SciELO - Scientific Electronic Library Online

 
vol.71 número1The association between area level socio-economic position and oral health-related quality of life in the South African adult populationContinuous education in sedation: Laryngospasm and management of the airway índice de autoresíndice de assuntospesquisa de artigos
Home Pagelista alfabética de periódicos  

Serviços Personalizados

Artigo

Indicadores

Links relacionados

  • Em processo de indexaçãoCitado por Google
  • Em processo de indexaçãoSimilares em Google

Compartilhar


South African Dental Journal

versão On-line ISSN 0375-1562
versão impressa ISSN 0011-8516

S. Afr. dent. j. vol.71 no.1 Johannesburg Fev. 2016

 

CLINICAL REVIEW

 

The emerging role of epigenetics in the pathogenesis of periodontitis - A review

 

 

PC Srinivasan

Professor, Periodontics, NSVK SV Dental College, Bangalore, Karnataka, India. Cell: 984 548 5963, e-mail: csprati@yahoo.com

 

 


ABSTRACT

Periodontitis is a chronic inflammatory disease affecting the supporting structures of the teeth. It is a complex disease with multifactorial etiology. Numerous studies have examined the role of the genetic factors in the etiology of periodontitis. Epigenetics is the study of the mitotically and meiotically heritable changes in the gene function that cannot be explained by changes in the DNA sequence. Studies have demonstrated that epigenetic alterations contribute to a number of diseases like cancer, metabolic and autoimmune disorders. An understanding of the epigenetic mechanisms helps to develop novel therapeutic aids which target the specific epigenetic sites. This article attempts to shed light on the role of epigenetic alterations in the pathogenesis of periodontitis. The role of the bacteria-induced epigenetic alterations in the host cell, the alterations in the cytokine profile and the role of the environmental factors like smoking on the epigenome are reviewed. Technological advances have enabled us to analyse and quantify the epigenetic changes on a large scale. Drugs which specifically target the epigenetic mechanisms may be used as valuable adjuncts to conventional periodontal therapy leading the way to personalized and preventive regimes.

Keywords: Periodontitis, inflammation, epigenetic mechanisms, pathogenesis, epigenome, DNA methyltrans-ferase.


 

 

INTRODUCTION

Periodontitis is one of the most common oral diseases in adult populations and is characterized by inflammation and destruction of the tooth supporting structures, ultimately leading to tooth loss in severe cases.1-2 Although periodontitis has a microbial etiology, its progression is multidimensional and can be influenced by several factors such as systemic diseases, environmental factors, and genetic factors.3 The individual variability in the susceptibility to periodontitis and in the response to the therapy as well can be attributed to intrinsic factors such as genetic4 and epigenetic factors.5 Epigenetics is the study of alterations in gene regulation not caused by changes in the DNA sequence. The term "Epigenetics" was coined by Conrad Waddington, who defined it as "the branch of biology that studies the causal interaction between genes and their product, which bring the phenotype into being."6 The Greek prefix 'epi' in epigenetics means 'on top of' or 'in addition to' genetics.7

 

EPIGENETIC MECHANISMS

Important epigenetic mechanisms include DNA methylation, post-transcriptional histone modifications (methylation, acetylation, ubiquitylation, and phosphorylation) that affect chromatin structure, RNA associated gene silencing and chromosome inactivation.8

A. DNA Methylation:

DNA methylation is the covalent transfer of a methyl group from S-adenosyl-L-methionine (SAM) to the 5th carbon atom of the cytosine residue in the Cytosine-phosphodiester-Guanine (CpG) dinucleotides. The methylation process occurs mostly in regions containing a high frequency of CpG dinucleotides, called "CpG islands" in the promoter region of a gene, and is associated with gene silencing in most cases.9 The process of DNA methylation is catalysed by a family of closely related DNA methyltransferases (DNMT1, DNMT3, and DNMT3b).10 The methyl groups block the binding of the transcription factors to DNA. This transcriptional repression leads to "gene silencing."11 The exposed methylation sites allow for interaction with methyl-binding proteins, such as methyl-CpG-binding domain proteins (MBDs).12 Additionally, these proteins are instrumental in assembling histone deacetylases (HDAC's) and thus influence chromatin condensation. HDAC's are enzymes which remove the acetyl group from histones. Thus the DNA gets wrapped more tightly around the histones.13 The closed chromatin configuration leads to gene silencing.14 Studies in the literature have shown that hypomethylation of DNA is associated with chromosomal instability and activation of transposable elements in human cancers.15 Thus, abnormal methylation patterns can lead to the development of diseases.

Histone modification

A nucleosome is the basic unit of the chromatin. It comprises DNA wrapped around an eight-member histone complex which consists of two copies each of H2A, H2B, H3, and H4. This composition provides a rigid structure to the chromatin. Histones have unstructured N-terminal tails, which undergo post-translational modifications including acetylation, methylation, ubiquitination, glycosylation, citrullination, ADP-ribosylation, carbonylation and sumoylation at certain positions.16 Post-translational modifications such as acetylation and methylation of conserved lysine residues on the amino terminal tail domains have been reported.17 Enzymes such as methyltransferases, demethylases, histone acetlytransferases (HAT's), and HDAC's either write or erase these modifications.18 Acetylation of core histones results in an "open" chromatin structure that facilitates gene transcription.19 Conversely, histone deacetylases remove the acetyl groups, causing the chromatin to become more condensed and thus gene transcription is repressed.20 Some histone marks, such as H3K27ac (H3 acetylated at lysine 27) and H3K9ac (H3 acetylated at lysine 9) are associated with active transcription of genes, whereas others like H3K27me3 (H3 methylated at lysine 27) and H4K27me3 (H4 methylated at lysine 27), are responsible for repression of gene activity. Thus acetylation is associated with activation of the gene, whereas methylation leads to silencing of gene.21 To summarize, post-translational histone modifications are powerful epigenetic mechanisms. These modifications can alter the chromatin structure. The chromatin is either open or condensed, thus regulating the gene transcription. This article mainly focuses on the acetylation of histones, the histone deacetylase inhibitors, their implications for the pathogenesis and therapeutic management of, periodontitis.

B. NON-CODING RNA:

The non-coding RNA's do not encode for a protein, but they are functionally relevant RNA molecules. These include transfer RNAs (tRNA's), ribosomal RNAs (rRNA's), micro-RNA's (mi-RNA's), and short-interfering RNA's (siRNA's).22 Studies have reported that these non-coding RNA's play a pivotal role in the development of oral cancer, specific syndromes, and exert influences on the immune mechanisms in the oral cavity.23,24 Micro-RNA's and short-interfering RNA's have been shown to regulate gene expression without altering the DNA sequence. Micro-RNA's have been shown to negatively regulate the expression of their target genes at the post-transcriptional level, thus leading to "gene silencing".20,25 This article attempts to review the role of micro-RNA's in the pathogenesis of periodontitis.

As our understanding of the pathogenesis of periodontal disease continues to grow, additional potential mechanisms linking the microbial biofilm to the disease process are being described.26 This review focuses on the intriguing role of epigenetic alterations in the pathogenesis of periodontitis.

The role of epigenetic mechanisms in periodontitis.

