IL-32 in EVs induces osteoclast differentiation in vitro and in vivo. (A-B) Preosteoclasts were treated with rhIL-32 (25 ng/mL), RANKL (50 ng/mL), or JJN3-derived EVs (5 μL) for 3 days before the cells were TRAP stained. (Original magnification ×200 in panel A.) Number of osteoclasts was determined by counting TRAP⁺ cells with >2 nuclei. Bars indicate relative number of osteoclasts (mean ± standard error of the mean [SEM]) from 5 independent experiments using peripheral blood mononuclear cells (PBMCs) from 5 different donors. (C-D) EVs (30 μL) from JJN3 cells cultured in normoxia (N) or hypoxia (H), rhIL-32 (3 μg in 30 μL of PBS), and PBS control (30 μL) were injected on top of the calvaria of NOD-SCID mice (n = 8 per group) every day for 5 days. After 3 more days, the mice were euthanized and the calvaria harvested. (Original magnification ×200 in panel C.) Amount TRAP⁺ bone surface (BS) was quantified using NIS-Elements BR software. (E) JJN3 cells in which IL-32 was silenced using CRISPR/Cas9 (KO) or control cells (WT) were cultured in normoxia or hypoxia for 48 hours. Cells and EVs obtained from the culture media were harvested and lysed before immunoblotting as indicated. (F) Preosteoclasts were treated with rhIL-32 (25 ng/mL), RANKL (50 ng/mL), or EVs (5 μL) obtained from JJN3 KO and JJN3 WT cells as indicated for 3 days. Number of osteoclasts was determined by counting TRAP⁺ cells with >2 nuclei. Bars indicate relative number of osteoclasts (mean + SEM) from 6 independent experiments using PBMCs from 6 different donors. (G-H) 100 000 JJN3 WT or JJN3 KO cells were injected intratibially in RAG2/GC KO mice. After 20 days, tibiae were harvested and examined by µCT. Representative images from the 2 groups are shown in panel G. Bars in panel H represents number of osteolytic lesions (mean + SEM). Significance was determined by Student t test. (I) NFκB p65 nuclear localization was examined in preosteoclast serum starved for 3 hours and then treated with EVs isolated from IL-32–expressing JJN3 cells (WT) or from IL-32 KO cells for 1 hour. p65 in red; Dapi nuclear stain in blue. (Original magnification ×400.) (J) Number of cells with NFκB p65 in the nucleus was counted to estimate percentage of nuclear localization. Data were obtained from 4 independent experiments using 4 different PBMC donors. For all experiments, unless otherwise stated, significance was determined by 1-way analysis of variance followed by Fisher’s least significant difference post hoc test. *P < .05, **P < .01, ***P < .001, ****P < .0001. TBS, total bone surface.