Human oral mucosa cell atlas reveals a stromal-neutrophil axis regulating tissue immunity

Abstract

The oral mucosa remains an understudied barrier tissue. This is a site of rich exposure to antigens and commensals, and a tissue susceptible to one of the most prevalent human inflammatory diseases, periodontitis. To aid in understanding tissue-specific pathophysiology, we compile a single-cell transcriptome atlas of human oral mucosa in healthy individuals and patients with periodontitis. We uncover the complex cellular landscape of oral mucosal tissues and identify epithelial and stromal cell populations with inflammatory signatures that promote antimicrobial defenses and neutrophil recruitment. Our findings link exaggerated stromal cell responsiveness with enhanced neutrophil and leukocyte infiltration in periodontitis. Our work provides a resource characterizing the role of tissue stroma in regulating mucosal tissue homeostasis and disease pathogenesis.

Trial registration: ClinicalTrials.gov NCT01568697.

Keywords: human oral mucosal single-cell atlas; mucosal immunity; oral atlas; oral mucosal immunity; periodontitis single-cell atlas; stromal -immune interaction.

Figure 1:. Major cell types in healthy adult oral mucosal tissues.

A. Schematic of the oral mucosal tissues biopsied with locations indicated (E-epithelium, LP- lamina propria). B. UMAP representation of major cell types identified by scRNA seq (n=21, 88,481 cells, left) and bar graph of relative cell proportions by tissue type (B-buccal, G-gingival) (right). C. Immunofluorescence depicting major cell types in healthy gingival and buccal mucosa. Scale bar: 41 μm. Stains indicated and color coded. D. Violin plots of selected markers for major cell populations and relative expression per tissue type. X-axis represents normalized average expression. E. t-SNE (left) and proportion plots (right) showing representation of major cell types in oral (n=21), skin (n=8), ileum (n=11), and lung (n=8) barrier tissues. All datasets were truncated to ensure equal number of cells (23,787) were compared. See also Table S1–S3, Figure S1.

Figure 2:. Stromal and epithelial subpopulations in healthy oral mucosa.

A,C,E. UMAP (left) and proportion plots (right) of endothelial (37,393 cells), epithelial (6,372 cells) and fibroblast (19,750 cells) populations, respectively, in gingival (G) and buccal (B) mucosa (n=13 and n=8, respectively). Refer to methods for statistical tests used. *p < 0.05, **p <0.01. B,D,F. Dot plots depicting the expression of cluster-defining genes and percentage of cells expressing each gene for endothelial, epithelial, and fibroblast populations, respectively. Expression values are normalized and scaled averages. Cartoon on the right illustrate cell subtypes associated with immune activation/recruitment. Proportion plots to the right of each illustration depict the contribution of each tissue type to each cell subtype. Clusters were generated using a resolution of 1 prior to subsetting into major cell types and only epithelial clusters were reclustered at a resolution of 0.1. See also Table S4, Figure S2.

Figure 3:. Immune populations in healthy oral mucosa.

A. UMAP (left) and proportion plots (right) of immune cell populations in oral mucosa (23,575 cells; gingival (G, n=13), buccal (B, n=8)). Immune cells were annotated using a combination of reference based (SingleR) and manual annotation. Refer to methods for statistical tests used. *p < 0.05, **p<0.01. B-D. Dot plots depicting expression of cell type marker genes. Expression values are normalized and scaled averages. E-H. Representative flow cytometry scatter plots from buccal (n=3–8) and gingival (n=8) biopsies. Cells were gated from Single/Live,CD45 (E) and CD45/HLADR (F), CD45⁺ cells were further gated for CD19/CD3 (G) and HLADR⁻SSC^highCD15/CD117 (H). Bar graphs (E-H) demonstrate % expression for each marker indicated, each dot represents an individual biopsy. Refer to methods for statistical tests used. p values indicated on each graph. See also Table S1, S3, S5, and Figure S3.

