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. 2019 Nov 26;10:2588.
doi: 10.3389/fimmu.2019.02588. eCollection 2019.

Spatial Distribution of Macrophages During Callus Formation and Maturation Reveals Close Crosstalk Between Macrophages and Newly Forming Vessels

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Free PMC article

Spatial Distribution of Macrophages During Callus Formation and Maturation Reveals Close Crosstalk Between Macrophages and Newly Forming Vessels

Jonathan Stefanowski et al. Front Immunol. .
Free PMC article

Abstract

Macrophages are essential players in the process of fracture healing, acting by remodeling of the extracellular matrix and enabling vascularization. Whilst activated macrophages of M1-like phenotype are present in the initial pro-inflammatory phase of hours to days of fracture healing, an anti-inflammatory M2-like macrophage phenotype is supposed to be crucial for the induction of downstream cascades of healing, especially the initiation of vascularization. In a mouse-osteotomy model, we provide a comprehensive characterization of vessel (CD31+, Emcn+) and macrophage phenotypes (F4/80, CD206, CD80, Mac-2) during the process of fracture healing. To this end, we phenotype the phases of vascular regeneration-the expansion phase (d1-d7 after injury) and the remodeling phase of the endothelial network, until tissue integrity is restored (d14-d21 after injury). Vessels which appear during the bone formation process resemble type H endothelium (CD31hiEmcnhi), and are closely connected to osteoprogenitors (Runx2+, Osx+) and F4/80+ macrophages. M1-like macrophages are present in the initial phase of vascularization until day 3 post osteotomy, but they are rare during later regeneration phases. M2-like macrophages localize mainly extramedullary, and CD206+ macrophages are found to express Mac-2+ during the expansion phase. VEGFA expression is initiated by CD80+ cells, including F4/80+ macrophages, until day 3, while subsequently osteoblasts and chondrocytes are main contributors to VEGFA production at the fracture site. Using Longitudinal Intravital Microendoscopy of the Bone (LIMB) we observe changes in the motility and organization of CX3CR1+ cells, which infiltrate the injury site after an osteotomy. A transient accumulation, resulting in spatial polarization of both, endothelial cells and macrophages, in regions distal to the fracture site, is evident. Immunofluorescence histology followed by histocytometric analysis reveals that F4/80+CX3CR1+ myeloid cells precede vascularization.

Keywords: CX3CR1 myeloid cells; H-type vessel; LIMB; bone regeneration; endothelial cell; intravital microscopy; macrophage.

