Chronic Porphyromonas gingivalis infection accelerates the occurrence of age-related granules in ApoE-/- mice brains

This study explored the origin of age-related granules in the apolipoprotein E gene knockout (ApoE−/−) B6 background mice brains following chronic gingival infection with Porphyromonas gingivalis for 24 weeks. Intracerebral localization of P. gingivalis was detected by fluorescence in situ hybridization (FISH) and its protease by immunohistochemistry. The age-related granules were observed by periodic acid–Schiff (PAS), silver impregnation, and immunostaining. FISH showed intracerebral dissemination of P. gingivalis cells (p = 0.001). PAS and silver impregnation demonstrated the presence of larger inclusions restricted to the CA1, CA2, and dentate gyrus sectors of the hippocampus. A specific monoclonal antibody to bacterial peptidoglycan detected clusters of granules with variable sizes in mice brains infected with P. gingivalis (p = 0.004), and also highlighted areas of diffuse punctate staining equating to physical tissue damage. Mouse immunoglobulin G was observed in the capillaries of the cerebral parenchyma of all P. gingivalis–infected brains (p = 0.001), and on pyramidal neurons in some severely affected mice, compared with the sham-infected mice. Gingipains was also observed in microvessels of the hippocampus in the infected mice. This study supports the possibility of early appearance of age-related granules in ApoE−/− mice following inflammation-mediated tissue injury, accompanied by loss of cerebral blood-brain barrier integrity

Identification of Specific Oral and Gut Pathogens in Full Thickness Colon of Colitis Patients: Implications for Colon Motility

Impaired colon motility is one of the leading problems associated with inflammatory bowel disease (IBD). An expanding body of evidence supports the role of microbiome in normal gut function and in progression of IBD. The objective of this work is to determine whether diseased full thickness colon specimens, including the neuromuscular region (critical for colon motility function), contain specific oral and gut pathogens. In addition, we compared the differences in colon microbiome between Caucasians (CA) and African Americans (AA). Thirty-nine human full thickness colon (diseased colon and adjacent healthy colon) specimens were collected from Crohn's Colitis (CC) or Ulcerative Colitis (UC) patients while they underwent elective colon surgeries. We isolated and analyzed bacterial ribosomal RNA (rRNA) from colon specimens by amplicon sequencing of the 16S rRNA gene region. The microbiome proportions were quantified into Operational Taxonomic Units (OTUs) by analysis with Quantitative Insights Into Microbial ecology (QIIME) platform. Two hundred twenty-eight different bacterial species were identified by QIIME analysis. However, we could only decipher the species name of fifty-three bacteria. Our results show that proportion of non-detrimental bacteria in CC or UC colon samples were altered compared to adjacent healthy colon specimens. We further show, for the first time in full thickness colon specimens, that microbiome of CC and UC diseased specimens is dominated by putative oral pathogens belonging to the Phyla Firmicutes (Streptococcus, Staphylococcus, Peptostreptococcus), and Fusobacteria (Fusobacterium). In addition, we have identified patterns of differences in microbiome levels between CA and AA specimens with potential implications for health disparities research. Overall, our results suggest a significant association between oral and gut microbes in the modulation of colon motility in colitis patients

Polymicrobial infection with major periodontal pathogens induced periodontal disease and aortic atherosclerosis in hyperlipidemic ApoE(null) mice.

Periodontal disease (PD) and atherosclerosis are both polymicrobial and multifactorial and although observational studies supported the association, the causative relationship between these two diseases is not yet established. Polymicrobial infection-induced periodontal disease is postulated to accelerate atherosclerotic plaque growth by enhancing atherosclerotic risk factors of orally infected Apolipoprotein E deficient (ApoE(null)) mice. At 16 weeks of infection, samples of blood, mandible, maxilla, aorta, heart, spleen, and liver were collected, analyzed for bacterial genomic DNA, immune response, inflammation, alveolar bone loss, serum inflammatory marker, atherosclerosis risk factors, and aortic atherosclerosis. PCR analysis of polymicrobial-infected (Porphyromonas gingivalis [P. gingivalis], Treponema denticola [T. denticola], and Tannerella forsythia [T. forsythia]) mice resulted in detection of bacterial genomic DNA in oral plaque samples indicating colonization of the oral cavity by all three species. Fluorescent in situ hybridization detected P. gingivalis and T. denticola within gingival tissues of infected mice and morphometric analysis showed an increase in palatal alveolar bone loss (p<0.0001) and intrabony defects suggesting development of periodontal disease in this model. Polymicrobial-infected mice also showed an increase in aortic plaque area (p<0.05) with macrophage accumulation, enhanced serum amyloid A, and increased serum cholesterol and triglycerides. A systemic infection was indicated by the detection of bacterial genomic DNA in the aorta and liver of infected mice and elevated levels of bacterial specific IgG antibodies (p<0.0001). This study was a unique effort to understand the effects of a polymicrobial infection with P. gingivalis, T. denticola and T. forsythia on periodontal disease and associated atherosclerosis in ApoE(null) mice

Periodontal disease parameters.