Upon a microbial attack, the host mounts an immune inflammatory response. Recent studies have demonstrated that bacteria can affect the chromatin structure and transcriptional program of host cells by influencing diverse epigenetic mechanisms.27 Epigenetic events determine gene expression and selective activation or inactivation of genes. These events modulate the production of inflammatory mediators, expression of cytokines and thus contribute to the pathogenesis of various infectious and inflammatory diseases.28

The role of microbial plaque-induced epigenetic changes in the pathogenesis of periodontitis.

The oral biofilm is a complex structure. Using whole ge-nomic probes and Checkerboard DNA-DNA hybridization methodology, Sockransky et al analysed 13, 261 plaque samples. About 40 different bacterial species were determined in the subgingival plaque and these bacteria were found to exist in complexes. Five major complexes (red, orange, yellow, green and purple complexes) were consistently observed. Of these, the red complex, comprised of Tannerella forsythia, Porphyromonas gingivalis and Treponema denticola is strikingly associated to the clinical measures of periodontal disease such as destruction-pocket depth and bleeding on probing. The orange complex consists of microbes like Fusobacterium nuclea-tum, Campylobacter rectus, Campylobacter showae etc. Plaque formation begins by early colonizers (streptococcus species) attaching to the pellicle-coated tooth structure. The early colonizers alter the local micro-environment and make it conducive for the intermediate (orange complex bacteria) and the late colonizers (red complex) to establish themselves and thrive in the area. Thus the plaque mass undergoes maturation.3

Studies in the literature point to the fact that putative periodontal pathogens like Porphyromonas gingivalis and Campylobacter rectus can induce epigenetic alterations in the gingival cells and tissues.29 The microbe-induced epigenetic alterations with the resultant disruption of the host innate immune mechanisms is a vital step in the disease progression.30

Plaque accumulation in the dento-gingival area elicits a host immune response in the gingival epithelium. The cells of the gingival epithelium make use of a myriad of signalling pathways to modulate the innate immune response to the various microorganisms.31 The toll-like receptors (TLRs) enable the gingival epithelial cells (GEC's) to recognize the pathogen-associated molecular patterns. The gingival epithelial cells then produce antimicrobial peptides such as human beta defensins and chemokines that activate the adaptive immune response.32-34 Yin and Chung demonstrated that Porphyromonas gingivalis perked up the expression of antimicrobial proteins human beta defensin and CC chemokine ligand 20 (CCL20). The gingival epithelial cells treated with this microbe showed decreased expression of histone deacetylase 1 and 2 (HDAC 1, HDAC 2), and DNA methyltransferase 1 (DNMT1). P. gingivalis also induced increased methylation of the promoter region of six genes, including the immune regulator CD276, elastase 2, toll-like receptor-2 (TLR2), interleukin-12 A(IL-12A), and two putative tumour-suppressor genes (TSG). The levels of the activating histone modification H3K4me3 were found to be reduced in GEC's incubated with Porphyromonas gingivalis. Fusobacterium nucleatum, a non-pathogen, did not induce such alterations in the GEC's. These findings corroborate the fact that P.gingivalis is capable of suppressing gene transcription.31 The lipopoly-saccharide (LPS) produced by Porphyromonas ginigivalis induces signalling of the toll-like receptor (TLR). The study demonstrated that the TLR signalling has far-flung effects on the epigenetic profiles of genes which respond to the TLR.35,36 Keratinocytes in the gingival epithelium when exposed to P.gingivalis LPS showed decreased expression of DNA methyltransferase 1 (DNMT1), Histone deacetylase 1 and 2 (HDAC1 & 2), the key enzymes involved in epigenetic mechanisms.37 Riggs et al. and Sealy et al. in their study demonstrated that butyric acid, a major short chain fatty acid produced by P.gingivalis, is a histone deacetylase inhibitor (HDACi).38,39 Thus, butyrate, a short chain fatty acid (SCFA) produced by P.gingivalis induces acetylation of histones in the cells of the periodontium.40-42

Recent evidence has also pointed to another interesting aspect in the bacteria-induced epigenetic alterations. This butyrate, an HDAC inhibitor produced by P.gingivalis, causes reactivation of human immunodeficiency virus (HIV) and Epstein-Barr virus (EBV).43-45 Morris et al. in their study showed that P.gingivalis metabolites like butyric acid enhanced the replication of Kaposi sarcoma-associated herpes virus (KSHV). The bacterial supernatant contained HDAC inhibition properties. This increased the global acetylation of H3 and H4 leading to the reactivation and replication of KSHV.42 Thus periodontal microbes like P.gingivalis and other opportunistic bacteria associated with the state of immunosuppression in HIV-positive individuals may collectively contribute to AIDS progression by reactivating the latent virus through HDAC inhibition.46,47 Oxidative stress is an imbalance between the production of a reactive oxygen species and the antioxidant defense, leading to tissue damage.48P.gingivalis induces oxidative stress, a process which is central to epigenetic modifications.49,50

Additional studies by Yin et al.51and Chung et al.52have demonstrated that epigenetic changes in P.gingivalis stimulated dendritic cells and GECs resulted in lower levels of cytokines and chemokines secreted by these cells. Uehara et al. reported that LPS derived from P.gingivalis inhibits osteoblastic cell differentiation. DNA hypermethylation was involved in the inhibitory effect of LPS on osteoblastic differentiation of fibroblasts derived from human periodontal ligament (HPDL).53

Campylobacter rectus, another putative periodontal pathogen may also induce epigenetic alterations in human cells. Bobetsis demonstrated that C. rectus downregulated the expression of insulin-like growth factor 2 (Igf2) gene via hypermethylation of Igf2 promoter in murine placenta.54 The reduced placental growth and foetal growth as a result of these epigenetic alterations may be involved in preterm births associated with C. rectus infection in humans.55 A study conducted by Miao et al. observed that Treponema denticola, another periodontal microbe, upregulated the matrix metalloproteinase-2 (MMP-2) gene. There was hypomethylation in the promoter region of the MMP-2 gene. MMP-2 is responsible for the matrix degradation and bone resorption in periodontitis. The authors suggested that adherence/internalization of T. denticola into the periodontal ligament cells may have contributed to the epigenetic alterations.56 Wu et al. suggested that DNA adenine methyltransferase (DAM) may regulate the genes required for the invasion process of Actinobacillus actinomycetemcomitans. Inactivation of DAM alters the virulence properties of this microbe.57

The role of microbe-induced epigenetic alterations in Herpesvirus Periodontitis.

