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Epigenetic characteristics in inflammatory candidate genes in aggressive periodontitis
Human Immunology xxx (2015) xxx–xxx
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Epigenetic characteristics in inflammatory candidate genes in aggressive periodontitis Susanne Schulz a,⇑, Uta Dorothee Immel b, Louise Just c, Hans-Günter Schaller a, Christiane Gläser d, Stefan Reichert a a
University School of Dental Medicine, Department of Operative Dentistry and Periodontology, Martin-Luther University Halle-Wittenberg, Germany Institute of Legal Medicine, Martin-Luther-University Halle-Wittenberg, Germany Clinic for Oral and Maxillofacial Plastic Surgery, Martin-Luther-University Halle-Wittenberg, Germany d Institute of Human Genetics and Medical Biology, Martin-Luther University Halle-Wittenberg, Germany b c
a r t i c l e
i n f o
Article history: Received 23 February 2015 Revised 29 June 2015 Accepted 12 October 2015 Available online xxxx Keywords: Periodontal inflammation Epigenetics CpG methylation Chemokine Cytokine
a b s t r a c t Background: Periodontitis is a chronic inflammatory disease triggered by the host immune response. Epigenetic modifications also affect the immune response. We assessed CpG methylation in 22 inflammatory candidate genes (ATF2, CCL25, CXCL14, CXCL3, CXCL5, CXCL6, FADD, GATA3, IL10RA, IL12A, IL12B, IL13, IL13RA1, IL15, IL17C, IL17RA, IL4R, IL6R, IL6ST, IL7, INHA, and TYK2) with respect to the occurrence of aggressive periodontitis (AgP). Patients and methods: In this study 15 AgP patients (53.3% males, 41.4 ± 10.5 years) and 10 controls (40.0% males, 36.9 ± 17.5 years) were included. The methylation patterns of gingival biopsies were quantified using EpiTectÒ Methyl Signature PCR Array Human Inflammatory Response. Results: In gingival biopsies taken from patients with AgP, CpG methylation of CCL25 (1.73% vs. 2.59%, p = 0.015) and IL17C (6.89% vs. 19.27%, p = 0.002) was significantly reduced as compared with periodontally healthy tissues. Discussion: We showed for the first time a differential methylation pattern for CCL25 and IL17C in periodontitis. CCL25 plays an important role in T-cell development, whereas IL17C regulates innate epithelial immune responses. The decrease in CpG methylation is presumably accompanied by an increase in gene expression. This could lead to a greater availability of CCL25 and interleukin 17C and support periodontal loss of attachment. Ó 2015 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.
1. Introduction Periodontitis is an infectious disease characterized by inflammation and destruction of tooth-supporting tissue [1]. A variety of extrinsic and intrinsic factors contribute to the manifestation and progression of the disease [2]. An individual’s ability to respond to a bacterial challenge plays a crucial role in disease progression. It is widely accepted that some individuals are more susceptible to periodontal disease than others. The same is obviously true for an individual’s response to medication. In determining susceptibility to periodontal inflammation intrinsic factors, in particular, need to be taken into consideration. In the last few decades, research focused on associating genetic variations with the ⇑ Corresponding author at: University School of Dental Medicine, Department of Operative Dentistry and Periodontology, Harz 42a, D-06097 Halle, Germany. E-mail address: [email protected] (S. Schulz).
etiology of periodontitis. Genetic variations may alter gene expression, which possibly influences an individual’s response to microbial load. A variety of case-control studies and genome-wide association studies have been performed in order to analyze the genetic influence on periodontitis [3–7]. An individual’s immune response is not only influenced by genetic characteristics, but there is a further level of gene regulation. Gene expression is also triggered by epigenetic modifications not based on an altered DNA sequence [8]. Such epigenetic modifications include distinct methylation of DNA or chemical alterations of DNA-associated proteins, the histones, and the nucleosomes [9]. The best investigated epigenetic modification is DNA methylation occurring at CpG islands, which are mostly located in the promoter regions of genes. Specific epigenetic alterations have been linked to the development of a variety of different diseases, such as cancer and also inflammatory diseases [10]. Initial studies revealed an epigenetic contribution to periodontitis, too
http://dx.doi.org/10.1016/j.humimm.2015.10.007 0198-8859/Ó 2015 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.
Please cite this article in press as: S. Schulz et al., Epigenetic characteristics in inflammatory candidate genes in aggressive periodontitis, Hum. Immunol. (2015), http://dx.doi.org/10.1016/j.humimm.2015.10.007
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[11]. Different methylation patterns were shown in TNF-a, Ecadherin, TLR2, and COX-2 [12–15] whereas for IFN-c and Il-6 no periodontitis-associated changes in methylation status were detected [16,17]. In these studies, mostly one gene was considered (only one study investigated two loci). Up to now, only one study was conducted as a genome wide study to compare a pooled sample of inflamed tissue (chronic periodontitis) with one pooled sample of healthy tissue [18]. Changes in the CpG methylation pattern and associated alterations in gene expression might account for the differences in an individual’s susceptibility to periodontal disease [19]. Knowledge of further markers of periodontitis, including epigenetic characteristics, would support the development of an individualized periodontal therapy and preventional regimens [19]. The present study was conducted in order to detect differentially methylated genes involved in immune response between patients with aggressive periodontitis and controls. We screened the promoter methylation status of a variety of genes, including chemokines, cytokines, inflammatory response, and autoimmunity genes (ATF2, CCL25, CXCL14, CXCL3, CXCL5, CXCL6, FADD, GATA3, IL10RA, IL12A, IL12B, IL13, IL13RA1, IL15, IL17C, IL15RA, IL4R, IL6R, IL6ST, IL7, INHA, TYK2) with respect to periodontal inflammation. Most of these genes have already been implicated in the etiology of periodontitis but up to now no investigations concerning epigenetic modifications of these genes in periodontal disease have been carried out. Here, we compared the methylation patterns in gingival biopsies of diseased sites (CAL P 6 mm) from patients suffering from AgP with sites without attachment loss (CAL 6 3 mm) taken from patients who had no periodontitis or only a mild chronically localized periodontitis in order to assess novel disease associated markers.
of the disease and episodes of acute gingivitis, abscess formation, tooth loosening or dental loss caused by the tooth loosening, and many unsuccessful attempts to heal the disease. Conversely to chronic periodontitis (ChP), the severity of periodontal tissue destruction was inconsistent with the amount of microbial deposits. In the radiographs angular bony defects were often visible. During periodontal surgery gingival biopsies were obtained during flap procedures at sites with CAL P 6 mm. Control individuals had no or only mild localized periodontitis. The percentage of sites with CAL P 4 mm of each control was 630%. The teeth from which gingival biopsies were taken did not have CAL values >3 mm. Gingival biopsies from control individuals were obtained, for example, while apically repositioning a flap to extend a clinical crown before restorative therapy or during surgical removal of wisdom teeth at adjacent teeth with CAL 6 3 mm. In general, we excluded persons who were pregnant or were nursing mothers, had a druginduced gingival hyperplasia, or had taken antibiotics in the last 6 months. Moreover, persons who chronically used antiinflammatory drugs or had a history of inflammatory diseases of the oral cavity (including herpes simplex infections) or diseases associated with periodontitis were excluded. The clinical assessment included determining the plaque index (PI), gingival index (GI), clinical probing depth (PDmm), and clinical attachment loss (CALmm) [20,21]. All parameters were assessed at the biopsy site. All participants gave their written consent to participate in this study. The study was approved by the ethics committee of the Medical School of the Martin-Luther University Halle. The investigations were carried out in accordance with the ethical guidelines of the ‘‘Declaration of Helsinki” and its amendment in ‘‘Tokyo and Venice” [22].
