Long-term αLy6G treatment significantly lowers marrow PreNeus in both control and Dmp1Cre.Socs3^f/f mice; PreNeus inhibit osteoclast formation in vitro; summary of findings and model of action. Shown are PreNeus (CD11b⁺Gr1⁺CXCR4^hic-kit^int) (A), immature neutrophils CD11b⁺Gr1⁺CXCR4^loc-kit^loCXCR2⁻) (B), and mature neutrophils (CD11b⁺Gr1⁺CXCR4^loc-kit^loCXCR2⁺) (C), each as a percentage of BM cells in control and Dmp1Cre.Socs3^f/f mice treated with IgG or αLy6G treatment for 6 weeks, as shown in Figure 4A. Data are mean ± SEM, with individual data for each animal shown; 2-way ANOVA results are shown below each graph including the interaction between both factors (genotype and treatment), with P values from Šidák post hoc tests of treatment effect shown above each genotype group. (D) Schematic of in vitro experiments. PreNeus, purified by FACS, from C57BL/6 mice were added to RAW264.7 cells differentiated to osteoclasts by addition of recombinant RANKL, for 1, 2, or 3 days at 3 × 10⁴ (+) or 6 × 10⁴ (++) cells per well. (E) Osteoclast numbers (tartrate-resistant acid phosphatase–positive [TRAP⁺] multinucleated cells) from experiment outlined in panel A. Data are mean + standard deviation of quadruplicate wells from a representative experiment, repeated 3 times. (F-G) Osteoclast numbers (TRAP⁺ multinucleated cells) (F) and representative images (G) of RAW264.7 cells differentiated to osteoclasts with RANKL, with addition of PreNeus for 2 days, as outlined in panel A, with and without the introduction of a Transwell to prevent direct cell-to-cell contact. (H) Summary of results from this and previous work, showing stage-specific effects of interventions on marrow neutrophil progenitors and osteoclasts in the presence and absence of Socs3 deletion. (I) Proposed model of action. RANK-expressing osteoclast progenitors extravasate from the marrow vasculature to the bone microenvironment where they differentiate into osteoclasts upon interaction with RANKL-expressing cells, possibly including CXCR12⁺ Adipo-CAR and Osteo-CAR cells. PreNeus, which cluster in this environment, inhibit osteoclast differentiation by release of soluble factors, such as LCN2, LTF, inhibin A or Hp, that act on osteoclast progenitors following the initial RANK-RANKL interaction. PreNeu, preneutrophil. Adipo-CAR cells, adiponectin-expressing Cxcl12-abundant reticular cells; Osteo-CAR cells, osteogenic marker-expressing Cxcl12-abundant reticular cells.