Gingival Transcriptome of Innate Antimicrobial Factors and the Oral Microbiome with Aging and Periodontitis

The epithelial barrier at mucosal sites comprises an important mechanical protective feature of innate immunity, and is intimately involved in communicating signals of infection/tissue damage to inflammatory and immune cells in these local environments. A wide array of antimicrobial factors (AMF) exist at mucosal sites and in secretions that contribute to this innate immunity. A non-human primate model of ligature-induced periodontitis was used to explore characteristics of the antimicrobial factor transcriptome (n = 114 genes) of gingival biopsies in health, initiation and progression of periodontal lesions, and in samples with clinical resolution. Age effects and relationship of AMF to the dominant members of the oral microbiome were also evaluated. AMF could be stratified into 4 groups with high (n = 22), intermediate (n = 29), low (n = 18) and very low (n = 45) expression in healthy adult tissues. A subset of AMF were altered in healthy young, adolescent and aged samples compared with adults (e.g., APP, CCL28, DEFB113, DEFB126, FLG2, PRH1) and were affected across multiple age groups. With disease, a greater number of the AMF genes were affected in the adult and aged samples with skewing toward decreased expression, for example WDC12, PGLYRP3, FLG2, DEFB128, and DEF4A/B, with multiple age groups. Few of the AMF genes showed a \u3e2-fold increase with disease in any age group. Selected AMF exhibited significant positive correlations across the array of AMF that varied in health and disease. In contrast, a rather limited number of the AMF significantly correlated with members of the microbiome; most prominent in healthy samples. These correlated microbes were different in younger and older samples and differed in health, disease and resolution samples. The findings supported effects of age on the expression of AMF genes in healthy gingival tissues showing a relationship to members of the oral microbiome. Furthermore, a dynamic expression of AMF genes was related to the disease process and showed similarities across the age groups, except for low/very low expressed genes that were unaffected in young samples. Targeted assessment of AMF members from this large array may provide insight into differences in disease risk and biomolecules that provide some discernment of early transition to disease

Oral Microbiome and Gingival Gene Expression of Inflammatory Biomolecules with Aging and Periodontitis

Although data describe the presence and increase of inflammatory mediators in the local environment in periodontitis vs. health in humans, details regarding how these responses evolve in the transition from health to disease, changes during disease progression, and features of a resolved lesion remain unknown. This study used a nonhuman primate model of ligature-induced periodontitis in young, adolescent, adult, and aged animals to document features of inflammatory response affected by age. Rhesus monkeys had ligatures tied and provided gingival tissue biopsy specimens at baseline, 0.5, 1, and 3 months of disease and at 5 months of the study, which was 2 months post-ligature removal for clinically resolved tissues. The transcriptome was assessed using microarrays for chemokine (n = 41), cytokine (n = 45), chemokine receptor (n = 21), cytokine receptor (n = 37), and lipid mediator (n = 31) genes. Limited differences were noted in healthy tissues for chemokine expression with age; however, chemokine receptor genes were decreased in young but elevated in aged samples. IL1A, IL36A, and IL36G cytokines were decreased in the younger groups, with IL36A elevated in aged animals. IL10RA/IL10RB cytokine receptors were altered with age. Striking variation in the lipid mediator genes in health was observed with nearly 60% of these genes altered with age. A specific repertoire of chemokine and chemokine receptor genes was affected by the disease process, predominated by changes during disease initiation. Cytokine/cytokine receptor genes were also elevated with disease initiation, albeit IL36B, IL36G, and IL36RN were all significantly decreased throughout disease and resolution. Significant changes were observed in similar lipid mediator genes with disease and resolution across the age groups. Examination of the microbiome links to the inflammatory genes demonstrated that specific microbes, including Fusobacterium, P. gingivalis, F. alocis, Pasteurellaceae, and Prevotella are most frequently significantly correlated. These correlations were generally positive in older animals and negative in younger specimens. Gene expression and microbiome patterns from baseline were distinctly different from disease and resolution. These results demonstrate patterns of inflammatory gene expression throughout the phases of the induction of a periodontal disease lesion. The patterns show a very different relationship to specific members of the oral microbiome in younger compared with older animals

