Osteocyte necrosis triggers osteoclast-mediated bone loss through macrophage-inducible C-type lectin
Darja Andreev, … , Georg Schett, Aline Bozec
Darja Andreev, … , Georg Schett, Aline Bozec
Published August 10, 2020
Citation Information: J Clin Invest. 2020;130(9):4811-4830. https://doi.org/10.1172/JCI134214.
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Research Article Bone biology Immunology

Osteocyte necrosis triggers osteoclast-mediated bone loss through macrophage-inducible C-type lectin

Abstract

Although the control of bone-resorbing osteoclasts through osteocyte-derived RANKL is well defined, little is known about the regulation of osteoclasts by osteocyte death. Indeed, several skeletal diseases, such as bone fracture, osteonecrosis, and inflammation are characterized by excessive osteocyte death. Herein we show that osteoclasts sense damage-associated molecular patterns (DAMPs) released by necrotic osteocytes via macrophage-inducible C-type lectin (Mincle), which induced their differentiation and triggered bone loss. Osteoclasts showed robust Mincle expression upon exposure to necrotic osteocytes in vitro and in vivo. RNA sequencing and metabolic analyses demonstrated that Mincle activation triggers osteoclastogenesis via ITAM-based calcium signaling pathways, skewing osteoclast metabolism toward oxidative phosphorylation. Deletion of Mincle in vivo effectively blocked the activation of osteoclasts after induction of osteocyte death, improved fracture repair, and attenuated inflammation-mediated bone loss. Furthermore, in patients with osteonecrosis, Mincle was highly expressed at skeletal sites of osteocyte death and correlated with strong osteoclastic activity. Taken together, these data point to what we believe is a novel DAMP-mediated process that allows osteoclast activation and bone loss in the context of osteocyte death.

Authors

Darja Andreev, Mengdan Liu, Daniela Weidner, Katerina Kachler, Maria Faas, Anika Grüneboom, Ursula Schlötzer-Schrehardt, Luis E. Muñoz, Ulrike Steffen, Bettina Grötsch, Barbara Killy, Gerhard Krönke, Andreas M. Luebke, Andreas Niemeier, Falk Wehrhan, Roland Lang, Georg Schett, Aline Bozec

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Figure 1

Osteocyte necrosis triggers osteoclastogenesis.

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Osteocyte necrosis triggers osteoclastogenesis. (A) Representative pictu...
(A) Representative pictures of hematoxylin and eosin (H&E) staining of tibial bones showing induced ablation of osteocytes in 9-week-old Dmp1-Cre+/iDTRfl/fl mice compared with Dmp1-Cre–/iDTRfl/fl littermate controls, 4 days after i.p. injection with 100 ng diphtheria toxin (DT) (n = 10/group). White arrows indicate filled lacunae, orange arrows dying osteocytes, and red arrows empty lacunae. Scale bars: 50 μm. (B) Representative micro–computed tomography (μCT) images of Dmp1-Cre+/iDTRfl/fl tibias compared with Dmp1-Cre–/iDTRfl/fl littermate controls and (C) quantification of bone volume per total volume (BV/TV), trabecular number (Tb.N), trabecular separation (Tb.Sp), and trabecular thickness (Tb.Th) (n = 9/group). Scale bar: 1 mm. (D) Representative TRAP staining in tibial sections of the aforementioned 2 groups. Dark blue arrows indicate the purple-stained osteoclasts. Scale bar: 50 μm. (E) Histomorphometric quantification of osteoclast surface per bone surface (Oc.S/BS) and osteoclast number per bone perimeter (Oc.N/B.Pm) in the tibia of the aforementioned 2 groups (n = 7–13/group). (F) Representative images of DMP1-GFP–positive (green) starved osteocytes (24 hours without fetal calf serum, FCS) compared with viable osteocytes (24 hours with FCS), stained for necrosis with PI (red) and Hoechst (blue). Scale bar: 100 μm. (G) Quantification of necrotic osteocytes after serum starvation (24 hours) compared with serum-supplemented controls (n = 10–12/group). (H) Representative pictures and (I) quantification of TRAP-positive polynucleated (≥5 nuclei) WT osteoclasts, supplemented with supernatant from viable (+FCS) or necrotic (–FCS) osteocytes (Ots) (1:2) on day 1 of culture for 24 hours, compared with a nonsupplemented control (n = 9/group). Scale bar: 200 μm. Data are shown as mean ± SD. P values were determined by 2-tailed Student’s t test for single comparisons (C, E, and G) or 1-way ANOVA for multiple comparisons (I).