Though periodontitis is a highly prevalent, chronic infectious disease afflicting the tooth-supporting structures, the etiology is still poorly understood.58 The major clinical characteristics of this enigmatic disease are perplexing. Findings such as the bilateral symmetrical pattern of the disease, spontaneous remission, why periodontitis affects only few teeth, with neighbouring teeth exhibiting much less attachment loss etc., cannot be explained solely on the basis of a microbial etiology. The co-infection of viruses with the microbes and their synergistic effect on the tissues has been suggested and may offer explanations for the confounding aspects of the disease.59

A number of studies have shown that herpes viruses, includingEpstein-Barr Virus(EBV),Human Cytomegalovirus (HCMV) and Herpes Simplex Virus-1 (HSV-1) are detected in high numbers in patients with periodontal and endodontic disease. They act synergistically with the periodontal bacteria.60-65 The herpes viruses may impair the local host defences and thus increase the pathogenic potential of the bacteria. The bacteria in turn may increase the virulence of the herpes viruses.66 The microbe-induced epigenetic modification of the viral genome may explain the link between viruses and bacteria in the pathogenesis pathway. Butyrate, produced by P.gingivalis, is a HDAC inhibitor. Bacteria-induced HDAC inhibition may reactivate the latent EBV and HIV virus.43,44,45 Reactivation of latent HIV virus through HDAC inhibition by P.gingivalis may contribute to AIDS progression.46,47

To conclude, studies strongly indicate that microbe-induced epigenetic alterations in the host cells and the viral genome may play an important role in the disease pathogenesis. These alterations have far-reaching implications. They can affect the prognostic and therapeutic outcomes of the disease. For example, butyrate, a metabolite of P.gingivalis can reactivate the latent HIV, EBV, and KSHV, thus leading to progression of virus-associated diseases. Bacteria and viruses have a synergistic action, thus increasing the pathogenicity. The use of anti-viral drugs as adjunct to conventional periodontal therapy may provide beneficial results and is a subject for further research.

Epigenetic changes and cytokines

Inflammation is the central component in the pathogenesis of periodontitis. Epigenetic alterations may have an effect on the cytokine profile, and thus can determine the outcome of the disease.

Epigenetic mechanisms have been evaluated in some cytokine genes. In periodontal disease, there is an overex-pression of pro-inflammatory cytokines (IL1, IL4, IL6 and IL-10).67-69 Epigenetic events such as hypomethylation and histone acetylation are associated with the inappropriate over-transcription of genes.70 Gomez et al. observed hy-pomethylation in the gene of cytokine interleukin-6 (IL-6) in the tissues of individuals with periodontitis, leading to an overexpression of this cytokine in the inflamed tissues.71 IL-6 is a key cytokine involved in bone resorption and has been detected in high levels on patients with periodontal disease.71,72 Babel et al. in their study showed overexpression of cytokine IL-6 in the inflamed tissues of subjects with chronic periodontitis.73

Interestingly, the overexpression of IL-6 might have an influence on the epigenetic changes in the cells. Studies conducted by Hodge et al., Stenvinkel et al., and Hodge et al., suggested that the overexpression of IL-6 might exert an epigenetic influence in the cells by regulating the DNMT gene or by maintaining it's methylation status.74-76 Long-standing persistent inflammation and bacterial infection may cause DNA methylation which in turn inactivates the suppressors of cytokine signalling and may thus contribute to exaggerated cytokine signalling.71,75 Wehbe et al. suggested that the over-expression of IL-6 may influence the expression and activity of DNMT, demethyl-ases or histone expression, which participates in regulation of gene methylation.77 A similar study by Hmadcha et al. showed that interleukin-1 beta causes activation of DNA methyltransferase and thus markedly supresses the genes which code for the interleukin.78

In periodontitis, the inflammatory response involves upregulation of transcription factors-nuclear factor kappa-B (NF-Kappa B) and signal transducers and activators of transcription (STAT).20 Oliveira et al. in their study evaluated the methylation status of DNA in the promoter region of interleukin-8 (IL-8, a chemokine) in gingival and oral mucosal cells, leukocytes in blood from healthy individuals, smokers and non-smoker subjects with chronic periodontitis. The methylation status was co-related with mRNA levels of IL-8. The study revealed that there was a higher percentage of hypomethylation of IL-8 gene in chronic periodontitis subjects in the DNA of oral mucosal cells.79

Cyclooxygenase-2 (COX-2) is an enzyme governing the production of prostaglandins that promote pain and inflammation. Zhang et al. evaluated the epigenetic changes in the promoter region of Prostaglandin synthase 2, the gene encoding for COX-2 in chronic periodontitis patients. The results revealed a hyper-methylation status of the gene and lower levels of COX-2 transcription in inflamed gingival biopsy cells.80 A study by Andia et al. in subjects with generalized aggressive periodontitis evaluated the DNA methylation status in the promoter region of IL-8 gene in oral and GEC's. The authors reported a hypomethylated status in oral and GEC's of these subjects.81 Zhang et al. evaluated the presence of epigenetic modifications in the promoter region of interferon gamma (IFNG) gene in gingival biopsy of chronic periodontitis subjects. Their study reported a significant hypomethylation and increased IFNG transcription.82 Corroborating this evidence, White et al, in their study, suggested that the expression of IFNG is regulated by the status of methylation in its promoter region.83 Sullivan et al. demonstrated that epigenetic alterations in the gene which codes for tumour necrosis factor alpha (TNF-α) actively regulates its expression and is present both consecutively and in response to acute stimulation of cells from the myeloid lineage.84 Another recent study by Zhang et al. showed that the Tumour necrosis factor alpha (TNF-α) promoter was hypermethylated at two CpG sites, resulting in decreased expression. Reversing the methylation by treatment with a demethylating agent in vitro, caused increased expression of TNF-α, indicating that the methylation indeed regulated the expression.85 De Souza et al., stated that variations in DNA methylation between healthy and periodontitis cases are higher in genes related to the immune-inflammatory process. DNA methylation must be modulating the chromatin regions, and consequently modulating the mRNA transcription of immune inflammatory genes associated with periodontitis. Thus DNA methylation can have an impact on the prognosis of the disease.86

Studies have revealed that in addition to DNA methylation, other epigenetic changes such as histone modifications also play a pivotal role in the pathogenesis of periodontitis. Gemmell et al. stated that the nature of the lymphocytic response determines the destructive periodontitis lesion. The progression from gingivitis to periodontitis is characterized by a transition from the Th1 to the Th2 subset of the T lymphocyte.87 Changes in the chromatin structure occur through epigenetic mechanisms like histone modification, DNA methylation and generation of DNAse I hypersensitive sites. These epigenetic modifications occur during the process of differentiation of the naïve T cells into the various lineages, thus determining the destructive characteristics of the lesions.88 Cantley et al. in their experimental mouse model study demonstrated that treatment by histone deacetylase inhibitors (HDACi) efficiently suppressed periodontal bone loss.89