2. Material and methods 2.2. Epigenetic studies 2.1. Study population and clinical investigations In all, 25 unrelated persons of the same Caucasian origin from Central Germany were involved in our pilot study. The patient group (n = 15) comprised AgP patients. The control group included 10 participants without or with mild periodontitis. The demographic data are given in Table 1. The study was performed at the Department of Operative Dentistry and Periodontology of the Martin-Luther University Halle-Wittenberg. Gingival biopsies were obtained from all participants and immediately frozen in liquid nitrogen. All patients and controls were assessed according to the classification system of periodontal diseases [1]. In particular, patients with generalized aggressive periodontitis were only included when there was evidence (from dental history and/or radiographs) that the onset of the disease occurred before the age of 35. The patients showed a clinical attachment loss of 4 mm or more in at least 30% of the teeth. The patients frequently reported a rapid progression
For epigenetic investigations, gingival biopsies were taken from each participant. Genomic DNA was prepared by using a QIAampÒ DNA Micro extraction kit (Qiagen, Hilden, Germany) according to the manufacturer’s manual. The epigenetic methylation pattern of each individual separately was assessed by using EpiTectÒ Methyl DNA Enzyme Kit (Qiagen, Hilden Germany) for the first step. The analyzed genes comprised chemokines, cytokines, cytokine receptors, and associated proteins as well as other inflammatory response and autoimmunity genes (Table 2). Four cleavage reactions (without enzyme, methylation-sensitive enzyme, methylation-dependent enzyme, and both enzymes) were carried out for each sample. For the restriction analyses, the MastercyclerÒ gradient (Eppendorf, Hamburg, Germany) was used (conditions: 37 °C for 6 h, 65 °C for 20 min, 4 °C hold). For assessing the CpG methylation pattern the EpiTectÒ Methyl II Signature PCR Array Human Inflammatory response (catalogue number: EAHS-521ZA-24, Qiagen, Hilden,
Table 1 Demographic and clinical periodontal characteristics.
Demographic data Age, years (mean ± SD) Male gender (%) Periodontal data Plaque index at site of biopsies (median, 25th/75th percentiles) Gingiva index at site of biopsies (mean ± SD) Clinical probing depth at site of biopsies (PD in mm mean ± SD) Clinical attachment loss at site of biopsies (CAL in mm mean ± SD) * ** ***
Patients with AgP n = 15
Controls n = 10
p-value
41.4 ± 10.5 53.3
36.9 ± 17.5 40.0
0.428* 0.806**
1.0 (0/1.0) 1.33 ± 0.7 7.20 ± 1.6 8.60 ± 2.7
1.0 (0/1.0) 0.50 ± 0.5 2.35 ± 0.67 2.35 ± 0.67
0.482*** 0.006* <0.001* <0.001*
Student’s t-test. Yates correction. Mann–Whitney-U test.
Please cite this article in press as: S. Schulz et al., Epigenetic characteristics in inflammatory candidate genes in aggressive periodontitis, Hum. Immunol. (2015), http://dx.doi.org/10.1016/j.humimm.2015.10.007
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Table 2 Analyzed genes, including gene symbols and function. Gene description
Gene symbol
Gene function
Activating transcription factor 2 Chemokine (C–C motif) ligand 25 Chemokine (C–X–C motif) ligand 14 Chemokine (C–X–C motif) ligand 3 Chemokine (C–X–C motif) ligand 5 Chemokine (C–X–C motif) ligand 6 Fas-associated via death domain GATA binding protein 3 Interleukin 10 receptor a
ATF2 CCL25 CXCL14 CXCL3 CXCL5 CXCL6 FADD GATA3 IL10RA
Interleukin 12A (natural killer cell stimulatory factor 1) Interleukin 12B (natural killer cell stimulatory factor 2) Interleukin 13 Interleukin 13 receptor a1 Interleukin 15 Interleukin 17C Interleukin 17 receptor A Interleukin 4 receptor Interleukin 6 receptor Interleukin 6 signal transducer (gp130) Interleukin 7
IL12A
Transcription factor, histone acetyltransferase Thymus expressed cytokine, chemotactic for thymocytes, macrophages, dentritic cells Breast and kidney expressed cytokine, chemotactic for monocytes, dentritic cells, NK cells Controls migration and adhesion of monocytes Epithelial-derived neutrophil-activating peptide, chemotactic for neutrophils Granulocyte chemotactic protein, 2chemoattractant for neutrophilic granulocytes Adaptor protein, involved in formation of the death-inducing signaling complex (DISC) during apoptosis T-cell-specific transcription factor involved in epithelial cell differentiation Subunit of IL10R, mediates immunosuppressive signal of IL10, inhibits synthesis of proinflammatory cytokines Subunit of IL12, influences T and natural killer cells, involved in Th1 and Th2 cell differentiation
Inhibin-a Tyrosin kinase 2
INHA TYK2
IL12B IL13 IL13RA1 IL15 IL17C IL17RA IL4R IL6R IL6ST IL7
Mediator of allergic inflammation Subunit of IL13R and IL4R, binds TYK2 Regulator of T cells and natural killer cells T cell-derived cytokine, involved in proinflammatory Th17 response Receptor for IL17A, involved in inflammatory and autoimmune diseases Receptor for IL13 and IL4, located on macrophages, implicated in regulation of inflammatory mediators Receptor for IL6, implicated in cell growth, differentiation and immune response Transmembrane protein, subunit of type I cytokine receptor within IL6 receptor family hematopoietic growth factor, stimulates differentiation into lymphoid progenitor cells, important for B and T cell development Regulates gonadal stromal cell proliferation Associated with type I and type II cytokine receptors and mediates phosphorylation
Germany) was applied. Real-time PCR was carried out using SYBR-green (RT2 SYBR Green ROX qPCR Mastermix, Qiagen, Hilden, Germany) in the 7500 Real-Time PCR system (Applied Biosystems, Darmstadt, Germany). The thermal cycler was programmed according to the manufacturer’s instructions, using the PCR cycling protocol (1 cycle: 95 °C for 10 min, hot start for activation of DNA polymerase; 3 cycles: 99 °C for 30 s, 72 °C for 1 min; 40 cycles: 97 °C for 15 s, 72 °C for 1 min, detection and recording SYBR Green fluorescence from each well during the annealing step of each cycle, melting curve according to instrument recommendations). The gene methylation status is provided as the percentages of unmethylated and methylated fractions of input DNA. Data were assessed by using a DCT method and evaluating the percentages with the software released by Qiagen Version 2.0, 02/03/2012. Each array includes specific control assays for monitoring the cutting efficiencies of methylation-sensitive and methylationdependent enzymes and ensuring reliable and reproducible results. 2.3. Statistical evaluation Statistical analyses were performed using the program SPSS 19.0 (SPSS Inc., Chicago, USA). P-values of 60.05 were considered significant. Categorical variables were plotted in contingency tables and evaluated by Chi-squared analysis and Yates continuity correction. If n < 5, Fisher’s exact test was performed. Metric parameters were analyzed using the Kolmogorov–Smirnov test (test of normal distribution). For statistical evaluation, Student’s t-test (normally distributed values) and Mann–Whitney U-test (values not distributed normally) were employed. The Holm–Bonferroni correction was applied for multiple testing.
3. Results 3.1. Clinical evaluation of the study groups All study participants were assessed according to demographic and periodontal parameters (Table 1). When comparing the patient
group with controls, no statistically significant differences in gender (p = 0.806) and age (p = 0.428) could be detected. In comparison to the controls, the clinical parameters of periodontitis such as GI (p = 0.006), PDmm (p < 0.001), and CALmm (p < 0.001) were significantly elevated in the patient group. With respect to PI no statistically significant difference (p = 0.482) could be detected. 3.2. Epigenetic methylation pattern associated with periodontal diagnosis The status of CpG methylation obtained using ‘‘EpiTectÒ Methyl II Signature PCR Array Human Inflammatory response” is presented in Fig. 1. Overall, the percentage of promoter methylation of inflammatory candidate genes was obviously low in gingival biopsies regardless of the periodontal diagnosis. Only IL13RA1 (% CpG methylation, mean ± SD: 18.1 ± 22.2) and IL17C (% CpG methylation, mean ± SD: 16.7 ± 19.5) showed a total methylation rate of over 10%. Investigating the CpG methylation associated with the periodontal diagnosis of the participants, significant differences were found. Controls showed a significantly higher percentage of methylation of both the chemokine (C–C motif) ligand 25 (CCL25, 1.5fold, p = 0.015) and interleukin 17C (IL17C, 2.8fold, p = 0.002). 4. Discussion The manifestation and progression of periodontitis is characterized by subgingival bacterial infection and an individual’s immune response. Exogenous and endogenous risk markers have been approved to modulate disease initiation and development. Furthermore, epigenetic characteristics, e.g., CpG methylation, have been shown to have an impact on the etiology of inflammatory diseases, including periodontitis [11,23,24]. However, up to now, only a few studies were undertaken to examine the influence of epigenetic modifications on promoter methylation in periodontal disease [12–17]. Since it could be shown that epigenetic changes are tissue specific, epigenetic
Please cite this article in press as: S. Schulz et al., Epigenetic characteristics in inflammatory candidate genes in aggressive periodontitis, Hum. Immunol. (2015), http://dx.doi.org/10.1016/j.humimm.2015.10.007
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Fig. 1. Percentage of CpG methylation in inflammatory candidate genes in dependence of periodontal status.
