This investigation compared the microbiomes colonizing teeth during the initiation, progression, and resolution of periodontitis in nonhuman primates (Macaca mulatta) at different ages. Subgingival plaque samples were collected at baseline; 0.5, 1, and 3 months following ligature-induced periodontitis; and following naturally occurring disease resolution at 5 months. Samples were analyzed using 16S amplicon sequencing to identify bacterial profiles across age groups: young (<3 years of age), adolescent (3 to 7 years), adult (12 to 15 years), and aged (17 to 23 years). α-Diversity of the microbiomes was greater in the adult/aged samples than in the young/adolescent samples. β-Diversity of the samples demonstrated clear age group differences, albeit individual variation in microbiomes between animals within the age categories was noted. Phylum distributions differed between the young/adolescent animals and the adult/aged animals at each of the time points, showing an enrichment of the phyla Spirochetes, Fusobacteria, and Bacteroidetes associated with periodontitis. Major differences in the top 50 operational taxonomic units (OTUs) were noted in the young and adolescent microbiomes during initiation and progression postligation compared to the adult and aged animals. The proportions of a large number of species in the top 50 OTUs were lower at baseline and in resolved disease microbiomes in the young samples, while profiles in adolescent animals were more consistent with the disease microbiomes. Microbiome profiles for resolution for adults and aged animals appeared more resilient and generally maintained a pattern similar to that of disease. Use of the model can expand our understanding of the crucial interactions of the oral microbiome and host responses in periodontitis.

Evidence has shown activation of T and B cells in gingival tissues in experimental models and in humans diagnosed with periodontitis. The results of this adaptive immune response are noted both locally and systemically with antigenic specificity for an array of oral bacteria, including periodontopathic species, e.g., Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. It has been recognized through epidemiological studies and clinical observations that the prevalence of periodontitis increases with age. This report describes our studies evaluating gingival tissue transcriptomes in humans and specifically exploiting the use of a non-human primate model of naturally occurring periodontitis to delineate gingival mucosal tissue gene expression profiles focusing on cells/genes critical for the development of humoral adaptive immune responses. Patterns of B cell and plasmacyte genes were altered in aging healthy gingival tissues. Substantial increases in a large number of genes reflecting antigen-dependent activation, B cell activation, B cell proliferation, and B cell differentiation/maturation were observed in periodontitis in adults and aged animals. Finally, evaluation of the relationship of these gene expression patterns with those of various tissue destructive molecules (MMP2, MMP9, CTSK, TNFα, and RANKL) showed a greater frequency of positive correlations in healthy tissues versus periodontitis tissues, with only MMP9 correlations similar between the two tissue types. These results are consistent with B cell response activities in healthy tissues potentially contributing to muting the effects of the tissue destructive biomolecules, whereas with periodontitis this relationship is adversely affected and enabling a progression of tissue destructive events.

This study focused on documenting characteristics of the gingival transcriptome during various stages of periodontitis targeting genes associated with apoptotic and autophagic pathways and changes that specifically associate with features of the oral microbiome. Methods: Macaca mulatta (n = 18; 12-23 years) were examined at baseline and 0.5, 1, and 3 months of disease progression, as well as 5 months with clinical disease resolution. 16S sequencing and microarray analyses examined changes in the microbiome and gingival transcriptome, respectively, at each time point from every animal. Results: Specific patterns of apoptotic and autophagic genes were identified related to the initiation and progression of disease. The analysis also provided insights on the principal bacteria within the complex microbiome whose abundance was significantly correlated with differences in apoptotic and autophagic gene expression. Bacteria were identified that formed associated complexes with similar effects on the host gene expression profiles. A complex of Leptotrichia_unclassifed, Capnocytophaga_unclassified, Prevotella sp. 317, and Veillonellaceae_[G-1] sp. 155 were significantly negatively correlated with both apoptosis and autophagy. Whereas, Veillonellaceae_[G-1], Porphyromonadaceae, and F. alocis 539 were significantly positively correlated with both pathways, albeit this relationship was primarily associated with pro-apoptotic genes. Conclusions: The findings provide evidence for specific bacteria/bacterial complexes within the oral microbiome that appear to have a more substantive effect on regulating apoptotic and autophagic pathways in the gingival tissues with periodontitis.

Summary Follicular helper T cells (Tfh) cells have been identified in the circulation and in tertiary lymphoid structures in chronic inflammation. Gingival tissues with periodontitis reflect chronic inflammation, so genomic footprints of Tfh cells should occur in these tissues and may differ related to aging effects. Macaca mulatta were used in a ligature‐induced periodontitis model [adult group (aged 12–23 years); young group (aged 3–7 years)]. Gingival tissue and subgingival microbiome samples were obtained at matched healthy ligature‐induced disease and clinical resolution sites. Microarray analysis examined Tfh genes (n = 54) related to microbiome characteristics documented using 16S MiSeq. An increase in the major transcription factor of Tfh cells, BCL6, was found with disease in both adult and young animals, while master transcription markers of other T cell subsets were either decreased or showed minimal change. Multiple Tfh‐related genes, including surface receptors and transcription factors, were also significantly increased during disease. Specific microbiome patterns were significantly associated with profiles indicative of an increased presence/function of Tfh cells. Importantly, unique microbial complexes showed distinctive patterns of interaction with Tfh genes differing in health and disease and with the age of the animals. An increase in Tfh cell responsiveness occurred in the progression of periodontitis, affected by age and related to specific microbial complexes in the oral microbiome. The capacity of gingival Tfh cells to contribute to localized B cell activation and active antibody responses, including affinity maturation, may be critical for controlling periodontal lesions and contributing to limiting and/or resolving the lesions.

Follicular helper T cells (Tfh) cells are generally considered critical in secondary lymphoid tissues; however, they are also identified in the circulation and in tertiary lymphoid structures in chronic inflammation. Gingival tissues with periodontitis reflect chronic inflammation so genomic footprints of Tfh cells should occur in these tissues and may differ related to aging effects. Methods: Macaca mulatta monkeys were used in a model of ligature-induced periodontitis [adult group (12-23 years of age); young group (3-7 years)]. Gingival tissue and subgingival microbiome samples were obtained at matched healthy sites, sites during ligature-induced disease, and in samples after clinical resolution. Microarray analysis examined Tfh genes (n=40) and the microbiome samples were examined using 16S MiSeq. Results: An apparent increase in the major transcription factor of Tfh cells, BCL6, was found with disease in both adult and young animals, while the master transcription markers of other T cell subsets were either decreased or showed minimal change. A number of the Tfh related genes, including surface receptors, secreted products and transcription factors were also significantly increased during disease. Unique microbial complexes showed patterns of interactions with Tfh genes that differed in health and disease. Conclusions: An increase in Tfh cell responsiveness occurred later in the progression of periodontitis, affected by age and strongly related to specific microbial complexes. The capacity of gingival Tfh cells to contribute to localized B cell activation and active antibody responses, including affinity maturation may be critical for controlling periodontal lesions and contributing to limiting and/or resolving the lesions.