The role of the microbiota in periodontal disease.

The last decade has witnessed unparalleled advances in our understanding of the complexity of the oral microbiome and the compositional changes that occur in subgingival biofilms in the transition from health to gingivitis and to destructive periodontal disease. The traditional view, which has held sway for the last 2 decades, that disease is characterized by the outgrowth of a consortium, or consortia, of a limited number of potentially pathogenic organisms, has given way to an alternative paradigm. In this new view, the microbiological changes associated with disease represent whole-scale alterations to the overall microbial population structure and to the functional properties of the entire community. Thus, and in common with other microbially mediated diseases of the gastrointestinal tract, the normally balanced, symbiotic, and generally benign commensal microbiome of the tooth-associated biofilm undergoes dysbiosis to a potentially deleterious microbiota. Coincident with progress in defining the microbiology of these diseases, there have been equally important advances in our understanding of the inflammatory systems of the periodontal tissues, their control, and how inflammation may contribute both to the development of dysbiosis and, in a deregulated state, the destructive disease process. One can therefore speculate that the inflammatory response and the periodontal microbiome are in a bidirectional balance in oral health and a bidirectional imbalance in periodontitis. However, despite these clear insights into both sides of the host/microbe balance in periodontal disease, there remain several unresolved issues concerning the role of the microbiota in disease. These include, but are not limited to, the factors which determine progression from gingivitis to periodontitis in a proportion of the population, whether dysbiosis causes disease or results from disease, and the molecular details of the microbial stimulus responsible for driving the destructive inflammatory response. Further progress in resolving these issues may provide significant benefit to diagnosis, treatment, and prevention.

Commensal bacteria-dependent select expression of CXCL2 contributes to periodontal tissue homeostasis.

The oral and intestinal host tissues both carry a heavy microbial burden. Although commensal bacteria contribute to healthy intestinal tissue structure and function, their contribution to oral health is poorly understood. A crucial component of periodontal health is the recruitment of neutrophils to periodontal tissue. To elucidate this process, gingival tissues of specific-pathogen-free and germ-free wild-type mice and CXCR2KO and MyD88KO mice were examined for quantitative analysis of neutrophils and CXCR2 chemoattractants (CXCL1, CXCL2). We show that the recruitment of neutrophils to the gingival tissue does not require commensal bacterial colonization but is entirely dependent on CXCR2 expression. Strikingly, however, commensal bacteria selectively upregulate the expression of CXCL2, but not CXCL1, in a MyD88-dependent way that correlates with increased neutrophil recruitment as compared with germ-free conditions. This is the first evidence that the selective use of chemokine receptor ligands contributes to neutrophil homing to healthy periodontal tissue.

Dropping anchor: attachment of peptidylarginine deiminase via A-LPS to secreted outer membrane vesicles of Porphyromonas gingivalis.

The periodontal pathogen Porphyromonas gingivalis has been invoked in the autoimmune disease rheumatoid arthritis (RA). This association relates to the peptidylarginine deiminase of P. gingivalis (PPAD), an enzyme capable of citrullinating human proteins and potentially contributing to loss of tolerance to citrullinated proteins in RA. PPAD is both retained in the outer membrane (OM) of P. gingivalis cells and secreted into the extracellular milieu, where it is detected in a soluble form and in association with outer membrane vesicles (OMVs). Recent studies showed that certain P. gingivalis proteins are retained in the OM through modification with an A-type lipopolysaccharide (A-LPS). Here, we investigated the possible involvement of A-LPS modification in the association of PPAD to the OM and OMVs. The results indicate that the OM- and OMV-associated PPAD is A-LPS-modified. The modified PPAD species is of low abundance in particular clinical isolates of P. gingivalis, which is not due to defects in the overall synthesis of A-LPS-modified proteins but, rather, to particular traits of the respective PPAD proteins. Lastly, we show that OMV association protects the A-LPS-modified PPAD from proteolytic degradation. Altogether, our observations show that A-LPS modification contributes to OM(V) sorting and 'protective secretion' of PPAD.

Protease-activated receptor 2 mediates human beta-defensin 2 and CC chemokine ligand 20 mRNA expression in response to proteases secreted by Porphyromonas gingivalis.

The oral pathogen Porphyromonas gingivalis secretes proteases such as Arg-gingipain B (RgpB) that activate protease-activated receptors (PARs). Human beta-defensins (hBDs) and the macrophage inflammatory protein 3alpha/CC chemokine ligand 20 (CCL20) produced by epithelial cells are antimicrobial peptides that provide cytokine function and play an important role in innate immunity. The aim of the present study was to determine whether specific members of the PAR family mediate the expression of these innate immunity markers in gingival epithelial cells (GECs) when exposed to P. gingivalis cell-free culture supernatant or purified RgpB. hBD-2 mRNA in GECs was induced in response to supernatant and purified RgpB from P. gingivalis (P = 0.02 and P = 0.016, respectively). This effect was abrogated by the protease inhibitor tosyl-l-lysine chloromethyl ketone (TLCK) (P < 0.05). In response to P. gingivalis supernatant and to purified RgpB, the hBD-2 mRNA expression was significantly decreased in PAR-2 gene knockdown cells, whereas no change was detected in PAR-1 gene knockdown cells. CCL20 mRNA expression also increased in response to the supernatant of P. gingivalis, and this effect was blocked by the protease inhibitor, TLCK (P = 0.05 and P = 0.024, respectively), and was blocked in PAR-2 gene knockdown cells. Our data indicate that hBD-2 and CCL20 mRNA up-regulation by P. gingivalis supernatant and purified RgpB was mediated via PAR-2, but not via PAR-1, and that proteases play a role in the regulation of innate immune responses in GECs. GECs use PARs to recognize P. gingivalis and mediate cell responses involved in innate immunity.