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.

Comparative Analysis of Gene Expression Patterns for Oral Epithelium-Related Functions with Aging.

Epithelial cells and functions of the epithelium are critical to the health of the oral cavity. We used a nonhuman primate model to profile the transcriptome of gingival tissues in health across the lifespan and hypothesized that in older animals, epithelial-related transcriptome patterns would reflect epithelial cells that are aggressively responsive to the surrounding environment and less able to modulate and resolve the noxious challenge from the bacteria. Rhesus monkeys (n = 34) with a healthy periodontium were distributed into four groups: ≤3 years (young), 3-7 years (adolescent), 12-16 years (adult), and 18-23 years (aged), and a buccal gingival sample from the premolar/molar region of each animal was obtained. RNA was subjected to a microarray analysis (GeneChip® Rhesus Macaque Genome Array, Affymetrix), and 336 genes examined that are linked to epithelium and epithelial cell functions categorized into 9 broad functional groups: extracellular matrix and cell structure; extracellular matrix remodeling enzymes; cell adhesion molecules, cytoskeleton regulation; inflammatory response; growth factors; kinases/cell signaling; cell surface receptors; junction associated molecules; autophagy/apoptosis; antimicrobial peptides; and transcription factors. Total of 255 genes displayed a normalized signal >100, and differences across the age groups were observed primarily in extracellular matrix and cell structure, cell adhesion molecules, and cell surface receptor gene categories with elevations in the aged tissues. Keratins 2, 5, 6B, 13, 16, 17 were all significantly increased in healthy-aged tissues versus adults, and keratins 1 and 2 were significantly decreased in young animals. Approximately 15 integrins are highly expressed in the gingival tissues across the age groups with only ITGA8, ITGAM (CD11b), and ITGB2 significantly increased in the aged tissues. Little impact of aging on desmosomal/hemidesmosomal genes was noted. These results suggest that healthy gingival aging has a relatively limited impact on the broader functions of the epithelium and epithelial cells, with some effects on genes for extracellular matrix and cell adhesion molecules (e.g., integrins). Thus, while there is a substantial impact of aging on immune system targets even in healthy gingiva, it appears that the epithelial barrier remains reasonably molecularly intact in this model system.

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.

Differential Gene Expression Profiles Reflecting Macrophage Polarization in Aging and Periodontitis Gingival Tissues.

Recent evidence has determined a phenotypic and functional heterogeneity for macrophage populations. This plasticity of macrophage function has been related to specific properties of subsets (M1 and M2) of these cells in inflammation, adaptive immune responses and resolution of tissue destructive processes. This investigation hypothesized that targeted alterations in the distribution of macrophage phenotypes in aged individuals, and with periodontitis would be skewed towards M1 inflammatory macrophages in gingival tissues. The study used a non-human primate model to evaluate gene expression profiles as footprints of macrophage variation in healthy and periodontitis gingival tissues from animals 3-23 years of age and in periodontitis tissues in adult and aged animals. Significant increases in multiple genes reflecting overall increases in macrophage activities were observed in healthy aged tissues, and were significantly increased in periodontitis tissues from both adults and aged animals. Generally, gene expression patterns for M2 macrophages were similar in healthy young, adolescent and adult tissues. However, modest increases were noted in healthy aged tissues, similar to those seen in periodontitis tissues from both age groups. M1 macrophage gene transcription patterns increased significantly over the age range in healthy tissues, with multiple genes (e.g. CCL13, CCL19, CCR7 and TLR4) significantly increased in aged animals. Additionally, gene expression patterns for M1 macrophages were significantly increased in adult health versus periodontitis and aged healthy versus periodontitis. The findings supported a significant increase in macrophages with aging and in periodontitis. The primary increases in both healthy aged tissues and, particularly periodontitis tissues appeared in the M1 phenotype.

Comparative analysis of expression of microbial sensing molecules in mucosal tissues with periodontal disease.