The role of Micro-RNA's (mi-RNA) in the pathogenesis of periodontitis have only been recently reviewed. Comparison of the mi RNA profiles in the healthy and the periodontitis tissues revealed that the mi RNA levels were increased in the latter group.90-92 In a recent study, Ogata et al. used mi-RNA microarray profiling and real time PCR analysis to determine the micro-RNA expression in the inflamed and healthy periodontal tissues. The results of their study revealed that the three most overexpressed miRNA's were hsa-miR-150, hsa-miR-223, and hsa-miR-200b, and the three most under expressed mi-RNA's were hsa-miR-379, hsa-miR-199a-5p, and hsa-miR-214. The overexpressed mi-RNA's are associated with inflammatory disease, organismal injury, abnormalities, urological disease, and cancer.93 Micro-RNA's have been implicated in controlling the TLR pathway which connects the innate and the adaptive pathways of the immune response. In their experimentally induced periodontitis in apolipoprotein E-deficient (ApoE-/-) mice, Nahid et al. reported that polymicrobial infection with periodontal pathogens like P.gingivalis, T. denticola, T. forsythia enhanced the levels of miR-146a. In the same study, human monocytic leukemia cell line (THP1) cultures were stimulated with a combination of periodontal pathogens. The results revealed that there was an increase in the mi-RNA levels in a time-dependent manner and this co-related with the downregulation of adaptor kinases IL-1 receptor associated kinase 1 (IRAK-1), tumour necrosis factor receptor-associated factor (TRAF-6), and TNF-α production by these cells. The authors concluded that the elevated levels of miR-146a downregulates IRAK-1 and TRAF-6. This may have been the reason for endotoxin tolerance. Hence, miR-146a may represent a target for therapeutic intervention.94 Similar results were obtained in the study of Xie et al. miRNA-146 inhibited the secretion of pro-inflammatory cytokine through IL-1 receptor-associated kinase 1 in human gingival fibroblasts.95 Ceppi et al. examined the levels of miR-155 in monocyte-derived dendritic cells exposed to endotoxin lipopolysaccharide. An elevated level of mi-R-155 was demonstrated and it was shown to downregulate TAK-1 binding protein-2 (TAB2), which plays an important role in IL-1 signalling pathways. The authors concluded that this negative feedback loop helped modulate the inflammatory responses of dendritic cells.96 Perri et al. in their study determined the expression profile of micro-RNA in obese individuals with and without periodontitis. They found that there was upregulated expression of several mi-RNA's in the inflamed gingival tissues of their patients with periodontitis and obesity. The findings suggest that inflamed periodontal tissues and obesity may share the inflammatory mi-RNA targets.97 Abnormal micro-RNA expression has been implicated in several inflammatory diseases and cancer. The micro-RNA targets may be amenable to intervention by drugs which target the specific epigenetic sites.

Tout ensemble, experimental studies have shed light on the impact of altered epigenetic patterns on the cytokine profile in patients with periodontitis. Long-standing chronic inflammation and bacterial infection may have an effect on the enzymes involved in the epigenetic mechanisms. The alterations in the cytokine profile may affect the prognostic outcome of the disease.

Role of the environmental factors in the patho-epi-genetics of periodontitis.

Periodontitis is a chronic inflammatory disease. Microbial plaque, the primary etiological agent, is essential to cause the disease in a susceptible host. There are several other factors like nutrition, toxic components in the environment, tobacco smoke, alcohol and different infectious agents that can affect the disease outcome. These factors can induce epigenetic alterations in the host cells.98,99 Studies have observed associations between smoking and global DNA methylation, linked with poor prognosis in lung cancer.100,101 A study conducted by Oliveira et al.,79revealed that there was a higher percentage of hypomethylation of IL-8 gene in the DNA of oral mucosal cells of subjects with chronic periodontitis. But there was no difference in the methylation status of IL-8 promoter in smokers and non-smoker subjects with periodontitis in this study. Smoking is an important risk factor for periodontal disease. Further studies evaluating the methylation in smokers with periodontal disease may be of interest.

Studies have elicited the role of diet and nutritional influences on the pathogenesis of periodontitis.102,103 Okano et al.104stated that folate deficiency during pregnancy leads to a lack of S-adenosylmethionine, a substrate required for the enzyme DNMT (DNA methyltransferase) to methylate CpG residues during embryonic development. Increase in the methylation rate in older individuals leads to gene silencing and could thereby contribute to the development of chronic diseases.105 Ohi et al.106in their study demonstrated hypermethylation of CpG in the promoter of the collagen-alpha 1 gene in the aged periodontal ligament.

The pathogenesis of periodontitis is complex. The above mentioned studies point to the multiple confounding factors that might play a role in the epigenetic mechanisms and thereby affect the disease outcome.

Epigenetic therapy in the management of periodontitis : personalized periodontal therapy.

Epigenetic changes occur more frequently than the genetic changes and are rendered reversible by treatment with pharmacological agents.107 Research in the use of pharmaceutical agents targeting the "epigenetic sites" is ongoing. Histone deacetylase inhibitors and DNA methyltransferase inhibitors have been in the vanguard of these approaches. Histone deacetylase inhibitors help in supressing bone re-sorption by osteoclasts.89 The deacetylase inhibitors help in promoting osteoblast maturation.108 In their study on P.gingivalis - induced experimental periodontitis in mice, Cantley et al. evaluated the bone volume changes after administering novel compounds targeting both Class I & II HDACs (1179.4b) and MS-275, which targets specifically the Class I HDAC. The results of the study revealed that Class I and II histone deacetylase inhibitor, 1179.b, significantly reduced P.gingivalis- induced bone loss.109

The above mentioned studies suggest that agents targeting the specific "epigenetic sites" can be considered as useful adjuncts in the management of periodontal disease.

Methods for analysing epigenetic mechanisms.

Technological advances have enabled the analysis of epigenetic analysis on a large scale.21

DNA methylation can be detected and quantified by the following techniques:

a. Bisulphite conversion.-In this technique sodium bisulphite modification of DNA enables the conversion of unmethylated cytosines to uracil, while the methylated cytosines remain unchanged.110

b. Global DNA Methylation Analysis-High Performance Liquid Chromatography (HPLC) is a classical method to quantify global DNA methylation.111

c. Gene-specific methylation analysis-can be characterized as either "candidate gene" or "genome-wide" approach.

The candidate gene approaches can be further divided into "sensitive" and "quantitative" approaches.

Methods for genome-wide analysis:

a. Microarray-based genome-wide analysis. Three main classes of microarray methods have been developed to map the 5-methylcytosine patterns in genomes :

1. Methods which enrich the highly methylated regions using an antibody specific for 5-methylcytosine or methyl-binding proteins.

2. Methods based on bisulphite modification.

3. Methods using methylation-sensitive restriction enzymes.112

Methylated DNA Immuno-precipitation-MeDIP

The DNA is immuno-precipitated using antimethylcytosine antibody and this immuno-precipitated DNA is hybridized to microarrays.113

Analysis of histone modifications:

The histone modification signals can be captured by chromatin immuno-precipitation (ChIP), in which an antibody is used to enrich DNA fragments from modification sites. ChIP-chip, ChIP-PET, ChIP-SAGE are some of the several ChIP based techniques.114-116 Ultrahigh-throughput sequencing technologies such as Illumina/Solexa sequencing has enabled the use of a new technique called ChIP-seq. ChiP-seq is becoming one of the main approaches due to its high coverage, high resolution and low cost. In ChIP-seq, the sequence of one end of a ChIP-enriched DNA fragment is read, and it is followed by mapping the short read, called tag, to the genome assembly in order to And the genome location of the fragment.117-119

Thus technological advances have enabled geneticists to analyse and quantify the epigenetic modifications on a large scale. The use of bioinformatics to compute the epigenetic alterations is very exciting and promising. These data are of enormous use in research and can be used in the development of drugs that target the epigenetic sites with greater specificity.