patterns in blood might be different from those in gingival tissue [9]. Therefore, the preferred approach for investigating the impact of epigenetics in periodontal disease should be to assess gingival tissue [12,15–17,23]. Up to now, no complex approach has been undertaken that took into consideration the multitude of different pathways involved in an individual’s immune response to periodontopathogens. In the present clinical study, the methylation status of a selected panel of important inflammatory genes associated with periodontal diagnosis was investigated for the first time (EpiTectÒ Methyl II Signature PCR Array Human Inflammatory response (Qiagen, Hilden, Germany). These genes comprised chemokines (CCL25, CXCL14, CXCL3, CXCL5, CXCL6), cytokines (IL12A, IL12B, IL17C, INHA), cytokine receptors and associated proteins (IL10RA, IL12B, IL13RA1, IL15, IL17RA, IL4R, IL6R, IL6ST), as well as other genes involved in inflammatory response and autoimmunity (AFT2, FADD, GATA3, IL13, IL7, TYK2). For most of the genes an impact on the etiology of periodontitis has been reported. We assessed differences in CpG methylation pattern corresponding to periodontal status for CCL25 and IL17C. Both genes exhibited a significant decrease in promoter methylation in gingival tissue of AgP patients in contrast to periodontal tissues obtained from individuals without or only mild periodontitis. Because in test group only sites with CAL P 6 mm and among the controls sites with CAL 6 3 mm were chosen a sufficient contrast between periodontal severe diseased and no pathologically CAL was reached. However, the decrease in CCL25 methylation (1.5fold) was less pronounced as for IL17C (2.8fold). Both changes were in the range of methylation differences found in genome wide epigenetic study by Barros and Offenbacher (2.19fold to 1.9fold) [18]. CCL25 and IL17C have been implicated in the pathways important for host reactions to bacterial infections. Both cytokines play an important role in Th17 cell mediated immune response. CCL25 is able to induce Th17 pathway, whereas IL17 is secreted by Th17 cells and promote the expression of further inflammatory mediators [25]. The chemokine CCL25 is selectively expressed in the thymus, the intestinal epithelium as well as the oral mucosa [26–28]. It plays a key role in synchronizing the inflammatory response as it is involved in leukocyte trafficking and T-cell development [29]. An induction of CCL25 gene expression due to Porphyromonas gingivalis infection and co-stimulation with its lipopolysaccharide was
shown in vitro [30]. The increase in CCL25 gene expression in gingival tissue might result in enhanced leukocyte recruitment, including polymorphonuclear leukocytes and monocytes to site of inflammation [30]. Taken our results into account the upregulation of CCL25 might be at least partially due to epigenetic changes in promoter methylation associated with gingival inflammation. IL17C belongs to the interleukin 17 family, which comprises T cell-derived cytokines involved in the initiation or maintenance of the proinflammatory response [31,32]. IL17C is highly expressed in inflammatory conditions [32]. However, a lot of studies are conducted to investigate the biological role of IL17 only little is known about IL17C. IL17 are shown to induce a variety of proinflammatory cytokines. Beklen et al. demonstrated that IL17 significantly increase Il1b and TNFa expression in human macrophages in vitro [33]. Furthermore, IL17 has been linked to the formation of bone-resorbing osteoclasts [34]. Periodontitis associated bone destruction is also mediated by osteoclasts and may therefore be influenced by IL17 expression, too [35]. Actually, IL17 was assumed to be involved in periodontitis-associated bone resorption by influencing RANKL and osteoprotegerin expression [36]. LPS of P. gingivalis enhanced IL17 expression in peripheral mononuclear cells in vitro [37]. Indeed, IL17 expression was shown to be upregulated in the gingival tissue and gingival crevicular fluid of periodontitis patients as compared with healthy tissue [33,38– 39]. Based on its crucial role at site of inflammation an upregulation in IL17C in periodontitis lesions is conceivable. The decrease in CpG methylation measured for CCL25 and IL17C could be indicative of an increase in promoter activity, possibly leading to an enhancement in gene expression. Epigenetic characteristics could contribute to differences in expression patterns of inflammatory cytokines in periodontitis. However, epigenetic modifications might be triggered by a variety of different extrinsic and intrinsic factors. In periodontal disease, the actual gingival inflammation and the bacterial load, but also demographic or socioeconomic factors could influence the methylation pattern. 4.1. Limitations of the study The present investigation was performed as a case-control study.
Please cite this article in press as: S. Schulz et al., Epigenetic characteristics in inflammatory candidate genes in aggressive periodontitis, Hum. Immunol. (2015), http://dx.doi.org/10.1016/j.humimm.2015.10.007
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Both epigenetic modifications and periodontal disease are known to be triggered by a variety of different factors. The complex nature of this interaction was not considered in the study. This study was conducted to establish possible bidirectional associations between the epigenetic CpG methylation pattern of selected inflammatory candidate genes and periodontal disease only. Potential false-positive and false-negative associations could not be excluded. The inclusion of only 25 highly selected individuals in this pilot study provides no power for statistically sufficient analyses. However, the advantage of a pilot study is that it helps to saves resources and offers the opportunities to improve the design of a comprehensive large scale study including functional analyses. For the control group [40] development of severe periodontitis at a later time cannot be excluded. Therefore, the epigeneticdependent effect due to this possible selection bias may likely be underestimated. The present data only apply to Caucasians of Central Germany and must therefore be interpreted with caution. It would not be reasonable to extrapolate the results to the general population. 4.2. In conclusion Our results show a difference in epigenetic methylation in inflammatory candidate genes (CCL25 an IL17C) according to periodontal status. This research will help to develop new approaches in periodontal research and gain new insights into the etiology of periodontal disease. Acknowledgements The study was supported by the German Society of Periodontology (DG PARO) and the Martin-Luther University of Halle, Germany, University School of Dental Medicine, Department of Operative Dentistry and Periodontology. The authors declare that they have no conflict of interest. We would like to thank all patients and healthy controls for their cooperation in this study. References [1] G.C. Armitage, Development of a classification system for periodontal diseases and conditions, Ann. Periodontol. 4 (1999) 1–6. [2] R.J. Genco, W.S. Borgnakke, Risk factors for periodontal disease, Periodontology 2000 (62) (2013) 59–94. [3] M.L. Laine, W. Crielaard, B.G. Loos, Genetic susceptibility to periodontitis, Periodontology 2000 (58) (2012) 37–68. [4] J. Zhang, X. Sun, L. Xiao, C. Xie, D. Xuan, G. Luo, Gene polymorphisms and periodontitis, Periodontology 2000 (56) (2011) 102–124. [5] A.S. Schäfer, G.M. Richter, M. Nothnagel, T. Manke, H. Dommisch, G. Jacobs, A. Arlt, P. Rosenstiel, B. Noack, B. Groessner-Schreiber, S. Jepsen, B.G. Loos, S. Schreiber, A genome-wide association study identifies GLT6D1 as a susceptibility locus for periodontitis, Hum. Mol. Genet. 19 (2010) 553–562. [6] K. Divaris, K.L. Monda, K.E. North, A.F. Olshan, L.M. Reynolds, W.C. Hsueh, E.M. Lange, K. Moss, S.P. Barros, R.J. Weyant, Y. Liu, A.B. Newman, J.D. Beck, S. Offenbacher, Exploring the genetic basis of chronic periodontitis: a genomewide association study, Hum. Mol. Genet. 22 (2013) 2312–2324. [7] P. Feng, X. Wang, P.L. Casado, E.C. Küchler, K. Deeley, J. Noel, H. Kimm, J.H. Kim, A.N. Haas, V. Quielato, L.L. Bonato, J.M. Granjeiro, C. Susin, A.R. Vieira, Genome wide association scan for chronic periodontitis implicates novel locus, BMC Oral Health 84 (2014), http://dx.doi.org/10.1186/1472-6831-14-84. [8] A. Bird, DNA methylation patterns and epigenetic memory, Genes Dev. 16 (2002) 6–21. [9] A. Sadakierska-Chudy, R.M. Kostrzewa, M. Filip, A comprehensive view of the epigenetic landscape part I: DNA methylation, passive and active DNA demethylation pathways and histone variants, Neurotox. Res. (2014), http:// dx.doi.org/10.1007/s12640-014-9497-5. [10] M. Ngollo, A. Dagdemir, S. Karsli-Ceppioglu, G. Judes, A. Pajon, F. PenaultLlorca, J.P. Boiteux, Y.J. Bignon, L. Guy, D.J. Bernard-Gallon, Epigenetic modifications in prostate cancer, Epigenomics 6 (2014) 415–425. [11] A.M. Lindroth, Y.J. Park, Epigenetic biomarkers: a step forward for understanding periodontitis, J. Periodontal Implant Sci. 43 (2013) 111–120.
