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Published in final edited form as: J Clin Periodontol. 2019 Jun 25;46(8):819–829. doi: 10.1111/jcpe.13151

Epigenetic and inflammatory events in experimental periodontitis following systemic microbial challenge

Daniela B Palioto 1,2,†, Livia S Finoti 1,†, Denis F Kinane 3, Manjunatha Benakanakere 1,*
PMCID: PMC6641985  NIHMSID: NIHMS1032551  PMID: 31131910

Abstract

Aim:

The purpose of this study was to determine inflammatory and epigenetic features following induction of oral and gut dysbiosis in experimental periodontitis in order to examine the interplay between oral and systemic infection.

Materials and Methods:

Periodontitis was induced in 6- to 8-wk-old C57BL/6 mice by: 1) ligature placement (Lig group) (oral challenge); 2) P. gingivalis gavage (Pg group) (systemic challenge) and; 3) the combination of the two models oral and systemic challenge (Pg+Lig). The duration of the experiment was 60 days, and the animals were then sacrificed for analyses. Alveolar bone-loss was assessed and a multiplex-Procarta Immunoassay was performed. Maxillae and gut tissues were immunostained for DNMT3b (de novo methylation marker), BTLA and IL-18R1 (inflammation markers).

Results:

Pg and Pg+Lig groups exhibited higher bone-loss when compared to Sham. BAFF, VEGF, RANKL, RANTES and IP-10 were significantly higher with Pg gavage. Likewise, DNMT3b was over-expressed in both gut and maxilla after the Pg administration. The same pattern was observed for BTLA and IL-18R1 in gut tissues.

Conclusions:

The systemic microbial challenge either alone or in combination with local challenge leads to distinct patterns of inflammatory and epigenetic features when compared to simply locally induced experimental periodontitis.

Keywords: Inflammation, epigenetics, experimental models of periodontitis, chronic periodontal disease, systemic disease

Clinical Relevance

Scientific rationale for study:

Local stimulation alone may not reflect natural periodontal disease development, and may be insufficient to drive systemic inflammation and epigenetic modifications.

Principal findings:

Bone-loss induced in mice that includes microbial gavage, better reflects the chronic periodontitis in humans.

Practical implications:

Local short-term stimulation in the mouse is not sufficient to create the dysbiotic conditions for chronic periodontitis. Systemic stimulation is necessary to induce the pathology associated with periodontitis. This experiment forces an interesting change in paradigm that chronic periodontitis needs a systemic perturbation and not just a local-challenge to develop the complex pathology of periodontal disease.

INTRODUCTION:

Periodontal disease is a multifactorial chronic immuno-inflammatory disease, where both the protective and supportive tissues of the periodontium are affected. The disease is not just confined to the oral cavity but has been strongly associated with other systemic chronic inflammatory conditions, such as metabolic syndrome (Daudt et al., 2018; Hajishengallis, 2015; Lamster & Pagan, 2016) and diabetes (Miguel‐Infante et al., 2018), cardiovascular disease (Lockhart et al., 2012; Tonetti & Van Dyke, 2013), and Alzheimer’s disease (Olsen & Singhrao, 2015), with varying degrees of causal corroboration. In contrast to the previous paradigm where attention was on the microbial composition of the periodontal biofilm, the host response is now being recognized as the major contributor driving the development of chronic periodontal destruction. The imbalance in biofilm composition and architecture, driven from commensals to pathobionts, as well as a non-resolving inflammation, sustains the tissue disease chronicity (Hajishengallis & Lamont, 2012; Meyle & Chapple, 2015).

The concept of a break-down of the mucosal immune system and “keystone” pathogen hypothesis might explain the host susceptibility to a disbiotic state (Hajishengallis et al., 2012). It is well documented that the innate inflammatory response initiated by epithelial mucosa transduces the signals to activate adaptive immune responses to infection (Benakanakere et al., 2015; Benakanakere et al., 2019; Kinane et al., 2017), and extensive signaling pathways are activated to provide appropriate responses.

