Periodontitis (deep seated gum disease) is the most prevalent chronic inflammatory disease to affect humans. As a major cause of tooth loss, severe periodontitis afflicts 8% of UK adults with over 50% having the disease to some extent. However, disease prevalence increases with age, and 85% of the UK population exhibit some periodontal destruction by 65-years-of-age. The cost to the UK economy is substantial and in 2008 was estimated to be £2.78 billion. Moreover, human costs are also high, with tooth loss creating serious issues for speech, diet/nutrition, appearance, and a substantial negative impact on oral health and quality of life. There is also mounting evidence that periodontitis has systemic consequences due to a “spill over” effect of the gum inflammation into the circulation. The impact of such low grade peripheral inflammation appears to be significant, with “all cause” mortality rates over a 25-year period being 85% higher in males with periodontal bone loss. Periodontitis is also recognised as an independent risk factor for cardiovascular disease and stroke. It also has an impact on diabetes control and is independently associated with rheumatoid arthritis.
Why periodontitis prevalence increases with age is uncertain, and research into this important issue is desperately needed, given our ageing population and the chronic disease burden on the individual patient as well as the economy. There are several areas in which the periodontal research group at Birmingham University are exploring why periodontitis becomes more prevalent as we get older and what mechanisms may be involved. Understanding such mechanisms provides opportunities for prevention and novel therapeutic approaches.
These studies include:
- Analysing the way in which the skin cells of the gums (epithelium) react to oral bacteria.
- Studying the behaviour of white blood cells (neutrophils) which can be very destructive to periodontal tissues.
- Determining what can be learned from comprehensively mapping the composition of saliva, as a diagnostic fluid.
Analysing the oral epithelium
Epithelium was traditionally perceived as a simple inert physical barrier, whose role was to prevent microbial entry into the connective tissues, thereby protecting the host’s vital systems from the external environment. However, contemporary research has revealed that epithelial cells play an active role in the host response to bacterial infections. Gingival (gum) epithelium is intimately related to the bacterial plaque biofilm and research at the Birmingham Dental School has demonstrated that gingival epithelial stimulation by key periodontal pathogens results in activation of cellular transcription factors, which regulate production of pro-inflammatory cytokines. This finding suggests a key role for epithelium in the initiation and propagation of periodontal inflammation. However to date, research investigating how the responses and behaviour of oral epithelial cells change as we age, and thus impacts on these important mechanisms is almost completely lacking, and may help explain the increasing prevalence of periodontitis in older patients.
The Periodontal Research Group (PRG) at Birmingham proposes studies to explore age-related changes in oral epithelium in terms of:
- Changes in efficiency of bacterial recognition (via pattern recognition receptors).
- Activation of redox sensitive transcription factors (NF-kB, Nrf-2, & AP-1).
- Gene transcription profiles.
- Levels of cytokine production.
- Levels of certain antioxidant micronutrients.
- Changes in epithelial permeability and barrier function and how these may be manipulated using novel micronutrient approaches.
Studying neutrophil behaviour
Evidence suggests that periodontitis occurs in individuals who have an abnormal neutrophil response to subgingival plaque bacteria and that neutrophil-mediated oxidant injury is an important feature of the disease. Peripheral blood neutrophils from periodontitis patients are hyper-reactive, after Fc?R and toll-like receptor (TLR) stimulation, as well as being hyperactive in respect of unstimulated, extracellular release of reactive oxygen species (ROS). Although Fc?R hyper-reactivity is reduced by therapy, baseline, unstimulated-ROS release is not, suggesting that reactive and constitutive mechanisms underlie the “hyper-inflammatory” phenotype. Priming with GM-CSF or the periodontal pathogens Porphyromonas gingivalis and Fusobacterium nucleatum (1 bacterial cell per neutrophil) reduces the Fc?R-mediated hyper-reactivity but not hyperactive, baseline unstimulated ROS release.
Hyper-reactive neutrophils differentially express 25, type I interferon-stimulated genes and IFN?, which can enhance Fc?R-mediated ROS generation in vitro, and is increased in plasma from periodontitis patients. Recent studies from this group have demonstrated that the ability of periodontitis plasma to prime for f-MLP-mediated ROS generation is, in part, due to the presence of GM-CSF, IL-8 and IFN?, supporting the idea that peripheral neutrophils in periodontitis patients are in a cytokine-primed state. Interestingly, transcripts for GM-CSF and IL-8 are up-regulated in gingival epithelial cells after stimulation with P. gingivalis and F. nucleatum, (see above), perhaps indicating that local tissue responses to plaque bacteria may contribute to peripheral neutrophil hyper-reactivity. There is currently, no data on the behaviour of PMNLs from older patients with respect to ROS release in response to different priming agents or to periodontal bacteria, and such studies are planned in order to shed light on this potentially important cause of periodontal tissue damage in older patients.
Recent reports of neutrophil hyper-reactivity in terms of ROS release and the involvement of IFN? in disease pathogenesis, have also, for the first time, raised the spectre of an autoimmune component to the pathogenesis of periodontitis. Moreover, type-1 interferon’s and ROS are associated with neutrophil extracellular trap (NET) formation. Whilst normally neutrophils combat periodontal pathogens by intracellular (phagocytosis) and extracellular (degranulation) mechanisms, NET killing has recently been described as an important new paradigm in neutrophil biology. NETs consist of a web of extracellular fibres of DNA plus histones (chromatin) and granular proteins, which bind to Gram-positive and Gram-negative bacteria. These chromatin structures immobilise high concentrations of extracellular antimicrobial peptides, including cathepsins and myeloperoxidase along with histones, leading to physical entrapment and killing of pathogenic bacteria. Lack of NET production (e.g. in Chronic Granulomatous Disease) or presence of bacteria that express DNAses which breakdown NETs, results in increased pathogenic activity and potentially catastrophic infections in certain patient subsets. The DNA-histone backbone of NETs acts as a target for endogenous or bacterial peptidylarginine deiminases (PADs), which citrullinate histones as well as other proteins, by deimidating arginine residues, potentially rendering them auto-immunogenic. Further studies are currently ongoing in this area to determine whether this mechanism of generating auto-antigens links periodontitis to other age-related systemic diseases, such as rheumatoid arthritis, and whether decreased efficiency in NET production associates with immune-senescence.
Characterising the saliva proteome
As we age our bodies change and leave behind markers of these changes. Powerful profiling techniques such as “proteomics” can determine these changes. Recent studies have already identified changes between young and older women by analysing their saliva. This fluid contains many proteins that are involved in immunological protection as well as serum derived proteins. The group is currently employing highly accurate and sensitive mass spectrometry techniques such as FT-ICR MS/MS to profile changes in the saliva proteome in healthy, diseased (gingivitis and periodontitis) and edentulous patients. As the oral microflora changes with age, as well as with disease, using sensitive meta-proteomic techniques will help us to unravel biomarkers for healthy ageing and non-invasive techniques for disease diagnosis. Higher levels of biomarkers for dental caries (decay) associated proteins (Cystatin S) have already been found to be elevated in elderly patients with caries and many more discoveries are being made.