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DEL-1 restrains osteoclastogenesis and inhibits inflammatory bone loss in nonhuman primates

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Science Translational Medicine  30 Sep 2015:
Vol. 7, Issue 307, pp. 307ra155
DOI: 10.1126/scitranslmed.aac5380
  • Fig. 1. DEL-1 is expressed by human and mouse osteoclasts.

    (A) Tissue sections from ligature-induced mouse periodontitis were stained for DEL-1, cathepsin K, and nuclei [4′,6-diamidino-2-phenylindole (DAPI)]. The fluorescence and differential interference contrast (DIC) images were merged and shown alongside TRAP staining of the same section. All images involve the same tissue section, which were processed for immunofluorescence followed by TRAP staining. Scale bars, 50 μm. (B) Undifferentiated mouse OCPs (−RANKL) and RANKL-differentiated osteoclasts (+RANKL) were assayed for expression of the indicated mRNA by real-time reverse transcription polymerase chain reaction (RT-PCR). Data were normalized to those of Gapdh mRNA and expressed as fold change in transcript levels relative to OCPs, which were assigned an average value of 1. (C) DEL-1 expression in whole-cell lysates from mouse (Mo) OCPs (−RANKL) and osteoclasts (+RANKL) detected by immunoblotting. (D) Fluorescent images of RANKL-differentiated mouse osteoclasts stained for DEL-1 and nuclei (DAPI). Scale bars, 100 μm. (E) DEL-1 mRNA (left) and protein (right) expression by undifferentiated human (Hu) CD14+ osteoclast precursors (−RANKL) and RANKL-differentiated osteoclasts (+RANKL) determined by RT-PCR and immunoblotting, respectively. Data were normalized to GAPDH mRNA and expressed as fold change in transcript levels relative to OCPs, which were assigned an average value of 1. β-Actin was used as a loading control. Data in (B) and (E) are means ± SD (B, n = 4; E, n = 3). P values were determined by unpaired t test.

  • Fig. 2. DEL-1 regulates mouse osteoclast differentiation and function.

    (A to C) Mouse RAW264.7 macrophages were stimulated with RANKL and treated with control or Del-1–specific siRNA. (A) Enumeration of TRAP-positive MNCs after 3 days. (B) After 3 days, the cells were assayed for the indicated mRNA. Data were normalized to Gapdh mRNA and presented relative to those of undifferentiated RAW264.7 cells (−RANKL), with the average set as 1. (C) Resorptive pit formation (dark spots in representative images) after culture under osteoclastogenic conditions on Ca3(PO4)2-coated wells. Data are relative to the control siRNA–treated group, set as 1. (D) (Left) RAW264.7 cells were cultured under osteoclastogenic conditions on Ca3(PO4)2-coated wells in the presence of DEL-1–Fc or Fc control (2 μg/ml). (Right) RAW264.7 cells were differentiated to osteoclasts in plastic plates in the absence of DEL-1–Fc, and the mature osteoclasts were transferred to Ca3(PO4)2-coated plates, where they were incubated for 12 hours together with DEL-1–Fc or Fc control (2 μg/ml). In both experiments, the total resorption area in each culture was measured and expressed relative to the control siRNA–treated group, set as 1. (E) Effect of Del-1 siRNA on NFAT response (luciferase activity) of RANKL-stimulated RAW264.7 cells. (F and G) Effect of DEL-1–Fc (2 μg/ml) on Nfatc1 (F) and Bcl6 (G) mRNA expression in RANKL-induced RAW264.7 osteoclasts that were treated with the indicated siRNA. Bcl6 and Nfatc1 expression were determined at 24 and 12 hours, respectively. Data were normalized to Gapdh mRNA and are relative to undifferentiated controls. All data are means ± SD (A, n = 5; B and E, n = 6; C, n = 5 to 8; D, n = 3; F and G, n = 5). P values were determined by one-way analysis of variance (ANOVA) or unpaired t test (for D). NS, not significant.

  • Fig. 3. DEL-1 dose-dependently inhibits mouse and human osteoclastogenesis and resorption pit formation.

