Resolving Macrophages Counter Osteolysis by Anabolic Actions on Bone Cells

Abstract

Progression of inflammatory osteolytic diseases, including rheumatoid arthritis and periodontitis, is characterized by increased production of proinflammatory mediators and matrix-degrading enzymes by macrophages and increased osteoclastic activity. Phenotypic changes in macrophages are central to the healing process in virtually all tissues. Using a murine model of periodontitis, we assessed the timing of macrophage phenotypic changes and the impact of proresolving activation during inflammatory osteolysis and healing. Proinflammatory macrophage activation and TNF-α overproduction within 3 wk after induction of periodontitis was associated with progressing bone loss. Proresolving activation within 1 wk of stimulus removal and markers of resolving macrophages (IL-10, TGF-β, and CD206) correlated strongly with bone levels. In vivo macrophage depletion with clodronate liposomes prevented bone resorption but impaired regeneration. Induction of resolving macrophages with rosiglitazone, a PPAR-γ agonist, led to reduced bone resorption during inflammatory stimulation and increased bone formation during healing. In vitro assessment of primary bone marrow-derived macrophages activated with either IFN-γ and LPS (proinflammatory activation) or IL-4 (proresolving activation) showed that IL-4-activated cells have enhanced resolving functions (production of anti-inflammatory cytokines; migration and phagocytosis of aged neutrophils) and exert direct anabolic actions on bone cells. Cystatin C secreted by resolving but not inflammatory macrophages explained, in part, the macrophage actions on osteoblasts and osteoclasts. This study supports the concept that therapeutic induction of proresolving functions in macrophages can recouple bone resorption and formation in inflammatory osteolytic diseases.

Keywords: bone regeneration; inflammation resolution; innate immunity; osteoblast; osteoclasts; periodontitis.

Figure 1.

Resolving macrophage markers correlate strongly with bone levels during progression and healing of periodontitis lesions. (A) Periodontitis was induced with a biofilm-retentive silk suture (ligature) placed inside the gingival sulcus of maxillary left molars for 21 d. In a subset of mice, ligatures were removed and lesions left to heal for another 21 d. Bone loss and gain in inflammation and healing (solid line) were measured as the distance between the cementoenamel junction (arrowheads) and alveolar bone (dotted line) crest on Masson’s trichrome–stained frontal sections. Scale bars, 0.2 mm. (B) Bone level changes during the inflammatory and healing phases of periodontitis were calculated relative to nonligated contralateral internal control molars (n = 4 mice/time point, *P < 0.05 vs. nonligated, paired t test). (C) mRNA levels for macrophage activation markers relative to the HPRT housekeeping gene in periodontal explants from diseased sites during peak inflammation (day 21) and healing (day 28), normalized to contralateral internal control (n = 4 mice/group, *P < 0.05 healing vs. inflammation, paired t tests). Dashed line separates proinflammatory (top) from anti-inflammatory (bottom) markers. (D) Representative immunohistochemistry micrographs of healthy (day 0), inflamed (day 21), and healing (day 28) periodontal tissues of maxillary second molars identifying macrophage markers F4/80 (pan-macrophage), TNFα (proinflammatory), and CD206 (proresolving). Arrows indicate areas of high expression in the superficial layer of lamina propria (F4/80) and bone crest (F4/80, TNFα, CD206), around the ligature (TNFα), and in perivascular areas of lamina propria (TNF-α, CD206). Magnification: 40×. Scale bar, 50 μm. (E) Correlations between bone level and gene expression of IL-10, TGF-β, and CD206, measured as the ratio between day 28 and day 21 at ligated and nonligated sites (*P < 0.05, Pearson). (F) Tissue expression of F4/80, TNFα, and CD206 in periodontal tissues surrounding diseased and healing second molars (n = 3 samples/group, 6 fields/sample *P < 0.05 vs. day 0, 1-way analysis of variance with multiple comparisons as unpaired t tests). Values are presented as mean ± SEM.

Figure 2.

Proresolving macrophage activation reduces in vivo bone resorption and enhances regeneration. (A) Bone loss prevention study: To test the impact of macrophage depletion and M2 activation on inflammatory bone loss, mice were treated with clodronate (macrophage depletion) or rosiglitazone (M2 activation) starting at 4 d prior to ligature placement and continuing for 3 wk. (B) Bone regeneration study: To test the impact of clodronate and rosiglitazone on bone regeneration following inflammatory resorption, mice were treated with the same regimen as in panel A starting at 4 d prior to ligature removal (day 21) and throughout the first week of healing. Micro–computed tomography volumetric analysis of bone levels in the (C) prevention and (D) regeneration studies (n = 3 mice/group, *P < 0.05, **P < 0.01, ***P < 0.001, 1-way analysis of variance with multiple comparisons as unpaired t tests). Data represent 1 of 2 similar experiments. Insets: Representative 3-dimensional renderings of micro–computed tomography scans of maxillae on day 21 (prevention study) and day 28 (regeneration study) indicating root exposure at the second molar (arrows). (E) Representative coronal sections of diseased maxillary second molars on day 21 stained for TRAP (arrowheads, positive cells). PDL, periodontal ligament; T, tooth. Magnification: 60×. Scale bars: 10 μm. (F) Quantification of TRAP-positive area in the area of ligated versus nonligated molars on day 21 (n = 3 mice/group, 10 micrographs/sample, *P < 0.05, 1-way analysis of variance with Tukey’s honestly significant difference post hoc). (G) Representative coronal sections of healing maxillary second molars on day 28 stained for osterix (arrowheads, positive cells along inner cortex). PDL, periodontal ligament; T, tooth. Magnification: 60×. Scale bars: 10 μm. (H) Quantification of CD206-positive tissue area on coronal sections of ligated versus nonligated second molars on days 21 and 28 (n = 3 mice/group, 10 micrographs/sample, *P < 0.05, day 21 vs. 28, unpaired t test). Values are presented as mean ± SEM.

