Gene Expression Profiles in Paired Gingival Biopsies from Periodontitis-Affected and Healthy Tissues Revealed by Massively Parallel Sequencing

<div><p>Periodontitis is a chronic inflammatory disease affecting the soft tissue and bone that surrounds the teeth. Despite extensive research, distinctive genes responsible for the disease have not been identified. The objective of this study was to elucidate transcriptome changes in periodontitis, by investigating gene expression profiles in gingival tissue obtained from periodontitis-affected and healthy gingiva from the same patient, using RNA-sequencing. Gingival biopsies were obtained from a disease-affected and a healthy site from each of 10 individuals diagnosed with periodontitis. Enrichment analysis performed among uniquely expressed genes for the periodontitis-affected and healthy tissues revealed several regulated pathways indicative of inflammation for the periodontitis-affected condition. Hierarchical clustering of the sequenced biopsies demonstrated clustering according to the degree of inflammation, as observed histologically in the biopsies, rather than clustering at the individual level. Among the top 50 upregulated genes in periodontitis-affected tissues, we investigated two genes which have not previously been demonstrated to be involved in periodontitis. These included interferon regulatory factor 4 and chemokine (C-C motif) ligand 18, which were also expressed at the protein level in gingival biopsies from patients with periodontitis. In conclusion, this study provides a first step towards a quantitative comprehensive insight into the transcriptome changes in periodontitis. We demonstrate for the first time site-specific local variation in gene expression profiles of periodontitis-affected and healthy tissues obtained from patients with periodontitis, using RNA-seq. Further, we have identified novel genes expressed in periodontitis tissues, which may constitute potential therapeutic targets for future treatment strategies of periodontitis.</p> </div

H&E and CD3-stained paraffin-embedded gingival biopsies obtained from one representative patient with periodontitis.

<p>A. H&E staining of inflammatory cells in periodontitis-affected sections. B. H&E staining of inflammatory cells in healthy gingival sections. C. Staining of the T-cell marker CD3 in periodontitis-affected sections. D. Staining of the T-cell marker CD3 in healthy sections. E, epithelium, C, connective tissue.</p

Clustering dendrogram and heatmap of periodontitis-affected and healthy biopsies.

<p>Clustering of all samples was based on gene transcripts with a median read above three times the background noise. The length of the branch between two biopsies and the colors of the heatmap correspond to degree of similarity between the gene expression profiles. Colors can be interpreted using the scale bar. Numbers in parentheses denote the inflammation scores of the biopsies after H&E histological evaluation.</p

Gene ontology (GO) analysis of differentially expressed genes.

<p>All significant (<i>p</i><0.05) Biological processes (GO categories) and their parent terms are shown. The color of each node illustrates the significance and can be interpreted using the scale bar, which displays the <i>p</i> value. Each node is also marked with the number of significantly regulated genes mapped to the GO category.</p

Immunohistochemical stainings of IRF4 and CCL18 in the connective tissue of periodontitis-affected gingival sections.

<p>A. Immunohistochemical staining of IRF4. B. Immunohistochemical staining of CCL18.</p

Enriched regulated (KEGG) biological pathways among unique genes in healthy tissues.

a<p>Lists of uniquely expressed genes within the enriched pathways can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046440#pone.0046440.s001" target="_blank">Table S1</a>.</p>b<p>adj <i>p</i> value indicates the significance of the enrichment, (adj <i>p</i><0.05).</p

Volcano plot displaying differential expression.

<p>Differential gene expression (adj <i>p</i><0.01) between periodontitis-affected and healthy gingival tissues. The <i>y</i> axis corresponds to the log₂ fold change value (<i>M</i> value), and the <i>x</i> axis displays the mean expression value.</p

Expression of the inflammatory mediators in periodontitis-affected and healthy tissues obtained by RNA-seq.

<p>The bars show the expression (log₂ fold change) pattern based on RNA-Seq reads of IL-1β, IL-6, IL-8, TNFα, RANTES and MCP-1.</p

Venn diagram of mRNA transcripts.

<p>Venn diagram showing genes that were uniquely expressed in periodontitis-affected (1375) and healthy (511) gingival tissues. The intersection of the two circles refers to transcripts, which are expressed in both periodontitis-affected and healthy gingival tissues (20 236).</p

Enriched regulated (KEGG) biological pathways among unique genes in periodontitis-affected tissues.

a<p>Lists of uniquely expressed genes within the enriched pathways can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046440#pone.0046440.s001" target="_blank">Table S1</a>.</p>b<p>adj <i>p</i> value indicates the significance of the enrichment, (adj <i>p</i><0.05).</p