    (A to F) RANKL-induced osteoclastogenesis and resorption pit formation were determined in mouse bone marrow–derived OCPs (A to C) and human CD14+ monocytes (D to F) in the presence of increasing concentrations of DEL-1–Fc or Fc control (2 μg/ml). (A and D) Osteoclasts were assayed for mRNA expression of the indicated molecules by RT-PCR. Results were normalized to those of GAPDH mRNA and are presented relative to those of undifferentiated (−RANKL) OCPs, set as 1. (B and E) Cells were stained for TRAP to detect osteoclasts, and TRAP-positive MNCs were counted. Representative images are shown. Scale bars, 200 μm. (C and F) OCPs on Ca3(PO4)2-coated wells were cultured under osteoclastogenic conditions, and resorptive areas (dark spots) were visualized by light microscopy. The total resorbed area in each culture was measured and expressed relative to the Fc control group, set as 1 (left). All data are means ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 compared to Fc control (one-way ANOVA).

  • Fig. 4. DEL-1 components involved in regulation of bone loss and neutrophil transmigration.

    (A) Periodontal bone loss was induced for 5 days in mice by ligating a maxillary second molar and leaving the contralateral tooth unligated (baseline control). The mice were microinjected in the gingiva with the indicated proteins 1 day before placing the ligature and every day thereafter until the day before sacrifice (day 5). DEL-1–Fc and DEL-1[RGE]–Fc (“RGE”), as well as the Fc protein control, were used at 1 μg. DEL-1[E1–E3]–Fc (“E1–3”) and DEL-1[E1–E2]–Fc (“E1–2”) were used at 0.54 and 0.49 μg, respectively (at molar equivalents to the intact DEL-1 protein, 12.3 pmol). These proteins were used also at higher quantities (2.0 and 2.5 μg for E1–3high and E1–2high, respectively) to test them at the same amount of DEL-1 protein (0.63 μg) as present in 1 μg of intact DEL-1–Fc molecule. Change in bone height was measured relative to unligated baseline. Data are means ± SD (n = 8, except for E1–2 and E1–2high with n = 5). **P < 0.01, ***P < 0.001 versus Fc control, unless otherwise indicated, one-way ANOVA. (B) Similar to (A), TRAP-positive MNCs were enumerated from eight random coronal sections of the ligated sites from each mouse. Data are averages ± SD (n = 5 mice, total 40 sections per group). ***P < 0.001 versus Fc control, unless otherwise indicated, one-way ANOVA. (C) Transmigration of human neutrophils through an endothelial cell monolayer (human umbilical vein endothelial cells) toward IL-8 in the absence or presence of the indicated molecules [all used at 5 μg/ml except for anti–LFA-1 mAb and IgG1 isotype control (IC) at 10 μg/ml]. Fc control was set to 100%, and data for other groups are relative to this value. Data are means ± SD (n = 14 to 16 sets of neutrophil cultures from four pooled experiments, except for isotype control, anti–LFA-1, and LFA878 with n = 5). **P < 0.01, ***P < 0.001 compared to Fc control, one-way ANOVA. P values for indicated groups were determined by unpaired t test.

  • Fig. 5. Differential capacities of DEL-1 and LFA-1 antagonist to inhibit bone loss.

    (A) Gingiva were dissected from mice subjected to ligature-induced periodontitis at the indicated times. The amount of myeloperoxidase (MPO) in gingival tissue homogenates was determined by enzyme-linked immunosorbent assay and normalized to total protein. Data are means ± SD (n = 4). (B) Similar bone loss inhibition experiment as in Fig. 4A with the exception that, in addition to the standard daily administration schedule (starting from day −1), the inhibitors were microinjected in other mice starting on day 3, when neutrophil recruitment had already taken place. Bone loss was determined at day 5. Fc control, DEL-1–Fc, and the LFA-1 antagonist LFA878 were used at 2 μg. Change in bone height was measured relative to unligated baseline. (C) mRNA expression at day 5 in dissected gingiva from mice in (B). Data were normalized to Gapdh mRNA and are presented as fold change in the transcript levels in ligated sites relative to those of unligated healthy tissue, set as 1. Data in (B) and (C) are means ± SD (n = 5 mice). *P < 0.05, **P < 0.01, ***P < 0.001 compared to Fc control, one-way ANOVA.

