Gingival and periodontal ligament fibroblasts are functionally distinct cell types within the dento-gingival unit that participate in host immune response. Their microenvironment influences the behavior and immune response to microbial challenge. We developed three-dimensional gingival and periodontal connective tissue equivalents (CTEs) using human fibrin-based matrix. The CTEs were characterized, and the heterogeneity in their innate immune response was investigated. The CTEs demonstrated no to minimal response to planktonic Streptococcus mitis and Streptococcus oralis, while their biofilms elicited a moderate increase in IL-6 and IL-8 production. In contrast, Fusobacterium nucleatum provoked a substantial increase in IL-6 and IL-8 production. Interestingly, the gingival CTEs secreted significantly higher IL-6, while periodontal counterparts produced higher IL-8. In conclusion, the gingival and periodontal CTEs exhibited differential responses to various bacterial challenges. This gives insights into the contribution of tissue topography and fibroblast heterogeneity in rendering protective and specific immune responses toward early biofilm colonizers.

In periodontal health, oral streptococci constitute up to 80% of the plaque biofilm. Yet, destructive inflammatory events of the periodontium are rare. This observation suggests that oral streptococci may possess mechanisms to co-exist with the host. However, the mechanisms employed by oral streptococci to modulate the innate immune response have not been well studied. One of the key virulence factors produced by oral streptococci is hydrogen peroxide (H2O2). In mammalian cells, H2O2 triggers the activation of nuclear factor erythroid 2-related factor 2 (Nrf2), a key pathway mediating antioxidant defence. This study aimed to determine (1) if H2O2 producing oral streptococci activated the Nrf2 pathway in macrophages, and (2) if the activation of Nrf2 influenced the innate immune response. We found that oral streptococci downregulated the innate immune response in a H2O2 dependent manner through the activation of the Nrf2. The activation of the Nrf2 signalling pathway led to the inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (NFĸB), the key transcription factor regulating pro-inflammatory response. This study showed for the first time that oral streptococci are unlikely passive bystanders but could play an active role in the maintenance of periodontal health by preventing overt inflammation.

Objectives: To investigate longitudinally the activation of Toll-like receptor-4 (TLR-4) by subgingival biofilm samples before and after nonsurgical periodontal therapy (NSPT).Materials and Methods: Forty periodontitis patients received NSPT and were reviewed 3 and 6 months post-treatment. Subgingival biofilm was sampled from 4 teeth per patient, at baseline and each followup time point. TLR-4 activation was determined using the HEK-BLUE™/hTLR4 system. Changes in TLR-4 activation and probing pocket depths (PPDs) were evaluated using generalised linear models, and the association between TLR-4 activation and pocket elimination (defined as 6-month PPDs ≤ 3mm) was determined using generalised estimating equations.Results: At 6 months, the mean TLR-4 activation by subgingival biofilm samples was significantly reduced from 11.2AU (95%CI 7.1AU, 15.4AU) to 3.6AU (95%CI 2.3AU, 4.8AU, p < 0.001), paralleling significant reductions in mean PPDs at sampled sites. The response to NSPT was associated with longitudinal TLR-4 activation profiles, with significantly higher TLR-4 activation by subgingival biofilm obtained from sites that did not achieve pocket elimination, compared to sites at which pocket elimination was achieved.Conclusions: The activation of TLR-4 by subgingival biofilm samples was reduced after NSPT and this reduction was significantly associated with the clinical improvements (PPD reductions) at sampled sites.Clinical Relevance: This study demonstrated an association between the longitudinal profile of TLR-4 activation by subgingival biofilm and periodontal treatment response. Longitudinal monitoring of TLR-4 activation by subgingival biofilm may potentially identify non-responsive sites, enabling targeted additional treatment.