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. 2022 Feb 4:13:835986.
doi: 10.3389/fimmu.2022.835986. eCollection 2022.

CCR2+ Macrophages Promote Orthodontic Tooth Movement and Alveolar Bone Remodeling

Hao Xu  1   2   3 , Shuting Zhang  1   2   4 , Adwait Amod Sathe  5 , Zhichun Jin  1   2   3 , Jiani Guan  1   2   3 , Wen Sun  2 , Chao Xing  5   6   7 , Hanwen Zhang  8   9 , Bin Yan  1   2   3
Affiliations

CCR2+ Macrophages Promote Orthodontic Tooth Movement and Alveolar Bone Remodeling

Hao Xu et al. Front Immunol. .

Abstract

During mechanical force-induced alveolar bone remodeling, macrophage-mediated local inflammation plays a critical role. Yet, the detailed heterogeneity of macrophages is still unknown. Single-cell RNA sequencing was used to study the transcriptome heterogeneity of macrophages during alveolar bone remodeling. We identified macrophage subclusters with specific gene expression profiles and functions. CellChat and trajectory analysis revealed a central role of the Ccr2 cluster during development, with the CCL signaling pathway playing a crucial role. We further demonstrated that the Ccr2 cluster modulated bone remodeling associated inflammation through an NF-κB dependent pathway. Blocking CCR2 could significantly reduce the Orthodontic tooth movement (OTM) progression. In addition, we confirmed the variation of CCR2+ macrophages in human periodontal tissues. Our findings reveal that mechanical force-induced functional shift of the Ccr2 macrophages cluster mediated by NF-κB pathway, leading to a pro-inflammatory response and bone remodeling. This macrophage cluster may represent a potential target for the manipulation of OTM.

