perilipin (Rabbit monoclonal)Cell Signaling TechnologyCat# 9349
RRID:AB_10829911
IF(1:100)
AntibodyAnti-P38alpha (Rabbit polyclonal)Santa Cruz BiotechnologyCat# sc-535
RRID:AB_632138
WB(1:1000)
AntibodyAnti-Phospho-NF-κB p65 (Ser536)
(Rabbit monoclonal)
Cell Signaling TechnologyCat# 3033
RRID:AB_331284
WB(1:1000)
AntibodyAnti-p44/42 MAP kinase (Rabbit polyclonal)Cell Signaling TechnologyCat# 9101
RRID:AB_331646
WB(1:1000)
AntibodyAnti-Phospho-SAPK/JNK (Thr183/Tyr185) (Rabbit polyclonal)Cell Signaling TechnologyCat# 9251
RRID:AB_331659
WB(1:1000)
Commercial assay or kiteBioscience BrdU Staining BufferThermo Fisher ScientificCat#: 00-5525-00
Peptide, recombinant proteinRecombinant Human sRANK LigandPeproTechCat# 310–0140 ng/mL
Peptide, recombinant proteinMurine M-CSFPeproTech315–0220 ng/ml
Chemical compound, drug5-Bromo-2′-deoxyuridine (Brdu)Sigma AldrichCat#: B5002200 mg/Kg
Commercial assay or kitLIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitationThermo Fisher ScientificL231051:1000
Commercial assay or kitMouse Tartrate Resistant Acid Phosphatase (TRAP) ELISA KitMyBioSource.com.MBS1601167
Software, algorithmSeuratPMID:29608179RRID:SCR_016341https://satijalab.org/seurat/get_started.html
Software, algorithmGraphpad Prism 8GraphPad SoftwareRRID:SCR_002798
Software, algorithmFlowjo V10.7.1FlowjoRRID:SCR_008520https://www.flowjo.com/solutions/flowjo
Software, algorithmZEN (blue edition) version 3.4ZEN (blue edition)RRID:SCR_013672https://www.zeiss.com/microscopy/en/products/software/zeiss-zen.html
OtherDAPI stainBD BiosciencesCat# 564907, RRID:AB_2869624FACS (1 ug/ml)

Additional files

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Kazuki Inoue
  2. Yongli Qin
  3. Yuhan Xia
  4. Jie Han
  5. Ruoxi Yuan
  6. Jun Sun
  7. Ren Xu
  8. Jean X Jiang
  9. Matthew B Greenblatt
  10. Baohong Zhao
(2023)
Bone marrow Adipoq-lineage progenitors are a major cellular source of M-CSF that dominates bone marrow macrophage development, osteoclastogenesis, and bone mass
eLife 12:e82118.
https://doi.org/10.7554/eLife.82118