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Single-cell analysis reveals immune cellular components in odontogenic keratocysts
Qi-Wen Man1,2, Rui-Fang Li1, Jian-Feng Liu1
1The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST), Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China.
Insights
Odontogenic keratocyst (OKC) fibroblasts, particularly myofibroblasts, release chemokines that attract immune cells. This interaction, involving myofibroblasts and immune cells, may drive OKC
Area of Science:
- Oral pathology and immunology
- Cell biology and molecular mechanisms
- Biomedical research
Background:
- Odontogenic keratocysts (OKCs) involve diverse cell populations.
- Immune and non-immune cells communicate via cytokines, chemokines, and direct contact.
- Understanding the immune microenvironment is crucial for OKC biology.
Purpose of the Study:
- To characterize the immune cell ecosystem within OKCs.
- To investigate the chemotactic role of OKC fibroblasts in immune cell migration.
- To elucidate the communication networks between OKC cells.
Main Methods:
- Mass cytometry for cell classification.
- Single-cell RNA sequencing and immunofluorescence for fibroblast analysis.
- ELISA, immunofluorescence, and chemotaxis assays for chemokine detection and cell migration studies.
- Cell communication network construction.
Main Results:
- OKCs are enriched with macrophages, neutrophils, and B cells.
- Myofibroblasts constitute a significant portion of OKC fibroblasts and are enriched in chemokine ligands.
- HIF-1α pathway activation in fibroblasts correlates with chemokine release, promoting peripheral blood mononuclear cell migration.
- Close interactions between myofibroblasts and immune cells were observed, with increased RANKL expression in fibroblasts.
Conclusions:
- OKC immune microenvironment characterized by chemokine-producing myofibroblasts.
- OKC fibroblasts exhibit chemotactic effects on immune cells.
- Interactions between immune cells and fibroblasts may contribute to OKC osteoclastogenic potential.
Objectives:
Various types of cells comprising a complex and diverse cell population are required for the biological activities of odontogenic keratocyst (OKC). Immune and non-immune cells collaborate via cytokine- or chemokine-mediated communication and direct cell-cell interactions. This study aimed to characterize the immune ecosystem and understand the potential chemotactic role of OKC fibroblasts in immune cell migration.
Materials And Methods:
Mass cytometry of 41 markers was employed for the classification of OKC cells from six OKC samples. Immunofluorescence staining and single-cell RNA sequencing (GSE176351) were used for the detection of fibroblast subpopulations. Enzyme-linked immunosorbent assay and immunofluorescence staining were employed for chemokine detection in hypoxia- and/or HIF-1α inhibitor-treated OKC fibroblasts and tissues. Chemotaxis assay was employed to determine the chemotactic effect of fibroblasts via co-culture with peripheral blood mononuclear cells. A cell communication network was constructed based on the single-cell RNA sequencing data.
Results:
The characterization of the immune cell types of OKC evidenced the enrichment of macrophages, neutrophils and B cells. The majority (41.5%) of fibroblast subsets consisted of chemokine ligand-enriched myofibroblasts. The activation of the HIF-1α signaling pathway in fibroblasts was associated with chemokine release. The chemokines released by OKC fibroblasts remarkably promoted the migration of peripheral blood mononuclear cells in the co-culture system. Close interactions between myofibroblasts and immune cells were validated by cell-cell interaction analysis. Increased RANKL expression was detected in OKC fibroblasts in the co-culture system with peripheral blood mononuclear cells.
Conclusions:
Our results provided deep insights into the immune ecosystem and highlighted the potential chemotactic effects of chemokine-enriched myofibroblasts within OKCs. The close interaction between immune cells and fibroblasts demonstrated in this study may be responsible for the osteoclastogenic effects of OKC fibroblasts.
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