Figure 4:. Periodontal disease is an inflammatory disease at the gingival mucosa

A. Illustration of the gingival mucosa in health (H, top) and periodontal disease (P, bottom). A schematic of methods for tissue analysis is presented (right). B. H&E staining of representative gingival health (left) and periodontitis (right) sections. E-epithelium, S-stroma. Scale bar: 100μm. C. Immunofluorescence staining for CD45⁺ lymphocytes. Scale bar: 50μm. D. Representative flow cytometry scatter plots (left). Cells were gated from Single/Live and stained with CD45, graph indicates CD45 % expression (one dot per patient, n=6/group. Refer to methods for statistical tests used, p value indicated). E. UMAP representation of major cell populations in health and periodontal disease (left; H: 53,739 cells, n=13; P: 34,401 cells, n=8). Graph demonstrates the proportion of each cell type in health and disease (right). Refer to methods for statistical tests used. *p < 0.05, ***p<0.001. See also Table S1.

Figure 5:. Immune cell subpopulations in periodontal disease.

A. UMAP (left) showing immune cell subpopulations from both health (n=13) and disease (n=8) divided spatially into three main categories (T/NK, B/Plasma and Myeloid/Granulocyte). Proportion plot (middle). Colors indicate the cell type annotated using SingleR and manual annotation and validated by cell-type specific gene expression (right). B. Bar graph depicts proportion of B, plasma cells and neutrophils in health (H) and periodontitis (P). Refer to methods for statistical tests used. ***p<0.001. C,D. Normalized average expression of plasma cell- (C) and neutrophil- (D) specific markers visualized in low-dimensional space with schex. Each area containing cells on the UMAP was divided into hexagonal areas and gene expression values within each area were averaged across cells present. E-H. Representative flow cytometry scatter plots from an independent health and periodontitis cohort. Cells were gated from Single/Live cells and CD45/CD3 (E), CD45/HLADR (F), CD45/CD38 (G), CD45/CD15 (H). Bar graphs demonstrate % expression (one dot per individual, n=3–9/group). Refer to methods for statistical tests used. P values are indicated on each graph. See also Table S1, Figure S4, S5.

Figure 6:. Stromal cell populations and stromal- immune interactome in periodontitis.

A-C. UMAP representation (top) and proportion plots (bottom) for endothelial (A; 29,050 cells), epithelial (B; 5,355 cells) and fibroblast (C; 16,705 cells) populations, respectively, in the combined health (n=13) and periodontitis (n=8) dataset. Gene labels indicate cluster-defining genes. Refer to methods for statistical tests used. D. Dot plots of pathways enriched in periodontal disease per major cell population. Some GO terms truncated for brevity, original terms and gene list in Table S6. The size of each dot represents the number of genes from each pathway. E. Alluvial plot showing selected ligand-receptor pairs significantly over-represented in periodontal disease determined by NicheNetR. For this analysis, only interactions of immune cells (receptors) with all other cell types, and only ligands and receptors that are differentially expressed (p<0.05), are considered. F. Chemokine/chemokine receptor interactions in health and periodontal disease. Dot plot indicating expression of chemokines in major cell types (top) and the immune cell(s) in the scRNA seq dataset that express cognate receptors (bottom). Expression values are normalized and scaled averages. Refer to Figure S6 for expression data of chemokine receptors. Clusters were generated using a resolution of 1 prior to sub-setting into major cell types and were not reclustered. See also Table S6, Figure S5, S6.

Figure 7:. Genes associated with periodontal disease susceptibility, microbe and damage sensing in the oral mucosa.

A. Dot plot depicting the normalized and scaled average expression of genes and percentage of expressing cells for genes associated with Mendelian forms of periodontitis (left) and genes identified by GWAS to be associated with periodontitis (right). B. Summary of select pattern recognition receptors and damage-associated molecular pattern and associated receptors expression in buccal and gingival healthy mucosa (top) and expression in gingival health and periodontitis (bottom). Expression is shown by major tissue type (Endothelial, Fibroblast, Epithelial, Immune). A solid white border indicates statistical significance and the tissue that had higher expression. Significance determined by non-parametric Wilcoxon rank sum test. See also Table S7, Figure S7.