5
[12] S. Zhang, S.P. Barros, A.J. Moretti, N. Yu, J. Zhou, J.S. Preisser, M.D. Niculescu, S. Offenbacher, Epigenetic regulation of TNFA expression in periodontal disease, J. Periodontol. 84 (2013) 1606–1616. [13] S. Zhang, S.P. Barros, M.D. Niculescu, A.J. Moretti, J.S. Preisser, S. Offenbacher, Alteration of PTGS2 promoter methylation in chronic periodontitis, J. Dent. Res. 89 (2010) 133–137. [14] W.T. Loo, L. Jin, M.N. Cheung, M. Wang, L.W. Chow, Epigenetic change in Ecadherin and COX-2 to predict chronic periodontitis, J. Transl. Med. 8 (2010) 100. [15] S.A. De Faria Amormino, T.C. Arao, A.M. Saraiva, R.S. Gomez, W.O. Dutra, J.E. da Costa, J. de Fatima Correia Silva, P.R. Moreira, Hypermathylation and low transcription of TLR2 gene in chronic periodontitis, Hum. Immunol. 74 (2013) 1231–1236. [16] F.A. Stefani, M.B. Viana, A.C. Dupim, J.A. Brito, R.S. Gomez, J.E. da Costa, P.R. Moreira, Expression, polymorphism and methylation pattern of interleukin-6 in periodontal tissue, Immunobiology 128 (2013) 1012–1017. [17] S. Zhang, A. Crivella, S. Offenbacher, A. Moretti, D.W. Paquette, S.P. Barros, Interferon-gamma promoter hypomethylation and increased expression in chronic periodontitis, J. Clin. Periodontol. 37 (2010) 953–961. [18] S.P. Barros, S. Offenbacher, Modifiable risk factors in periodontal disease: epigenetic regulation of gene expression in the inflammatory response, Periodontology 2000 (64) (2013) 95–100. [19] L. Larsson, R.M. Castilho, W.V. Giannobile, Epigenetics and its role in periodontal disease: a state-of-the-art review, J. Periodontol. 86 (2015) 556– 568. [20] H. Loe, J. Silness, Periodontal disease in pregnancy. I. Prevalence and severity, Acta Odontol. Scand. 21 (1963) 533–551. [21] J. Silness, H. Loe, Periodontal disease in pregnancy II. Correlation between oral hygiene and periodontal condition, Acta Odontol. Scand. 22 (1964) 121–135. [22] World medical Association, World Medical Association Declaration of Helsinki, ethical principles for medical research involving human subjects, JAMA 310 (2013) 2191–2194. [23] D. Bayarsaihan, Epigenetic mechanisms in inflammation, J. Dent. Res. 90 (2011) 9–17. [24] S. Lod, T. Johansson, K.H. Abrahamsson, L. Larsson, The influence of epigenetics in relation to oral health, Int. J. Dent. Hyg. 12 (2014) 48–54. [25] M.F.S. Costa, V.U. Bornstein, A.L. Candea, A. Henriques-Pons, M.G. Henriques, C. Penido, CCL25 induces a4b7 integrin dependent migration of IL-17+cd T lymphocytes during an allergic reaction, Eur. J. Immunol. 42 (2012) 1250– 1260. [26] D. Kabelitz, D. Wesch, Features and functions of cdT lymphocytes: focus on chemokines and their receptors, Crit. Rev. Immunol. 23 (2003) 339–370. [27] E.J. Kunkel, D.J. Campbell, E.C. Butcher, Chemokines in lymphocyte trafficking and intestinal immunity, Microcirculation 10 (2003) 313–323. [28] K. Otten, J. Dragoo, H.C. Wang, J.R. Klein, Antigen-induced chemokine activation in mouse buccal epithelium, Biochem. Biophys. Res. Commun. 304 (2003) 36–40. [29] A.P. Vicari, D.J. Figueroa, J.A. Hedrick, J.S. Foster, K.P. Singh, S. Meone, N.G. Copeland, D.J. Gilbert, N.A. Jenkins, K.B. Bacon, A. Zlotnik, TACK: a novel CC chemokine specifically expressed by thymic dentritic cells and potentially involved in T cell development, Immunity 7 (1997) 291–301. [30] S. Ekhlassi, L.Y. Crruggs, T. Garza, D. Montufar-Solis, A.J. Moretti, J.R. Klein, Porphyromonas gingivalis lipopolysaccharide induces tumor necrosis factor-a and interleukin-6 secretion, and CCL25 gene expression, in mouse primary gingival cell lines: interleukin-6-driven activation of CCL2, J. Periodont. Res. 43 (2008) 431–439. [31] H. Li, J. Chen, A. Huang, J. Stinson, S. Heldens, J. Foster, P. Dowd, A.L. Gurney, W. I. Wood, Cloning and characterization of IL-17b and IL-17C, two new members of IL-17 cytokine family, Proc. Natl. Acad. Sci. 97 (2000) 7736–7778. [32] S. Aggarwal, A.L. Gurney, IL-17: prototype member of an emerging cytokine family, J. Leukoc. Biol. 71 (2002) 1–8. [33] A. Beklen, M. Ainola, M. Hukkanen, C. Gurgan, T. Sorsa, Y.T. Konttinen, MMPs, IL-1, and TNF are regulated by IL-17 in periodontitis, J. Dent. Res. 86 (2007) 347–351. [34] Y. Lee, The role of interleukin-17 in bone metabolism and inflammatory skeletal disease, BMB Rep. 46 (2013) 479–483. [35] P.M. Bartold, M.D. Cantley, D.R. Haynes, Mechanisms and control of pathologic bone loss in periodontitis, Periodontology 2000 (53) (2010) 55–69. [36] D. Lin, L. Li, Y. Sun, W. Wang, X. Wang, Y. Ye, X. Chen, Y. Xu, IL-17 regulates the expression of RNAKL and OPG in human periodontal ligament cells via TRAF6/ TBK1-JNK/NF-kB pathways, Immunology (2014), http://dx.doi.org/10.1111/ imm.12395. [37] T. Oda, H. Yoshie, K. Yamazaki, Porphyromonas gingivalis antigen preferentially stimulates T cells to express IL-17 but not receptor activator of NF-kappaB ligand in vitro, Oral Micorbiol. Immunol. 18 (2013) 30–36. [38] A. Mitani, W. Niedbala, T. Fujimura, M. Mogi, S. Miyamae, N. Higuchi, A. Abe, T. Hishikawa, M. Mizutani, Y. Ishihara, H. Nakamura, K. Kurita, N. Ohno, Y. Tanaka, M. Hattori, T. Noguchi, Increased expression of interleukin-35 and -17, but not -27, in gingival tissues with chronic periodontitis, J. Periodontol. (2014), http://dx.doi.org/10.1902/jop.2014.140293. [39] O.G. Shaker, N.A. Ghallab, IL-17 and IL-11 GCF levels in aggressive and chronic periodontitis patients: relation to PCR bacterial detection, Mediators Inflamm. (2012), http://dx.doi.org/10.1155/2012/174764. [40] WHO Scientific Group, Epidemiology, etiology, and prevention of periodontal diseases, World Health Org. Tech. Rep. Ser. 1–60 (1978).
