Polymicrobial Oral Infection with Four Periodontal Bacteria Orchestrates a Distinct Inflammatory Response and Atherosclerosis in ApoE null Mice.

Periodontal disease (PD) develops from a synergy of complex subgingival oral microbiome, and is linked to systemic inflammatory atherosclerotic vascular disease (ASVD). To investigate how a polybacterial microbiome infection influences atherosclerotic plaque progression, we infected the oral cavity of ApoE null mice with a polybacterial consortium of 4 well-characterized periodontal pathogens, Porphyromonas gingivalis, Treponema denticola, Tannerealla forsythia and Fusobacterium nucleatum, that have been identified in human atherosclerotic plaque by DNA screening. We assessed periodontal disease characteristics, hematogenous dissemination of bacteria, peripheral T cell response, serum inflammatory cytokines, atherosclerosis risk factors, atherosclerotic plaque development, and alteration of aortic gene expression. Polybacterial infections have established gingival colonization in ApoE null hyperlipidemic mice and displayed invasive characteristics with hematogenous dissemination into cardiovascular tissues such as the heart and aorta. Polybacterial infection induced significantly higher levels of serum risk factors oxidized LDL (p < 0.05), nitric oxide (p < 0.01), altered lipid profiles (cholesterol, triglycerides, Chylomicrons, VLDL) (p < 0.05) as well as accelerated aortic plaque formation in ApoE null mice (p < 0.05). Periodontal microbiome infection is associated with significant decreases in Apoa1, Apob, Birc3, Fga, FgB genes that are associated with atherosclerosis. Periodontal infection for 12 weeks had modified levels of inflammatory molecules, with decreased Fas ligand, IL-13, SDF-1 and increased chemokine RANTES. In contrast, 24 weeks of infection induced new changes in other inflammatory molecules with reduced KC, MCSF, enhancing GM-CSF, IFNγ, IL-1β, IL-13, IL-4, IL-13, lymphotactin, RANTES, and also an increase in select inflammatory molecules. This study demonstrates unique differences in the host immune response to a polybacterial periodontal infection with atherosclerotic lesion progression in a mouse model

Polybacterial infection-induced alteration of serum inflammatory markers in ApoE^null mice.

<p>Sera from polybacterial-infected mice (n = 6) and sham-infected mice (n = 6) at both 12 and 24 weeks was analyzed as described in Materials and Methods.</p><p>Polybacterial infection-induced alteration of serum inflammatory markers in ApoE^null mice.</p

Polybacterial infection elicits a distinct splenic T cell response.

<p>Representative images of the gating scheme are shown in panels 2 and 3, with panel 2 showing the lymphocyte gate, panel 3 showing the CD3⁺ CD4⁺ lymphocyte gate, and panel 4 showing the histogram of CD3⁺ CD4⁺ Receptor⁺ lymphocytes. (A). CD3⁺ CD4⁺ IFNγR⁺ cells indicate Th1 response. (B). CD3⁺ CD4⁺ IL4R⁺ cells indicate Th2 response. (C). CD3⁺ CD4⁺ IL17R⁺ cells indicate Th17 response. Poly—polybacterial infection. ** p < 0.01.</p

Distribution of <i>P</i>. <i>gingivalis</i>, <i>T</i>. <i>denticola</i>, <i>T</i>. <i>forsythia</i>, <i>F</i>. <i>nucleatum</i> genomic DNA in ApoE^null mouse tissue by PCR.

<p>Hematogenous dissemination and invasion of four periodontal pathogens in systemic organs were detected by presence of bacterial genomic DNA by PCR.</p><p>Distribution of <i>P</i>. <i>gingivalis</i>, <i>T</i>. <i>denticola</i>, <i>T</i>. <i>forsythia</i>, <i>F</i>. <i>nucleatum</i> genomic DNA in ApoE^null mouse tissue by PCR.</p

Polymicrobial infection-induced altered expression of atherosclerosis related genes in ApoE^null mouse.

<p>Twenty-four week polybacterial-infected and sham-infected aortic tissue samples were processed and analyzed as described in Methods.</p><p>Polymicrobial infection-induced altered expression of atherosclerosis related genes in ApoE^null mouse.</p

Viable bacteria were recovered from infected mouse heart and aorta.

