Insights into the Human Oral Microbiome and Alzheimer’s Disease

Abstract

INTRODUCTION: The reason why some individuals develop sporadic Alzheimer’s disease (AD) and others are unaffected remains unresolved. One theory still to be elucidated is the role of the oral microbiome in neurodegeneration. The effects of key pathogens and virulence factors associated with gum disease such as Porphyromonas gingivalis (P. gingivalis) on neuropathology is of interest and discoveries in this field could lead to development of novel therapeutics and preventative strategies. Chronic infections and inflammation are known to weaken the first line of defence for the brain; the blood brain barrier (BBB).

AIM: The aim of this study was to investigate the potential contribution from pathogens of the periodontal microbiome to the development of AD and whether the mediators produced during periodontal disease (PD), such as P. gingivalis outer membrane vesicles (OMV) and Lipopolysaccharides (LPS), were capable of inducing changes associated with inflammatory BBB damage.

RESULTS: The association between a microbial presence in the brain and AD pathology was confirmed by high throughput genomic analysis and PCR analysis of human post-mortem tissues from AD patients and age matched controls. Brain microbiome analysis was carried out by next generation sequencing (NGS) to genus level. Bacterial reads in AD patients (n=9) were 9-fold higher compared to controls (n=6), with a dominance of Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes. PCR amplification at UCLan was applied to the post-mortem brain samples using bacterial and fungal primers. These investigations provided limited data and limitations of the utilised methods were acknowledged.

An in vitro BBB model, composed of primary-derived human astrocytes (HA), pericytes (HBPC) and brain microvascular endothelial cells (HBMEC), cultured in a transwell system representing the neurovascular unit of the BBB, was utilised to evaluate the effects of P. gingivalis virulence factors (LPS and OMVs). The integrity of the BBB after application of P. gingivalis LPS (unconjugated and fluorescein isothiocyanate (FITC) -conjugated) and OMVs were investigated and correlated with transport of LPS across the barrier. A significant decrease (p ≤ 0.05) in transendothelial electrical resistance (TEER) was observed when unconjugated P. gingivalis LPS (0.3, 10, 100 μg/ml) and P. gingivalis OMVs (0.1, 0.3, 50 and 100 μg/ml) were applied to the BBB relative to the control (FITC-alone), but the response was not concentration dependent (no significant difference between groups).

The TEER values did not recover to pre-treatment baseline as measured by a significant deficit in TEER (p ≤ 0.05) after application of unconjugated P. gingivalis LPS and OMVs.
The application of P. gingivalis LPS-FITC conjugate in conjunction with 10 μg/ml P. gingivalis OMVs showed a similar pattern in terms of TEER response. Application of P. gingivalis OMVs to a monoculture of HBMEC was evaluated using immunofluorescence microscopy and showed disruption of the tight junction zona occludens protein (ZO-1) after addition of 0.3 μg/mL P. gingivalis OMVs compared to controls (media only).

CONCLUSION: These findings show that the integrity of tight junctions of the human BBB could be weakened by association with P. gingivalis virulence factors LPS and OMVs containing proteolytic enzymes (gingipains). P. gingivalis LPS act through toll-like receptor 4 (TLR4) and it has been shown that rodent cells are less sensitive to this than human cells. This highlights the importance of developing models as close to human anatomy and physiology as possible. The BBB and monolayer models utilised in this study were found to be valuable tools for further cell-bacteria interaction studies.

FUTURE WORK: The recognition that no PD bacterial cell has been found in the brain emphasises the need to learn more about the virulence factors produced in PD, such as LPS and gingipains and to gain information about how a chronic oral infection affects the BBB. The findings in this study provide additional information of the processes involved in virulence factor-CNS interactions, potentially suggesting how chronic periodontal disease in middle to later life could increase the risk of late onset dementia such as AD.