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Epigenetic characteristics in inflammatory candidate genes in aggressive periodontitis Susanne Schulz a,⇑, Uta Dorothee Immel b, Louise Just c, Hans-Günter Schaller a, Christiane Gläser d, Stefan Reichert a a
University School of Dental Medicine, Department of Operative Dentistry and Periodontology, Martin-Luther University Halle-Wittenberg, Germany Institute of Legal Medicine, Martin-Luther-University Halle-Wittenberg, Germany Clinic for Oral and Maxillofacial Plastic Surgery, Martin-Luther-University Halle-Wittenberg, Germany d Institute of Human Genetics and Medical Biology, Martin-Luther University Halle-Wittenberg, Germany b c
a r t i c l e
i n f o
Article history: Received 23 February 2015 Revised 29 June 2015 Accepted 12 October 2015 Available online xxxx Keywords: Periodontal inflammation Epigenetics CpG methylation Chemokine Cytokine
a b s t r a c t Background: Periodontitis is a chronic inflammatory disease triggered by the host immune response. Epigenetic modifications also affect the immune response. We assessed CpG methylation in 22 inflammatory candidate genes (ATF2, CCL25, CXCL14, CXCL3, CXCL5, CXCL6, FADD, GATA3, IL10RA, IL12A, IL12B, IL13, IL13RA1, IL15, IL17C, IL17RA, IL4R, IL6R, IL6ST, IL7, INHA, and TYK2) with respect to the occurrence of aggressive periodontitis (AgP). Patients and methods: In this study 15 AgP patients (53.3% males, 41.4 ± 10.5 years) and 10 controls (40.0% males, 36.9 ± 17.5 years) were included. The methylation patterns of gingival biopsies were quantified using EpiTectÒ Methyl Signature PCR Array Human Inflammatory Response. Results: In gingival biopsies taken from patients with AgP, CpG methylation of CCL25 (1.73% vs. 2.59%, p = 0.015) and IL17C (6.89% vs. 19.27%, p = 0.002) was significantly reduced as compared with periodontally healthy tissues. Discussion: We showed for the first time a differential methylation pattern for CCL25 and IL17C in periodontitis. CCL25 plays an important role in T-cell development, whereas IL17C regulates innate epithelial immune responses. The decrease in CpG methylation is presumably accompanied by an increase in gene expression. This could lead to a greater availability of CCL25 and interleukin 17C and support periodontal loss of attachment. Ó 2015 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.
1. Introduction Periodontitis is an infectious disease characterized by inflammation and destruction of tooth-supporting tissue [1]. A variety of extrinsic and intrinsic factors contribute to the manifestation and progression of the disease [2]. An individual’s ability to respond to a bacterial challenge plays a crucial role in disease progression. It is widely accepted that some individuals are more susceptible to periodontal disease than others. The same is obviously true for an individual’s response to medication. In determining susceptibility to periodontal inflammation intrinsic factors, in particular, need to be taken into consideration. In the last few decades, research focused on associating genetic variations with the ⇑ Corresponding author at: University School of Dental Medicine, Department of Operative Dentistry and Periodontology, Harz 42a, D-06097 Halle, Germany. E-mail address: [email protected] (S. Schulz).
etiology of periodontitis. Genetic variations may alter gene expression, which possibly influences an individual’s response to microbial load. A variety of case-control studies and genome-wide association studies have been performed in order to analyze the genetic influence on periodontitis [3–7]. An individual’s immune response is not only influenced by genetic characteristics, but there is a further level of gene regulation. Gene expression is also triggered by epigenetic modifications not based on an altered DNA sequence [8]. Such epigenetic modifications include distinct methylation of DNA or chemical alterations of DNA-associated proteins, the histones, and the nucleosomes [9]. The best investigated epigenetic modification is DNA methylation occurring at CpG islands, which are mostly located in the promoter regions of genes. Specific epigenetic alterations have been linked to the development of a variety of different diseases, such as cancer and also inflammatory diseases [10]. Initial studies revealed an epigenetic contribution to periodontitis, too
http://dx.doi.org/10.1016/j.humimm.2015.10.007 0198-8859/Ó 2015 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.
Please cite this article in press as: S. Schulz et al., Epigenetic characteristics in inflammatory candidate genes in aggressive periodontitis, Hum. Immunol. (2015), http://dx.doi.org/10.1016/j.humimm.2015.10.007
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[11]. Different methylation patterns were shown in TNF-a, Ecadherin, TLR2, and COX-2 [12–15] whereas for IFN-c and Il-6 no periodontitis-associated changes in methylation status were detected [16,17]. In these studies, mostly one gene was considered (only one study investigated two loci). Up to now, only one study was conducted as a genome wide study to compare a pooled sample of inflamed tissue (chronic periodontitis) with one pooled sample of healthy tissue [18]. Changes in the CpG methylation pattern and associated alterations in gene expression might account for the differences in an individual’s susceptibility to periodontal disease [19]. Knowledge of further markers of periodontitis, including epigenetic characteristics, would support the development of an individualized periodontal therapy and preventional regimens [19]. The present study was conducted in order to detect differentially methylated genes involved in immune response between patients with aggressive periodontitis and controls. We screened the promoter methylation status of a variety of genes, including chemokines, cytokines, inflammatory response, and autoimmunity genes (ATF2, CCL25, CXCL14, CXCL3, CXCL5, CXCL6, FADD, GATA3, IL10RA, IL12A, IL12B, IL13, IL13RA1, IL15, IL17C, IL15RA, IL4R, IL6R, IL6ST, IL7, INHA, TYK2) with respect to periodontal inflammation. Most of these genes have already been implicated in the etiology of periodontitis but up to now no investigations concerning epigenetic modifications of these genes in periodontal disease have been carried out. Here, we compared the methylation patterns in gingival biopsies of diseased sites (CAL P 6 mm) from patients suffering from AgP with sites without attachment loss (CAL 6 3 mm) taken from patients who had no periodontitis or only a mild chronically localized periodontitis in order to assess novel disease associated markers.
of the disease and episodes of acute gingivitis, abscess formation, tooth loosening or dental loss caused by the tooth loosening, and many unsuccessful attempts to heal the disease. Conversely to chronic periodontitis (ChP), the severity of periodontal tissue destruction was inconsistent with the amount of microbial deposits. In the radiographs angular bony defects were often visible. During periodontal surgery gingival biopsies were obtained during flap procedures at sites with CAL P 6 mm. Control individuals had no or only mild localized periodontitis. The percentage of sites with CAL P 4 mm of each control was 630%. The teeth from which gingival biopsies were taken did not have CAL values >3 mm. Gingival biopsies from control individuals were obtained, for example, while apically repositioning a flap to extend a clinical crown before restorative therapy or during surgical removal of wisdom teeth at adjacent teeth with CAL 6 3 mm. In general, we excluded persons who were pregnant or were nursing mothers, had a druginduced gingival hyperplasia, or had taken antibiotics in the last 6 months. Moreover, persons who chronically used antiinflammatory drugs or had a history of inflammatory diseases of the oral cavity (including herpes simplex infections) or diseases associated with periodontitis were excluded. The clinical assessment included determining the plaque index (PI), gingival index (GI), clinical probing depth (PDmm), and clinical attachment loss (CALmm) [20,21]. All parameters were assessed at the biopsy site. All participants gave their written consent to participate in this study. The study was approved by the ethics committee of the Medical School of the Martin-Luther University Halle. The investigations were carried out in accordance with the ethical guidelines of the ‘‘Declaration of Helsinki” and its amendment in ‘‘Tokyo and Venice” [22].