 

CONCLUSION

Periodontitis is a chronic inflammatory disease involving the supporting structures of the tooth. It is a polymicrobial infectious disease. The host tissue mounts an immune inflammatory response to combat the bacterial attack. A number of studies reveal the role of epigenetic mechanisms in the pathogenesis of periodontitis. Bacteria cause epigenetic alterations in the gingival cells and tissues. These epigenetic changes can cause "silencing/ shut down" of genes involved in local defences and so the chances of survival of the microbes in the local microenvironment is significantly enhanced. They may also cause rapid re-establishment of virulent flora, thereby giving rise to refractory/resistant forms of periodontal disease.26 This emphasizes the importance of thorough surgical debridement and complete removal of infected granulation tissue which may act as a reservoir of bacteria. It is also logical to use antimicrobials as adjuncts to non-surgical/surgical periodontal therapy to eliminate the microbes which have tissue-invasive properties. Studies have also revealed that epigenetic changes in the cytokine genes have a crucial role to play in the pathogenesis of this inflammatory disease. However it needs to be ascertained whether these epigenetic alterations lead to increased susceptibility to the disease or whether they are a consequence of the long-standing chronic inflammatory response.51 It is a "chicken or egg" scenario which is most perplexing. Periodontitis has a complex multifactorial etiology. Though microbial plaque is the primary etiologic factor, systemic diseases like diabetes, age and environmental factors such as smoking may affect the disease outcome. These confounding factors also have a role to play on the epig-enome. Hence studies evaluating the epigenetic events in the pathogenesis of periodontitis should be viewed with caution. Geneticists have made terrific progress and have developed drugs that target the "epigenetic sites." These drugs can be used as valuable adjuncts to conventional periodontal therapy. This type of therapeutic approach is showing great promise in the treatment of other diseases affected by aberrant epigenetic marks like cancer, lupus, and asthma, neurological disorders like Huntington's chorea, Alzheimer's disease and diabetes. The challenge with this approach is to specifically target the epigenetic marks which have negatively influenced the gene, leaving alone the beneficial ones that help maintain health. It has always been thought that "our genes are set in stone" and are beyond our influence. The concept that the epigenome can be altered by pharmacologic intervention is very profound and empowering. Technological advances have enabled analysis and quantification of the epigenetic changes and have been instrumental in the development of drugs which target the epigenetic sites with greater specificity.

 

ACRONYMS

ChIP: chromatin immuno-precipitation

COX-2: Cyclooxygenase-2

CpG: Cytosine-phosphodiester-Guanine

DAM: DNA adenine methyltransferase

DNMT1, DNMT3, and DNMT3b: DNA methyltransferases

EBV: Epstein-Barr virus

GEC's: gingival epithelial cells

HAT's: histone acetlytransferases

HDAC's: histone deacetylases

HDACi: Histone deacetylase inhibitors

HIV: human immunodeficiency virus

HPDL: human periodontal ligament

IFNG: interferon gamma gene

IRAK-1: receptor associated kinase 1

KSHV: Kaposi sarcoma-associated herpes virus

LPS: lipopolysaccharide

MMP-2: matrix metalloproteinase-2

MBDs: methyl-CpG-binding domain proteins

mi-RNA Micro-RNA's

THP1: monocytic leukemia cell line

NF-Kappa B: nuclear factor kappa-B

SAM: S-adenosyl-L-methionine

SCFA: short chain fatty acid

siRNA's: short-interfering RNA's

STAT: signal transducers and activators of transcription

TAB2: TAK-1 binding protein-2

TLRs: Toll-like receptors

RNA's: Transfer RNAs (tRNA's), ribosomal RNAs (rRNA's), micro-RNA's (mi-RNA's

TRAF-6: tumour necrosis factor receptor-associated factor

TSG: tumour-suppressor genes

 

References

1. Petersen PE, Ogawa H. The global burden of periodontal disease: towards integration with chronic disease prevention and control. Periodontology 2000, 2012; 60: 15-39.         [ Links ]

2. Petersen PE, Ogawa H. Strengthening the prevention of periodontal disease: the WHO approach. J Periodontol 2005; 76:2187-93.         [ Links ]

3. Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL Jr. Microbial complexes in subgingival plaque. J Clin Periodontol 1998; 25:134-44.         [ Links ]

4. Michalowicz BS, Diehl SR, Gunsolley JC, Sparks BS, Brooks CN, Koertge TE, et al. Evidence of a substantial genetic basis for risk of adult periodontitis. J Periodontol 2000; 71:1699-707.         [ Links ]

5. Offenbacher S, Barros SP, Beck JD. Rethinking periodontal inflammation. J Periodontol 2008; 79:1577-84.         [ Links ]

6. Goldberg AD, Allis CD, Bernstein E. Epigenetics: a landscape takes shape. Cell 2007; 128:635-8.         [ Links ]

7. Berger SL, Kouzarides T, Sheikhattar r, Shilatifard A. An operational definition of epigenetics. Genes Dev 2009; 23:783.         [ Links ]

8. Waggoner D. Mechanisms of disease: epigenesist. Semin Pediatr Neurol 2007; 14:7-14.         [ Links ]

9. Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 2003; 349:2042-54.         [ Links ]

10. Hermann A, Gowher H, Jeltsch A. Biochemistry and biology of mammalian DNA methyltransferases. Cell Mol Life Sci 2004; 61:2571-87.         [ Links ]

11. Hark AT, Schoenherr CJ, Katz DJ, Ingram RS, Levorse JM, Tilgh-man SM. CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus. Nature 2000; 405:486-9.         [ Links ]

12. Loenen WA. S-adenosylmethionine: jack of all trades and master of everything? Biochem Soc Trans 2006; 34: 330-3.         [ Links ]

13. Vucic EA, Brown CJ, Lam WL. Epigenetics of cancer progression. Pharmacogenomics 2008; 9:215-34.         [ Links ]

14. Bird AP, Wolffe AP. Methylation-induced repression belts, braces, and chromatin. Cell 1999; 99:451-4.         [ Links ]

15. Cheung HH, Lee TL, Rennert OM, Chan WY. DNA methyla-tion of cancer genome. Birth Defects Res C Embryo Today 2009; 87:335-50.         [ Links ]

16. Fuchs J, Demidov D, Houben A, Schubert I. Chromosomal histone modification patterns-from conservation to diversity. Trends Plant Sci 2006; 11:199-208.         [ Links ]

17. Egger G, Liang G, Aparicio A, Jones PA. Epigenetics in human disease and prospects for epigenetic therapy. Nature. 2004 May 27; 429(6990):457-63.         [ Links ]

18. Doolittle JM, Webster-Cyriaque J. Polymicrobial infection and bacterium mediated epigenetic modification of DNA tumor viruses contribute to pathogenesis. 2014,.mBio 5(3):e01015-14.         [ Links ]

19. Campos EI, Reinberg D. Histones: annotating chromatin. Annu Rev Genet 2009; 43:559-99.         [ Links ]

20. Bayarsaihan D. Epigenetic mechanisms in inflammation. J Dent Res 2011; 90:9-17.         [ Links ]

21. Portela A, Esteller M. 2010. Epigenetic modifications and human disease. Nat. Biotechnol. 28:1057-68.         [ Links ]

22. Seo JY, Park YJ, Yi YA, Hwang JY, Lee IB, Cho BH, Son HH, Seo DG. Epigenetics: general characteristics and implications for oral health. Restor Dent Endod. 2015; 40(1):14-22.         [ Links ]

23. Sun Q, Liu H, Chen Z. The fine tuning role of microRNA interaction in odontoblast differentiation and disease. Oral Dis 2014 Mar 22.         [ Links ]

24. Perez P, Jang SI, Alevizos I. Emerging landscape of non-coding RNAs in oral health and disease. Oral Dis 2014; 20:226-35.         [ Links ]

