Tannerella forsythia infection-induced calvarial bone and soft tissue transcriptional profiles

Abstract

Tannerella forsythia is associated with subgingival biofilms in adult periodontitis, although the molecular mechanisms contributing to chronic inflammation and loss of periodontal bone remain unclear. We examined changes in the host transcriptional profiles during a T. forsythia infection using a murine calvarial model of inflammation and bone resorption. Tannerella forsythia was injected into the subcutaneous soft tissue over calvariae of BALB/c mice for 3 days, after which the soft tissues and calvarial bones were excised. RNA was isolated and Murine GeneChip (Affymetrix, Santa Clara, CA) array analysis of transcript profiles showed that 3226 genes were differentially expressed in the infected soft tissues (P < 0.05) and 2586 genes were differentially transcribed in calvarial bones after infection. Quantitative real-time reverse transcription-polymerase chain reaction analysis of transcription levels of selected genes corresponded well with the microarray results. Biological pathways significantly impacted by T. forsythia infection in calvarial bone and soft tissue included leukocyte transendothelial migration, cell adhesion molecules (immune system), extracellular matrix-receptor interaction, adherens junction, and antigen processing and presentation. Histologic examination revealed intense inflammation and increased osteoclasts in calvariae compared with controls. In conclusion, localized T. forsythia infection differentially induces transcription of a broad array of host genes, and the profiles differ between inflamed soft tissues and calvarial bone.

Figure 1

Leukocyte transendothelial migration pathway containing genes differentially regulated by Tannerella forsythia in calvarial bone compared with sham-infected controls at P ≤ 0.05, adapted from PATHWAY EXPRESS and using the Kyoto Encyclopedia of Genes and Genomes nomenclature. Genes shown in red are upregulated, genes shown in blue are downregulated, and green indicates no change in gene expression at the P < 0.05 significance level. +p, phosphorylation event; −p, dephosphorylation event; ?, receptors that are yet to be identified; O, other molecule. An arrow indicates a molecular interaction resulting in transendothelial migration, leukocyte activation, regulation of actin cytoskeleton and a line without an arrowhead indicates a molecular interaction resulting in inhibition.

Figure 2

Leukocyte transendothelial migration pathway containing genes differentially regulated by Tannerella forsythia in calvarial soft tissue compared with sham-infected controls at P ≤ 0.05, adapted from PATHWAY EXPRESS and using the Kyoto Encyclopedia of Genes and Genomes nomenclature. Red indicates induction, blue indicates repression, and green indicates no change in gene expression. +p, phosphorylation event; −p, dephosphorylation event; ?, receptors that are yet to be identified; O, other molecule. An arrow indicates a molecular interaction resulting in transendothelial migration, leukocyte activation, regulation of actin cytoskeleton and a line without an arrowhead indicates a molecular interaction resulting in inhibition.

Figure 3

Cell adhesion molecules pathway (immune system) containing genes differentially regulated by Tannerella forsythia in calvarial bone and soft tissue compared with sham-infected controls at P ≤ 0.05, adapted from PATHWAY EXPRESS and using the Kyoto Encyclopedia of Genes and Genomes nomenclature. Red indicates induction, blue indicates repression, and green indicates no change in gene expression. An arrow indicates a molecular interaction resulting in activation of dendritic cells, macrophages, T cells (T-cell receptor signaling pathway), and a line without an arrowhead indicates a molecular interaction resulting in inhibition.

Figure 4

Extracellular membrane receptor interaction pathway containing genes differentially regulated by Tannerella forsythia in calvarial bone compared with sham-infected controls at P ≤ 0.05, adapted from PATHWAY EXPRESS and using the Kyoto Encyclopedia of Genes and Genomes nomenclature. Red indicates induction, blue indicates repression, and green indicates no change in gene expression. An arrow indicates a molecular interaction resulting in extracellular matrix receptor activation, regulation of integrin (VLA proteins, leukoproteins, cytoadhesin, focal adhesion, proteoglycan, glycoprotein).

Figure 5

Focal adhesion pathway containing genes differentially regulated by Tannerella forsythia in calvarial bone compared with sham-infected controls at P ≤ 0.05, adapted from PATHWAY EXPRESS and using the Kyoto Encyclopedia of Genes and Genomes nomenclature. Red indicates induction, blue indicates repression, and green indicates no change in gene expression. An arrow indicates a molecular interaction resulting in extracellular matrix-receptor interaction, cytokine–cytokine receptor interaction, regulation of actin cytoskeleton, cell proliferation, cell cycle, and three signaling pathway systems (phosphatidyl inositol, mitogen-activated protein kinase, Wnt).

Figure 6

Effects of Tannerella forsythia local injection on mouse calvaria. Live T. forsythia bacteria (1.5 × 10⁹) were injected once daily for 3 days into the subcutaneous tissues overlying the calvaria of mice. All photomicrographs are of sections stained with hematoxylin & eosin. (A) Lack of edema and inflammation in the calvarial soft tissue of a sham-infected control mouse (10 ×). (B) Numerous osteoclasts (black arrows) are seen throughout the inner aspects of the calvarial bone mainly in the suture area. (20 ×). (C) Activated osteoclasts at higher magnification (40 ×). Also note the increase in marrow space size, as a result of the increased bone resorption in (B, C).

Figure 6

Effects of Tannerella forsythia local injection on mouse calvaria. Live T. forsythia bacteria (1.5 × 10⁹) were injected once daily for 3 days into the subcutaneous tissues overlying the calvaria of mice. All photomicrographs are of sections stained with hematoxylin & eosin. (A) Lack of edema and inflammation in the calvarial soft tissue of a sham-infected control mouse (10 ×). (B) Numerous osteoclasts (black arrows) are seen throughout the inner aspects of the calvarial bone mainly in the suture area. (20 ×). (C) Activated osteoclasts at higher magnification (40 ×). Also note the increase in marrow space size, as a result of the increased bone resorption in (B, C).

Figure 6

Effects of Tannerella forsythia local injection on mouse calvaria. Live T. forsythia bacteria (1.5 × 10⁹) were injected once daily for 3 days into the subcutaneous tissues overlying the calvaria of mice. All photomicrographs are of sections stained with hematoxylin & eosin. (A) Lack of edema and inflammation in the calvarial soft tissue of a sham-infected control mouse (10 ×). (B) Numerous osteoclasts (black arrows) are seen throughout the inner aspects of the calvarial bone mainly in the suture area. (20 ×). (C) Activated osteoclasts at higher magnification (40 ×). Also note the increase in marrow space size, as a result of the increased bone resorption in (B, C).