Oral Microbial Biofilm Stimulation of Epithelial Cell Responses

Oral bacterial biofilms trigger chronic inflammatory responses in the host that can result in the tissue destructive events of periodontitis. However, the characteristics of the capacity of specific host cell types to respond to these biofilms remain ill-defined. This report describes the use of a novel model of bacterial biofilms to stimulate oral epithelial cells and profile select cytokines and chemokines that contribute to the local inflammatory environment in the periodontium. Monoinfection biofilms were developed with Streptococcus sanguinis, Streptococcus oralis, Streptococcus gordonii, Actinomyces naeslundii, Fusobacterium nucleatum, and Porphyromonas gingivalis on rigid gas-permeable contact lenses. Biofilms, as well as planktonic cultures of these same bacterial species, were incubated under anaerobic conditions with a human oral epithelial cell line, OKF4, for up to 24 h. Gro-1α, IL1α, IL-6, IL-8, TGFα, Fractalkine, MIP-1α, and IP-10 were shown to be produced in response to a range of the planktonic or biofilm forms of these species. P. gingivalis biofilms significantly inhibited the production of all of these cytokines and chemokines, except MIP-1α. Generally, the biofilms of all species inhibited Gro-1α, TGFα, and Fractalkine production, while F. nucleatum biofilms stimulated significant increases in IL-1α, IL-6, IL-8, and IP-10. A. naeslundii biofilms induced elevated levels of IL-6, IL-8 and IP-10. The oral streptococcal species in biofilms or planktonic forms were poor stimulants for any of these mediators from the epithelial cells. The results of these studies demonstrate that oral bacteria in biofilms elicit a substantially different profile of responses compared to planktonic bacteria of the same species. Moreover, certain oral species are highly stimulatory when in biofilms and interact with host cell receptors to trigger pathways of responses that appear quite divergent from individual bacteria

Gingival transcriptomic patterns of macrophage polarization during initiation, progression, and resolution of periodontitis.

Phenotypic and functional heterogeneity of macrophages is clearly a critical component of their effective functions in innate and adaptive immunity. This investigation hypothesized that altered profiles of gene expression in gingival tissues in health, disease, and resolution would reflect changes in macrophage phenotypes occurring in these tissues. The study used a nonhuman primate model to evaluate gene expression profiles as footprints of macrophage variation using a longitudinal experimental model of ligature-induced periodontitis in animals from 3 to 23 years of age to identify aging effects on the gingival environment. Significant differences were observed in distribution of expressed gene levels for M0, M1, and M2 macrophages in healthy tissues with the younger animals showing the least expression. M0 gene expression increased with disease in all but the aged group, while M1 was increased in adult and young animals, and M2 in all age groups, as early as disease initiation (within 0.5 months). Numerous histocompatibility genes were increased with disease, except in the aged samples. An array of cytokines/chemokines representing both M1 and M2 cells were increased with disease showing substantial increases with disease initiation (e.g. IL1A, CXCL8, CCL19, CCL2, CCL18), although the aged tissues showed a more limited magnitude of change across these macrophage genes. The analytics of macrophage genes at sites of gingival health, disease, and resolution demonstrated distinct profiles of host response interactions that may help model the disease mechanisms occurring with the formation of a periodontal lesion

Oral Microbiome and Gingival Tissue Apoptosis and Autophagy Transcriptomics

Objective: 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

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 on Apoptosis Gene Expression in Oral Mucosal Tissues

Apoptotic processes are important for physiologic renewal of an intact epithelial barrier and contribute some antimicrobial resistance for bacteria and viruses, as well as anti-inflammatory effects that benefits the mucosa. The oral cavity presents a model of host-bacterial interactions at mucosal surfaces, in which a panoply of microorganisms colonizes various niches in the oral cavity and creates complex multispecies biofilms that challenge the gingival tissues. This report details gene expression in apoptotic pathways that occur in oral mucosal tissues across the lifespan, using a nonhuman primate model. Macaca mulatta primates from 2 to 23 years of age (n = 23) were used in a cross-sectional study to obtain clinical healthy gingival tissues specimens. Further, mRNA was prepared and evaluated using the Affymetrix Rhesus GeneChip and 88 apoptotic pathway genes were evaluated. The results identified significant positive correlations with age in 12 genes and negative correlations with an additional five genes. The gene effects were predicted to alter apoptosis receptor levels, extrinsic apoptotic pathways through caspases, cytokine effects on apoptotic events, Ca+2-induced death signaling, cell cycle checkpoints, and potential effects of survival factors. Both the positively and negatively correlated genes within the apoptotic pathways provided evidence that healthy tissues in aging animals exhibit decreased apoptotic potential compared to younger animals. The results suggested that decreased physiologic apoptotic process in the dynamic septic environment of the oral mucosal tissues could increase the risk of aging tissues to undergo destructive disease processes through dysregulated inflammatory responses to the oral microbial burden

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

Transcriptome Analysis of B Cell Immune Functions in Periodontitis: Mucosal Tissue Responses to the Oral Microbiome in Aging

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

Epithelial Interleukin-8 Responses to Oral Bacterial Biofilms

An in vitro model of bacterial biofilms on rigid gas-permeable contact lenses (RGPLs) was developed to challenge oral epithelial cells. This novel model provided seminal data on oral biofilm-host cell interactions, and with selected bacteria, the biofilms were more effective than their planktonic counterparts at stimulating host cell responses