25. Kaikkonen MU, Lam MT, Glass CK. Non-coding RNAs as regulators of gene expression and epigenetics. Cardiovasc Res 2011; 90:430-40.         [ Links ]

26. Iacopino AM. Epigenetics: New explanations for old problems? J Can Dent Assoc 2010; 76:a76.         [ Links ]

27. Bierne H, Hamon M, Cossart P. Epigenetics and bacterial infections. Cold Spring Harb Perspect Med 2012.         [ Links ]

28. Nile CJ, Read RC, Akil M, Duff GW, Wilson AG. Methylation status of a single CpG site in the IL6 promoter is related to IL6 messenger RNA levels and rheumatoid arthritis. Arthritis Rheum 2008; 58:2686-93.         [ Links ]

29. Barros SP and Offenbacher S. Epigenetics: connecting environment and genotype to phenotype and disease. J Dent Res 2009; 88(5):400-8.         [ Links ]

30. Darveau RP. The oral microbial consortium's interaction with the periodontal innate defense system. NA Cell Biol 2009; 28:389-95.         [ Links ]

31. Yin L, Chung WO. Epigenetic regulation of human beta de-fensin 2 and CC chemokine ligand 20 expression in gingival epithelial cell in response to oral bacteria. Mucosal Immunol 2011; 4:409-19.         [ Links ]

32. Pedra JH, Cassel SI, Sutterwala FS. Sensing pathogens and danger signals by the inflammasome. Curr Opin Immunol 2009; 21:10-6.         [ Links ]

33. Manavalan B, Basith S, Choi S. Similar structures but different roles-An updated perspective on TLR structures. Front Physiol 2011; 2:41.         [ Links ]

34. Philbin VI, Levy O. Developmental biology of the innate immune response: implications for neonatal and infant vaccine development. Pediatr Res 2009; 65:98R-105R.         [ Links ]

35. Winder DM, Pett MR, Foster N,Shivji MK, Herdman MT, Stanley MA, Venkitaraman AR, Coleman N.2007. An increase in DNAdouble-strand breaks, induced by Ku70 depletion, is associated with human papilloma virus 16 episome loss and de novo viral integration events. J. Pathol. 213: 27-34.         [ Links ]

36. Foster SL, Hargreaves DC, Medzhitov R. 2007. Gene-specific control of inflammation by TLR-induced chromatin modifications. Nature 447: 972-8.         [ Links ]

37. De Camargo Pereira G, Guimarães GN, Planello AC, Santamaria MP, de Souza AP, Line SR, Marques MR, Guimarães GN. 2013. Porphyromonasgingivalis LPS stimulation downregulates DNMT1, DNMT3a, and JMJD3 gene expression levels in human HaCaT keratinocytes. Clin. Oral Investig. 17:1279-85.         [ Links ]

38. Riggs MG, Whittaker RG, Neumann JR, Ingram VM. N-Butyrate causes histone modification in HeLa and Friend erythroleukemia cells. Nature 1977; 268:462-4.         [ Links ]

39. Sealy L, Chalkley R. The effect of sodium butyrate on histone modification. Cell 1978; 14:115-21.         [ Links ]

40. Takahashi N, Sato T, Yamada T. 2000. Metabolic pathways for cytotoxic end product formation from glutamate- and aspartate-containing peptides by Porphyromonas gingivalis. J. Bacteriol. 182:4704-10.         [ Links ]

41. Kepler GM, Nguyen HK, Webster-Cyriaque J, Banks HT. 2007. A dynamic model for induced reactivation of latent virus. J. Theor. Biol. 244:451-62.         [ Links ]

42. Morris TL, Arnold RR, Webster-Cyriaque J.. Signaling cascades triggered by bacterial metabolic end products during reactivation of Kaposi's sarcoma-associated herpesvirus. J. Virol. 2007; 81:6032-42.         [ Links ]

43. Imai K, Inoue H, Tamura M, Cueno ME, Inoue H, Takeichi O, Kusama K, Saito I, Ochiai K. 2012. The periodontal pathogen Porphyromonas gingivalis induces the Epstein-Barr virus lytic switch transactivator ZEBRA by histone modification. Bio-chimie 94:839-46.         [ Links ]

44. Imai K, Yamada K, Tamura M, Ochiai K, Okamoto T.. Reactivation of latent HIV-1 by a wide variety of butyric acid-producing bacteria. Cell. Mol. Life Sci. 2012; 69:2583-92.         [ Links ]

45. Kantor B, Ma H, Webster-Cyriaque J, Monahan PE, Kafri T. Epigenetic activation of unintegrated HIV-1 genomes by gut-associated short chain fatty acids and its implications for HIV infection. Proc. Natl. Acad. Sci. U. S. A. 2009;106:18786-91.         [ Links ]

46. Imai K, Ochiai K, Okamoto T. Microbial interaction between HIV-1 and anaerobic bacteria producing butyric acid: its potential implication in AIDS progression. Future Virol.7: 2012; 1005-14.         [ Links ]

47. Imai K, Victoriano AF, Ochiai K, Okamoto T. Microbial interaction of periodontopathic bacterium Porphyromonas gingivalis and HIV possible causal link of periodontal diseases to AIDS progression-. Curr. HIV Res. 2012.;10:238-44.         [ Links ]

48. Janaina de Cássia Orlandi Sardi. Oxidative stress in diabetes and periodontitis. N Am J Med Sci. 2013 Jan; 5(1): 58-9.         [ Links ]

49. Sawamoto Y, Sugano N, Tanaka H, Ito K.. Detection of periodontopathic bacteria and an oxidative stress marker in saliva from periodontitis patients. Oral Microbiol. Immunol. 2005; 20:216-20.         [ Links ]

50. Licciardi PV, Wong SS, Tang ML, Karagiannis TC.. Epigenome targeting by probiotic metabolites. Gut Pathog. 2010; 2:24.         [ Links ]

51. Yin L, Sanson B, An J, Hacker BM, Silverman GA, Dale BA, et al. Differential effects of peripopathogens on host protease inhibitors SLP1, elafin, SCCA1, SCCA 2. J Oral Microbiol 2010; 2:1-12.         [ Links ]

52. Chung WO, An JY, Yin L, Hacker BM, Rohani MG, Dommisch H, et al. Interplay of protease-activated receptors and NOD pattern recognition receptors in epithelial innate immune responses to bacteria. Immunol Lett 2010; 131:113-9.         [ Links ]

53. Uehara O, Abiko Y, Saitoh M, Miyakawa H, Nakazawa F. Lipopolysaccharide extracted from Porphyromonas gingivalis induces DNA hypermethylation of runt-related transcription factor 2 in human periodontal ligament fibroblasts. Journal of Micrbiology, Immunology and Infection 2014; 47:176-81.         [ Links ]

54. Bobetsis YA, Barros SP, Lin DM, Weidman JR, Dolinoy DC, Jittle RL, et al. Bacterial infection promotes DNA hypermethylation. J Dent Res 2007; 86:169-74.         [ Links ]

55. Offenbacher S, Lieff S, Boggess KA, Murtha AP, Madianos PN, Champagne CM, McKaig RG, Jared HL, Mauriello SM, Auten RL Jr, Herbert WN, Beck JD. Maternal periodontitis and prematurity. Part I: Obstetric outcome of prematurity and growth restriction. Ann Periodontol. 2001; 6(1):164-74.         [ Links ]