  • Fig. 6. DEL-1–Fc decreases inflammatory clinical parameters and bone loss in NHP model of periodontitis.

    Starting 3 days after initiation of ligature-induced periodontitis in NHPs, DEL-1–Fc or Fc control was injected locally into the maxillary interdental papillae from the first premolar to the second molar (50 μg of each compound per papilla), two times weekly, in opposites sides of the mouth. (A) Clinical parameters of inflammation over time. Data are means ± SD (n = 3 monkeys). *P < 0.05, compared with Fc control (paired t test). (B) At baseline and at week 6, standardized x-ray dental images were taken and the maxillary bone heights (CEJ-ABC distance) were measured at six points (specified in Materials and Methods). Data are the six-site averages for each monkey at baseline and at week 6. For each pair of Fc control and DEL-1–Fc treatments, bone loss was calculated as bone height at baseline minus bone height at 6 weeks (right panel). P value was determined by paired t test. (C) TRAP-positive MNCs (osteoclasts) were enumerated in 10 random sections for each bone biopsy specimen taken between the second premolar and first molar, from Fc control– or DEL-1–Fc–treated sites of each of the three animals. The numbers of osteoclasts were averaged for each Fc control– or DEL-1–Fc–treated specimen. Data are individual sites with means ± SD (n = 3 monkeys, 10 data points per site). P value was determined by paired t test.

  • Fig. 7. Decreased levels of proinflammatory cytokines in the GCF and periodontal tissue of DEL-1–Fc–treated monkeys.

    (A) During clinical examinations in Fig. 6A, GCF was collected from the monkeys to assay cytokine levels. Data are means ± SD (n = 3 monkeys). *P < 0.05, **P < 0.01 compared with Fc control (paired t test). (B and C) Immunohistochemistry of gingival and bone biopsies from Fc control– or DEL-1–Fc–treated sites at week 6. (B) Overlays of DIC and fluorescent images. (C) Overlays of DIC and fluorescent images from an Fc control–treated site stained for RANKL and cathepsin K. All images in (C) involve the same tissue section, which was processed for immunofluorescence followed by TRAP staining. Scale bars, 100 μm.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/307/307ra155/DC1

    Methods

    Fig. S1. Linear structure and functions of the DEL-1 protein.

    Fig. S2. Inhibition of DEL-1 expression by siRNA treatment.

    Fig. S3. β2 integrin expression is inhibited by siRNA treatment.

    Fig. S4. DEL-1 up-regulates BCL6 in human osteoclast precursors undergoing RANKL-induced differentiation.

    Fig. S5. DEL-1 deficiency enhances osteoclastogenesis.

    Fig. S6. Features of DEL-1 involved in regulation of osteoclastogenesis and resorption pit formation.

    Fig. S7. The RGD motif of DEL-1 is involved in its ability to inhibit the resorptive activity of mature osteoclasts.

  • Supplementary Material for:

    DEL-1 restrains osteoclastogenesis and inhibits inflammatory bone loss in nonhuman primates

    Jieun Shin, Tomoki Maekawa, Toshiharu Abe, Evlambia Hajishengallis, Kavita Hosur, Kalyani Pyaram, Ioannis Mitroulis, Triantafyllos Chavakis, George Hajishengallis*

    *Corresponding author. E-mail: geoh{at}upenn.edu

    Published 30 September 2015, Sci. Transl. Med. 7, 307ra155 (2015)
    DOI: 10.1126/scitranslmed.aac5380

    This PDF file includes:

    • Methods
    • Fig. S1. Linear structure and functions of the DEL-1 protein.
    • Fig. S2. Inhibition of DEL-1 expression by siRNA treatment.
    • Fig. S3. β2 integrin expression is inhibited by siRNA treatment.
    • Fig. S4. DEL-1 up-regulates BCL6 in human osteoclast precursors undergoing RANKL-induced differentiation.
    • Fig. S5. DEL-1 deficiency enhances osteoclastogenesis.
    • Fig. S6. Features of DEL-1 involved in regulation of osteoclastogenesis and resorption pit formation.
    • Fig. S7. The RGD motif of DEL-1 is involved in its ability to inhibit the resorptive activity of mature osteoclasts.

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