Interestingly, the susceptibility to local changes in the periodontium may not be directly related to oral biofilm composition or quantity, but rather, could be related to the subtle shifts on the systemic level, for instance, changes that occurs in the gut’s mucosal immune system. The gut and the oral cavity share similar features associated with a profuse microbial flora, and relatively common susceptibility for microbial-driven chronic inflammation (Garlet & Santos, 2014). Hence, knowledge of whether the oral infection manifests systemic inflammation or vice versa has not been understood in detail. It is well established that B and T-lymphocytes predominate the inflammatory infiltrate of human chronic periodontitis tissues similar to that of systemic inflammatory condition (Abe et al., 2015; Hajishengallis, 2015). Interestingly, B and T-lymphocyte attenuator (BTLA), a co-inhibitory receptor expressed by both B and T-lymphocytes regulate lymphocyte activation on infection and inflammation, and BTLA expression levels could potentially identify patients with chronic systemic infection (Shubin et al., 2013). A pro-inflammatory cytokine IL-18 belongs to the IL-1 cytokine superfamily is involved in a wide variety of inflammatory diseases and potentiates both innate and adaptive immunity (Chen et al., 2012; Enoksson et al., 2011; Imaoka et al., 2013). This cytokine binds to its receptor IL-18R1 and activates inflammatory signaling pathway (Azam et al., 2003). The pro-inflammatory cytokines such as TNF and IL-1 up-regulates IL-18R1 expression in epithelial cells and enhance IL-8 secretion (Krásná et al., 2005).

The identification of how pathogens influence local and systemic inflammation mediated gene-specific epigenetic changes may represent exciting opportunities for the development of novel strategies for preventing and treating periodontal disease. Epigenetic changes such as DNA methylation could occur as a result of the activity of DNA methyltransferases (DNMTs). It is well known that DNMT3a and DNMT3b accounts for de novo cytosine methylation at previously unmethylated CpG sites (Benakanakere et al., 2015; Benakanakere et al., 2019). Moreover, the experimental periodontitis model induced by ligature placement, an acute reaction, may not reflect the chronicity and persistent dysbiotic state observed in periodontitis of humans. Keeping in view of this disparity, we sought to address the inflammatory and epigenetic features after the induction of host gut/oral dysbiosis in experimental periodontitis mouse model to understand whether systemic infection is driven by, or exacerbates periodontal disease.

MATERIAL AND METHODS

Animals and study design:

The animals (C57BL/6) were procured from the Jackson Laboratories. All animal procedures described here were approved by the University of Pennsylvania Institutional Animal Care and Use Committee. Five mice each were grouped as Control (SHAM), Ligature (Ligature-induced periodontitis), oral gavage of P. gingivalis, Ligature + P. gingivalis oral gavage. Oral gavage: The oral gavage was according to our previously published procedure (Benakanakere et al., 2015). The total duration of the experiment lasted for 60 days starting with 10 days of antibiotic (sulfatrim pediatric suspension) treatment (Figure 1A). After 3 days of antibiotic free period, oral gavage in mice was performed every other day for a total of 6 times with oral inoculation of 109 P. gingivalis in 200 μl of 2% carboxymethilcellulose. After 42 days of first oral gavage, the animals were sacrificed for analyses. Ligature model: The ligature model was according to previously published method (Abe et al., 2016). Briefly, after 10 days of antibiotic treatment and 3 of antibiotic free period, the ligature was placed for 5 days and the animals in this group were sacrificed for analysis. For the group that received Ligature + P. gingivalis, the ligature was placed and P. gingivalis oral gavage was initiated similar to that of oral gavage model. After 5 days, the ligature was removed and the animals were allowed to heal and then sacrificed after 42 days.

Figure 1:

Figure 1:

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6–8 week-old mice were compared for alveolar bone loss. Ligature was placed around the maxillary left second molar (ligature side) and right second molar was used as a control side. A: Diagrammatic illustration of animal experiment. B: Representative figures from experimental periodontal disease as indicated by alveolar bone loss. C: Quantification analysis of the alveolar bone resorption and periodontal lesion. The ligature placement, oral gavage and combination of oral gavage and ligature induced significant bone loss compared to control animals. *p<0.05

Analysis of alveolar bone:

For micro-computed tomography (micro-CT) analysis, the maxilla was dissected, fixed for 24 h in cold 10% formalin, and immersed in RNAlater solution (Thermofisher, CA) for micro-CT analysis on micro-CT-35 equipment (Scanco Medical AG, Bassersdorf, Switzerland). The percentage of bone was measured as the remaining bone volume around second molar using ITK-SNAP software. The percentage of bone volume was measured as area occupied by bone between two adjacent teeth (Mesial – between the first and the second molar; Distal – between the second and the third molar). Bone volume fraction was calculated from the bone volume (BV) and total volume (TV) as BV/TV. The alveolar bone loss was calculated by applying the distance transformation by filling maximal spheres in the bone structures using the calculation based on the density of bone as determined by the micro-CT according to the manufacturer’s recommended protocol and as per the published article (Dai et al., 2016).

