Fig 1: Infiltrating ATMs in eWAT are related to bone marrow metabolism.a Immunofluorescence staining of macrophages (F4/80) in eWAT after mice were injected with clodronate liposomes (CL) or control liposomes (Ctrl) into bilateral eWAT for 8 weeks. Green, F4/80; blue, DAPI stain for cell nuclei. Scale bar: 75 µm. b Quantification of the F4/80-positive area percentage with immunofluorescence staining from A (n = 5 biologically independent samples). c, d Representative µCT image (c) and quantification (n = 5 biologically independent samples) (d) of proximal tibiae after mice were injected with control liposomes or CL for 8 weeks. Scale bar: 500 µm. e µCT quantification of rBMAT in tibiae after mice were injected with control liposomes or CL for 8 weeks (n = 4 biologically independent samples). f Heatmap summarizing the fold changes in mRNA expression of different biomarkers in eWAT from NFD- and HFD-fed mice at week 12 (n = 4 biologically independent samples). g Relative expression of Spp1 in eWAT (n = 4 biologically independent samples) and iWAT (n = 4 biologically independent samples) over the course of feeding. Images are representative of 3 independent experiments. All data are presented as mean ± SD. Two-way ANOVA with Tukey’s post-hoc test (b, d, e) and two-way ANOVA with Sidak’s post-hoc test (g) were used. Source data are provided as a Source Data file. See also Supplementary Tables 4 and 5.
Fig 2: OPN induces lysosomal-dependent lipid metabolism in BMDMs.a Representative western blots (left) and quantification (right, n = 3 biologically independent samples) of OPN receptor av and ß3 expression in BMDMs and metabolically activated macrophages (MMes) after culture for 4 days in vitro. b Schematic diagram illustrating the co-culture system as indicated below (c). c, d Representative images (c) and quantification (n = 5 biologically independent samples) (d) of crystal violet staining of BMDMs (ctrl) and MMes treated with or without rOPN for 24 h in a Transwell system. Scale bar: 50 µm. e Immunofluorescence images of buoyant BMDMs (F4/80) containing lipid vesicles (Perilipin 1) isolated from bone marrow supernatant at week 12. Scale bar: 15 µm. f FACS analysis of FC + (F4/80+ and CD11b+ , top) and Perilipin1+ (bottom) BMDMs from NFD- (left) and HFD-fed (right) mice. P9, FC + and Perilipin1+ BMDM population from Q2. g Percentage of FC + and Perilipin1+ BMDMs (n = 5 biologically independent samples). h Lactate concentration in bone marrow supernatant (n = 5 biologically independent samples). i Schematic diagram illustrating the co-culture system as described below (j). j Immunofluorescence staining of the co-cultured BMDMs for 4 days in vitro; the eWAT from mice fed the HFD for 8 weeks was placed in the upper chamber; Neu Ab (2.0 µg/ml) was added into the lower chamber. Scale bar: 50 µm. The right-most image is an enlarged view of the field in the white dashed box. Scale bar: 12.5 µm. k Total intracellular ATP concentration (left, co-culture for 0.5 h) and lactate concentration (right, co-culture for 10 h) in BMDMs with the treatments indicated in j (n = 4 biologically independent samples). l Relative expression of different V-ATPase subunits in ctrl (BMDMs), MMes, and MMes+ rOPN after culture for 24 h (n = 3 biologically independent samples). rOPN, 0.5 µg/ml. Images are representative of 3 independent experiments. All data are presented as mean ± SD. Two-tailed Welch’s t-test (a), one-way ANOVA with Tukey’s post-hoc test (d, k), two-way ANOVA with Sidak’s post-hoc test (g, h), and two-way ANOVA with Tukey’s post-hoc test (l) were used. Source data are provided as a Source Data file.