Figure 3.

Profiling of differentially activated macrophages. (A) Primary bone marrow macrophages—nonactivated (M0) and activated (M1, IFNγ and LPS; M2, IL-4)—were profiled by flow cytometry and mass spectrometry. M1 cells were F4/80^posCD80^highCD026^neg and M2 cells F4/80^posCD80^lowCD026^pos. Inset: Morphologic characteristics of M1 and M2 cells assessed by confocal microscopy of activated eGFP Raw264.7 cells. (B) Heat map representing the relative abundance (expressed as Z scores) in supernatants of M0, M1, and M2 macrophages. The area provided by PEAKS label-free quantification search from samples separately collected, prepared, and analyzed by mass spectrometry was log transformed for Z-score calculation. The Z-score values were hierarchically clustered. Cluster 3 includes 77 proteins highly expressed in M2-activated cells relative to M0 and M1. Inset: Proteases, antiproteases, and bone anabolic factors identified in cluster 3.

Figure 4.

IL-4-activated macrophages inhibit osteoclast differentiation and stimulate mineralization by osteoblasts. (A) Representative micrographs of osteoclasts differentiated in the presence of factors secreted by differentially activated macrophages. Bone marrow–derived osteoclast precursors were plated on glass chamber plates (2 × 10⁵ cells/well) and incubated with M-CSF and RANKL for 8 d. On day 4, supernatants of primary activated macrophages (M0, nonactivated; M1, IFN-γ and LPS activation; M2, IL-4 activation) were added to differentiating osteoclasts and trained for TRAP/Hoechst. Scale bars: top, 50 μm; bottom, 10 μm. (B) Primary osteoclast precursors were incubated with different ratios (a, 1:1; b, 1:5; c, 1:10) of activated macrophages or their supernatants for 4 d. Relative TRAP absorbance was measured as optical density at 405 nm (n = 3 separate experiments run in triplicate, *P < 0.05, 1-way analysis of variance with Tukey’s honestly significant difference post hoc). (C) MC3T3-E1 cells (7 × 10⁵ cells/well) were cultured to induce osteoblast differentiation (ascorbic acid, β-glycerophosphate) and then incubated with different ratios (1:10, 1:5, 1:1) of activated macrophage supernatant every 3 d for 21 d. Representative micrographs of calcium deposition by primary osteoblasts incubated at 1:10 ratio with supernatants of activated macrophages assessed by alizarin red S staining. Positive and negative controls: MC3T3-E1 with and without differentiation factors, respectively. Left: alizarin red–stained calcification; right: thresholding for positive area of calcification. (D) ImageJ quantification of mineral coverage area of the culture dish (n = 3, *P < 0.05, 1-way analysis of variance with Tukey’s honestly significant difference post hoc). Values are presented as mean ± SEM.

Figure 5.

Cystatin C mediates the regulatory actions of resolving macrophages on bone cells. (A) Western blot analysis of cystatin C in cell lysates of primary bone marrow cells activated to M1- and M2-like phenotype. (B) Band intensity was measured in ImageJ and normalized with β-actin (n = 3 experiments, *P < 0.05, unpaired t test vs. M0). (C) Representative images of cystatin C staining in inflamed (day 21) and healing (day 28) periodontitis lesions of mice treated with rosiglitazone, indicating increased expression with treatment at inner cortex and periodontal blood vessels (arrowheads). L, ligature. Magnification: 60×; rosiglitazone (right): 80×. Scale bars: 10 μm. (D) Primary mouse calvarial stromal cells were differentiated to osteoblasts (50 μg/mL of ascorbic acid, 10mM β-glycerophosphate) and incubated with different ratios (1:10, 1:5, 1:1) of supernatants from M1, M2, or cystatin C–immunodepleted M2 cells for 10 d. Mineral coverage area was assessed by alizarin red S staining. Negative control: osteoblast precursors without differentiation factors (n = 3 separate experiments run in triplicate, *P < 0.05, 1-way analysis of variance with Tukey’s honestly significant difference post hoc). (E) Bone marrow mononuclear osteoclast precursors cells were plated with different ratios (1:10, 1:5, 1:1) of supernatant from M1, M2, or cystatin C–immunodepleted M2 cells activated for 8 d. TRAP activity was measured as optical density at 405 nm (n = 3 separate experiments run in triplicate, *P < 0.05, 1-way analysis of variance with Tukey’s honestly significant difference post hoc). Values are presented as mean ± SEM.