Decalcification:

After micro-CT scanning the samples were subjected to decalcification using 5mL of 10% ethylene-diamine-tetraacetic acid (EDTA) in RNAlater (Thermofisher, CA) pH 5.2 at room temperature for 14 days (Belluoccio et al., 2013). Following decalcification, maxillae were washed with PBS, embedded in OCT and stored at −80°C until further use.

Multiplex Immunoassay:

Peripheral blood was collected before the animals were sacrificed, and the levels of serum cytokines (BAFF, GM-CSF, IFN-gamma, IL-1 beta, IL-10, IL-12p70, IL-18, IL-2, IL-23, IL-25, IL-33, IL-6, IL-7, IP-10, M-CSF, MIP-1α, RANTES, sRANKL, TNF, VEGF) were measured using Procarta Multiplex Cytokine kit (Thermofisher, CA) according to manufacturer’s instructions. These cytokines were chosen based on their significance with periodontal disease and/or systemic inflammation or repair.

Immunofluorescence staining:

Maxillae (frozen decalcified sections) and gut tissues (frozen sections) were immunestained according to (Abe et al., 2015) by using antibodies against DNMT3b (de novo methylation marker), BTLA and IL-18R1 (inflammation markers).

Statistical analysis:

Experimental data were reported as mean +/− standard deviation (SD). The Shapiro-Wilk test was used to assess the normality of the quantitative data distribution. Because the data were distributed normally, statistical analysis were determined by one-way ANOVA followed by Tukey test. All the analyses were performed using GraphPad Prism 5 (San Diego, CA) and a minimal level of p<0.05 was considered significant.

RESULTS:

Combination of ligature and oral gavage induced experimental periodontitis suppress inflammatory mediators:

The objective of this study was to address the inflammatory and epigenetic features after induction of host gut/oral dysbiosis using experimental periodontitis models. First, we sought to compare two different models of experimental periodontitis using C57BL/6 mice in order to select the best possible infection model to pursue the epigenetic studies in vivo. We utilized ligature induced periodontitis with or without P. gingivalis oral gavage. As expected, all three scenarios (ligature, gavage and ligature + gavage) induced significant bone loss compared to sham animals. Overtime, after the period allowed for the chronification of the lesions, we found that the alveolar bone loss was greater in the gavage and in the combination model. Although, the ligature group experienced significant bone loss, at the end of the healing period it was not statistically different than sham group, demonstrating a tendency to return to the original condition (Figure 1B & C). Further, in order to examine the systemic inflammatory markers in these animals, the serum samples were isolated and subjected to Procarta multiplex cytokine analysis. Mice with ligature and oral gavage groups induced significantly higher serum BAFF, IP-10, RANTES, RANKL and VEGF (Figure 2). Surprisingly, ligature + gavage treated mice induced significantly less serum BAFF, IP-10, RANTES, RANKL and VEGF (Figure 2).

Figure 2:

Figure 2:

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6–8 week-old mice were compared for serum cytokine levels: BAFF, IP-10, Rantes, RANKL and VEGF were measured in serum samples using ProCarta multiplex ELISA. The pattern of increased inflammatory markers and reduced levels was observed in gavage + Ligature group. *p<0.05

Inflammatory and Epigenetic markers are elevated in the periodontium of mice treated with ligature and oral gavage combination:

Since we observed significant bone resorption and reduction in the serum cytokine levels in mice with ligature and oral gavage, we further tested the inflammatory and epigenetic markers in the periodontium. After decalcification, the maxillae were subjected to microtome sectioning and immunostaining with DNMT3b (de novo methylation marker), BTLA and IL18R1 (inflammation markers) antibodies. We observed higher numbers of positively stained cells on the surface of alveolar bone and within the gingiva in the gavage and ligature + gavage group than in the ligature group alone (Figure 3). Similarly, higher numbers of positively stained cells were noted for BTLA and IL18R1 on the surface of alveolar bone and within the gingiva in ligature + gavage group (Figure 4).

Figure 3:

Figure 3:

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Inflammatory and epigenetic markers in a local and systemic microbial challenge. Immunofluorescence of DNMT3b in the gingival tissues sections: Higher numbers of positively stained cells for DNMT3b were noted on the surface of alveolar bone in gavage model (Pink (DNMT3b); Blue (DAPI)).

