DAMPs and alarmin gene expression patterns in aging healthy and diseased mucosal tissues.

Introduction: Periodontitis is delineated by a dysbiotic microbiome at sites of lesions accompanied by a dysregulated persistent inflammatory response that undermines the integrity of the periodontium. The interplay of the altered microbial ecology and warning signals from host cells would be a critical feature for maintaining or re-establishing homeostasis in these tissues. Methods: This study used a nonhuman primate model (Macaca mulatta) with naturally-occurring periodontitis (n = 34) and experimental ligature-induced periodontitis (n = 36) to describe the features of gene expression for an array of damage-associate molecular patterns (DAMPs) or alarmins within the gingival tissues. The animals were age stratified into: ≤3 years (Young), 7-12 years (Adolescent), 12-15 years (Adult) and 17-23 years (Aged). Gingival tissue biopsies were examined via microarray. The analysis focused on 51 genes representative of the DAMPs/alarmins family of host cell warning factors and 18 genes associated with tissue destructive processed in the gingival tissues. Bacterial plaque samples were collected by curette sampling and 16S rRNA gene sequences used to describe the oral microbiome. Results: A subset of DAMPs/alarmins were expressed in healthy and naturally-occurring periodontitis tissues in the animals and suggested local effects on gingival tissues leading to altered levels of DAMPs/alarmins related to age and disease. Significant differences from adult healthy levels were most frequently observed in the young and adolescent animals with few representatives in this gene array altered in the healthy aged gingival tissues. Of the 51 target genes, only approximately ⅓ were altered by ≥1.5-fold in any of the age groups of animals during disease, with those increases observed during disease initiation. Distinctive positive and negative correlations were noted with the DAMP/alarmin gene levels and comparative expression changes of tissue destructive molecules during disease across the age groups. Finally, specific correlations of DAMP/alarmin genes and relative abundance of particular microbes were observed in health and resolution samples in younger animals, while increased correlations during disease in the older groups were noted. Conclusions: Thus, using this human-like preclinical model of induced periodontitis, we demonstrated the dynamics of the activation of the DAMP/alarmin warning system in the gingival tissues that showed some specific differences based on age.

Periodontitis-resistant and -susceptible matriline regulation of gingival transcriptome in nonhuman primates.

OBJECTIVE: This report identified if gingival gene expression transcriptomes demonstrated unique profiles that discriminated periodontitis-susceptible (PDS) and periodontitis-resistant (PDR) animals in health and disease. BACKGROUND: Nonhuman primates generally organize their social groups based upon matriline origin. We have used a multi-generational colony of rhesus macaques to identify matrilines presenting with significant differences in periodontitis (e.g., earlier age onset, greater prevalence, and severity). METHODS: Animals from 12 to 23 years of age (n = 17; 8 - PDR, 9 - PDS) were entered into a ligature-induced periodontitis trial. Gingival biopsies were taken at baseline and 0.5, 1, 3, and 5 months post-ligation, and microarray analysis was used to quantify gene expression in samples at each time point. RESULTS: Over 1000 genes showed significant (p < .01) differences in the PDR versus PDS animals at baseline. The frequency of differences generally decreased during the disease process, and increased with resolution (i.e., 5 months). A nearly 2:1 ratio of elevated gene levels was noted in baseline PDR samples that included up-regulated MMPs, Fc receptors, chemokines, interleukins, and innate immune receptors, and down-regulated genes particularly related to epithelial biology. Most dramatically, there was a skewed differential expression of adaptive immune response genes in the PDR and epithelial cell structure/function genes in PDS samples. CONCLUSIONS: The results demonstrate substantive differences in gingival tissue response capacity/programming in PDR and PDS samples that may contribute to the differences in clinical outcomes related to the heritability of disease risk through matrilines.

Sex and Age Effects on Healthy Gingival Transcriptomic Patterns.

