Single-Cell Atlas of Immune Microenvironment in Orthodontic Tooth Movement

Orthodontic treatment depends on periodontal tissue remodeling. While the immune system has been found to regulate this process, previous studies mostly focused on the static molecular changes in conventional immune cell sets. A recent study used single-cell RNA sequencing (scRNA-seq) to analyze macrophages under orthodontic tooth movement (OTM), introducing new subclusters and functions. However, changes in other immune cells and their interactions with surrounding stromal cells are yet unclarified. Therefore, we performed scRNA-seq in mice, aiming to describe a more comprehensive immune landscape during OTM, while further exploring the dynamic changes, functions, and cellular interactions of immune cells at a higher resolution. We first confirmed the dynamic activation of osteoclasts, osteoblasts, and immune cells at various time points. The scRNA-seq analysis identified 7 cell lineages and 18 major cell types, with immune cells forming the largest proportion. Monocytic cells, granulocytes, and lymphocytes were selected for individual reclustering, followed by analysis of specific gene expression, functional enrichment, and distribution changes during OTM. Pseudotime analysis was applied to monocytic cells and granulocytes. We identified 4 developmental pathways in monocytic cells toward dendritic cells, different subsets of macrophages, and osteoclasts. Monocytic cells tended to be more differentiated during OTM. Meanwhile, in granulocytes, neutrophil subclusters were all highly differentiated. Additionally, we assessed the cellular interactions during OTM, revealing enhanced signaling from macrophages toward osteoclasts, especially in Ccl, Tnf, and Spp1 pathways. We identified that the C3ar1⁺ macrophage subcluster expressed these cytokines at a high level and was enriched in positive regulation of the mitogen-activated protein kinase cascade, indicating its positive regulation toward osteoclast activity. In conclusion, this study revealed the complex immune microenvironment during OTM, providing a detailed perspective on the diverse immune cell types, their specific functions, and cellular interactions.