Single-cell RNA sequencing reveals rebalancing of immunological response in patients with periodontitis after non-surgical periodontal therapy

Abstract

Background: Periodontitis is a major inflammatory disease of the oral mucosa that is not limited to the oral cavity but also has systemic consequences. Although the importance of chronic periodontitis has been emphasized, the systemic immune response induced by periodontitis and its therapeutic effects remain elusive. Here, we report the transcriptomes of peripheral blood mononuclear cells (PBMCs) from patients with periodontitis.

Methods: Using single-cell RNA sequencing, we profiled PBMCs from healthy controls and paired pre- and post-treatment patients with periodontitis. We extracted differentially expressed genes and biological pathways for each cell type and calculated activity scores reflecting cellular characteristics. Intercellular crosstalk was classified into therapy-responsive and -nonresponsive pathways.

Results: We analyzed pan-cellular differentially expressed genes caused by periodontitis and found that most cell types showed a significant increase in CRIP1, which was further supported by the increased levels of plasma CRIP1 observed in patients with periodontitis. In addition, activated cell type-specific ligand-receptor interactions, including the BTLA, IFN-γ, and RESISTIN pathways, were prominent in patients with periodontitis. Both the BTLA and IFN-γ pathways returned to similar levels in healthy controls after periodontal therapy, whereas the RESISTIN pathway was still activated even after therapy.

Conclusion: These data collectively provide insights into the transcriptome changes and molecular interactions that are responsive to periodontal treatment. We identified periodontitis-specific systemic inflammatory indicators and suggest unresolved signals of non-surgical therapy as future therapeutic targets.

Keywords: CRIP1; Chronic periodontitis; Immune response; RESISTIN; Single-cell RNA sequencing; Therapeutic target.

Fig. 1

Composition of peripheral blood mononuclear cells (PBMCs) from healthy donors and pre- and post-treatment periodontitis patients A. The graphical abstract of this study B. Bar graph showing cell numbers in each sample. Blue represents healthy donor samples, orange represents pre-treatment patient samples, and yellow represents post-treatment patient samples C. UMAP plot of 111,213 PBMCs from all subjects, colored according to the major cell lineages D. Scatter plot of canonical marker genes for 13 major lineages projected onto the UMAP plot. The red and gray spectra indicate the expression levels of each gene E. Bar graph showing the proportion of the major cell types for each participant, colored according to the cell type F. PCA plot of the participants using six clinical variables: ESR, CRP, clinical attachment level, probing pocket depth, plaque index, and gingival index

Fig. 2

Differentially expressed genes in innate immune cells, monocytes, dendritic cells, and NK cells A. UMAP plot of the three monocyte subsets: classical, non-classical, and intermediate monocytes B–D. Dot plot showing differential expression levels of genes in each monocyte subtype (B), NK cells (C) and mDCs (D). Displayed genes showed similar expression in control and post-treatment groups, but inverse expression patterns in the pre-treatment group. The color of the dots represents the expression levels of the gene, whereas dot size represents the percent of cells expressing the gene E. Activity score of positive regulation of lipopolysaccharide-mediated signaling pathway in mDCs. Enrichment of genes with GO term GO:0031666 was calculated using the Wilcoxon rank-sum test. p ≤ 0.1 (•), p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****), p ≥ 0.05 (ns)

Fig. 3

Periodontitis-induced transcriptional alterations in adaptive immune, B, CD4T, and CD8T cells A. UMAP plot of the four B cell subsets: exhausted B cells, switched memory B (switched MB) cells, non-switched memory B (non-switched MB) cells, and plasmablasts B. UMAP plot of the four CD4⁺ T-cell subsets: T helper 1 (Th1), T helper 17 (Th17), follicular helper T (Tfh), and regulatory T (Treg) C. UMAP plot of the three CD8⁺ T-cell subsets: central memory T (TCM), effector memory T (TEM), and terminal effector T (TTE) cells D-F. Dot plot showing differential expression levels of genes in the B cell subtypes (D), CD4T cell subtypes (E), and CD8T cell subtypes (F). Figure descriptions are the same as Fig. 2B-D. G. The plasma levels of CRIP1, IFITM1, TNF-α, CRP, and ESR in healthy controls and patients with periodontitis were measured using ELISA. P-values were calculated using the Wilcoxon rank-sum test to compare the two groups H. Relationship between CRIP1 and TNF-α, CRP, and ESR measurements. The Pearson correlation coefficient and p-values were calculated from the correlation test I. Receiver operating characteristic (ROC) curves of CRIP1 and IFITM1 ROC curve analysis showed a clear distinction between the healthy and periodontitis groups

Fig. 4

BTLA signaling pathway and proportion of BTLA⁺ pDCs A–C. Circle plot of BTLA signaling in healthy controls (A), pre-treatment (B), and post-treatment (C) groups. The direction of the arrow indicates the signal sender and receiver and the color of the arrow is the same as that of the sender cell. The edge width corresponds to the strength of the ligand-receptor pairs D. Proportion of BTLA⁺ pDCs in healthy, pre- and post-treatment groups E. Violin plot for the tolerance score of Tregs. Tolerance was calculated using the gene set from GO:0002645 (termed ‘positive regulation of tolerance induction’). Asterisks denote the significance of the differences between the groups calculated using the Wilcoxon rank-sum test. p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****), p ≥ 0.05 (ns)

Fig. 5

Circle plot and expression level of IFNG signaling pathway A. Circle plot of IFNG signaling in the pre-treatment group, similar to Fig. 4 A‒C. The edge width corresponds to the strength of the ligand-receptor pairs B. Violin plot of IFNG expression levels. The horizontal line indicates the average in each group, and the asterisks denote the significance of the difference between groups calculated using the Wilcoxon rank-sum test. p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****), p ≥ 0.05 (ns) C. Proportion of IFNG⁺ NK cells in the healthy, pre- and post-treatment groups D. Average expression of cytokines including IL1, IL12, IL23, and TNF-α in dendritic cells by condition and IFNG receptor expression. The horizontal line indicates the average in each group, and asterisks denote the significance of the difference between groups calculated using the Wilcoxon rank-sum test. p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****), p ≥ 0.05 (ns)

Fig. 6

Circle plot and expression level of RESISTIN signaling pathway A-C. Circle plot of RESISTIN signaling in the healthy (A), pre-treatment (B), and post-treatment (C) groups, similar to Fig. 4 A‒C. The edge width corresponds to the strength of the ligand‒receptor pairs D. Relative contribution of ligand-receptor pairs for RESISTIN signaling pathway E. Violin plot of RETN expression levels in monocytes. The horizontal line indicates the average in each group, and the asterisks denote the significance of the difference between groups calculated using the Wilcoxon rank-sum test. p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****), p ≥ 0.05 (ns) F. Proportion of RETN⁺ monocytes in the healthy, pre- and post-treatment groups G. Violin plot of RETN expression levels in mDCs. The horizontal line indicates the average in each group, and the asterisks denote the significance of the difference between groups calculated using the Wilcoxon rank-sum test. p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****), p ≥ 0.05 (ns) H. Proportion of RETN⁺ mDCs in the healthy, pre- and post-treatment groups