Figure 4:

Figure 4:

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Higher numbers of positively stained cells for BTLA and IL-18R1 were noted on the surface of alveolar bone in gavage model. (Red: BTLA; Green: IL-18R1; Blue: nuclei (DAPI)).

Experimental periodontitis in mice alters gut inflammation:

The altered serum inflammatory cytokines and inflammatory markers such as BTLA, IL18R1, and de novo DNA methylation marker DNMT3b upregulation in the periodontium of mice treated with ligature and oral gavage combination prompted us to investigate the gut tissue to examine whether the combination of ligature and gavage induced significantly higher gut inflammation elevating serum cytokine levels. After the experiment, gut tissues were dissected, embedded and 12μ sections were subjected to immunostaining with DNMT3b, BTLA and IL18R1 antibodies. Interestingly, we observed higher numbers of positively stained cells for DNMT3b on the epithelial tissue of the gut in the oral gavage model compared to the ligature or ligature + gavage (Figure 5). The expression of BTLA and IL-18R1 staining was also similar where higher numbers of positively stained cells were noted in oral gavage model (Figure 6). These findings suggested that oral gavage model may result in elevated systemic inflammation than in the ligature model.

Figure 5:

Figure 5:

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Immunofluorescence of DNMT3b in gut tissue. Higher numbers of positively stained cells for DNMT3b were noted on the epithelial tissue in gavage model (Pink (DNMT3b); Blue (DAPI)).

Figure 6:

Figure 6:

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Immunofluorescence of inflammatory markers in the gut tissue: Higher numbers of positively stained cells for BTLA and IL-18R1 were noted in gavage model. (Red: BTLA; Green: IL-18R1; Blue: nuclei (DAPI)).

DISCUSSION:

The objective of this study was to determine the immune-inflammatory pattern and also evaluate de novo DNA methylation marker after local and systemic dysbiosis induction. The experimental periodontal disease was induced locally (ligature), systemically (Pg gavage) and a combination of local and systemic induction (Pg gavage + Ligature). As expected, all animals in our experimental groups developed significant levels of alveolar bone loss. However, the chronicity expected for a periodontal lesion was not observed in the ligature-induced only periodontitis. Our data indicates that, when gavage was administered, on its own or in combination with oral challenge, a chronic periodontitis was observed. This resolution of inflammation in mice occurred during the healing period when the stimulus was withdrawn. This could be due to elevated endogenous inflammation resolving factors such as developmental endothelial locus-1 protein (DEL-1) and/or resolvins (Hasturk & Kantarci, 2015; Maekawa et al., 2015; Shin et al., 2015; Van Dyke, 2017). On the other hand, at the end of the 42 days period, we observed a consistent bone loss in the group where the microbial gavage was used. The same was not observed in the ligature only procedure without microbial challenge. Serum cytokine levels were analyzed in all the groups but we found that BAFF, VEGF, RANKL, Rantes and IP-10 were elevated only in the ligature and gavage groups. Due to methodological constraints, we were not able to show these cytokines in the gingival tissue and correlate results with the gut tissue. Nonetheless, the tissues of the intestine also showed a similar pattern of BTLA and IL-18R1 expression as that of the gingiva but the staining was more pronounced in the gut.

The term epigenetics relates to changes in gene expression that include chemical alterations of DNA and its associated proteins such as histones (Barros et al., 2018; Benakanakere et al., 2015; Jośko-Ochojska et al., 2018). These changes lead to remodeling of the chromatin and subsequent activation or inactivation of a gene (Barros & Offenbacher, 2009). Epigenetic mechanisms have been found to contribute to the disease, including cancer and autoimmune or inflammatory diseases. Recent evidence suggests that epigenetic alterations are possibly triggered by the host microbiota and environmental cues (Benakanakere et al., 2015). These epigenetic alternations (DNA methylation and histone modifications) can have long-term effect on host’s immune homeostasis (Feil & Fraga, 2012). The relationship between microbiota and DNA methylation to chronic inflammation has been clearly established by Helicobacter pylori infection. In this case, DNA methylation in the genes of gastric mucosa were closely associated with the risk of gastric cancer which was linked to H. pylori infection (Nakajima et al., 2009). The constant and direct contact of the microbiota and host cells in various organs in the body, especially the gut, may lead to the idea that microbiota can be a primary mechanism of regulating the host epigenome (Qin & Wade, 2017). Furthermore, recent investigations suggest that there seems to be a complex, pertinent and effectual relationship between the intestinal microbiota, the immune system, and epigenetic modifications (Obata et al., 2014).

