Creating an atlas of the bone microenvironment during oral inflammatory-related bone disease using single-cell profiling
- Sichuan University, China
- Sun Yat-sen University, China
- Maine Medical Center Research Institute, United States
Abstract
Oral inflammatory diseases such as apical periodontitis are common bacterial infectious diseases that may affect the periapical alveolar bone tissues. A protective process occurs simultaneously with the inflammatory tissue destruction, in which mesenchymal stem cells (MSCs) play a primary role. However, a systematic and precise description of the cellular and molecular composition of the microenvironment of bone affected by inflammation is lacking. In this study, we created a single cell atlas of cell populations that compose alveolar bone in healthy and inflammatory disease states. We investigated changes in expression frequency and patterns related to apical periodontitis, as well as the interactions between MSCs and immunocytes. Our results highlight an enhanced self-supporting network and osteogenic potential within MSCs during apical periodontitis-associated inflammation. MSCs not only differentiated towards osteoblast lineage cells, but also expressed higher levels of osteogenic related markers, including Sparc and Col1a1. This was confirmed by lineage tracing in transgenic mouse models and human samples from oral inflammatory-related alveolar bone lesions. In summary, the current study provides an in-depth description of the microenvironment of MSCs and immunocytes in both healthy and disease states. We also identified key apical periodontitis-associated MSC subclusters and their biomarkers, which could further our understanding of the protective process and the underlying mechanisms of oral inflammatory-related bone disease. Taken together, these results enhance our understanding of heterogeneity and cellular interactions of alveolar bone cells under pathogenic and inflammatory conditions. We provide these data as a tool for investigators not only to better appreciate the repertoire of progenitors that are stress responsive but importantly to help design new therapeutic targets to restore bone lesions caused by apical periodontitis and other inflammatory-related bone diseases.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting file; Source Data files have been provided for Figures 1, 2, 3, 4, 5, 6, 7, Figure1-figure supplement 1 and Figure5-figure supplement 3.
Article and author information
Author details
Funding
National Natural Science Foundation of China (81800928)
- Yi Fan
National Natural Science Foundation of China (81901040)
- Chenchen Zhou
National Natural Science Foundation of China (82171001)
- Chenchen Zhou
Young Elite Scientist Sponsorship Program by CAST (2020QNRC001)
- Chenchen Zhou
Young Elite Scientist Sponsorship Program by CAST (2018QNR001)
- Yi Fan
Sichuan Science and Technology Program (2019YJ0054)
- Yi Fan
Research Funding from West China School/Hospital of Stomatology Sichuan University (RCDWJS2021-1)
- Yi Fan
State Key Laboratory of Oral Diseases Open Funding Grant (SKLOD-R013)
- Yi Fan
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All animal experiments were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee at the State Key Laboratory of Oral Diseases, Sichuan University (Permit Number: WCHSIRB-D-2021-339).
Human subjects: This study was permitted by the Ethical Committees of the West China Hospital of Stomatology, Sichuan University (Permit Number: WCHSIRB-D-2021-292). Written informed consent was obtained from all patients.
Copyright
© 2023, Fan et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Creating an atlas of the bone microenvironment during oral inflammatory-related bone disease using single-cell profiling
eLife 12:e82537.
https://doi.org/10.7554/eLife.82537
Further reading
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- Cell Biology
Distal appendages are nine-fold symmetric blade-like structures attached to the distal end of the mother centriole. These structures are critical for formation of the primary cilium, by regulating at least four critical steps: ciliary vesicle recruitment, recruitment and initiation of intraflagellar transport (IFT), and removal of CP110. While specific proteins that localize to the distal appendages have been identified, how exactly each protein functions to achieve the multiple roles of the distal appendages is poorly understood. Here we comprehensively analyze known and newly discovered distal appendage proteins (CEP83, SCLT1, CEP164, TTBK2, FBF1, CEP89, KIZ, ANKRD26, PIDD1, LRRC45, NCS1, CEP15) for their precise localization, order of recruitment, and their roles in each step of cilia formation. Using CRISPR-Cas9 knockouts, we show that the order of the recruitment of the distal appendage proteins is highly interconnected and a more complex hierarchy. Our analysis highlights two protein modules, CEP83-SCLT1 and CEP164-TTBK2, as critical for structural assembly of distal appendages. Functional assays revealed that CEP89 selectively functions in RAB34+ ciliary vesicle recruitment, while deletion of the integral components, CEP83-SCLT1-CEP164-TTBK2, severely compromised all four steps of cilium formation. Collectively, our analyses provide a more comprehensive view of the organization and the function of the distal appendage, paving the way for molecular understanding of ciliary assembly.
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- Cell Biology
The primary cilium is a microtubule-based organelle that cycles through assembly and disassembly. In many cell types, formation of the cilium is initiated by recruitment of ciliary vesicles to the distal appendage of the mother centriole. However, the distal appendage mechanism that directly captures ciliary vesicles is yet to be identified. In an accompanying paper, we show that the distal appendage protein, CEP89, is important for the ciliary vesicle recruitment, but not for other steps of cilium formation (Tomoharu Kanie, Love, Fisher, Gustavsson, & Jackson, 2023). The lack of a membrane binding motif in CEP89 suggests that it may indirectly recruit ciliary vesicles via another binding partner. Here, we identify Neuronal Calcium Sensor-1 (NCS1) as a stoichiometric interactor of CEP89. NCS1 localizes to the position between CEP89 and a ciliary vesicle marker, RAB34, at the distal appendage. This localization was completely abolished in CEP89 knockouts, suggesting that CEP89 recruits NCS1 to the distal appendage. Similarly to CEP89 knockouts, ciliary vesicle recruitment as well as subsequent cilium formation was perturbed in NCS1 knockout cells. The ability of NCS1 to recruit the ciliary vesicle is dependent on its myristoylation motif and NCS1 knockout cells expressing a myristoylation defective mutant failed to rescue the vesicle recruitment defect despite localizing properly to the centriole. In sum, our analysis reveals the first known mechanism for how the distal appendage recruits the ciliary vesicles.