Keywords: CCR2; bone remodeling; inflammatory; macrophage; single-cell sequencing.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Orthodontic tooth movement induces inflammation in the periodontal tissues. (A) Representative 3D micro-CT reconstruction of murine OTM model. (White arrow: direction of force and tooth movement). (B) The distance of OTM and (C) the ratio of CD11b+F4/80+ macrophages at day 0 and 7. Values are mean ± SD. n = 5. ***P < 0.001. (C, D) Representative flow cytometric analysis of CD11b+F4/80+ macrophages at day 0 and 7. Values are mean ± SD. n = 4 or 5. *P < 0.05. (E) mRNA expression of inflammatory factors of macrophages in murine alveolar bone at day 0 and 7. Values are mean ± SD. n = 5. ***P < 0.001. (F) Representative immuno-fluorescence staining and quantification of CD68+ macrophages in human periodontal tissue. Scale bar = 50μm. Values are mean ± SD. n = 5. **P < 0.01. (G) The flow chart of experimental design.
Figure 2
Figure 2
Single-cell analyses show macrophage subsets in murine alveolar bone. (A) UMAP plot of cells in control group. 12 (0 to 11) clusters were identified. Dimensional reduction was performed with principal component analysis (PCA) and visualization using UMAP plots. (B) Dot plot of differentially expressed genes in each cluster. (C) Feature plots depicting single-cell gene expression of individual genes. (D) Proportions of 12 different macrophage clusters. (E) The top 60-65 markers (adj. p-value < 0.05) per cluster were used to identify the functional enrichment categories using Metascape. Pathway enrichment is expressed as the -log10(P) adjusted for multiple comparison.
Figure 3
Figure 3
CellChat and Single-cell trajectories reveal cell communications and developmental relationships among macrophage clusters in the steady-state. (A) Heatmap shows the relative strength of each signal pathway network for each cluster with both incoming and outgoing signaling patterns. (B) Inferred CCL Signaling pathway network of steady-state clusters. (C) Heatmap of the CCL Signaling network displaying relative importance of each cell group ranked according to the computed four network centrality measures. (D) Relative contribution of each ligand-receptor pair as it affects the overall communication network of the CCL signaling pathway. (E) Differentially expressed genes between clusters were used to generate hypothetical developmental relationships using Dynverse. (F) Representative gene expression plotted as a function of pseudotime. (G) Heatmap of differentially expressed genes ordered based on their simple kinetics through pseudotime using Dynverse.
Figure 4
Figure 4
Single-cell analyses show macrophage subsets in murine alveolar bone after orthodontic force application. (A) UMAP plot of cells in OTM group. 8 (0 to 7) clusters were identified. Dimensional reduction was performed with principal component analysis (PCA) and visualization using UMAP plots. (B) Dot plot of conserved differentially expressed genes in each cluster. (C) Feature plots depicting single-cell gene expression of individual genes. (D) Proportions of 8 different macrophage clusters. (E) The top 60-65 markers (adj. p-value < 0.05) per cluster were used to identify the functional enrichment categories using Metascape. Pathway enrichment is expressed as the -log10(P-adjusted) for multiple comparison.
Figure 5
Figure 5
CellChat and Single-cell trajectories reveal cell communications and developmental relationships of macrophages after orthodontic force application. (A) Heatmap shows the relative strength of each signal pathway network for each cluster with both incoming and outgoing signaling patterns. (B) Inferred CCL Signaling pathway network of case clusters. (C) Heatmap of the CCL Signaling network displaying relative importance of each cell group ranked according to the computed four network centrality measures. (D) Relative contribution of each ligand-receptor pair as it affects the overall communication network of the CCL signaling pathway. (E) Differentially expressed genes between clusters were used to generate hypothetical developmental relationships using Dynverse. (F) Representative gene expression plotted as a function of pseudotime. (G) Heatmap of differentially expressed genes ordered based on their simple kinetics through pseudotime using Dynverse.
Figure 6
Figure 6
High-dimensional analyses reveal both unique and shared macrophage genes and functions after force application. (A) Bar chart of the relative proportions of clusters from two groups. (B) Heatmap of the top 30 differentially expressed genes in control only, OTM only, and overlapping populations. (C) Heatmap of the top 15 differentially expressed genes in Ccr2 cluster between two groups. (D) Pathway analysis (gProfiler) of commonly and differentially expressed genes in Ccr2 cluster. (E) Representative immunofluorescence staining and quantification of CD68+CCR2+ macrophages in the human periodontal tissue. Scale bar = 20μm. Values are mean ± SD. n = 5. **P < 0.01.
Figure 7
Figure 7
CCR2/CCL2 axis promoted pro-inflammatory macrophages through the phosphorylation of p65. (A) Relative mRNA expression of Nos2 in untreated, CCL2 treated and LPS treated macrophages. Data is from 3 independent experiments. Values are mean ± SD. *P < 0.05. **P <0.01. (B) Representative immunofluorescence staining of iNOS in untreated, CCL2 treated and LPS treated macrophages. (C) Relative mRNA expression of inflammatory factors in macrophages treated with LPS or IL-4, and RS504393. Data is from 3 independent experiments. Values are mean ± SD. *P < 0.05. ns, no significance. (D) Relative mRNA expression of Ccr2 in macrophages treated with CCL2 or not, and with Ccr2 siRNA. Data is from 3 independent experiments. Values are mean ± SD. *P < 0.05. (E, F) Relative mRNA expression of pro-inflammatory (E) and anti-inflammatory factors (F) in macrophages treated with CCL2 or not, and with Ccr2 siRNA. Data is from 3 independent experiments. Values are mean ± SD. *P < 0.05. ns, no significance. (G) Representative immunofluorescence staining of p65 in macrophages in untreated, treated with CCL2, and CCL2+RS504393. Data is from 3 independent experiments. Values are mean ± SD. *P < 0.05. **P <0.01. (H) Relative mRNA expression of inflammatory factors Il6, Il1β and nos2 treated with CCL2 or p65 inhibitor. Data is from 3 independent experiments. Values are mean ± SD. *P < 0.05, ns, no significance.
Figure 8
Figure 8
CCL2 treatment promoted the phosphorylation of p65 in macrophages. (A) Western blot analysis of p-p65 and p65 expression in macrophages treated with CCL2 for 0, 30, 60 and 360 minutes. Data is from 3 independent experiments. Densitometric quantitation with Image J. Values are mean ± SD. *P < 0.05. (B) Western blot analysis of p-p65 and p65 expression in macrophages treated with CCL2 and RS504393. Data is from 3 independent experiments. Densitometric quantitation with Image J. Values are mean ± SD. *P < 0.05. ns, no significance. (C, D) Western bolt analysis of p-p65 and p65 expression in macrophages treated with CCL2 or not, and with Ccr2 siRNA. Data is from 3 independent experiments. Densitometric quantitation with Image J. Values are mean ± SD. *P < 0.05. (E, F) The distance of OTM in TM and TM+CCR2i group and representative 3D micro-CT reconstruction. White arrow: direction of force and tooth movement. Values are mean ± SD. n = 5. ***P < 0.001. (G) Graphic abstract of this study.
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References

    1. Garlet TP, Coelho U, Silva JS, Garlet GP. Cytokine Expression Pattern in Compression and Tension Sides of the Periodontal Ligament During Orthodontic Tooth Movement in Humans. Eur J Oral Sci (2007) 115:355–62. doi: 10.1111/j.1600-0722.2007.00469.x - DOI - PubMed
    1. Zhang S, Zhang H, Jin Z, Wang S, Wang Y, Zhu L, et al. . Fucoidan Inhibits Tooth Movement by Promoting Restorative Macrophage Polarization Through the STAT3 Pathway. J Cell Physiol (2020) 235:5938–50. doi: 10.1002/jcp.29519 - DOI - PubMed
    1. Sima C, Viniegra A, Glogauer M. Macrophage Immunomodulation in Chronic Osteolytic Diseases-the Case of Periodontitis. J Leukoc Biol (2019) 105:473–87. doi: 10.1002/JLB.1RU0818-310R - DOI - PMC - PubMed
    1. Hienz SA, Paliwal S, Ivanovski S. Mechanisms of Bone Resorption in Periodontitis. J Immunol Res (2015) 2015:615486. doi: 10.1155/2015/615486 - DOI - PMC - PubMed
    1. He D, Kou X, Yang R, Liu D, Wang X, Luo Q, et al. . M1-Like Macrophage Polarization Promotes Orthodontic Tooth Movement. J Dent Res (2015) 94:1286–94. doi: 10.1177/0022034515589714 - DOI - PubMed

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