Figures

Figure 1
Figure 1
Histological characterization of tissue formation and distribution of mesenchymal progenitor cells, osteoblasts and endothelial cells during bone regeneration in a mouse-osteotomy model. (A) Movat's Pentachrome staining indicates the phases of bone regeneration over 21 days. Scale bars = 500 μm. (B) Sox9 and Runx2 immunofluorescence staining for progenitor cells undergoing differentiation at day 3, 7, 14, and 21 indicating double-positive cells at day 7 and high numbers of Runx2+ cells within the fracture gap at day 14 and 21. Exemplary images of n = 3–6 per time point. Scale bars = 200 μm. Insets show enlarged representative areas marked by white frames. (C) Exemplary images at day 3, 7, 14, and 21 displaying the distribution of CD31+ ECs and Osx+ osteoprogenitors/pre-osteoblasts revealing close proximity between CD31+ endothelium and Osx+ cells. Scale bars = 200 μm. (D) Exemplary images for Sox9 or Runx2 and CD31 staining at day 7. Insets show enlarged representative areas marked by white frames. While Runx2+ cells can be found closed to endothelial cells, Sox9+ cells can be found in not yet vascularized areas. (E) Quantification of cellular compartments present in the fracture gap during regeneration. Endothelial cells (ECs; CD31+) and osteoprogenitors/pre-osteoblasts (Osx+) were quantified based on the relative presence of positive pixels with the respective markers in immunofluorescence images supporting the descriptive analysis on the spatiotemporal distribution of progenitor cells, osteoblasts and endothelial cells over time. Data show mean ± SD for n = 3–6.
Figure 2
Figure 2
Vessels formed during fracture healing show characteristics of type H endothelium and are embedded in mineralized tissue. (A) Intensity analysis of Emcn and/or CD31 in the fracture gap. Mean intensities were normalized to the maximum intensity present in each image. Mean intensities were normalized to 780 the maximum intensity present in each image. Data are shown as scatter dot plot with mean ± SD while one dot is representative for one slide of one individual animal. Friedman test with Dunn's post-hoc test were performed to determine statistical differences; p-values are indicated with *p < 0.05, **p < 0.01. (B) Emcnhi CD31hi endothelium shows spatial differences in abundance throughout the bone. Exemplary image showing the definition for the osteotomy gap, the gap adjacent regions and the bone marrow area, as used in our analyses. Scale bar = 200 μm. (C) Images of Emcnhi CD31hi endothelium at day 3, 7, 14, and 21. Data are representative for n = 3 (d3/7) and n = 6 (d14/21). (D) Quantification of areas occupied by Emcnhi CD31hi type H endothelium during the time course of bone healing. Data are shown as Mean ± SD. (E) Emcn and CD31 staining combined with phase contrast images (DIC) in the soft to hard callus transition of fracture healing highlight morphological characteristics also present in growth plate bone growth. Upper panels: invasion of vessels in cartilaginous tissue and type H vessel-like budding is indicated by arrows. Lower panels: CD31+ only vessels are assumed to be arterioles (arrows) (7). Scale bars = 200 μm.
Figure 3
Figure 3
Distinct localization of macrophage subsets during bone regeneration indicates location-dependence of their functions. (A) Immunofluorescence staining of F4/80, CD206, and Emcn shows the abundance of F4/80+ cells in the osteotomy gap. They localize close to Emcnhi endothelium. F4/80+CD206+ M2-like macrophages primarily localize in extramedullar areas (blue inset). F4/80hi cells localize in proximity to Emcnhi endothelium (yellow inset). Endothelium shows CD206-positivity in proximity to the gap (orange inset). Scale bars = 500 μm. (B) Immunofluorescence staining reveals spatiotemporal distribution of CD206+ and Mac-2+ cells. CD206+ M2-like macrophages were Mac-2+ at d3 and single-positive by d21. Scale bars = 500 μm. (C) Pixel-based area analysis of immunofluorescence images of CD31, F4/80, and TRAP show a reduction of CD31 and F4/80 signals over time and an increase in osteoclasts. (D) Object-based analysis for (E,F), based on regions within the osteotomy gap which show high or low expression of Emcn (Emcnhi vs. Emcnlo). Scale bar = 500 μm. (E) F4/80+ cell frequency in the Emcnhi region decreases between d3 and d21 to levels of the Emcnlo region. Mann Whitney test, two-tailed, *p = 0.0476. (F) Proximity analysis performed by object-based quantification in two distances from the endothelium. Data was normalized to the overall number of the object population in the respective region. Compared to the Emcnlo region, more objects (F4/80+ or nuclei) localize in proximity to the endothelium in the Emcnhi region. Cells were considered in proximity to each other when their distance amounted to that equivalent to less than half of a nucleus diameter (<3.5 μm), in order to include cells which are either in contact with or in the direct vicinity of vessels. Cells were considered distant were located further than one cell layer (>7 μm) apart from each other. Data are representative for n = 3 (d3/7) and n = 6 (d14/21).
Figure 4
Figure 4
Expression of VEGFA in CD80+ cells and bone forming areas at the osteotomy site. Immunofluorescence staining over the course of regeneration shows (A) pro-inflammatory CD80+ cells adjacent to the osteotomy gap, which are in part F4/80+ in the early phase (at day 1 and 2). (B) F4/80+ cells were predominantly VEGFA. (C) CD80+VEGFA+ cells localize at the interface between the hematoma and surrounding tissue. (D) In areas of bone formation VEGFA expression is high, but F4/80+ cells re VEGFA during progressing bone regeneration until day 21. Scale bars = 500 μm. n(day 1) = 3, n(day 2) = 1.
Figure 5
Figure 5
Quantitative analysis and LIMB imaging demonstrate high abundance of the myeloid CX3CR1+ F4/80+ cell subset preceding vascularization. (A) Overview image of a section from a whole bone taken at day 4 after LIMB osteotomy. The LIMB osteotomy-model model uses a gradient refractive index lens which is removed before bone sectioning thus a lens void remains. Vessels and macrophages polarize distally in this example. (B) Scoring of CD31 and F4/80 allows quantification of polarization in proximal or distal orientation with respect to the fracture gap. Scoring of DAPI staining was used as internal control. Wilcoxon matched-pairs signed rank test. Data are representative for n = 7 (d4), n = 6 (d6), n = 4 (d8), and n = 3 (d14). (C) Immunofluorescence images of sections from bones showing the presence of myeloid subsets at various time points after LIMB osteotomy. CX3CR1:GFP+ and F4/80+ cells negative for Ly6C/Ly6G (Gr-1), accumulate at and invade into the fracture gap. Scale bars = 200 μm. (D) Inset of the area of d8. The majority of myeloid cells are CX3CR1+F4/80+ and localize in distance from Gr-1+ cells. Scale bars = 200 μm. (E) Quantitative analysis is performed in the rectangular region of the fracture gap between corticalis, osteotomy cuts, and lens void (osteotomy gap). Scale bar = 100 μm. (F) Quantitative, object-based analysis of the osteotomy gap for Gr-1, F4/80, CX3CR1:GFP show a decrease of Gr-1+, an increase of F4/80+ and massive increase of CX3CR1+ cells. Results of 20 pooled samples. (G) Object-analysis among the CX3CR1+ cells reveals that Gr-1F4/80+ account for the majority of cells present in the gap over the whole time course of regeneration (left panel). Of those, the majority were non-round cells (right panel). (H) Intravital two-photon LIMB-microscopy of the osteotomy gap in an individual CX3CR1:GFP mouse. CX3CR1:GFP+ cells invade the osteotomy gap in the displayed field of view (250 × 250 μm) at day 2–3 and fully populate the region by day 3–4. Simultaneously, numerous CX3CR1+ cells precede the vascularization in space and time. Vasculature was made visible using intravenous injection of Qtracker 655. Scale bars = 100 μm. Data are representative of 5 mice analyzed.

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