Fig. 2: Neutrophils inhibit osteoblast differentiation from MPCs and promote osteoclast formation through TGFβ.

a Mesenchymal progenitor cells (MPCs) co-cultured with Ly6G⁺ BM cells from 3- and 21-mon-old mice for 4 d and b ALP⁺ OB area. Mean ± SD (n = 3 biologically independent samples/group). c Active TGFβ1 protein levels in lysates of Ly6G⁺ BM cells magnetically-isolated from 4- and 22-mon-old mice tested using ELISA. Mean ± SD (n = 5 biologically independent male mice/group). d MPCs from 3-mon-old C57 mice co-cultured with TGFβ1⁺ neutrophils (TNs) sorted from BM of 3- and 20-mon-old C57 mice and treated with vehicle or the TGFβ neutralizing Ab, 1D11. ALP staining performed for osteoblast measurement. Mean±SD (n = 4 biologically independent samples/group). e MPCs from TGFβRII^fl/fl (WT) and Prx1^Cre;TGFβRII^fl/fl (cKO) male mice co-cultured with TNs sorted from 20-mon-old C57 mice (A-TN), and ALP⁺ stained osteoblasts. Mean ± SD (n = 4 biologically independent samples/group). f, g Osteoclast precursors from 3-mon-old C57 mice co-cultured with TNs from 20-mon-old mice (A-TN) plus addition of vehicle or 1D11, and TRAP-stained. Bar = 100 μm. Mean ± SD (n = 4 biologically independent samples/group). h–k MPCs from TGFβRII^fl/fl (WT) and Prx1^Cre;TGFβRII^fl/fl (cKO) male mice were implanted subcutaneously into NSG mice along with TNs sorted from 3- (Y) and 24-mon-old (A) mTmG mice. Implants were harvested 1 month later, processed through paraffin, and H&E- and TRAP-stained. G: implanted GelFoam (red). B: newly-formed bone (yellow). h, i Newly generated bone area. Bar = 50 μm. j, k Osteoclast (red) number in newly generated bone. G: implanted GelFoam (blue). B: newly-formed bone (light pink). Bar = 25 μm. Mean ± SD (n = 4 biologically independent male mice/group). Analyses: Student’s two-sided unpaired t test in (c); one-way ANOVA with Tukey’s post hoc test in all others. Source data are provided as a Source data file.