<p>(<b>A</b>) Serum IgG antibody levels from ApoE^null mice collected after 16 weeks of oral polymicrobial infection with <i>P. gingivalis</i>, <i>T. denticola,</i> and <i>T. forsythia</i> (n = 10−15). The graph shows the results for IgG antibody reactive with each of the three bacterial species. The bars indicate mean antibody levels in serum from mice infected with consortia or from controls. Significantly higher levels of IgG antibody were seen in polymicrobial-infected mice when compared to sham-infected controls. (<b>B</b>) Area of horizontal alveolar bone resorption in mandibular and maxillary palatal surfaces in ApoE^null mice. Each bar indicates the mean alveolar bone resorption for three molars in each quadrant of infected (n = 15) and sham-infected mice (n = 10). The vertical line denotes standard deviation from mean. *** Asterisks indicate significantly different (<i>P</i><0.0001) than the sham-infected controls. The IgG antibody levels are expressed as log₁₀. <i>Pg</i> indicates <i>P. gingivalis</i>; <i>Td</i> indicates <i>T. denticola</i>; <i>Tf</i> indicates <i>T. forsythia</i>; Cont indicates sham-infected control mice. <i>Pg</i>/<i>Td</i>/<i>Tf</i> indicates polymicrobial infection with <i>P. gingivalis</i>, <i>T. denticola</i> and <i>T. forsythia</i>. (<b>C, D</b>) Fluorescent <i>in situ</i> hybridization of formalin-fixed paraffin-embedded right mandible section from a polymicrobial-infected mouse using Alexafluor-568 labeled 16S RNA species specific (<i>P. gingivalis, T. dentcola</i> and <i>T. forsythia</i>) oligonucleotide probes. Red fluorescence indicates the presence of bacterial 16S RNA. The white arrow in the zoomed in square points to <i>T. dentcola</i> spirochete morphotype. The white arrow heads point to <i>P. gingivalis</i> coccobacilli morphotype. (<b>E, F</b>) Representative images of maxilla and mandible palatal surfaces showing extensive interproximal intrabony defects in mice infected with <i>Pg/Td/Tf</i>. M1 indicates first molar, M2 indicates second molar and M3 indicates third molar. Black arrows indicates intrabony defects. (<b>G</b>) Sham-infected mouse mandible-palatal showing no intrabony defects.</p

Atherosclerotic vascular disease parameters.

<p>(<b>A</b>) The total aortic plaque area was measured and an increase in plaque area (mm²) was found for polymicrobial-infected ApoE^null mice when compared to sham-infected control mice (<i>P</i><0.05). (<b>B</b>) Intimal/medial thickness ratios were determined and an increase was observed in the polymicrobial-infected mice compared to sham-infected mice. Polymicrobial infection-induced macrophage infiltration in ApoE^null mice. 4 µm thick paraffin embedded sections of the aortic arch were cut and stained with H&E; (<b>C</b>) Number of macrophage per total adventitial area. (<b>D</b>) Number of macrophage per total intimal area. Control indicates sham-infected ApoE^null mice and <i>Pg</i>+<i>Td</i>+<i>Tf</i> indicate polymicrobial-infected mice. An increase in macrophages was seen in both the adventitial and intimal layers in the polymicrobial-infected mice when compared to the sham-infected mice. Histologic evaluation of 4 µm thick paraffin embedded aortic arch sections stained using H? plaque area is flanked with black arrows, macrophage infiltration is indicated using white arrow with open head and cholesterol crystals are indicated using white arrows with filled heads. (<b>E</b>) Sham-infected control mice tissue (Magnification 40×); (<b>F</b>) <i>Pg, Td,</i> and <i>Tf</i> infection mice tissue (Magnification 40×) containing larger plaque area, increased macrophage infiltration and increased presence of cholesterol crystals, suggesting polymicrobial infection-induced aortic plaque and aortic wall thickness in ApoE^null mice.</p

Schematic diagram.

<p>Schematic diagram of the experimental design illustrating the length of the study and highlighting important time points. Eight polymicrobial infections (Infection I through Infection VIII) are indicated at 12, 14, 16, 18, 20, 22, 24 and 26 weeks of age. Oral plaque samplings are indicated by PCR at week 2, 8, 14 and 16 following the initial infection. Blood and tissue specimen collection was performed at euthanasia following 16 weeks of infection.</p

Distribution of ApoE^null mice oral microbial samples positive for bacterial genomic DNA by PCR.

a<p>Indicate time points at which oral microbial samples were collected (2, 8, 14, and 16 weeks) following polymicrobial infection for determination of microbial colonization by species specific PCR analysis.</p>b<p>Indicate mice were infected for 8 alternate weeks with <i>P. gingivalis</i>, <i>T. denticola</i>, and <i>T. forsythia</i>. Samples were analyzed using appropriate specific PCR primers with positive and negative controls.</p>c<p>Oral microbial samples were collected from sham-infected control mice periodically and examined for <i>P. gingivalis</i>, <i>T. denticola</i>, and <i>T. forsythia</i> using bacteria specific primers and all mice were negative. - Indicate oral microbial samples negative by PCR analysis. <i>Pg</i>-indicate <i>P. gingivalis</i>; <i>Td</i>-indicate <i>T. denticola</i>; and <i>Tf</i>-indicate <i>T. forsythia</i>.</p

Polymicrobial infection-induced periodontal intrabony defects in ApoE^null mouse.

*<p>^aThe frequency was calculated by tooth surfaces containing periodontal intrabony defects out of total tooth surfaces. Calculation of total tooth surface: polymicrobial-infected group has 90 tooth surfaces [15 mice×3 molars×2 sides (buccal, palatal)] and control has 60 (10 mice×3 molars×2 sides).</p>**<p>^bNumbers in the parenthesis indicate the percentage of tooth surface with periodontal intrabony defects.</p

Determination of polymicrobial oral Infection-induced risk factors for atherosclerosis.

<p>Values are mean±SD;</p>*<p>indicates <i>P</i><0.05;</p>**<p>indicates <i>P</i><0.001;</p>a<p>n = 13 mice per group;</p>b<p>n = 15 mice per group;</p>c<p>n = 5 mice per group.</p