Research ArticleHEALTH AND MEDICINE

Enhancing the regenerative effectiveness of growth factors by local inhibition of interleukin-1 receptor signaling

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Science Advances  12 Jun 2020:
Vol. 6, no. 24, eaba7602
DOI: 10.1126/sciadv.aba7602
  • Fig. 1 Bone regeneration driven by BMP-2 and PDGF-BB is enhanced in Il1r1−/− mice.

    (A and B) Critical-size calvarial defects (4.5-mm diameter) in wild-type (wt) or Il1r1−/− mice were treated with BMP-2 or PDGF-BB (1 μg) delivered by a fibrin matrix. Eight weeks after treatment, bone regeneration was measured by micro–computed tomography (microCT). Representative calvarial reconstructions (average of the individual samples) are shown in (A). Original defect area is shaded with a dashed outline. Coverage of defects and volume of new bone formed are shown in (B). Data are means ± SEM. n = 6. Student’s t test. **P < 0.01; ***P < 0.001.

  • Fig. 2 IL-1β inhibits the morphogenic activity of BMP-2 and PDGF-BB.

    (A and B) MSCs were cultured in normal medium or osteogenesis induction medium containing BMP-2 and IL-1β. Matrix mineralization was detected with alizarin red after 21 days. Representative wells (2 cm2) are shown in (A). Expression of osteoblast-specific genes was determined by quantitative polymerase chain reaction (PCR). Fold changes in gene expression relative to MSCs cultured in normal medium are shown in (B). Alpl, alkaline phosphatase; Runx2, runt-related transcription factor 2; Ibsp, integrin-binding sialoprotein. n = 4. (C and D) MSCs were seeded for 10 days with PDGF-BB and IL-1β. Representative wells (9 cm2) are shown in (C). Graphs in (D) show CFU-F and average size of colonies. n = 6. (E) MSC proliferation in response to PDGF-BB and IL-1β after 72 hours. n = 6. (F) MSC Transwell migration induced by PDGF-BB and IL-1β (increase over basal migration after 6 hours). n = 6. For all panels, data are means ± SEM. For (B), Student’s t test. For (D) to (F), one-way ANOVA with Bonferroni post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001.

  • Fig. 3 IL-1β makes cells less responsive to BMP-2 and PDGF-BB signaling.

    (A) Phospho-Smad levels in MSCs pretreated with IL-1β or phosphate-buffered saline (PBS) 30 min after BMP-2 stimulation. n = 4. (B to D) MSCs were treated with IL-1β or PBS. Smad1/5/8 levels after 24 hours in (B). n = 6. Smurf1 and Smurf2 relative expression in (C). n = 3. Smurf2 levels after 24 hours in (D). n = 4. (E) MSC matrix mineralization in response to BMP-2, IL-1β, and heclin (21 days, representative wells of 2 cm2). (F) Smad1/5/8 levels in MSCs 24 hours after stimulation with IL-1β and heclin. n = 6. (G) Phospho-Akt (S473, solid squares/circles, left y axis) and total Akt (open squares/circles, right y axis) in IL-1β–conditioned MSCs stimulated with PDGF-BB. n = 4. (H and I) MSCs were incubated with IL-1β. Phlpp1 and Phlpp2 relative expression in (H). n = 6. PHLPP1 levels after 24 hours in (I). n = 4. (J) MSC proliferation in response to PDGF-BB, IL-1β, and NSC-45586 after 72 hours. n = 6. (K) MSC Transwell migration induced by PDGF-BB, IL-1β, and NSC-45586 (increase over basal migration after 6 hours). n = 6. (L) Phospho-Akt (S473) levels 120 min after PDGF-BB stimulation of MSCs pretreated with NSC-45586 and IL-1β. n = 4. (M and N) SA-β-gal activity assessed with senescence green probe (SGP) against forward scatter area (FSC-A) in MSCs cultured with IL-1β. Representative flow cytometry plots in (M) and median fluorescence intensities in (N). n = 3. For all panels, data are means ± SEM. For (A), (C), (F), (H), and (J) to (L), one-way ANOVA with Bonferroni post hoc test. For (B), (D), (I), and (N), Student’s t test. For (G), two-way ANOVA with Bonferroni post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001. n.s., not significant.

