Identification of a periodontal pathogen and bihormonal cells in pancreatic islets of humans and a mouse model of periodontitis.

Results from epidemiological and prospective studies indicate a close association between periodontitis and diabetes. However the mechanisms by which periodontal pathogens influence the development of prediabetes/diabetes are not clear. We previously reported that oral administration of a periodontal pathogen, Porphyromonas gingivalis (Pg) to WT mice results in insulin resistance, hyperinsulinemia, and glucose intolerance and that Pg translocates to the pancreas. In the current study, we determined the specific localization of Pg in relation to mouse and human pancreatic α- and ß-cells using 3-D confocal and immunofluorescence microscopy and orthogonal analyses. Pg/gingipain is intra- or peri-nuclearly localized primarily in ß-cells in experimental mice and also in human post-mortem pancreatic samples. We also identified bihormonal cells in experimental mice as well as human pancreatic samples. A low percentage of bihormonal cells has intracellular Pg in both humans and experimental mice. Our data show that the number of Pg translocated to the pancreas correlates with the number of bihormonal cells in both mice and humans. Our findings suggest that Pg/gingipain translocates to pancreas, particularly ß-cells in both humans and mice, and this is strongly associated with emergence of bihormonal cells.

Chronic oral application of a periodontal pathogen results in brain inflammation, neurodegeneration and amyloid beta production in wild type mice.

BACKGROUND: The results from cross sectional and longitudinal studies show that periodontitis is closely associated with cognitive impairment (CI) and Alzhemer's Disease (AD). Further, studies using animal model of periodontitis and human post-mortem brain tissues from subjects with AD strongly suggest that a gram-negative periodontal pathogen, Porphyromonas gingivalis (Pg) and/or its product gingipain is/are translocated to the brain. However, neuropathology resulting from Pg oral application is not known. In this work, we tested the hypothesis that repeated exposure of wild type C57BL/6 mice to orally administered Pg results in neuroinflammation, neurodegeneration, microgliosis, astrogliosis and formation of intra- and extracellular amyloid plaque and neurofibrillary tangles (NFTs) which are pathognomonic signs of AD. METHODS: Experimental chronic periodontitis was induced in ten wild type 8-week old C57BL/6 WT mice by repeated oral application (MWF/week) of Pg/gingipain for 22 weeks (experimental group). Another 10 wild type 8-week old C57BL/6 mice received vehicle alone (control group) MWF per week for 22 weeks. Brain tissues were collected and the presence of Pg/gingipain was determined by immunofluorescence (IF) microscopy, confocal microscopy, and quantitative PCR (qPCR). The hippocampi were examined for the signs of neuropathology related to AD: TNFα, IL1ß, and IL6 expression (neuroinflammation), NeuN and Fluoro Jade C staining (neurodegeneration) and amyloid beta1-42 (Aß42) production and phosphorylation of tau protein at Ser396 were assessed by IF and confocal microscopy. Further, gene expression of amyloid precursor protein (APP), beta-site APP cleaving enzyme 1 (BACE1), a disintegrin and metalloproteinase domain-containing protein10 (ADAM10) for α-secretase and presenilin1 (PSEN1) for É£-secretase, and NeuN (rbFox3) were determined by RT-qPCR. Microgliosis and astrogliosis were also determined by IF microscopy. RESULTS: Pg/gingipain was detected in the hippocampi of mice in the experimental group by immunohistochemistry, confocal microscopy, and qPCR confirming the translocation of orally applied Pg to the brain. Pg/gingipain was localized intra-nuclearly and peri-nuclearly in microglia (Iba1+), astrocytes (GFAP+), neurons (NeuN+) and was evident extracellularly. Significantly greater levels of expression of IL6, TNFα and IL1ß were evident in experimental as compared to control group (p<0.01, p<0.00001, p<0.00001 respectively). In addition, microgliosis and astrogliosis were evident in the experimental but not in control group (p <0.01, p<0.0001 respectively). Neurodegeneration was evident in the experimental group based on a fewer number of intact neuronal cells assessed by NeuN positivity and rbFOX3 gene expression, and there was a greater number of degenerating neurons in the hippocampi of experimental mice assessed by Fluoro Jade C positivity. APP and BACE1 gene expression were increased in experimental group compared with control group (p<0.05, p<0.001 respectively). PSEN1 gene expression was higher in experimental than control group but the difference was not statistically significant (p = 0.07). ADAM10 gene expression was significantly decreased in experimental group compared with control group (p<0.01). Extracellular Aß42 was detected in the parenchyma in the experimental but not in the control group (p< 0.00001). Finally, phospho-Tau (Ser396) protein was detected and NFTs were evident in experimental but not in the control group (p<0.00001). CONCLUSIONS: This study is the first to show neurodegeneration and the formation of extracellular Aß42 in young adult WT mice after repeated oral application of Pg. The neuropathological features observed in this study strongly suggest that low grade chronic periodontal pathogen infection can result in the development of neuropathology that is consistent with that of AD.