Creating an atlas of the bone microenvironment during oral inflammatory-related bone disease using single-cell profiling

  1. Yi Fan
  2. Ping Lyu
  3. Ruiye Bi
  4. Chen Cui
  5. Ruoshi Xu
  6. Clifford J Rosen
  7. Quan Yuan
  8. Chenchen Zhou

  • Curated by eLife

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    In this important project, the authors used single-cell RNA-sequencing (scRNA-Seq) technology to profile the transcriptome of alveolar bone marrow single cells and demonstrated the protective role of mesenchymal stem cells (MSCs) during apical periodontitis. With comprehensive data, the authors identified new inflammatory biomarkers associated with the pathogenesis of oral inflammatory diseases. Their study suggests that certain MSC subsets may have a potential role in healing bone lesions.

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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 toward 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.

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  1. Version published to 10.7554/elife.82537 on eLife
  2. eLife

    Author Response

    Reviewer #3 (Public Review):

    This is an interesting study to examine how alveolar bone responds to oral infection using unbiased scRNA-seq. The manuscript is well-written and the results are convincing.

    1. The authors should revise the abstract. The study did nothing with the understanding of healing. The whole conditions were performed under infection and inflammation which actually induce bone loss, but not healing.

    Thank you for raising this point. We have revised the manuscript accordingly.

    1. Since periapical inflammation causes progressive bone loss, how MSC with increasing osteogenic potentials contributes to bone loss? The authors should discuss it.

    We would like to thank the reviewer for this important comment. Although AP is an inflammatory disease with periapical bone loss, the progression of AP is usually self-limiting in which a new equilibrium has been established between root canal pathogens and anti-infective defense mechanisms (Wang, Zhang, Xiong, & Peng, 2011). Animal experiments revealed that the bone lesion size reached to stable 21 days after establishing AP, which was resulted from a balance of bone remodeling (Márton & Kiss, 2014; Wang et al., 2011). Previous studies have shown that human apical granulation tissues contain osteogenic cells (Maeda, Wada, Nakamuta, & Akamine, 2004). A population of MSCs were isolated from human periapical cysts, which tended to be directed to differentiate toward the osteogenesis lineage (Marrelli, Paduano, & Tatullo, 2013, 2015; Tatullo et al., 2015). Activated by inflammatory bone destruction, these MSCs with increased osteogenic potentials may rescue the bone resorption process, which reach the equilibrium between bone formation and resorption then drive the progression of AP into stable states (Márton & Kiss, 2014). Since the pathologic stimuli exists constantly, the protective actions can alleviate the bone loss to some extent. In clinical practice, root canal therapy (RCT) aims to disinfect and remove the pathogenic factors, which makes the protective activities overweigh the destructive ones (L. M. Lin, Ricucci, Lin, & Rosenberg, 2009). The bone lesions of AP patients receiving RCT usually fully recovered with resolution of radiolucency after the inflammation is controlled in apical area (Soares, Santos, Silveira, & Nunes, 2006). The healing of AP lesion is highly correlated with the osteogenic potential of inflamed MSCs (L. M. Lin et al., 2009).

    We added the related contents in the discussion section.

    1. Did the authors detect osteoclasts by scRNA-seq? If not, are there any precursors of osteoclasts identified in inflammatory alveolar bones? 1) I suggest that the authors provide a more detailed analysis of inflammation since this is a unique model to study oral bone inflammation.

    Thank you for this valuable point. Bone destruction is a major pathological factor in chronic inflammatory diseases such as AP. Various cytokines including TNF-α, IL-1α, IL-6 were released by immunocytes to recruit the osteoclast precursors and induce the maturation of osteoclasts. We detected osteoclast markers including Ctsk, Acp5, Mmp9 and Nfatc1 by scRNA-seq. Moreover, Csfr1, Cx3cr1, Itgam, and Tnfrs11a were used to identify osteoclast precursors. The expression pattern of these osteoclast-related markers in all clusters were presented in Figure 3A. Markers of osteoclast and osteoclast precursors were highly expressed in the clusters of monocyte and macrophage. The expression levels of these markers were analyzed in all clusters (Figure 3B). The GO analysis showed that inflammation related immune reactions and bone resorption activity were significantly enriched in macrophage cluster (Figure 3C). Moreover, pseudotime analysis was performed for the clusters of macrophage and monocyte. Two independent branch points were determined and five monocyte/macrophage subclusters scattered at different branches in the developmental tree (Figure 3D, G). The results showed that the monocyte cluster differentiated into the macrophage cluster (Figure 3E). During this trajectory, the gene expression pattern across pseudotime showed that osteoclastic genes, such as Ctsk, Acp5, Mmp9, Atp6v0d2, and Dcstamp were progressively elevated (Figure 3F). Of note, we have observed a branch which was highly positive for Ctsk and Acp5 (Figure 3H), indicating the mature osteoclasts were differentiated from monocyte/macrophage lineage and contributed to inflammatory bone resorption during AP. We have also analyzed the expression of osteoclast related genes using the bulk RNA-seq library built on mandibular samples extracted from mice with AP. Markers of osteoclast and osteoclast precursors were significantly upregulated, confirming the osteoclasts activity in the inflammatory-related bone lesion (Figure 3I). Please see page 9 and figure 3.

