Invasion of oral and aortic tissues by oral spirochete Treponema denticola in ApoE(-/-) mice causally links periodontal disease and atherosclerosis

Abstract

Treponema denticola is a predominantly subgingival oral spirochete closely associated with periodontal disease and has been detected in atherosclerosis. This study was designed to evaluate causative links between periodontal disease induced by chronic oral T. denticola infection and atherosclerosis in hyperlipidemic ApoE(-/-) mice. ApoE(-/-) mice (n = 24) were orally infected with T. denticola ATCC 35404 and were euthanized after 12 and 24 weeks. T. denticola genomic DNA was detected in oral plaque samples, indicating colonization of the oral cavity. Infection elicited significantly (P = 0.0172) higher IgG antibody levels and enhanced intrabony defects than sham infection. T. denticola-infected mice had higher levels of horizontal alveolar bone resorption than sham-infected mice and an associated significant increase in aortic plaque area (P ≤ 0.05). Increased atherosclerotic plaque correlated with reduced serum nitric oxide (NO) levels and increased serum-oxidized low-density lipoprotein (LDL) levels compared to those of sham-infected mice. T. denticola infection altered the expression of genes known to be involved in atherosclerotic development, including the leukocyte/endothelial cell adhesion gene (Thbs4), the connective tissue growth factor gene (Ctgf), and the selectin-E gene (Sele). Fluorescent in situ hybridization (FISH) revealed T. denticola clusters in both gingival and aortic tissue of infected mice. This is the first study examining the potential causative role of chronic T. denticola periodontal infection and vascular atherosclerosis in vivo in hyperlipidemic ApoE(-/-) mice. T. denticola is closely associated with periodontal disease and the rapid progression of atheroma in ApoE(-/-) mice. These studies confirm a causal link for active oral T. denticola infection with both atheroma and periodontal disease.

FIG 1

T. denticola infection schedule and spirochete-specific serum antibody levels in ApoE^−/− mice. (A) Schematic diagram of the experimental design illustrating T. denticola infections, oral plaque sampling, euthanasia, and collection of blood and tissue specimens. (B) Serum IgG and IgM antibody levels in T. denticola-infected mice (Td) and sham-infected mice (Cont). Each bar represents the group mean (n = 12 mice) antibody level. T. denticola-infected mice at both 12 and 24 weeks had statistically significant higher levels of IgG than the sham-infected controls (*, P ≤ 0.05). All the tests were performed in triplicates. Data points and error bars represent means and standard deviations from three independent experiments.

FIG 2

Horizontal alveolar bone resorption of ApoE^−/− mice following T. denticola infection. (A and B) Morphometric analysis of 12- and 24-week total horizontal alveolar bone resorption in mice (n = 12) (***, P < 0.001; **, P < 0.005). Each bar indicates the mean horizontal alveolar bone resorption. Measurements were made between the cementoenamel junction (CEJ) and alveolar bone crest (ABC) of three molar teeth by three independent individuals blinded to the treatment group. Error bars indicate standard deviations. Panels C to F are representative images of measureable bone resorption denoted by the area inside the red line. Mandible-lingual view of T. denticola-infected (C) and sham-infected (D) mice at 12 weeks; mandible lingual view of 24-week T. denticola-infected (E) and sham-infected (F) mice; (G) representative images of mandible palatal surfaces showing extensive interproximal intrabony defects (shown by black arrows) in mice infected with T. denticola at 24 weeks. M1, first molar; M2, second molar; M3, third molar; H, sham-infected mice showing no visible intrabony defects.

FIG 3

T. denticola infection induced an increase in atherosclerotic plaque growth in the ascending aorta of ApoE^−/− mice in comparison to sham-infected mice. (A) Bar graph of total aortic plaque area (mm²) in T. denticola-infected ApoE^−/− mice at 12 weeks (*, P < 0.05); (B) intimal thickness (mm) (*, P < 0.05); (C) intimal/medial thickness ratios in T. denticola-infected mice; (D) medial thickness (mm); (E) representative cross section of T. denticola-infected mouse aorta at 12 weeks at the level of the aortic valve showing a larger plaque; (F) ascending aortic cross section at the level of aortic valve from a sham-infected mouse at 12 weeks; (G) ascending aorta with large globular plaque in a T. denticola-infected mouse at 24 weeks with increased macrophage infiltration and large cholesterol crystals; (H) ascending aortic cross section from a control mouse at 24 weeks with smaller aortic plaque. Black arrowheads indicate plaque margins; thin arrows indicate plaque with cholesterol crystals. The adventitia is indicated by A, media by M, intima by I, and lumen by L. (I) Total aortic plaque area (mm²) in T. denticola-infected ApoE^−/− mice at 24 weeks (**, P < 0.01); (J) intimal thickness (mm); (K) intimal/medial thickness ratios (*, P < 0.05); (L) medial thickness (mm) in the 24-week T. denticola-infected mice. Six animals were examined in each group at both 12 and 24 weeks of infection. Multiple cross sections were stained for measurement, and analysis was performed three independent times (2 to 3 sections per aortic area per mouse) by two individuals blinded to the treatment group. Magnification, ×100 for panels E and F and ×200 for panels G and H.

FIG 4

Fluorescent in situ hybridization (FISH) of T. denticola-infected mouse gingival and aortic tissue sections. (A) T. denticola-infected mouse gingival connective tissue at 12 weeks; (B) T. denticola-infected mouse gingival epithelium at 12 weeks; (C) T. denticola-infected gingival epithelial matrix at 12 weeks; (D) T. denticola-infected heart tissue (endothelium and smooth muscle) at 12 weeks; (E) T. denticola-infected aorta at 12 weeks; (F) T. denticola-infected aortic plaque at 24 weeks; (G) fluorescent image of T. denticola-infected aortic plaque tissue; (H) bright-field view (same location as panel G); (I) overlaid image (panels G and H) of T. denticola-infected aortic plaque tissue. P, plaque; L, lumen; RBC, red blood cells in the lumen. White arrows point to the presence of T. denticola in the infected aortic plaque tissue. Brightly fluorescent bacteria (white arrows) are seen with the rRNA species-specific T. denticola probe conjugated to Alexa Fluor 568. The procedure was repeated several times on different sections for confirmation, and a representative few were chosen.

FIG 5

T. denticola infection induced changes in serum of ApoE^−/− mice. (A) Effects of oral infection with T. denticola on serum levels of serum amyloid A (SAA) (n = 6 mice); (B) alteration in serum lipoprotein (chylomicrons, VLDL [**, P < 0.01], LDL, HDL) levels in T. denticola-infected mice compared to those in controls (n = 6 mice); (C) significant increase in serum total cholesterol (**, P < 0.01) and serum total triglycerides levels in T. denticola-infected mice (n = 6); (D) significant changes in serum nitric oxide levels (*, P < 0.05) in T. denticola-infected mice compared to control mice (n = 6 mice in each group); (E) changes in serum-oxidized low-density lipoprotein (OxyLDL) levels after infection with T. denticola and sham-infected ApoE^−/− mice (n = 6 in each group). All the tests were run in triplicates. Data points and error bars represent means and standard deviations for infected compared to control mice.