Oral neutrophil transcriptome changes result in a pro-survival phenotype in periodontal diseases

Abstract

Background: Periodontal diseases are inflammatory processes that occur following the influx of neutrophils into the periodontal tissues in response to the subgingival bacterial biofilm. Current literature suggests that while neutrophils are protective and prevent bacterial infections, they also appear to contribute to damage of the periodontal tissues. In the present study we compare the gene expression profile changes in neutrophils as they migrate from the circulation into the oral tissues in patients with chronic periodontits and matched healthy subjects. We hypothesized that oral neutrophils in periodontal disease patients will display a disease specific transcriptome that differs from the oral neutrophil of healthy subjects.

Methods: Venous blood and oral rinse samples were obtained from healthy subjects and chronic periodontitis patients for neutrophil isolation. mRNA was isolated from the neutrophils, and gene expression microarray analysis was completed. Results were confirmed for specific genes of interest by qRT-PCR and Western Blot analysis.

Results and discussion: Chronic periodontitis patients presented with increased recruitment of neutrophils to the oral cavity. Gene expression analysis revealed differences in the expression levels of genes from several biological pathways. Using hierarchical clustering analysis, we found that the apoptosis network was significantly altered in patients with chronic inflammation in the oral cavity, with up-regulation of pro-survival members of the Bcl-2 family and down-regulation of pro-apoptosis members in the same compartment. Additional functional analysis confirmed that the percentages of viable neutrophils are significantly increased in the oral cavity of chronic periodontitis patients.

Conclusions: Oral neutrophils from patients with periodontal disease displayed an altered transcriptome following migration into the oral tissues. This resulted in a pro-survival neutrophil phenotype in chronic periodontitis patients when compared with healthy subjects, resulting in a longer-lived neutrophil. This is likely to impact the severity and length of the inflammatory response in this oral disease.

Figure 1. Oral chronic inflammation increases neutrophil recruitment to the site of infection.

The proportion of neutrophils recruited to the oral cavity was counted in healthy patients, patients with chronic periodontits. * p ≤ 0.02; vs. Healthy. All data are mean ± SEM. Healthy subjects (n = 14); Generalized Periodontitis (n = 62).

Figure 2. The oral neutrophil in chronic periodontitis has high transcriptional activity compared to the oral neutrophil in a healthy patient.

(A) Graphic representation of the number of genes that are either up regulated (red) or down regulated (green), when blood neutrophils (PMN-B) were compared with oral neutrophils (PMN-O) in healthy individuals and chronic periodontitis patients FC ≥2; p-value ≤0.05. (B) Heatmap of genes with FC ≥5. Genes shown in red are up-regulated and those shown in green are down-regulated in Oral samples of Chronic Periodontitis patients (n = 4). The complete list of genes can be found at the file S1.

Figure 3. Multi analysis of Gene Ontology (GO) annotations demonstrates that some key functions are preserved during inflammatory process in the mouth.

The GO terms grouped into Cellular Components that are uniquely enriched in healthy patients (green) and chronic periodontits patients (red). Significantly enriched GO terms in both comparisons are marked yellow. The degree of color saturation of each node is positively correlated with the significance of enrichment of the corresponding GO term. Red arrows stand for relationship between two enriched GO terms, black solid arrows stand for relationship between enriched and unenriched terms.

Figure 4. Cluster analysis of Gene Ontology (GO) annotations reveals apoptosis regulation as one of the main changes in the neutrophil transcriptome pattern in an inflamed site.

The GO terms assigned to healthy patients (black) and chronic periodontits patients (striped) were grouped into clusters. In general, a similar percentage of genes were found in most categories. The apparently significant difference between the number of genes from healthy patients and chronic periodontitis patients falls into the apoptosis cluster. Differences in demonstrated categories are significant.

Figure 5. Graphic representation of Death Receptor Pathway and Apoptosis Signaling Pathway in neutrophils demonstrates that they are altered in neutrophils in chronic inflammation.

IPA canonical pathway analysis of Death Receptor Signaling Pathway in neutrophils from (A) healthy individuals and (B) chronic periodontitis patients. Red represents up-regulated genes, green are down-regulated genes and white symbols depict neighbouring genes in this analysis.

Figure 6. Validation of microarray data with qRT-PCR.

(A) Quantitative real time PCR was used to quantify gene expression of selected genes from neutrophils isolates from blood and oral rinse. Results are expressed as fold vs. Blood expression used as internal control. Gene expression was normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a reference gene. (B) Correspondence between mean fold change (FC) values obtained by microarray (X) vs. qRT-PCR (y) analysis. The diagonal line represents the ideal correspondence trend (Pearson correlation r = 0.8712, p-value ≤0.01). All data are mean ± SEM from 3 independent experiments (n = 4).

Figure 7. Functional confirmation of microarray data with Western Blotting and by Flow Cytometry – Presenting up-regulation of anti-apoptosis and down-regulation of pro-apoptosis proteins.

(A) Western blotting quantification; 15 µg of protein from total cell lysate of PMN-B and PMN-O from chronic periodontitis patients were loaded, expression was normalized to β-actin used as internal control (n = 4). (B) Representative Western Blot of members of apoptosis pathway Bcl-xl (anti-apoptosis), Bim and Bax (pro-apoptosis). (C) Summary of relative changes in BCL-2 family (Ratio of expression in PMN-O compared to PMN-B). (D) Percentages of viable cells were analyzed by Flow cytometry in oral neutrophils (n = 3). Cells that were negative for Annexin V and PI were considered viable. * p ≤ 0.05 vs. Healthy. All data are mean ± SEM from 3 independent experiments.