2. Material and methods 2.2. Epigenetic studies 2.1. Study population and clinical investigations In all, 25 unrelated persons of the same Caucasian origin from Central Germany were involved in our pilot study. The patient group (n = 15) comprised AgP patients. The control group included 10 participants without or with mild periodontitis. The demographic data are given in Table 1. The study was performed at the Department of Operative Dentistry and Periodontology of the Martin-Luther University Halle-Wittenberg. Gingival biopsies were obtained from all participants and immediately frozen in liquid nitrogen. All patients and controls were assessed according to the classification system of periodontal diseases [1]. In particular, patients with generalized aggressive periodontitis were only included when there was evidence (from dental history and/or radiographs) that the onset of the disease occurred before the age of 35. The patients showed a clinical attachment loss of 4 mm or more in at least 30% of the teeth. The patients frequently reported a rapid progression
For epigenetic investigations, gingival biopsies were taken from each participant. Genomic DNA was prepared by using a QIAampÒ DNA Micro extraction kit (Qiagen, Hilden, Germany) according to the manufacturer’s manual. The epigenetic methylation pattern of each individual separately was assessed by using EpiTectÒ Methyl DNA Enzyme Kit (Qiagen, Hilden Germany) for the first step. The analyzed genes comprised chemokines, cytokines, cytokine receptors, and associated proteins as well as other inflammatory response and autoimmunity genes (Table 2). Four cleavage reactions (without enzyme, methylation-sensitive enzyme, methylation-dependent enzyme, and both enzymes) were carried out for each sample. For the restriction analyses, the MastercyclerÒ gradient (Eppendorf, Hamburg, Germany) was used (conditions: 37 °C for 6 h, 65 °C for 20 min, 4 °C hold). For assessing the CpG methylation pattern the EpiTectÒ Methyl II Signature PCR Array Human Inflammatory response (catalogue number: EAHS-521ZA-24, Qiagen, Hilden,
Table 1 Demographic and clinical periodontal characteristics.
Demographic data Age, years (mean ± SD) Male gender (%) Periodontal data Plaque index at site of biopsies (median, 25th/75th percentiles) Gingiva index at site of biopsies (mean ± SD) Clinical probing depth at site of biopsies (PD in mm mean ± SD) Clinical attachment loss at site of biopsies (CAL in mm mean ± SD) * ** ***
Patients with AgP n = 15
Controls n = 10
p-value
41.4 ± 10.5 53.3
36.9 ± 17.5 40.0
0.428* 0.806**
1.0 (0/1.0) 1.33 ± 0.7 7.20 ± 1.6 8.60 ± 2.7
1.0 (0/1.0) 0.50 ± 0.5 2.35 ± 0.67 2.35 ± 0.67
0.482*** 0.006* <0.001* <0.001*
Student’s t-test. Yates correction. Mann–Whitney-U test.
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Table 2 Analyzed genes, including gene symbols and function. Gene description
Gene symbol
Gene function
Activating transcription factor 2 Chemokine (C–C motif) ligand 25 Chemokine (C–X–C motif) ligand 14 Chemokine (C–X–C motif) ligand 3 Chemokine (C–X–C motif) ligand 5 Chemokine (C–X–C motif) ligand 6 Fas-associated via death domain GATA binding protein 3 Interleukin 10 receptor a
ATF2 CCL25 CXCL14 CXCL3 CXCL5 CXCL6 FADD GATA3 IL10RA
Interleukin 12A (natural killer cell stimulatory factor 1) Interleukin 12B (natural killer cell stimulatory factor 2) Interleukin 13 Interleukin 13 receptor a1 Interleukin 15 Interleukin 17C Interleukin 17 receptor A Interleukin 4 receptor Interleukin 6 receptor Interleukin 6 signal transducer (gp130) Interleukin 7
IL12A
Transcription factor, histone acetyltransferase Thymus expressed cytokine, chemotactic for thymocytes, macrophages, dentritic cells Breast and kidney expressed cytokine, chemotactic for monocytes, dentritic cells, NK cells Controls migration and adhesion of monocytes Epithelial-derived neutrophil-activating peptide, chemotactic for neutrophils Granulocyte chemotactic protein, 2chemoattractant for neutrophilic granulocytes Adaptor protein, involved in formation of the death-inducing signaling complex (DISC) during apoptosis T-cell-specific transcription factor involved in epithelial cell differentiation Subunit of IL10R, mediates immunosuppressive signal of IL10, inhibits synthesis of proinflammatory cytokines Subunit of IL12, influences T and natural killer cells, involved in Th1 and Th2 cell differentiation
Inhibin-a Tyrosin kinase 2
INHA TYK2
IL12B IL13 IL13RA1 IL15 IL17C IL17RA IL4R IL6R IL6ST IL7
Mediator of allergic inflammation Subunit of IL13R and IL4R, binds TYK2 Regulator of T cells and natural killer cells T cell-derived cytokine, involved in proinflammatory Th17 response Receptor for IL17A, involved in inflammatory and autoimmune diseases Receptor for IL13 and IL4, located on macrophages, implicated in regulation of inflammatory mediators Receptor for IL6, implicated in cell growth, differentiation and immune response Transmembrane protein, subunit of type I cytokine receptor within IL6 receptor family hematopoietic growth factor, stimulates differentiation into lymphoid progenitor cells, important for B and T cell development Regulates gonadal stromal cell proliferation Associated with type I and type II cytokine receptors and mediates phosphorylation
Germany) was applied. Real-time PCR was carried out using SYBR-green (RT2 SYBR Green ROX qPCR Mastermix, Qiagen, Hilden, Germany) in the 7500 Real-Time PCR system (Applied Biosystems, Darmstadt, Germany). The thermal cycler was programmed according to the manufacturer’s instructions, using the PCR cycling protocol (1 cycle: 95 °C for 10 min, hot start for activation of DNA polymerase; 3 cycles: 99 °C for 30 s, 72 °C for 1 min; 40 cycles: 97 °C for 15 s, 72 °C for 1 min, detection and recording SYBR Green fluorescence from each well during the annealing step of each cycle, melting curve according to instrument recommendations). The gene methylation status is provided as the percentages of unmethylated and methylated fractions of input DNA. Data were assessed by using a DCT method and evaluating the percentages with the software released by Qiagen Version 2.0, 02/03/2012. Each array includes specific control assays for monitoring the cutting efficiencies of methylation-sensitive and methylationdependent enzymes and ensuring reliable and reproducible results. 2.3. Statistical evaluation Statistical analyses were performed using the program SPSS 19.0 (SPSS Inc., Chicago, USA). P-values of 60.05 were considered significant. Categorical variables were plotted in contingency tables and evaluated by Chi-squared analysis and Yates continuity correction. If n < 5, Fisher’s exact test was performed. Metric parameters were analyzed using the Kolmogorov–Smirnov test (test of normal distribution). For statistical evaluation, Student’s t-test (normally distributed values) and Mann–Whitney U-test (values not distributed normally) were employed. The Holm–Bonferroni correction was applied for multiple testing.
3. Results 3.1. Clinical evaluation of the study groups All study participants were assessed according to demographic and periodontal parameters (Table 1). When comparing the patient
group with controls, no statistically significant differences in gender (p = 0.806) and age (p = 0.428) could be detected. In comparison to the controls, the clinical parameters of periodontitis such as GI (p = 0.006), PDmm (p < 0.001), and CALmm (p < 0.001) were significantly elevated in the patient group. With respect to PI no statistically significant difference (p = 0.482) could be detected. 3.2. Epigenetic methylation pattern associated with periodontal diagnosis The status of CpG methylation obtained using ‘‘EpiTectÒ Methyl II Signature PCR Array Human Inflammatory response” is presented in Fig. 1. Overall, the percentage of promoter methylation of inflammatory candidate genes was obviously low in gingival biopsies regardless of the periodontal diagnosis. Only IL13RA1 (% CpG methylation, mean ± SD: 18.1 ± 22.2) and IL17C (% CpG methylation, mean ± SD: 16.7 ± 19.5) showed a total methylation rate of over 10%. Investigating the CpG methylation associated with the periodontal diagnosis of the participants, significant differences were found. Controls showed a significantly higher percentage of methylation of both the chemokine (C–C motif) ligand 25 (CCL25, 1.5fold, p = 0.015) and interleukin 17C (IL17C, 2.8fold, p = 0.002). 4. Discussion The manifestation and progression of periodontitis is characterized by subgingival bacterial infection and an individual’s immune response. Exogenous and endogenous risk markers have been approved to modulate disease initiation and development. Furthermore, epigenetic characteristics, e.g., CpG methylation, have been shown to have an impact on the etiology of inflammatory diseases, including periodontitis [11,23,24]. However, up to now, only a few studies were undertaken to examine the influence of epigenetic modifications on promoter methylation in periodontal disease [12–17]. Since it could be shown that epigenetic changes are tissue specific, epigenetic
Please cite this article in press as: S. Schulz et al., Epigenetic characteristics in inflammatory candidate genes in aggressive periodontitis, Hum. Immunol. (2015), http://dx.doi.org/10.1016/j.humimm.2015.10.007
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Fig. 1. Percentage of CpG methylation in inflammatory candidate genes in dependence of periodontal status.
