Doxorubicin causes bone loss in male mice.

(A, B) Femurs from WT male mice were analyzed by VivaCT before (baseline) and 4 weeks after a single intraperitoneal injection of 5 mg/kg doxorubicin. (A) Cross sections of 3D reconstructions. Scale bar: 500 μm. (B) Bone parameters. (C-E) Femurs from WT male mice, analyzed 4 weeks after a single intraperitoneal injection of vehicle or doxorubicin. Specimens were stained for TRAP activity. (C) Representative images. Scale bar: 500 μm. (D) N.Oc/BS. (E) Oc.S/BS. N=5 mice/group. Data are mean ± SEM. Student t test was used. *P < 0.05; **P < 0.01; ***P < 0.001. BMD, bone mineral density; BV/TV, bone volume/total volume; Dox, doxorubicin; N.Oc/BS, OC number/bone surface; Oc.S/BS, OC surface/bone surface; OC, osteoclast; ns, not significant; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation; WT, wild-type.

Doxorubicin causes cytokinemia, leukopenia, release of DAMPs, and NETosis in vivo.

Twelve-week-old WT mice were exposed to a single dose of vehicle or 5 mg/kg doxorubicin. Serum samples were harvested 3 days (A-D) or 2 days (F-J) later and analyzed by MSD (IL-1β, IL-6, and TNF-α) or ELISA (IL-18). (E) Blood was collected for cell counts at the indicated time-points after a single dose of 5 mg/kg doxorubicin injection. cfDNA was measured using Qubit (I) or SYBR green (J). Data are mean ± SEM. N=5-12 mice/group. *P < 0.05; **P < 0.01; ***P < 0.001 vs. 0 hour; ##P < 0.01 vs. 2 or 24 hours. Student t test (A-D, F-J) and One-way ANOVA (E) were used. cfDNA, cell free DNA; Cit-H3, citrullinated histone H3; Dox, doxorubicin; IL, interleukin; MPO, myeloperoxidase; WBC, white blood cells.

Doxorubicin activates inflammasomes and causes macrophage pyroptosis.

WT BMMs were left untreated or primed with LPS for 3 hours, then treated with various doxorubicin concentrations for 16 hours. IL-1β (A), LDH (B), and ATP (C) in the conditioned media were measured by ELISA, the cytotoxicity detection Kit or ATP detection Kit, respectively. (D-F) WT BMMs from Asc-citrine mice were primed with 100 ng/ml LPS for 3 hours and treated or not with 15 µM nigericin for 30 minutes or 10 µM doxorubicin for 16 hours. (G) Non-primed cells were also treated with 10 µM doxorubicin for 16 hours. Scale bar: 50 µm. ASC specks were visualized under fluorescence microscopy and quantified using imageJ. (H) Quantitative data. Data are mean ± SEM from experimental triplicates and are representative of at least 2 independent experiments. ***P < 0.001 vs. untreated- or LPS-treated cultures. One-way ANOVA. ASC, apoptosis-associated speck-like protein containing a CARD; ATP, adenosine triphosphate; Dox, doxorubicin; IL-1β, interleukin-1β; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; WT, wild-type.

Doxorubicin activates inflammasome –dependent and –independent pathways in macrophages.

WT BMMs were left untreated or primed with LPS for 3 hours, then treated with various doxorubicin concentrations for 16 hours. Whole cell lysates were analyzed by immunoblotting. Data are representative of at least 3 independent experiments. AIM2, absent in melanoma 2; cCasp, cleaved caspase; cGSDM, cleaved gasdermin; LPS, lipopolysaccharide; Dox, doxorubicin; WT, wild type.

Doxorubicin activates inflammasome –dependent and –independent pathways in neutrophils.

WT mouse bone marrow neutrophils were left untreated or primed with LPS for 3 hours, then treated with various doxorubicin concentrations for 16 hours. (A) Whole cell lysates were analyzed by immunoblotting. Blots are representative of at least 3 independent experiments. IL-1β (B) and LDH (C) in the conditioned media were measured by ELISA and by the cytotoxicity detection Kit, respectively. Data are mean ± SEM from experimental triplicates and are representative of at least 2 independent experiments. ***P < 0.001 vs. LPS; ##P < 0.01, ###P < 0.001 vs. untreated cultures. One-way ANOVA was used. cCasp, cleaved caspase; cGSDM, cleaved gasdermin; IL-1β, interleukin-1β LDH, lactate dehydrogenase; LPS, lipopolysaccharide; Dox, doxorubicin.

Doxorubicin causes NETosis in vitro.

(A) WT mouse bone marrow neutrophils were left untreated or treated with 10 µM doxorubicin for 16 hours. Cit-H3 and MPO were analyzed by immunofluorescence. Scale bar: 50 μm. Images are representative of at least 3 independent experiments. (B) Neutrophils were left untreated or primed with LPS for 3 hours, then treated with 10 µM doxorubicin for 16 hours. cfDNA in the conditioned medium was extracted and quantified. (C) Neutrophils were left untreated or primed with LPS for 3 hours, then treated with 10 µM doxorubicin and/or DNase I for 16 hours. IL-1β in the conditioned media were measured by ELISA. Data are mean ± SEM from experimental triplicates and are representative of at least 2 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 vs. LPS; #P < 0.05, ###P < 0.001 vs. LPS + Dox. One-way ANOVA was used. Dox, doxorubicin; cfDNA, cell free DNA; Cit-H3, citrullinated histone H3; MPO, myeloperoxidase.

AIM2 and NLRP3 inflammasomes are involved in bone-damaging effects of doxorubicin.

WT, Aim2-/-, Nlrp3-/-, Aim2-/-;Nlrp3-/- or Casp1-/- BMMs (A, B) and neutrophils (C, D) were left untreated or primed with LPS for 3 hours, then exposed or not to 10 µM doxorubicin for 16 hours. IL-1β (A, C) and LDH (B, D) in the conditioned media were measured by ELISA and by the cytotoxicity detection Kit, respectively. (E-G) Femurs from male mice were analyzed by VivaCT before (baseline) and 4 weeks after a single intraperitoneal injection of 5 mg/kg doxorubicin. (E) Cross sections of 3D reconstructions. Scale bar: 500 μm. (F) BV/TV changes. (G) BMD changes. (H, I) Femurs harvested from different genotypes male mice were analyzed 4 weeks after a single intraperitoneal injection of vehicle or doxorubicin. Specimens were stained for TRAP activity. (H) N.Oc/BS. (I) Oc.S/BS. Data are mean ± SEM from experimental triplicates and are representative of at least 2 independent experiments (A-D); n=5-8 mice/group (E-I). Data are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 vs. control, LPS or vehicle; ##P < 0.01, ###P < 0.001 vs. other genotypes or WT treated with Dox. Two-way ANOVA (A-D, H-I) and One-way ANOVA (F-G) were used. AIM2, absent in melanoma 2; BMD bone mineral density; BV/TV, bone volume/total volume; casp1, caspase-1; Cont, control; Dox, doxorubicin; IL-1β, interleukin-1β; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; N.Oc/BS, OC number/bone surface; Oc.S/BS; OC, osteoclast; WT, wild-type.