56. Miao D, Godovikova V, Qian X, Seshadrinathan S, Kapila YL, Fenno JC. Treponema denticola upregulates MMP-2 activation in periodontal ligament cells: interplay between epigenetics and periodontal infection. Arch Oral Biol. 2014; 59(10):1056-64.         [ Links ]

57. Wu H, Lippmann JE, Oza JP, Zeng M, Fives-Taylor P, Reich NO. Inactivation of DNA adenine methyltransferase alters virulence factors in Actinobacillus actinomycetemcomitans. Oral Microbiol Immunol. 2006; 21:238-44.         [ Links ]

58. Loe H, Anerud A, Boysen H. The natural history of periodontal disease in man: prevalence, severity, and extent of gingival recession. J Periodontol. 1992; 63:489-95.         [ Links ]

59. Slots J. Human viruses in periodontitis. Periodontol 2000. 2010; 53:89-110.         [ Links ]

60. Slots J. Herpesviruses in periodontal diseases. Periodontol. 2000. 2005; 38:33-62.         [ Links ]

61. Kubar A, Saygun I, Ozdemir A, Yapar M, Slots J. Real-time polymerase chain reaction quantification of human cytome-galovirus and Epstein-Barr virus in periodontal pockets and the adjacent gingiva of periodontitis lesions. J. Periodontal Res. 2005; 40:97-104.         [ Links ]

62. Li H, Chen V, Chen Y, Baumgartner JC, Machida CA. Herpes-viruses in endodontic pathoses: association of Epstein-Barr virus with irreversible pulpitis and apical periodontitis. J. Endod. 2009; 35:23-9.         [ Links ]

63. Sabeti M, Valles Y, Nowzari H, Simon JH, Kermani-Arab V, Slots J. Cytomegalovirus and Epstein-Barr virus DNA transcription in endodontic symptomatic lesions. Oral Microbiol. Immunol. 2003; 18:104-8.         [ Links ]

64. Slots J, Kamma JJ, Sugar C. The herpes-virus-Porphyromonas gingivalis-periodontitis axis. J. Periodontal Res. 2003; 38:318-23.         [ Links ]

65. Saygun I, Kubar A, Ozdemir A, Yapar M, Slots J. Herpes viral bacterial interrelationships in aggressive periodontitis. J. Periodontal Res. 2004; 39:207-12.         [ Links ]

66. Slots J. Herpes-virus periodontitis: infection beyond biofilm. J Calif Dent Assoc. 2011 Jun; 39(6):393-9.         [ Links ]

67. Kinane DF, Hart TC. Genes and gene polymorphisms associated with periodontal disease. Crit Rev Oral Biol Med. 2003; 14(6): 430-49.         [ Links ]

68. Shapira L, Wilensky A, Kinane DF. Effect of genetic variability on the inflammatory response to periodontal infection. J Clin Periodontol. 2005; 32:72-86.         [ Links ]

69. Moreira PR, Lima PMA, Sathler KOB, Imanishi SA, Costa JE, Gomes RS, et al. Interleukin-6 expression and gene polymorphism are associated with severity of periodontal disease in a sample of Brazilian individuals. Clin Exp Immunol. 2007; 148:119-26.         [ Links ]

70. Mi X, Zeng F. Hypomethylation of interleukin-4 and 6 promoters in T cells from systemic lupus erythematosus. Acta Pharmacol Sin. 2008; 29:105-12.         [ Links ]

71. Gomez RS, Dutra Wo, Moreira PR. Epigenetics and periodontal disease: future perspectives. Inflamm Res. 2009; 58:625-9.         [ Links ]

72. Costa PP, Trevisan GL, Macedo GO, Palioto DB, Souza SL, Grisi MF, et al. Salivary interleukin-6, matrix metalloproteinase-8, and osteoprotegerin in patients with periodontitis and diabetes. J Periodontol. 2010; 81(3):384-91.         [ Links ]

73. Babel N, Cherepnev G, Babel D, Tropmann A, Hammer M, Volk HD, et al. Analysis of tumour necrosis factor-alpha, transforming growth factor-beta, interleukin-10, IL-6, and in-terferon-gamma gene polymorphisms in patients with chronic periodontitis. J Periodontol 2006; 77:1978-83.         [ Links ]

74. Hodge DR, Xiao W, Clausen PA, Heidecker G, Szyf M, Farrar WL. Interleukin-6 regulation of the human DNA methyltransferase (HDNMT) gene in human erythroleukemia cells. J Biol Chem. 2001; 276:39508-11.         [ Links ]

75. Stenvinkel P, Karimi M, Johansson S, Axelsson J, Suliman M, Lindholm B, et al. Impact of inflammation on epigenetic DNA methylation: a novel risk factor for cardiovascular disease? J Intern Med. 2007; 261:488-99.         [ Links ]

76. Hodge DR, Peng B, Cherry JC, Hurt EM, Fox SD, Kelley JA, et al. Interleukin 6 supports the maintenance of p53 tumour suppressor gene promotor methylation. Cancer Res. 2005; 65: 4673-82.         [ Links ]

77. Wehbe H, Henson R, Meng F, Mize-Berge J, Patel T. Inter-leukin-6 contributes to growth in cholangiocarcinoma cells by aberrant promoter methylation and gene expression. Cancer Res. 2006; 66:10517-24.         [ Links ]

78. Hmadcha A, Bedoya FJ, Sobrino F, Pintado E. Methylation-dependent gene silencing induced by interleukin 1 beta via nitric oxide production. J Exp Med. 1999; 190:1595-603.         [ Links ]

79. Oliveira NF, Damm GR, Andia DC, Salmon C, Nociti FH Jr, Line SR, et al. DNA methylation status of the IL8 gene promoter in oral cells of smokers and non-smokers with chronic periodontitis. J Clin Periodontol 2009; 36:719-25.         [ Links ]

80. Zhang S, Barros SP, Niculescu MD, Moretti AJ, Preisser JS, Offenbacher S. Alteration of PTGS2 promoter methylation in chronic periodontitis. J Dent Res 2010; 89(2):133-7.         [ Links ]

81. Andia DC, de Oliveira NF, Casarin RC, Casati MZ, Line SR, de Souza AP. DNA methylation status of the IL8 gene promoter in aggressive periodontitis. J Periodontol 2010; 81(9):1336-41.         [ Links ]

82. Zhang S, Crivello A, Offenbacher S, Moretti A, Paquette DW, Barros SP. Interferon-gamma promoter hypomethylation and increased expression in chronic periodontitis. J Clin Period-ontol 2010; 37(11):953-61.         [ Links ]

83. White GP, Watt PM, Holt BJ, Holt PG. Differential patterns of methylation of the IFN-gamma promoter at CpG and non-CpG sites underlie differences in IFN-gamma gene expression between human neonatal and adult CD45RO-T cells. J Immunol. 2002; 168:2820-7.         [ Links ]

84. Sullivan KE, Reddy ABM, Dietzmann K, Suriano AR, Kocieda VP, Stewart M. et al. Epigenetic regulation of tumour necrosis alpha. Mol Cell Biol. 2007; 27:5147-60.         [ Links ]