patterns in blood might be different from those in gingival tissue [9]. Therefore, the preferred approach for investigating the impact of epigenetics in periodontal disease should be to assess gingival tissue [12,15–17,23]. Up to now, no complex approach has been undertaken that took into consideration the multitude of different pathways involved in an individual’s immune response to periodontopathogens. In the present clinical study, the methylation status of a selected panel of important inflammatory genes associated with periodontal diagnosis was investigated for the first time (EpiTectÒ Methyl II Signature PCR Array Human Inflammatory response (Qiagen, Hilden, Germany). These genes comprised chemokines (CCL25, CXCL14, CXCL3, CXCL5, CXCL6), cytokines (IL12A, IL12B, IL17C, INHA), cytokine receptors and associated proteins (IL10RA, IL12B, IL13RA1, IL15, IL17RA, IL4R, IL6R, IL6ST), as well as other genes involved in inflammatory response and autoimmunity (AFT2, FADD, GATA3, IL13, IL7, TYK2). For most of the genes an impact on the etiology of periodontitis has been reported. We assessed differences in CpG methylation pattern corresponding to periodontal status for CCL25 and IL17C. Both genes exhibited a significant decrease in promoter methylation in gingival tissue of AgP patients in contrast to periodontal tissues obtained from individuals without or only mild periodontitis. Because in test group only sites with CAL P 6 mm and among the controls sites with CAL 6 3 mm were chosen a sufficient contrast between periodontal severe diseased and no pathologically CAL was reached. However, the decrease in CCL25 methylation (1.5fold) was less pronounced as for IL17C (2.8fold). Both changes were in the range of methylation differences found in genome wide epigenetic study by Barros and Offenbacher (2.19fold to 1.9fold) [18]. CCL25 and IL17C have been implicated in the pathways important for host reactions to bacterial infections. Both cytokines play an important role in Th17 cell mediated immune response. CCL25 is able to induce Th17 pathway, whereas IL17 is secreted by Th17 cells and promote the expression of further inflammatory mediators [25]. The chemokine CCL25 is selectively expressed in the thymus, the intestinal epithelium as well as the oral mucosa [26–28]. It plays a key role in synchronizing the inflammatory response as it is involved in leukocyte trafficking and T-cell development [29]. An induction of CCL25 gene expression due to Porphyromonas gingivalis infection and co-stimulation with its lipopolysaccharide was
shown in vitro [30]. The increase in CCL25 gene expression in gingival tissue might result in enhanced leukocyte recruitment, including polymorphonuclear leukocytes and monocytes to site of inflammation [30]. Taken our results into account the upregulation of CCL25 might be at least partially due to epigenetic changes in promoter methylation associated with gingival inflammation. IL17C belongs to the interleukin 17 family, which comprises T cell-derived cytokines involved in the initiation or maintenance of the proinflammatory response [31,32]. IL17C is highly expressed in inflammatory conditions [32]. However, a lot of studies are conducted to investigate the biological role of IL17 only little is known about IL17C. IL17 are shown to induce a variety of proinflammatory cytokines. Beklen et al. demonstrated that IL17 significantly increase Il1b and TNFa expression in human macrophages in vitro [33]. Furthermore, IL17 has been linked to the formation of bone-resorbing osteoclasts [34]. Periodontitis associated bone destruction is also mediated by osteoclasts and may therefore be influenced by IL17 expression, too [35]. Actually, IL17 was assumed to be involved in periodontitis-associated bone resorption by influencing RANKL and osteoprotegerin expression [36]. LPS of P. gingivalis enhanced IL17 expression in peripheral mononuclear cells in vitro [37]. Indeed, IL17 expression was shown to be upregulated in the gingival tissue and gingival crevicular fluid of periodontitis patients as compared with healthy tissue [33,38– 39]. Based on its crucial role at site of inflammation an upregulation in IL17C in periodontitis lesions is conceivable. The decrease in CpG methylation measured for CCL25 and IL17C could be indicative of an increase in promoter activity, possibly leading to an enhancement in gene expression. Epigenetic characteristics could contribute to differences in expression patterns of inflammatory cytokines in periodontitis. However, epigenetic modifications might be triggered by a variety of different extrinsic and intrinsic factors. In periodontal disease, the actual gingival inflammation and the bacterial load, but also demographic or socioeconomic factors could influence the methylation pattern. 4.1. Limitations of the study The present investigation was performed as a case-control study.
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Both epigenetic modifications and periodontal disease are known to be triggered by a variety of different factors. The complex nature of this interaction was not considered in the study. This study was conducted to establish possible bidirectional associations between the epigenetic CpG methylation pattern of selected inflammatory candidate genes and periodontal disease only. Potential false-positive and false-negative associations could not be excluded. The inclusion of only 25 highly selected individuals in this pilot study provides no power for statistically sufficient analyses. However, the advantage of a pilot study is that it helps to saves resources and offers the opportunities to improve the design of a comprehensive large scale study including functional analyses. For the control group [40] development of severe periodontitis at a later time cannot be excluded. Therefore, the epigeneticdependent effect due to this possible selection bias may likely be underestimated. The present data only apply to Caucasians of Central Germany and must therefore be interpreted with caution. It would not be reasonable to extrapolate the results to the general population. 4.2. In conclusion Our results show a difference in epigenetic methylation in inflammatory candidate genes (CCL25 an IL17C) according to periodontal status. This research will help to develop new approaches in periodontal research and gain new insights into the etiology of periodontal disease. Acknowledgements The study was supported by the German Society of Periodontology (DG PARO) and the Martin-Luther University of Halle, Germany, University School of Dental Medicine, Department of Operative Dentistry and Periodontology. The authors declare that they have no conflict of interest. We would like to thank all patients and healthy controls for their cooperation in this study. References [1] G.C. Armitage, Development of a classification system for periodontal diseases and conditions, Ann. Periodontol. 4 (1999) 1–6. [2] R.J. Genco, W.S. Borgnakke, Risk factors for periodontal disease, Periodontology 2000 (62) (2013) 59–94. [3] M.L. Laine, W. Crielaard, B.G. Loos, Genetic susceptibility to periodontitis, Periodontology 2000 (58) (2012) 37–68. [4] J. Zhang, X. Sun, L. Xiao, C. Xie, D. Xuan, G. Luo, Gene polymorphisms and periodontitis, Periodontology 2000 (56) (2011) 102–124. [5] A.S. Schäfer, G.M. Richter, M. Nothnagel, T. Manke, H. Dommisch, G. Jacobs, A. Arlt, P. Rosenstiel, B. Noack, B. Groessner-Schreiber, S. Jepsen, B.G. Loos, S. Schreiber, A genome-wide association study identifies GLT6D1 as a susceptibility locus for periodontitis, Hum. Mol. Genet. 19 (2010) 553–562. [6] K. Divaris, K.L. Monda, K.E. North, A.F. Olshan, L.M. Reynolds, W.C. Hsueh, E.M. Lange, K. Moss, S.P. Barros, R.J. Weyant, Y. Liu, A.B. Newman, J.D. Beck, S. Offenbacher, Exploring the genetic basis of chronic periodontitis: a genomewide association study, Hum. Mol. Genet. 22 (2013) 2312–2324. [7] P. Feng, X. Wang, P.L. Casado, E.C. Küchler, K. Deeley, J. Noel, H. Kimm, J.H. Kim, A.N. Haas, V. Quielato, L.L. Bonato, J.M. Granjeiro, C. Susin, A.R. Vieira, Genome wide association scan for chronic periodontitis implicates novel locus, BMC Oral Health 84 (2014), http://dx.doi.org/10.1186/1472-6831-14-84. [8] A. Bird, DNA methylation patterns and epigenetic memory, Genes Dev. 16 (2002) 6–21. [9] A. Sadakierska-Chudy, R.M. Kostrzewa, M. Filip, A comprehensive view of the epigenetic landscape part I: DNA methylation, passive and active DNA demethylation pathways and histone variants, Neurotox. Res. (2014), http:// dx.doi.org/10.1007/s12640-014-9497-5. [10] M. Ngollo, A. Dagdemir, S. Karsli-Ceppioglu, G. Judes, A. Pajon, F. PenaultLlorca, J.P. Boiteux, Y.J. Bignon, L. Guy, D.J. Bernard-Gallon, Epigenetic modifications in prostate cancer, Epigenomics 6 (2014) 415–425. [11] A.M. Lindroth, Y.J. Park, Epigenetic biomarkers: a step forward for understanding periodontitis, J. Periodontal Implant Sci. 43 (2013) 111–120.