  • Fig. 4 Delivering BMP-2 and PDGF-BB triggers IL-1β release by macrophages.

    (A and B) Calvarial defects were treated with fibrin matrices containing saline (fibrin only), BMP-2, or PDGF-BB (1 μg). Fibrin matrices with bone tissue surrounding the defects were collected at different time points. Graph in (A) shows IL-1β concentration in the harvested samples (IL-1β concentration per milliliter of tissue lysate). n = 3 per time point. Graph in (B) shows IL-1β concentrations measured at day 6 in mice with macrophages (control liposomes) or depleted of macrophages (clodronate liposomes). n = 4. (C) Primary macrophages were treated with BMP-2 or PDGF-BB. IL-1β concentration in the media after 24 hours was measured. n = 4. Statistics are between no treatment (0 ng/ml) and treatments. For all panels, data are means ± SEM. For (A), two-way ANOVA with Bonferroni post hoc test. For (B), Student’s t test. For (C), one-way ANOVA with Bonferroni post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001.

  • Fig. 5 Proposed mechanism by which IL-1R1 signaling desensitizes bone-forming cells to growth factors.

    IL-1R1 signaling in bone-forming cells (MSCs and osteoblasts) stimulates the expression of PHLPPs and Smurf2. Higher levels of PHLPPs promote quicker dephosphorylation of Akt and dampen PDGF-BB signaling. Higher levels of Smurf2 impair the responsiveness of cells to BMP-2 by promoting ubiquitination and thus degradation of Smad1/5/8. IL-1R1 signaling also accelerates senescence likely via Smurf2. BMP-2 and PDGF-BB further stimulate IL-1β release by macrophages. Phosphorylation is indicated by small circles with the letter P.

  • Fig. 6 Super-affinity IL-1Ra enhances the regenerative capacity of BMP-2 and PDGF-BB.

    (A) Design of super-affinity PDGF-BB and IL-1Ra. Affinity for ECM is high for BMP-2, medium for PDGF-BB, and low for IL-1Ra. PlGF123–141 is added to the C terminus of PDGF-BB and IL-1Ra to confer super-affinity for ECM. (B) Dissociation constant (Kd) of wild-type and PlGF123–141-fused proteins for fibronectin (Fn), vitronectin (Vn), tenascin-C (Tnc), and fibrinogen (Fg). n = 3. (C) Fibrin matrices containing growth factor and IL-1Ra variants were incubated in buffer (containing or not plasmin) that was changed every day. Graphs show cumulative release of the recombinant proteins. n = 3. (D and E) Calvarial defects (4.5-mm diameter) were treated with growth factor (1 μg) and IL-1Ra (1 μg) variants delivered with fibrin. Regeneration was measured by microCT after 8 weeks. Representative calvarial reconstructions (average of the individual samples) in (D). Original defect area is shaded with a dashed outline. Defect coverage and new bone volume in (E). n = 6. (F and G) Femoral defects (2 mm wide, dashed boxes) were treated with growth factors (1 μg) and IL-1Ra/PlGF123–141 (1 μg) in fibrin. Regeneration was measured by microCT after 12 weeks. Representative femur reconstructions in (F). New bone volume formed in (G). n = 5. For all panels, data are means ± SEM. For (B), (E) (BMP-2 groups), and (G), Student’s t test. For (C), two-way ANOVA with Bonferroni post hoc test. For (E), one-way ANOVA with Bonferroni post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001.

Supplementary Materials

  • Supplementary Materials

    Enhancing the regenerative effectiveness of growth factors by local inhibition of interleukin-1 receptor signaling

    Ziad Julier, Rezvan Karami, Bhavana Nayer, Yen-Zhen Lu, Anthony J. Park, Kenta Maruyama, Gisela A. Kuhn, Ralph Müller, Shizuo Akira, Mikaël M. Martino

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