Host-derived pattern recognition receptors (PRRs) are necessary for effective innate immune engagement of pathogens that express microbial-associated molecular patterns (MAMP) ligands for these PRRs. This study used a nonhuman primate model to evaluate the expression of these sensing molecules in gingival tissues. Macaca mulatta aged 12-24 with a healthy periodontium (n = 13) or periodontitis (n = 11) provided gingival tissues for assessment of naturally-occurring periodontitis. An additional group of animals (12-23 years; n = 18) was subjected to a 5 month longitudinal study examining the initiation and progression of periodontitis, RNA was isolated and microarray analysis conducted for gene expression of the sensing PRRs. The results demonstrated increased expression of various PRRs in naturally-occurring established periodontitis. Selected PRRs also correlated with both bleeding on probing (BOP) and pocket depth (PD) in the animals. The longitudinal model demonstrated multiple TLRs, as well as selected other PRRs that were significantly increased by 2 weeks during initiation of the lesion. While gene expression levels of various PRRs correlated with BOP and PD at baseline and resolution of disease, few correlated with these clinical parameters during initiation and progression of the lesion. These findings suggest that the levels of various PRRs are affected in established periodontitis lesions, and that PRR expression increased most dramatically during the initiation of the disease process, presumably in response to the juxtaposed microbial challenge to the tissues and goal of reestablishing homeostasis.

Comparative analysis of microbial sensing molecules in mucosal tissues with aging.

Host-bacterial interactions at mucosal surfaces require recognition of the bacteria by host cells enabling targeted responses to maintain tissue homeostasis. It is now well recognized that an array of host-derived pattern recognition receptors (PRRs), both cell-bound and soluble, are critical to innate immune engagement of microbes via microbial-associated molecular patterns (MAMP). This report describes the use of a nonhuman primate model to evaluate changes in the expression of these sensing molecules related to aging in healthy gingival tissues. Macaca mulatta aged 3-24 years were evaluated clinically and gingival tissues obtained, RNA isolated and microarray analysis conducted for gene expression of the sensing pattern recognition receptors (PRRs). The results demonstrated increased expression of various PRRs in healthy aging gingiva including extracellular (CD14, CD209, CLEC4E, TLR4), intracellular (NAIP, IFIH1, DAI) and soluble (PTX4, SAA1) PRRs. Selected PRRs were also correlated with both bleeding on probing (BOP) and pocket depth (PD) in the animals. These findings suggest that aged animals express altered levels of various PRRs that could affect the ability of the tissues to interact effectively with the juxtaposed microbial ecology, presumably contributing to an enhanced risk of periodontitis even in clinically healthy oral mucosal tissues with aging.

Effects of aging in the expression of NOD-like receptors and inflammasome-related genes in oral mucosa.

The molecular changes underlying the higher risk of chronic inflammatory disorders during aging remain incompletely understood. Molecular variations in the innate immune response related to recognition and interaction with microbes at mucosal surfaces could be involved in aging-related inflammation. We developed an ontology analysis of 20 nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) and seven inflammasome-related genes (IRGs) in healthy and inflamed/periodontitis oral mucosal tissues from young, adolescent, adult, and aged non-human primates (Macaca mulatta) using the GeneChip(®) Rhesus Macaque Genome array. Validation of some of the significant changes was done by quantitative reverse transcription-polymerase chain reaction. The expression of NLRB/NAIP, NLRP12, and AIM2 increased with aging in healthy mucosa whereas NLRC2/NOD2 expression decreased. Although higher expression levels of some NLRs were generally observed with periodontitis in adult mucosal tissues (e.g. NLRB/NAIP, NLRP5, and NLRX1), various receptors (e.g. NLRC2/NOD2 and NLRP2) and the inflammasome adaptor protein ASC, exhibited a significant reduction in expression in aged periodontitis tissues. Accordingly, the expression of NLR-activated innate immune genes, such as HBD3 and IFNB1, was impaired in aged but not adult periodontitis tissues. Both adult and aged tissues showed significant increase in interleukin-1ß expression. These findings suggest that the expression of a subset of NLRs appears to change with aging in healthy oral mucosa, and that aging-related oral mucosal inflammation could involve an impaired regulation of the inflammatory and antimicrobial response associated with downregulation of specific NLRs and IRGs.