Many chronic inflammatory diseases demonstrate demographic associations such as sex, age, and race-ethnicity. Periodontitis has been found to be increased with age and in males. This study used nonhuman primates representing a human-like model for periodontitis and examined the gingival transcriptome stratified on sex and age. Thirty-six Macaca mulatta in 4 age groups-young (<3 y), adolescent (3-7 y), adult (12-15 y), and aged (>17 y)-with a healthy periodontium were used to characterize gene expression in healthy gingival tissues. Gene expression was compared to clinical measures of bleeding on probing (BOP) and probing pocket depth (PPD). The results demonstrated sex differences in number of up- and downregulated genes that increased with age. Female animals generally showed elevated expression of genes related to host immunoinflammatory responses, and males showed increased expression of tissue structural genes. Gene expression correlations with BOP and/or PPD showed minimal overlap between the sexes, while male animals demonstrated substantial overlap in genes that correlated with both BOP and PPD clinical features. A cluster analysis of genes significantly different between sexes showed a clear sex and age discrimination in the young and adolescent animals. In the older groups, the genes clustered predominately by sex, irrespective of age group. A pathway analysis identified that significant gene expression patterns were quite similar in adolescent and adult animals, while the young and aged samples were quite distinct. The results confirmed substantial sex related variations in gingival tissue biology that were affected by age and observed even in adolescent animals. This suggests that "programming" of the gingival tissues related to sex can occur rather early in life and presage variations in future risk for periodontitis.

Gingival transcriptomics of follicular T cell footprints in progressing periodontitis.

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.

Immune system transcriptome in gingival tissues of young nonhuman primates.

BACKGROUND AND OBJECTIVE: Young/adolescent humans harbor many microorganisms associated with periodontal disease in adults and show substantial gingival inflammatory responses. However, younger individuals do not demonstrate the soft- and hard-tissue destruction that hallmark periodontitis. MATERIAL AND METHODS: This study evaluated responses to the oral microbial ecology in gingival tissues from clinically healthy young Macaca mulatta (< 3 years of age) compared with older animals (5-23 years of age). RNA was isolated from the tissues and analyzed for the transcriptome using the Rhesus Macaque GeneChip (Affymetrix). RESULTS: Global transcriptional profiling of four age groups revealed a subset of 159 genes that were differentially expressed across at least one of the age comparisons. Correlation metrics generated a relevance network abstraction of these genes. Partitioning of the relevance network revealed seven distinct communities comprising functionally related genes associated with host inflammatory and immune responses. A group of genes was identified that were selectively increased/decreased or positively/negatively correlated with gingival profiles in the animals. A principal components analysis created metagenes of expression profiles for classifying the 23 animals. CONCLUSION: The results provide novel system-level insights into gene-expression differences in gingival tissues from healthy young animals, weighted toward host responses associated with anti-inflammatory biomolecules or those linked with T-cell regulation of responses. The combination of the regulated microenvironment may help to explain the apparent 'resistance' of younger individuals to developing periodontal disease.

Oral epithelial cell responses to multispecies microbial biofilms.

This report describes the use of a novel model of multispecies biofilms to stimulate profiles of cytokines/chemokines from oral epithelial cells that contribute to local inflammation in the periodontium. Streptococcus gordonii (Sg)/S. oralis (So)/S. sanguinis (Ss) and Sg/Fusobacterium nucleatum (Fn)/Porphyromonas gingivalis (Pg) biofilms elicited significantly elevated levels of IL-1α and showed synergistic stimulatory activity compared with an additive effect of the 3 individual bacteria. Only the Sg/Actinomyces naeslundii (An)/Fn multispecies biofilms elicited IL-6 levels above those of control. IL-8 was a primary response to the Sg/An/Fn biofilms, albeit the level was not enhanced compared with a predicted composite level from the monospecies challenges. These results represent some of the first data documenting alterations in profiles of oral epithelial cell responses to multispecies biofilms.

Optimizing qPCR for the Quantification of Periodontal Pathogens in a Complex Plaque Biofilm.

Quantitative PCR (qPCR) has recently been used to quantify microorganisms in complex communities, including dental plaque biofilms. However, there is variability in the qPCR protocols being used. This study was designed to evaluate the validity of two of these variables with the intent of developing a more standardized qPCR protocol. The two variables evaluated were (1) the use of DNA content versus actual cell counts to estimate bacterial numbers in mixed plaque samples and (2) the effectiveness of three different universal primers versus species specific primers in amplifying specific target pathogens in these samples. Results lead to the development of a standardized protocol that was shown to be highly reproducible as demonstrated by low coefficients of variation. The results also confirmed that this standardized qPCR protocol can be used as a sensitive method for quantifying specific bacterial species in human plaque samples.