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. 2023 Nov;29(8):3420-3432.
doi: 10.1111/odi.14293. Epub 2022 Jul 10.

Single-cell analysis reveals immune cellular components in odontogenic keratocysts

Qi-Wen Man  1   2 , Rui-Fang Li  1 , Jian-Feng Liu  1 , Yi Zhao  1   3 , Bing Liu  1   2
Affiliations

Single-cell analysis reveals immune cellular components in odontogenic keratocysts

Qi-Wen Man et al. Oral Dis. 2023 Nov.

Abstract

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.

Keywords: flow cytometry; genetic heterogeneity; immune system; single-cell analysis.

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