<p>(A). FISH reveals cluster of coccobacilli morphotype of <i>P</i>. <i>gingivalis</i> in mouse aortic arch at 12 weeks, indicated by white arrow heads. (B). Enlarged inset in D showing clusters of <i>P</i>. <i>gingivalis</i> coccobacilli morphotype indicated by white arrow heads. (C). No bacteria were detected in sham-infected mouse aortic tissues. Scale bar is 10 micrometers.</p

Polybacterial infection significantly increased atherosclerotic plaque growth in the ascending aorta and the aortic root.

<p>(A). Total plaque area of infected mice was elevated at both 12- and 24 weeks of infection, but was significant compared to controls at 24 weeks of infection (p < 0.05). (B). Intimal/medial layer thickness ratio of infected mice was increased relative to sham-infected mice at both 12- and 24 weeks of infection, although not significant (p = 0.1599 and 0.1339). (C). CD3⁺ T cell counts were significantly higher in 24 week–infected mice than sham-infected mice. (D). H & E stained aortic section illustrates large plaques in infected mice. (E). H & E stained aortic section showing small plaques in sham-infected mice. (F). Aortic Sections demonstrating numerous CD3⁺ T cells in 24-week polybacterial-infected mouse (20X magnification). (G). Aortic Sections demonstrating numerous CD3⁺ T cells in 24-week polybacterial-infected mouse (200X magnification). (H). Aortic section from sham-infected mice devoid of infiltrating CD3⁺ T cells. Black arrow heads indicate atherosclerotic plaque. Red arrows indicate infiltrated CD3⁺ T cells. L—artery lumen, I—intimal layer, M—medial layer, A—adventitial layer. * p < 0.05.</p

Polybacterial-infection induced bacterial-specific humoral immune response and alveolar bone resorption.

<p>(A). Serum IgG antibody response to the four bacteria used in periodontal infection at 12 and 24 weeks. (B). Serum IgM antibody response to the four bacteria used in periodontal infection at 12 and 24 weeks. Representative left maxilla lingual view of polybacterial-infected and sham-infected mice at both 12- and 24 weeks with area of bone resorption outlined from alveolar bone crest to cementoenamel junction. (C). Total alveolar bone resorption is significantly greater than controls at 24 weeks of polybacterial infection. (D). Representative left maxilla lingual view of polybacterial-infected and sham-infected mice at both 12- and 24 weeks with area of bone resorption outlined from alveolar bone crest to cementoenamel junction. Poly—polybacterial infection, Con—sham-infected control, <i>Pg/Td/Tf/Fn</i>–polybacterial infection <i>Pg</i>–<i>P</i>. <i>gingivalis</i>, <i>Td</i>–<i>T</i>. <i>denticola</i>, <i>Tf</i>–<i>T</i>. <i>forsythia</i>, <i>Fn</i>–<i>F</i>. <i>nucleatum</i>. * p < 0.05, ** p < 0.01, *** p < 0.001.</p

Polybacterial infection enhances atherogenic potential of infected ApoE^-/- mice.

<p>(A). Total serum cholesterol and total serum triglycerides are significantly elevated in infected mice. (B). Chylomicrons and VLDL are significantly elevated in infected mice. (C). Acute inflammatory marker SAA is not significantly elevated in infected mice. (D). Oxidized LDL is significantly elevated in infected mice. (E). Serum nitric oxide is significantly decreased in infected mice. Chol—cholesterol, Trigly—triglycerides, CM—chylomicrons, VLDL—very low-density lipoprotein, LDL—low density lipoprotein, HDL—high density lipoprotein, Cn—control, Ply—poly infection, <i>Pg</i>–<i>P</i>. <i>gingivalis</i> infection, <i>Td</i>–<i>T</i>. <i>denticola infection</i>, <i>Tf</i>–<i>T</i>. <i>forsythia</i> infection, <i>Fn</i>–<i>F</i>. <i>nucleatum</i> infection, OxyLDL—oxidized LDL. n = 6, * p < 0.05, ** p < 0.01.</p