85. Zhang S, Barros SP, Moretti AJ, Yu N, Zhou J, Preisser JS, et al. Epigenetic regulation of TNFA expression in periodontal disease. J Periodontol 2013 Epub Jan 31.         [ Links ]

86. Ana Paula De Souza, Aline Cristiane Planello, Marcelo Rocha Marques, Daniel Diniz De Carvalho, Sergio Roberto Peres Line et al. High-throughput DNA analysis shows the importance of methylation in the control of immune inflammatory gene transcription in chronic periodontitis. Clinical Epigenetics 2014; 6:15.         [ Links ]

87. Gemmell E, Yamazaki K, Seymour GJ. Destructive periodontitis lesions are determined by the nature of the lymphocytic response. Crit Rev Oral Biol Med 2002; 13:17-34.         [ Links ]

88. Wilson CB, Rowell E, Sekimata M. Epigenetic control of T-helper cell differentiation. Nat Rev Immunol 2009; 9:91-105.         [ Links ]

89. Cantley MD, Bartold PM, Marino V, Fairlie DP, Le GT, Lucke AJ, et al. Histone deacetylase inhibitors and periodontal bone loss. J Periodontal Res 2011; 46:697-703.         [ Links ]

90. Xie YF, Shu R, Jiang SY, Liu DL, Zhang XL. Comparison of microRNA profiles of human periodontal diseased and healthy gingival tissues. Int J Oral Sci 2011; 3:125-34.         [ Links ]

91. Lee YH, Hee SN, Jeong SY, Jeong SH, Park HR, Chung J. Comparison of inflammatory microRNA expression in healthy and periodontitis tissues. Biocell 2011; 35:42-9.         [ Links ]

92. Stoecklin-Wasmer C, Guarnieri P, Celenti R, Demmer RT, Kebschull M, Papapanou PN. MicroRNAs and their target genes in gingival tissues. J Dent Res 2012; 91:934-40.         [ Links ]

93. Ogata Y, Matsui S, Kato A, Zhou L, Nakayama Y, and Takai H. Original MicroRNA expression in inflamed and non-inflamed gingival tissues from Japanese patients. Journal of Oral Science 2014; 56, (4):253-60.         [ Links ]

94. Nahid MA, Rivera M, Lucas A, Chan EK, Kesavalu L. Polymicro-bial infection with periodontal pathogens specifically enhances microRNA miR-146a in ApoE-/- mice during experimental periodontal disease. Infect Immun 2011; 79:1597-605.         [ Links ]

95. Xie Y, Shu R, Jiang S, Liu D, Ni J, Zhang X. MicroRNA-146 inhibits pro-inflammatory cytokine secretion through IL-1 receptor-associated kinase 1 in human gingival fibroblasts. J Inflammation 2013; 10:20.         [ Links ]

96. Ceppi M, Pereira PM, Dunand-Santhier I, Barras E, Reith W, Santos MA, et al. MicroRNA-155 modulates the interleukin-1 signalling pathway in activated human monocyte-derived dendritic cells. Proc Natl Acad Sci USA 2009; 106:2735-40.         [ Links ]

97. Perri R, Nares S, Zhang S, Barros SP, Offenbacher A. Micro-RNA modulation in obesity and periodontitis. J Dent Res 2012; 91:33-8.         [ Links ]

98. Johnson IT, Belshaw NJ. Environment, diet and CpG island methylation: epigenetic signals in gastrointestinal neoplasia. Food Chem Toxicol. 2008; 46:1346-59.         [ Links ]

99. Vaissie 're T, Sawan C, Herceg Z. Epigenetic interplay between histone modifications and DNA methylation in gene silencing. Mutat Res. 2008; 659:40-8.         [ Links ]

100. Hillemacher T, Frieling H, Moskau S, Muschler MA, Semmler A, Kornhuber J, et al. Global DNA methylation is influenced by smoking behaviour. Eur Neuropsychopharmacol. 2008; 18:295-8.         [ Links ]

101. Kikuchi S, Yamada D, Fukami T, Maruyama T, Ito A, Asamura H, et al. Hypermethylation of the TSLC1/IGSF4 promoter is associated with tobacco smoking and a poor prognosis in primary non-small cell lung carcinoma. Cancer. 2006; 106:1751-8.         [ Links ]

102. an der Velden U, Kuzmanova D, Chapple IL. Micronutritional approaches to periodontal therapy. J Clin Periodontol 2011 Mar; 38 Suppl 11:142-58.         [ Links ]

103. Milward MR, Chapple IL. Dental health: the role of diet in periodontal disease. Dent Health 2013; 52:18-21.         [ Links ]

104. Okano M, Bell DW, Haber DA, Li E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 1999; 99:247-57.         [ Links ]

105. Sanchez-Pernaute O, Ospelt C, Neidhart M, Gay S. Epigenetic clues to rheumatoid arthritis. J Autoimmun. 2008; 30:12-20.         [ Links ]

106. Ohi T, Uehara Y, Takatsu M, Watanabe M, Ono T. Hypermethylation of CpG in the promoter of the COL1A1 gene in the aged periodontal ligament. J Dent Res. 2006; 85:245-50.         [ Links ]

107. Laird PW. The power and promise of DNA methylation markers. Nat Rev Cancer 2003; 3:253-66.         [ Links ]

108. Schroeder TM, Westendorf JJ. Histone deacetylase inhibitors promote osteoblast maturation. J Bone Miner Res 2005; 20:2254-63.         [ Links ]

109. Cantley MD, Bartold PM, Fairlie DP, Rainsford KD, Haynes DR. Histone deacetylase inhibitors as suppressors of bone destruction in inflammatory diseases. J Pharm Pharmacol 2012; 64:763-74.         [ Links ]

110. Clark SJ, Harrison J, Paul CL, Frommer M. High sensitivity mapping of methylated cytosines. Nucleic Acids Res 1994; 22:2990-7.         [ Links ]

111. Ehrlich M, Gama-Sosa MA, Huang LH, et al. Amount and distribution of 5-methylcytosine in human DNA from different types of tissues of cells. Nucleic Acids Res 1982; 10:2709-21.         [ Links ]

112. Shen L and Waterland AR. Methods of DNA methylation analysis. Curr Opin Clin Metab Care 2007; 10:576-81.         [ Links ]

113. Weber M, Davies JJ, Wittig D, et al. Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat Genet 2005; 37:853-62.         [ Links ]

114. Impey S et al. Defining the CREB regulon: a genome wide analysis of transcription factor regulatory regions, Cell 2004; 119:1041-54.         [ Links ]

115. Kim TH and Ren B. Genome-wide analysis of protein-DNA interactions. Annu. Rev. Genomics Hum. Genet. 2006; 7:81-102.         [ Links ]

116. Wei CL et al. A global mapping of p53 transcription factor binding sites in the human genome. Cell 2006; 124:207-19.         [ Links ]

117. Barski A et al. High-resolution profiling of histone methylation in the human genome. Cell 2007; 129:823-37.         [ Links ]

118. Johnson DS et al. Genome-wide mapping of in vivo protein-DNA interactions. Science 2007; 316:1497-1502.         [ Links ]

119. Mardis ER. ChIP-seq:; welcome to the new frontier. Nat. Methods 2007; 4:613-4.         [ Links ]