    1. It is known that macrophages can be classified into M1 and M2. Based on scRNA-seq, did the authors observe these two types?

    We appreciate this point raised by the reviewer. We used CD86, CD80, IL1β, and TNF as markers of M1-like macrophages. CD163, CD206, MSR1 and IL-10 were used as markers to detect M2 subset in the macrophage cluster. The analysis of macrophage cluster showed the M1-like macrophage accounted for the vast majority in AP lesions. The expression pattern of M2 markers were also presented in macrophage cluster (Figure 3-figure supplement 1A, B).

  3. eLife

    eLife assessment

    In this important project, the authors used single-cell RNA-sequencing (scRNA-Seq) technology to profile the transcriptome of alveolar bone marrow single cells and demonstrated the protective role of mesenchymal stem cells (MSCs) during apical periodontitis. With comprehensive data, the authors identified new inflammatory biomarkers associated with the pathogenesis of oral inflammatory diseases. Their study suggests that certain MSC subsets may have a potential role in healing bone lesions.

  4. eLife

    Reviewer #1 (Public Review):

    In this study, the authors collected mandibular alveolar bone samples from control mice and the mice with apical periodontitis (AP) and performed single-cell RNA sequencing (scRNA-Seq) experiment. Using the data from cell subsets of the mandibular alveolar bone, the authors compared the expression profiles of the mice with AP with those from control mice. They also determined the relationship between MSCs and immune cells and confirmed the role of a subset of MSCs in inflammation. In addition, the authors demonstrated MSC differentiation potential to mature osteoblasts during AP inflammation. Using transgenic mouse models and samples derived from patients with chronic AP, they further confirmed the findings of scRNA-Seq data. Taken together, these results reveal the heterogeneity and interactions of alveolar bone cells during periodontitis inflammation. One of their key findings is to identify a subset of MSC cells and their differentiation in the inflamed tissues.

  5. eLife

    Reviewer #2 (Public Review):

    Periodontal inflammation is a very common disease and constitutes a challenge for public health. Healing of periodontal tissue after an acute or chronic inflammation remains to be very difficult, if not possible. Although MSCs were known to be present and support periodontal physiological turnover, periodontal tissue hardly regenerates after the inflammation process. Therefore, learning the difference between periodontal MSCs under physiological or inflammation conditions is a fundamental issue. Due to the technical challenges, a comprehensive single-cell Seq analysis on periodontal tissue has never been performed. The current study made the first breakthrough on this issue.

    Overall, the manuscript provides important information on the response of periodontal tissue towards acute inflammation.Additional experiments are needed to support their major conclusions.

  6. eLife

    Reviewer #3 (Public Review):

    This is an interesting study to examine how alveolar bone responds to oral infection using unbiased scRNA-seq. The manuscript is well-written and the results are convincing.

    1. The authors should revise the abstract. The study did nothing with the understanding of healing. The whole conditions were performed under infection and inflammation which actually induce bone loss, but not healing.

    2. Since periapical inflammation causes progressive bone loss, how MSC with increasing osteogenic potentials contributes to bone loss? The authors should discuss it.

    3. Did the authors detect osteoclasts by scRNA-seq? If not, are there any precursors of osteoclasts identified in inflammatory alveolar bones? 1) I suggest that the authors provide a more detailed analysis of inflammation since this is a unique model to study oral bone inflammation.

    4. It is known that macrophages can be classified into M1 and M2. Based on scRNA-seq, did the authors observe these two types?

  7. Version published to 10.1101/2022.09.13.507807v1 on bioRxiv