Fig 3: OPN induced IL-6 and MMP production in TNF-stimulated FLSs and IgG from patients with anti-OPN antibody increased their production. A RT-qPCR analysis of inflammatory genes (normalized relative to GAPDH mRNA). FLSs were stimulated with TNF (5 ng/ml) and OPN, cit-OPN, Col II, a-ENO, or Vim for 24 h. Data represent the mean ± SEM of triplicates from one representative experiment of three independent donors. B Proliferation of FLSs. The FLSs were incubated with OPN or cit-OPN for 1 week. The number of cells was quantified using the MTT assay. Results are presented as the mean ± SD from three independent donors. C FLSs were stimulated with TNF (5 ng/ml) and OPN for 24 h. IgG from anti-cit-OPN-positive RA patients or anti-cit-OPN-negative RA patients were added simultaneously. Data represent the mean ± SEM of 10 independent donors. Data are shown as the mean ± SEM. * p < 0.05 and ** p < 0.01 by Tukey–Kramer test (B) or Mann–Whitney’s U test (C)
Fig 4: eWAT-secreted OPN regulates bone resorption.a Representative western blots (left) and quantification (right, n = 3 biologically independent samples) of av and ß3 in BMSCs, BMDMs, and osteoclasts (Oc) in vitro. b Immunohistochemical staining for OPN and cathepsin K (CTSK) in serial sections of tibiae from mice fed for 12 weeks. Scale bar: 50 µm. c Representative western blots (left) and quantification (right, n = 3 biologically independent samples) of the bands of JNK and p-JNK in BMDMs, Oc, and Oc+rOPN after culture for 4 days in vitro. rOPN, 0.5 µg/ml. d Representative images of the osteo-erosion surface (top) and crystal violet staining of the unwashed Osteo Assay surface with or without rOPN treatment under osteoclastogenic induction for 12 days (bottom). Scale bar: 50 µm. e Quantification of the bone resorption area from d (n = 3 biologically independent samples). f Relative expression of Mmp9 in BMDMs with or without rOPN treatment under osteoclastogenic induction for 3 and 6 days, respectively (n = 3 biologically independent samples). g Representative western blots of av, ß3, and MMP9 in the bone marrow of NFD- and HFD-fed mice at week 12. h Immunohistochemical staining of MMP9 from the proximal tibiae of mice after injection of saline or OPN-neutralizing antibody (Neu Ab) into bilateral eWAT for 8 weeks. Scale bar: 100 µm (the bottom row was magnified from the top row, scale bar: 50 µm). i, j Representative µCT images (i) and quantification (n = 5 biologically independent samples) (j) of proximal tibiae after mice were injected with saline or Neu Ab. Scale bar: 500 µm. k, l Representative µCT images (k) and quantification (n = 4 biologically independent samples) (l) of rBMAT of tibiae after mice were injected with saline or Neu Ab. Scale bar: 500 µm. Images are representative of 3 independent experiments. All data are presented as mean ± SD. One-way ANOVA with Tukey’s post-hoc test (a, c), two-tailed Welch’s t-test (e), by two-way ANOVA with Sidak’s post-hoc test (f), and two-way ANOVA with Tukey’s post-hoc test (j, l) were used. Source data are provided as a Source Data file. See also Supplementary Tables 7 and 8.
Fig 5: Positivity of autoantigen in sera of patients with rheumatoid arthritis (RA). A Serum IgG antibodies against osteopontin (OPN), fibronectin (FN), tenascin-C (TNC), α-enolase (α-ENO), bone sialoprotein (BSP), type 2 collagen (Col II), vimentin (Vim), and fibrinogen (Fgn) were quantified by enzyme-linked immunosorbent assay (ELISA). B Antibodies against citrullinated osteopontin (cit-OPN), citrullinated fibronectin (cit-FN), citrullinated tenascin-C (cit-TNC), citrullinated α-enolase (cit-α-ENO), citrullinated bone sialoprotein (cit-BSP), citrullinated type 2 collagen (cit-Col II), citrullinated vimentin (cit-Vim), and citrullinated fibrinogen (cit-Fgn) were tested. Serum samples from 30 RA patients and 7 healthy donors were used. The dashed line indicates the cutoff, defined as the mean plus two standard deviations (SDs), and the positivity of each antibody in RA patients is shown
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