Chemotherapy activates inflammasomes to cause inflammation-associated bone loss
- Division of Bone and Mineral Diseases, Washington University School of Medicine, United States
- Aclaris Therapeutics, Inc, United States
- Department of Orthopaedic Surgery, Washington University School of Medicine, United States
- Shriners Hospitals for Children, United States
Figures
Figure 1 with 1 supplement
Doxorubicin causes bone loss in male mice.
Femurs from WT male mice were analyzed by VivaCT before (baseline) and 4 weeks after a single intraperitoneal injection of 5 mg/kg doxorubicin. Cross sections of 3D reconstructions. Scale bars: 500 …
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Figure 1—source data 1
Micro-computed tomography (µCT) analysis in Figure 1B.
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig1-data1-v1.xlsx
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Figure 1—source data 2
Tartrate-resistant acidic phosphatase (TRAP) staining analysis in Figure 1D and E.
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig1-data2-v1.xlsx
Figure 1—figure supplement 1
Doxorubicin causes bone loss in female mice.
Femurs from 10-week-old WT female (A, C) and male (B, D) mice were analyzed by VivaCT before (baseline) and 4 weeks after a single i.p. injection of 5 mg/kg doxorubicin (A–C) or vehicle (D). For …
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Figure 1—figure supplement 1—source data 1
Micro-computed tomography (µCT) analysis in Figure 1—figure supplement 1C–D.
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig1-figsupp1-data1-v1.xlsx
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Figure 1—figure supplement 1—source data 2
Dynamic bone parameters analysis in Figure 1—figure supplement 1E.
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig1-figsupp1-data2-v1.xlsx
Figure 2
Doxorubicin causes cytokinemia, leukopenia, release of danger-associated molecular patterns (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) …
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Figure 2—source data 1
Serum samples analysis of inflammatory cytokines, ATP, citrullinated histone 3 (Cit-H3), myeloperoxidase (MPO), and cfDNA in Figure 2A–D and F–J.
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig2-data1-v1.xlsx
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Figure 2—source data 2
Complete blood count in Figure 2E.
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig2-data2-v1.xlsx
Figure 3
Doxorubicin activates inflammasomes and causes macrophage pyroptosis.
WT bone marrow-derived macrophages (BMMs) were left untreated or primed with LPS for 3 hr, then treated with various doxorubicin concentrations for 16 hr. IL-1β (A), LDH (B), and ATP (C) in the …
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Figure 3—source data 1
IL-1β, lactate dehydrogenase (LDH), and ATP analysis of bone marrow-derived macrophages (BMMs) supernatant in Figure 3A–C.
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig3-data1-v1.xlsx
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Figure 3—source data 2
ASC+ cells analysis in Figure 3H.
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig3-data2-v1.xlsx
Figure 4 with 2 supplements
Doxorubicin activates inflammasome-dependent and -independent pathways in macrophages.
WT bone marrow-derived macrophages (BMMs) were left untreated or primed with LPS for 3 hr, then treated with various doxorubicin concentrations for 16 hr. Whole cell lysates were analyzed by …
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Figure 4—source data 1
Original file for the western blot analysis in Figure 4 (NLRP3).
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig4-data1-v1.zip
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Figure 4—source data 2
Original file for the western blot analysis in Figure 4 (AIM2).
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig4-data2-v1.zip
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Figure 4—source data 3
Original file for the western blot analysis in Figure 4 (caspase-1).
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig4-data3-v1.zip
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Figure 4—source data 4
Original file for the western blot analysis in Figure 4 (cleaved caspase-1).
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig4-data4-v1.zip
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Figure 4—source data 5
Original file for the western blot analysis in Figure 4 (caspase-3/cleaved caspase-3).
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig4-data5-v1.zip
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Figure 4—source data 6
Original file for the western blot analysis in Figure 4 (gasdermin D [GSDMD]/cleaved GSDMD).
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig4-data6-v1.zip
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Figure 4—source data 7
Original file for the western blot analysis in Figure 4 (gasdermin D [GSDMD]/cleaved GSDMD [(p10])).
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig4-data7-v1.zip
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Figure 4—source data 8
Original file for the western blot analysis in Figure 4 (GSDME/cleaved GSDME).
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig4-data8-v1.zip
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Figure 4—source data 9
Original file for the western blot analysis in Figure 4 (β-actin).
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig4-data9-v1.zip
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Figure 4—source data 10
Original images of the relevant western blot analysis (NLRP3, AIM2, caspase-1/cleaved caspase-1, caspase-3/cleaved caspase-3, gasdermin D [GSDMD]/cleaved GSDMD, GSDME/cleaved GSDME, and β-actin) with highlighted bands and sample labels in Figure 4.
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig4-data10-v1.zip
Figure 4—figure supplement 1
Cleavage sites of gasdermin D (GSDMD) and GSDME.
Cleavage sites of murine GSDMD by caspase-1 and caspase-3 (A). Cleavage sites of murine GSDME by caspase-3 (B).
Figure 4—figure supplement 2
Doxorubicin activates inflammasome-dependent and -independent pathways in macrophages.
WT bone marrow-derived macrophages (BMMs) were left untreated or primed with lipopolysaccharide (LPS) for 3 hr, then treated with various doxorubicin concentrations for 16 hr. Bar graph represents …
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Figure 4—figure supplement 2—source data 1
Western blot analysis of bone marrow-derived macrophages (BMMs) in Figure 4—figure supplement 2.
