Gene expression profiling of periodontitis-affected gingival tissue by spatial transcriptomics

Abstract

Periodontitis is a highly prevalent chronic inflammatory disease of the periodontium, leading ultimately to tooth loss. In order to characterize the gene expression of periodontitis-affected gingival tissue, we have here simultaneously quantified and localized gene expression in periodontal tissue using spatial transcriptomics, combining RNA sequencing with histological analysis. Our analyses revealed distinct clusters of gene expression, which were identified to correspond to epithelium, inflamed areas of connective tissue, and non-inflamed areas of connective tissue. Moreover, 92 genes were identified as significantly up-regulated in inflamed areas of the gingival connective tissue compared to non-inflamed tissue. Among these, immunoglobulin lambda-like polypeptide 5 (IGLL5), signal sequence receptor subunit 4 (SSR4), marginal zone B and B1 cell specific protein (MZB1), and X-box binding protein 1 (XBP1) were the four most highly up-regulated genes. These genes were also verified as significantly higher expressed in gingival tissue of patients with periodontitis compared to healthy controls, using reverse transcription quantitative polymerase chain reaction. Moreover, the protein expressions of up-regulated genes were verified in gingival biopsies by immunohistochemistry. In summary, in this study, we report distinct gene expression signatures within periodontitis-affected gingival tissue, as well as specific genes that are up-regulated in inflamed areas compared to non-inflamed areas of gingival tissue. The results obtained from this study may add novel information on the genes and cell types contributing to pathogenesis of the chronic inflammatory disease periodontitis.

Figure 1

Gene activity within periodontitis-affected gingival tissue. The total numbers of transcripts per spot are visualized at their corresponding location within the gingival biopsy section. The spots are colored according to their log2 transformed total transcript content (yellow = low number of transcripts, red = high number of transcripts). CT: connective tissue; E: epithelium; IC: inflammatory cells.

Figure 2

Dimensionality reduction of the gene expression data obtained from the periodontal biopsy section. (a) t-distributed stochastic neighbor embedding (t-SNE) plot performed on the gene expression data from spots covered by tissue. K-means clustering analysis has identified three groups of spots, which have been assigned a color each. (b) The spots visualized at their original positions in the gingival tissue. The colors indicate the clusters each spot was assigned to in the t-SNE plot in (a).

Figure 3

Distribution of gene activity of the four genes up-regulated to the greatest extent in areas of inflammatory cells compared to areas without inflammatory cells. The gene expression distribution of immunoglobulin lambda-like polypeptide 5 (IGLL5), signal sequence receptor subunit 4 (SSR4), marginal zone B and B1 cell specific protein (MZB1) and X-box binding protein 1 (XBP1) are shown. Yellow spots correspond to low expression of IGLL5, SSR4, MZB1, or XBP1, whereas red spots correspond to high gene expression. All count values have been normalized and log2 transformed.

Figure 4

Gene expression validation in gingival tissue biopsies using qRT-PCR. The four most highly upregulated genes in connective tissue with inflammatory cells, compared to connective tissue without inflammatory cells, as identified by spatial transcriptomics, were validated using qRT-PCR in RNA from six gingival tissues from patients with periodontitis and four control samples without periodontitis. Gene expression is shown as log2 fold change (FC) of periodontitis samples relative to healthy controls and was calculated according to the ΔΔCt method, where each periodontitis sample was normalized to the control samples and to GAPDH (reference gene) of corresponding periodontitis sample. The reactions were run in duplicates and experiment was repeated with similar results. IGLL5: immunoglobulin lambda-like polypeptide 5; SSR4: signal sequence receptor subunit 4; MZB1: marginal zone B and B1 cell specific protein; XBP1: X-box binding protein 1.

Figure 5

Protein expression in gingival tissue biopsies by immunohistochemistry. Gingival tissue sections obtained from patients with periodontitis were stained for signal sequence receptor subunit 4 (SSR4), marginal zone B and B1 cell specific protein (MZB1) and X-box binding protein 1 (XBP1). The sections were counterstained with Mayer’s hematoxylin. Images were taken with x20 objective. Representative images of gingival biopsies from four patients with periodontitis are shown. Scale bar: 50 μm. CT: connective tissue; E: epithelium; IC: inflammatory cells.