Fusobacterium nucleatum Alters Atherosclerosis Risk Factors and Enhances Inflammatory Markers with an Atheroprotective Immune Response in ApoE(null) Mice

Abstract

The American Heart Association supports an association between periodontal disease (PD) and atherosclerotic vascular disease (ASVD) but does not as of yet support a causal relationship. Recently, we have shown that major periodontal pathogens Porphyromonas gingivalis and Treponema denticola are causally associated with acceleration of aortic atherosclerosis in ApoEnull hyperlipidemic mice. The aim of this study was to determine if oral infection with another significant periodontal pathogen Fusobacterium nucleatum can accelerate aortic inflammation and atherosclerosis in the aortic artery of ApoEnull mice. ApoEnull mice (n = 23) were orally infected with F. nucleatum ATCC 49256 and euthanized at 12 and 24 weeks. Periodontal disease assessments including F. nucleatum oral colonization, gingival inflammation, immune response, intrabony defects, and alveolar bone resorption were evaluated. Systemic organs were evaluated for infection, aortic sections were examined for atherosclerosis, and inflammatory markers were measured. Chronic oral infection established F. nucleatum colonization in the oral cavity, induced significant humoral IgG (P=0.0001) and IgM (P=0.001) antibody response (12 and 24 weeks), and resulted in significant (P=0.0001) alveolar bone resorption and intrabony defects. F. nucleatum genomic DNA was detected in systemic organs (heart, aorta, liver, kidney, lung) indicating bacteremia. Aortic atherosclerotic plaque area was measured and showed a local inflammatory infiltrate revealed the presence of F4/80+ macrophages and CD3+ T cells. Vascular inflammation was detected by enhanced systemic cytokines (CD30L, IL-4, IL-12), oxidized LDL and serum amyloid A, as well as altered serum lipid profile (cholesterol, triglycerides, chylomicrons, VLDL, LDL, HDL), in infected mice and altered aortic gene expression in infected mice. Despite evidence for systemic infection in several organs and modulation of known atherosclerosis risk factors, aortic atherosclerotic lesions were significantly reduced after F. nucleatum infection suggesting a potential protective function for this member of the oral microbiota.

Fig 1. Oral infection schedule and periodontal disease parameters.

(A) Oral infection schedule. (B) Twelve-week infection-induced horizontal alveolar bone resorption was statistically significant on the maxilla palatal and mandible lingual sides. (C) Twenty-four-week infection-induced horizontal ABR was statistically significant on the maxilla palatal, maxilla buccal, and mandible lingual sides. (D) Representative left maxilla lingual view images of horizontal ABR measurements, outlining the area between the cemento-enamel junction (CEJ) and alveolar bone crest (ABC) of molars 1, 2, and 3 of 12- and 24 week F. nucleatum-infected and sham-infected mice. (E) Representative images of gingival histology of 12- and 24 week-infected and sham-infected mice demonstrating minimal gingival inflammation. * P<0.05, ** P<0.01, *** P<0.001.

Fig 2. Chronic oral infection with F. nucleatum induced significant levels of serum F. nucleatum-specific antibodies.

Graphs represent the fold-increase in F. nucleatum-specific IgG or IgM antibody titer in infected mice over control mice at both 12 and 24 weeks of infection. (*** P<0.001).

Fig 3. Chronic oral infection results in an unexpected reduction in plaque area at 24 weeks.

(A) Infected mouse plaque area was significantly reduced when compared to sham-infected mice at 24 weeks of infection. Control mouse plaque area was significantly increased at 24 weeks relative to 12 weeks. (B) Intimal thickness at 12 and 24 weeks. (C) Medial thickness at 12 and 24 weeks. (D) Intimal/medial thickness ratios again indicate a significant reduction in plaque at 24 weeks in infected mice when compared to controls. (E) Twenty-four week-infected mouse aorta demonstrating plaque. Scale bar is 100μm. Arrowheads indicate plaque. (F) CD3⁺ T cell counts were significantly higher in 12 week-infected mice than controls and also higher than 24 week-infected mice. (G) Twelve-week infected mouse aorta stained for CD3⁺ T cells. Arrows define plaque margins, arrow heads point to CD3⁺ stained cells (brown staining). Scale bar is 100μm. (h) F4/80⁺ macrophage counts in 12 week-infected mice were unaffected. (I) Twenty-four week-infected mouse aorta stained for F4/80⁺ cells. Arrows define plaque margins, arrow heads point to F4/80⁺ stained cells. Scale bar is 50μm. Fn—F. nucleatum, Con—control, L—lumen, I, Int—intimal layer, M—medial layer, A, Adv—adventitial layer. * P<0.05, ** P<0.01, *** P<0.001.

Fig 4. Chronic oral infection with F. nucleatum significantly alters serum risk factors for atherosclerosis.

(A) Twenty four-week-infected mice (n = 6) developed significantly elevated levels of total serum cholesterol and total serum triglycerides relative to sham-infected mice (n = 6). (B) F. nucleatum infected mice developed significantly elevated levels of chylomicrons (CM), very low density lipoproteins (VLDL), low density lipoprotein (LDL) and high density lipoproteins (HDL) relative to sham-infected mice (n = 6). (C) Infection significantly increased the total serum oxyLDL in infected mice. (D) Infected mice exhibited significantly elevated acute phase inflammatory protein serum amyloid A at 24 weeks. (E) Infection with F. nucleatum did not significantly alter the concentration of serum nitric oxide at 24 weeks of infection. Fn—F. nucleatum infected mice, Con—control, Chol—cholesterol, Trigly—triglycerides, CM—chylomicrons, VLDL—very low density lipoprotein, LDL—low density lipoprotein, HDL—high density lipoprotein. * P<0.05, ** P<0.01, *** P<0.001.