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[12] S. Zhang, S.P. Barros, A.J. Moretti, N. Yu, J. Zhou, J.S. Preisser, M.D. Niculescu, S. Offenbacher, Epigenetic regulation of TNFA expression in periodontal disease, J. Periodontol. 84 (2013) 1606–1616. [13] S. Zhang, S.P. Barros, M.D. Niculescu, A.J. Moretti, J.S. Preisser, S. Offenbacher, Alteration of PTGS2 promoter methylation in chronic periodontitis, J. Dent. Res. 89 (2010) 133–137. [14] W.T. Loo, L. Jin, M.N. Cheung, M. Wang, L.W. Chow, Epigenetic change in Ecadherin and COX-2 to predict chronic periodontitis, J. Transl. Med. 8 (2010) 100. [15] S.A. De Faria Amormino, T.C. Arao, A.M. Saraiva, R.S. Gomez, W.O. Dutra, J.E. da Costa, J. de Fatima Correia Silva, P.R. Moreira, Hypermathylation and low transcription of TLR2 gene in chronic periodontitis, Hum. Immunol. 74 (2013) 1231–1236. [16] F.A. Stefani, M.B. Viana, A.C. Dupim, J.A. Brito, R.S. Gomez, J.E. da Costa, P.R. Moreira, Expression, polymorphism and methylation pattern of interleukin-6 in periodontal tissue, Immunobiology 128 (2013) 1012–1017. [17] S. Zhang, A. Crivella, S. Offenbacher, A. Moretti, D.W. Paquette, S.P. Barros, Interferon-gamma promoter hypomethylation and increased expression in chronic periodontitis, J. Clin. Periodontol. 37 (2010) 953–961. [18] S.P. Barros, S. Offenbacher, Modifiable risk factors in periodontal disease: epigenetic regulation of gene expression in the inflammatory response, Periodontology 2000 (64) (2013) 95–100. [19] L. Larsson, R.M. Castilho, W.V. Giannobile, Epigenetics and its role in periodontal disease: a state-of-the-art review, J. Periodontol. 86 (2015) 556– 568. [20] H. Loe, J. Silness, Periodontal disease in pregnancy. I. Prevalence and severity, Acta Odontol. Scand. 21 (1963) 533–551. [21] J. Silness, H. Loe, Periodontal disease in pregnancy II. Correlation between oral hygiene and periodontal condition, Acta Odontol. Scand. 22 (1964) 121–135. [22] World medical Association, World Medical Association Declaration of Helsinki, ethical principles for medical research involving human subjects, JAMA 310 (2013) 2191–2194. [23] D. Bayarsaihan, Epigenetic mechanisms in inflammation, J. Dent. Res. 90 (2011) 9–17. [24] S. Lod, T. Johansson, K.H. Abrahamsson, L. Larsson, The influence of epigenetics in relation to oral health, Int. J. Dent. Hyg. 12 (2014) 48–54. [25] M.F.S. Costa, V.U. Bornstein, A.L. Candea, A. Henriques-Pons, M.G. Henriques, C. Penido, CCL25 induces a4b7 integrin dependent migration of IL-17+cd T lymphocytes during an allergic reaction, Eur. J. Immunol. 42 (2012) 1250– 1260. [26] D. Kabelitz, D. Wesch, Features and functions of cdT lymphocytes: focus on chemokines and their receptors, Crit. Rev. Immunol. 23 (2003) 339–370. [27] E.J. Kunkel, D.J. Campbell, E.C. Butcher, Chemokines in lymphocyte trafficking and intestinal immunity, Microcirculation 10 (2003) 313–323. [28] K. Otten, J. Dragoo, H.C. Wang, J.R. Klein, Antigen-induced chemokine activation in mouse buccal epithelium, Biochem. Biophys. Res. Commun. 304 (2003) 36–40. [29] A.P. Vicari, D.J. Figueroa, J.A. Hedrick, J.S. Foster, K.P. Singh, S. Meone, N.G. Copeland, D.J. Gilbert, N.A. Jenkins, K.B. Bacon, A. Zlotnik, TACK: a novel CC chemokine specifically expressed by thymic dentritic cells and potentially involved in T cell development, Immunity 7 (1997) 291–301. [30] S. Ekhlassi, L.Y. Crruggs, T. Garza, D. Montufar-Solis, A.J. Moretti, J.R. Klein, Porphyromonas gingivalis lipopolysaccharide induces tumor necrosis factor-a and interleukin-6 secretion, and CCL25 gene expression, in mouse primary gingival cell lines: interleukin-6-driven activation of CCL2, J. Periodont. Res. 43 (2008) 431–439. [31] H. Li, J. Chen, A. Huang, J. Stinson, S. Heldens, J. Foster, P. Dowd, A.L. Gurney, W. I. Wood, Cloning and characterization of IL-17b and IL-17C, two new members of IL-17 cytokine family, Proc. Natl. Acad. Sci. 97 (2000) 7736–7778. [32] S. Aggarwal, A.L. Gurney, IL-17: prototype member of an emerging cytokine family, J. Leukoc. Biol. 71 (2002) 1–8. [33] A. Beklen, M. Ainola, M. Hukkanen, C. Gurgan, T. Sorsa, Y.T. Konttinen, MMPs, IL-1, and TNF are regulated by IL-17 in periodontitis, J. Dent. Res. 86 (2007) 347–351. [34] Y. Lee, The role of interleukin-17 in bone metabolism and inflammatory skeletal disease, BMB Rep. 46 (2013) 479–483. [35] P.M. Bartold, M.D. Cantley, D.R. Haynes, Mechanisms and control of pathologic bone loss in periodontitis, Periodontology 2000 (53) (2010) 55–69. [36] D. Lin, L. Li, Y. Sun, W. Wang, X. Wang, Y. Ye, X. Chen, Y. Xu, IL-17 regulates the expression of RNAKL and OPG in human periodontal ligament cells via TRAF6/ TBK1-JNK/NF-kB pathways, Immunology (2014), http://dx.doi.org/10.1111/ imm.12395. [37] T. Oda, H. Yoshie, K. Yamazaki, Porphyromonas gingivalis antigen preferentially stimulates T cells to express IL-17 but not receptor activator of NF-kappaB ligand in vitro, Oral Micorbiol. Immunol. 18 (2013) 30–36. [38] A. Mitani, W. Niedbala, T. Fujimura, M. Mogi, S. Miyamae, N. Higuchi, A. Abe, T. Hishikawa, M. Mizutani, Y. Ishihara, H. Nakamura, K. Kurita, N. Ohno, Y. Tanaka, M. Hattori, T. Noguchi, Increased expression of interleukin-35 and -17, but not -27, in gingival tissues with chronic periodontitis, J. Periodontol. (2014), http://dx.doi.org/10.1902/jop.2014.140293. [39] O.G. Shaker, N.A. Ghallab, IL-17 and IL-11 GCF levels in aggressive and chronic periodontitis patients: relation to PCR bacterial detection, Mediators Inflamm. (2012), http://dx.doi.org/10.1155/2012/174764. [40] WHO Scientific Group, Epidemiology, etiology, and prevention of periodontal diseases, World Health Org. Tech. Rep. Ser. 1–60 (1978).
Please cite this article in press as: S. Schulz et al., Epigenetic characteristics in inflammatory candidate genes in aggressive periodontitis, Hum. Immunol. (2015), http://dx.doi.org/10.1016/j.humimm.2015.10.007