Epigenetic modifications are clearly pertinent to the development of periodontal disease and present emerging therapeutic possibilities aimed at epigenetic targets (epidrugs) associated with the disruption of tissue homeostasis and the development of periodontitis (Benakanakere et al., 2019; Larsson et al., 2015). Larsson et al., tested the epigenetic and inflammatory signaling pathways to determine the systemic epigenetic changes influenced periodontal disease outcome in mice (Larsson et al., 2015). In our study, we observed that maxillae showed higher DNMT3b expression in animals with ligature and gavage, whereas, gavage group showed higher DNMT3b expression in the gut tissue samples. These observations suggest that the systemic inflammation instigated with the administration of P. gingivalis by gavage may alter the mucosal immune system with repercussion on oral bone tissues via de novo methylation altering the epigenetic landscape. Hence, we believe that local and systemic infection with epigenetic modifications in mice better represent periodontal inflammation similar to that of humans.

The connection between periodontal disease and systemic diseases has been studied in diabetic mice and mice with susceptibility to cardiovascular diseases using ligature induced periodontitis (Lourenςo et al., 2018; Page et al., 1997; Sanz et al., 2019; Tonetti & Van Dyke, 2013). However, mice do not develop natural periodontitis and there is a lack of evidence that prominent human pathogens related to periodontal disease pathogenesis are present in the oral cavity of mice (Abe & Hajishengallis, 2013; Hajishengallis et al., 2016; Jiao et al., 2013). Interestingly, rabbits do not develop periodontitis with ligature alone (Andrian et al., 2006; Graves et al., 2008; Hajishengallis et al., 2011; Malek et al., 1994; Rovin et al., 1966). Rabbits require the addition of human pathogen such as P. gingivalis to the oral cavity by gavage to develop periodontitis. Although there are many animal studies reported and many being conducted along with in vitro studies to elucidate the disease process, it is impossible to dissect the complex process of periodontal pathogenesis due to non-linearity of host inflammatory responses (Kantarci et al., 2015). The immune response to oral bacteria and the subsequent activation of inflammatory signaling is not only dependent on genetic factors but also on epigenetic mechanisms presenting additional regulatory pathways of genes involved in worsening inflammation, including gingivitis and periodontitis. Nevertheless, the connection between periodontitis, systemic disease and epigenetics has to be elucidated by using a proper animal model of experimental periodontitis. In this study, it is important to note that the experimental periodontitis was conducted in C57BL/6 mice that are considered a less susceptible for the oral gavage model of periodontitis induction (Graves et al., 2008). Hence, we modified the protocol and introduced P. gingivalis multiple times during the procedure of oral gavage than previously reported (de Molon et al., 2015). As observed, by increasing the period of microbial challenge, our method was successfully promoted significant bone loss, inflammatory and epigenetic changes both at the local as well as at the systemic level. A tendency to return to the normal condition (compared to the control ones), observed in the Ligature group after a healing period, leads to the rational of spontaneous restoration, reinforcing that, by itself, ligature would not drive lesions to a chronic process once the host overcome the mechanical stimuli, and thus suggest that the immune changes tend to be more resolvable. Nevertheless, there is still a need for further investigation in order to confirm our findings with greater sample size and genome wide epigenetic and metabolomics assays. Our data is consistent with the hypothesis that in humans a short term challenge will produce gingivitis but sustaining this challenge long term and which includes some systemic perturbation is needed for chronic periodontitis. These findings are relevant to future therapeutic and management approaches.

In summary, the inflammatory markers and epigenetic changes observed after oral and systemic microbial challenge were significantly higher when compared to local ligature induced periodontitis. The systemic microbial challenge or the combination of oral and systemic challenge may be a good strategy for studying inflammatory markers and epigenetic changes in experimental periodontal disease and better for modeling human periodontal disease.

ACKNOWLEDGEMENTS:

Authors would like to thank Dr. Carlota Danesi for the help with the Micro-CT analysis. The authors also thank the intellectual and technical inputs from Dr Tetsuhiro Kajikawa.

Conflict of Interest and Sources of Funding Statement

The authors declare that there are no conflicts of interest in this study.

This research was supported by National Institute of Dental and Craniofacial Research, NIH (R01DE024160). Daniela Palioto received fellowship support from FAPESP São Paulo Research Foundation # 2016/21471–0.

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