Fig 2: Systemic inflammation in TLR9−/− mice plays an important role in osteoclastic bone loss. a, b Rag1−/− BMNCs were cocultured with TLR9−/− (KO) or wildtype (WT) splenocytes and stimulated with RANKL and CSF1 in a transwell system. The OCLs were stained by TRAP (a) and numbers quantified (b). c–j Adoptive transfer of TLR9−/− splenocytes induced bone loss in Rag1−/− mice. c Representative 3D μCT images of femurs from each group. d Trabecular BMD, BV/TV, and Tb. N of femurs and L3 vertebrae in recipient mice (KO_Rag1−/−, n = 10; WT_Rag1−/−, n = 11). e Serum CTX and P1NP. KO_Rag1−/−, n = 7; WT_Rag1−/−, n = 8. f Spleens of KO_Rag1−/− and WT_Rag1−/− mice. g–h Spleen (g) and bone marrow (h) T cell populations analyzed by flow cytometry. n = 4–6 mice per group. The numbers represent the frequencies in total splenic or bone marrow cells. i Serum cytokine levels. KO_Rag1−/−, n = 7; WT_Rag1−/−, n = 8. j–p The bone marrow transplantation experiment. k Trabecular BMD, BV/TV, and Tb. N of femurs and L3 vertebrae in the recipient mice (KOchim, n = 6; WTchim, n = 7). l–m In vitro osteoclastogenesis assay using BMNCs from KOchim and WTchim mice. l TRAP-stained OCLs. m Quantification of OCL numbers. n Circulating CTX and P1NP levels. KOchim, n = 7; WTchim, n = 8. o Serum TNFα, IL6 and RANKL levels. KOchim, n = 7; WTchim, n = 8. p Spleens of KOchim and WTchim mice. Error bars represent the s.d. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000 1 and ns P > 0.05; statistical significance was determined using an unpaired two-tailed t-test

Fig 3: Osteoclast differentiation by conditioned media involves apoptotic bodies and receptor activator of nuclear factor κ-B ligand (RANKL). MLO-Y4 cells were treated with CBE for seven days and conditioned media were harvested and centrifuged to obtain apoptotic body and supernatant fractions. Osteoclast differentiation assays were performed in the presence of the fraction containing the apoptotic bodies (A) or the supernatant fraction (B). Experiments were carried out in the presence or absence of recombinant osteoprotegerin (OPG) to evaluate RANKL involvement. + cells with ≥3 nuclei were counted. Data are expressed as mean ± SD (n = 5) and are representative of three independent experiments performed. ** p < 0.01 *** p < 0.001 one-way ANOVA.

Fig 4: TLR9−/− mice had low bone mass, increased osteoclastogenesis and systemic inflammation. a Representative 3D μCT images of femurs and L3 vertebrae from TLR9−/− (KO) and wildtype (WT) mice. b Trabecular bone microarchitecture of femurs and L3 vertebrae showing bone mineral density (BMD), bone volume per tissue volume (BV/TV), number of trabeculae (Tb. N), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp). KO, n = 8; WT, n = 9. c Representative HE (upper panels) and TRAP (lower panels) stained images of distal femurs from each group. d Circulating CTX and P1NP levels. KO, n = 6; WT, n = 8. e Histomorphometric analysis of bone resorption indices. n = 8 per group. f–h In vitro osteoclastogenesis assay. f TRAP-stained osteoclast like cells (OCLs). g Quantification of number and area of TRAP+ multinucleated OCLs. h qPCR analysis of osteoclast signature genes’ expression at the end of assay. i Western blots showing expression of the indicated molecules in early osteoclast precursors. j–k In vitro osteoclastogenesis of wildtype BMNCs in the presence of TLR9 antagonist. j TRAP-stained OCLs. k Quantification of OCL numbers. l Spleens from 8-week-old male TLR9−/− and wildtype mice. m Levels of TNFα, IFNγ, IL1β, and RANKL in serum and bone marrow supernatants. n = 3 to 13 per group. n Splenic T cell populations analyzed by flow cytometry. n = 6–10 per group. o The bone marrow T cell populations. n = 5–10 per group. The numbers in n and o represent the frequencies in total splenic or bone marrow cells. Eight-week-old sex-matched mice were used in d, e and m–o. One-way ANOVA with Turkey’s multiple comparison tests was conducted in k. In other panels, statistical significance was determined using an unpaired two-tailed t-test. Error bars represent the s.d. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000 1 and ns P > 0.05

Fig 5: There are low levels of RANKL/RANK at the maternal–fetal interface during miscarriage. (a) Immunohistochemistry analysis of RANKL expression in villi and deciduas from women with normal pregnancy (n=12) or miscarriage (n=12) during the first trimester. RANKL expression was localized to the cell membrane and the cytoplasm (arrows) in the deciduas and villi. Original magnification: × 200. (b and c) FCM analysis of the percentage of RANK+ dMφ from women with normal pregnancy (n=11) or miscarriage (n=11) during the first-trimester. (d and e) FCM analysis of the percentage of CD163+, CD206+, CD80+ and CD86+dMφ from women with normal pregnancy (n=11) or miscarriage (n=11) during the first trimester. Normal: normal pregnant women; Miscarriage: SA women. Data are expressed as the mean±S.E.M. **P<0.01 and ***P<0.001 (Student’s t-test)