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig4-figsupp2-data1-v1.xlsx
Figure 5 with 1 supplement
Doxorubicin activates inflammasome-dependent and -independent pathways in neutrophils.
WT mouse bone marrow neutrophils were left untreated or primed with LPS for 3 hr, then treated with various doxorubicin concentrations for 16 hr. Whole cell lysates were analyzed by immunoblotting. …
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Figure 5—source data 1
Original file for the western blot analysis in Figure 5A (NLRP3).
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig5-data1-v1.zip
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Figure 5—source data 2
Original file for the western blot analysis in Figure 5A (caspase-1/cleaved caspase-1).
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig5-data2-v1.zip
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Figure 5—source data 3
Original file for the western blot analysis in Figure 5A (gasdermin D [GSDMD]/cleaved GSDMD).
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig5-data3-v1.zip
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Figure 5—source data 4
Original file for the western blot analysis in Figure 5A (GSDME/cleaved GSDME).
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig5-data4-v1.zip
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Figure 5—source data 5
Original file for the western blot analysis in Figure 5A (caspase-3/cleaved caspase-3).
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig5-data5-v1.zip
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Figure 5—source data 6
Original file for the western blot analysis in Figure 5A (β-actin).
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig5-data6-v1.zip
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Figure 5—source data 7
Original images for the western blot analysis (NLRP3, caspase-1/cleaved caspase-1, gasdermin D [GSDMD]/cleaved GSDMD, GSDME/cleaved GSDME, caspase-3/cleaved caspase-3, and β-actin) with highlighted bands and sample labels in Figure 5A.
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig5-data7-v1.zip
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Figure 5—source data 8
IL-1β and lactate dehydrogenase (LDH) analysis of bone marrow neutrophils supernatant in Figure 5B and C.
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig5-data8-v1.xlsx
Figure 5—figure supplement 1
Doxorubicin activates inflammasome-dependent and -independent pathways in neutrophils.
Wild-type (WT) neutrophils were left untreated or primed with lipopolysaccharide (LPS) for 3 hr, then treated with various doxorubicin concentrations for 16 hr. Bar graph represents the quantitative …
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Figure 5—figure supplement 1—source data 1
Western blot analysis of bone marrow neutrophils in Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig5-figsupp1-data1-v1.xlsx
Figure 6
Doxorubicin causes NETosis in vitro.
Wild-type (WT) mouse bone marrow neutrophils were left untreated or treated with 10 µM doxorubicin for 16 hr (A). Cit-H3 and MPO were analyzed by immunofluorescence. Scale bars: 50 μm. Images are …
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Figure 6—source data 1
Cell-free DNA (cfDNA) and IL-1β analysis of bone marrow neutrophils supernatant in Figure 6B–C.
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig6-data1-v1.xlsx
Figure 7 with 2 supplements
AIM2 and NLRP3 inflammasomes are involved in bone-damaging effects of doxorubicin.
WT, Aim2-/-, Nlrp3-/-, Aim2-/-;Nlrp3-/- or Casp1-/- bone marrow-derived macrophages (BMMs) (A, B) and neutrophils (C, D) were left untreated or primed with LPS for 3 hr, then exposed or not to 10 µM …
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Figure 7—source data 1
IL-1β and lactate dehydrogenase (LDH) analysis of different genotypes of bone marrow-derived macrophages (BMMs) and bone marrow neutrophils supernatant in Figure 7A–D.
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig7-data1-v1.xlsx
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Figure 7—source data 2
Micro-computed tomography (µCT) analysis of different genotypes of mice in Figure 7F and G.
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig7-data2-v1.xlsx
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Figure 7—source data 3
Tartrate-resistant acidic phosphatase (TRAP) staining analysis of different genotypes of mice in Figure 7H and I.
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig7-data3-v1.xlsx
Figure 7—figure supplement 1
AIM2 and NLRP3 inflammasomes are involved in bone-damaging effects of doxorubicin in male mice.
Femurs from WT, Aim2-/-, Nlrp3-/-, Aim2-/-; Nlrp3-/- or Casp1-/- male mice were analyzed by VivaCT before (baseline) and 4 weeks after a single intraperitoneal injection of 5 mg/kg doxorubicin (A, B)…
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Figure 7—figure supplement 1—source data 1
Micro-computed tomography (µCT) analysis of different genotypes of male mice in Figure 7—figure supplement 1B.
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig7-figsupp1-data1-v1.xlsx
Figure 7—figure supplement 2
AIM2 and NLRP3 inflammasomes are involved in bone-damaging effects of doxorubicin in female mice.
Femurs from WT, Aim2-/-, Nlrp3-/-, Aim2-/-; Nlrp3-/- or Casp1-/- female mice were analyzed by VivaCT before (baseline) and 4 weeks after a single intraperitoneal injection of 5 mg/kg doxorubicin. …
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Figure 7—figure supplement 2—source data 1
Micro-computed tomography (µCT) analysis of different genotypes of female mice in Figure 7—figure supplement 2B–D.
- https://cdn.elifesciences.org/articles/92885/elife-92885-fig7-figsupp2-data1-v1.xlsx
Figure 8
Graphical abstract.
Double line arrows: direct effects of doxorubicin on target cells. Solid line arrows: direct contribution to systemic inflammation. Broken line arrows: indirect contribution to systemic inflammation.
© 2024, BioRender Inc. Figure 8 was created using BioRender, and is published under a CC BY-NC-ND license. Further reproductions must adhere to the terms of this license.
