Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Periodontitis increases rheumatic factor serum levels and citrullinated proteins in gingival tissues and alter cytokine balance in arthritic rats

Periodontitis increases rheumatic factor serum levels and citrullinated proteins in gingival tissues and alter cytokine balance in arthritic rats

  • Mônica G. Corrêa, 
  • Silvana B. Sacchetti, 
  • Fernanda Vieira Ribeiro, 
  • Suzana Peres Pimentel, 
  • Renato Corrêa Viana Casarin, 
  • Fabiano Ribeiro Cirano, 
  • Marcio Z. Casati
PLOS
x

Abstract

This study investigated some immunological features by experimental periodontitis (EP) and rheumatoid arthritis (RA) disease interact in destructive processes in arthritic rats. Rats were assigned to the following groups: EP +RA; RA; EP; and Negative Control. RA was induced by immunizations with type-II collagen and a local immunization with Complete Freund’s adjuvant in the paw. Periodontitis was induced by ligating the right first molars. The serum level of rheumatoid factor (RF) and anti-citrullinated protein antibody (ACCPA) were measured before the induction of EP (T1) and at 28 days after (T2) by ELISA assay. ACCPA levels were also measured in the gingival tissue at T2. The specimens were processed for morphometric analysis of bone loss, and the gingival tissue surrounding the first molar was collected for the quantification of interleukin IL-1β, IL-4, IL-6, IL-17 and TNF-α using a Luminex/MAGpix assay. Paw edema was analyzed using a plethysmometer. Periodontitis increased the RF and ACCPA levels in the serum and in the gingival tissue, respectively. Besides, the level of paw swelling was increased by EP and remained in progress until the end of the experiment, when EP was associated with RA. Greater values of IL-17 were observed only when RA was present, in spite of PE. It can be concluded that periodontitis increases rheumatic factor serum levels and citrullinated proteins level in gingival tissues and alter cytokine balance in arthritic rats; at the same time, arthritis increases periodontal destruction, confirming the bidirectional interaction between diseases.

Introduction

Both rheumatoid arthritis (RA) and periodontitis are chronic inflammatory diseases that lead to tissue destruction. Rheumatoid arthritis is a chronic inflammatory polyarthritis with a prevalence of 0.5% to 1.0% in adults in industrialized countries [1]. The etiology is multifactorial and the pathogenesis is not fully understood. One possibility is autoimmunity to citrullinated proteins, which is highly specific for RA and may be related to the pathogenesis of the disease [2]. Periodontitis, per se, is a bacterial chronic inflammatory condition that leads to the occurrence of supporting tissue destruction and is host-mediated by the local production of immune-inflammatory mediators in response to periodontopathogens [3].

In both RA and periodontitis, inflammatory cell infiltration occurs (T lymphocytes, macrophages and polymorphonuclear), which leads to progressive tissue destruction [3, 4]. Moreover, the maintenance of the inflammatory process is mediated by cytokines in RA and also in periodontitis [3, 5, 6]. Moreover, based on these shared characteristics, there is some evidence of a bidirectional pathway of progression.

An important study of The third National Health and Nutrition Examination Survey (NHANES III), included 4461 patients aged 60 years or older, who had undergone both musculoskeletal and dental examinations and showed that rheumatic subjects were more likely to be edentulous [odds ratio (OR) 2.27, 95% confidence interval (CI) 1.56 3.31] and have periodontitis (OR 1.82, 95% CI 1.04 3.20) compared with non-RA subjects [7]. Some other studies reported poorer periodontal status in patients with rheumatoid arthritis compared to systemically healthy patients. Mercado et al. [8, 9] showed that RA patients had a greater level of tooth loss and higher percentage of pockets than the control group (healthy patients), with no differences in plaque index and bleeding on probing. Furthermore, these authors showed that the number of deep pockets (≥ 6 mm) in RA patients was significantly higher than in controls.

On the other hand, some studies showed periodontitis as a risk factor for the development or increasing severity of RA [10, 11]. There is evidence that the treatment of periodontitis can reduce the severity of arthritis [10, 12, 13] by reducing proinflammatory factors such as CRP-reactive protein and erythrocyte sedimentation. Moreover, Porphyromonas gingivalis (Pg), a well-known periodontal pathogen, may be important in the RA development process because of its capacity of citrullinate proteins by releasing peptidylarginine deiminase [14, 15] and has been related to RA worsening in animal studies [16, 17, 18, 19]. Some previous studies indicated that Gram-negative periodontal bacteria, in particular Pg, could be associated with an increase in rheumatic factor (RF) levels, by direct stimulation [20] or an immunological response against them [21].

In recent years, studies have been trying to investigate the ways in which both diseases interact and increase their aggressiveness. At the same time an experimental AR rat model showed the potential increase in gingival levels of gelatinase, collagenase, TNF-a and IL-1β in normoreactive rats [22], other studies showed that existing periodontitis significantly influenced the induction and severity of RA [19]. However, more clarification and basic data are still needed to better assess this bidirectional relationship between AR and PD. Considering the above evidence, this study evaluates the impact of experimental rheumatoid arthritis and experimental periodontitis in rats in regards of seric and local mediators that could contribute to the better understanding of RA-PD link.

Materials and methods

Animals

Forty adult male Wistar rats (200–300 g—Butantan Institute, Butantã, São Paulo, Brazil.) were used. The rats were acclimatized for 15 days before use and they were kept in temperature-controlled cages, exposed to a 24-h light–dark cycle of equal time, and had free access to water and food ad libitum (Labina, Purina, Paulínia, São Paulo, Brazil) in the Bioterium of Paulista University. The experimental procedure was approved by the Paulista University Institutional Animal Care and Use Committee (205/13 CEP/ICS/UNIP).

Experimental design

Treatment groups.

The animals were randomly assigned to one of the following treatment groups: 1- experimental periodontitis + and experimental arthritis (EP+RA) (N = 10); 2- experimental arthritis (RA) (N = 10); 3- experimental periodontitis (EP) (N = 10); 4- Control (n = 10) (Fig 1). This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The experimental procedure was approved by the Paulista University Institutional Animal Care and Use Committee (Permit Number: 205/13 CEP/ICS/UNIP). All surgery was performed general anesthesia by the intramuscular administration of ketamine hydrochloride (0.5 mL/kg; Dopalen, Agribrands, Paulínia, São Paulo, Brazil) and xylazine hydrochloride (10 mg/kg; Rompun, Bayer, São Paulo, São Paulo, Brazil), and all efforts were made to minimize suffering.

Rheumatoid arthritis model–RA model.

RA was induced by two immunizations of type-II collagen and a third immunization with Complete Freund’s adjuvant (CFA—F5881–10 mL; Sigma-Aldrich, São Paulo, São Paulo, Brazil). The first dose of type-II collagen (C9879-1G - Sigma-Aldrich, São Paulo, São Paulo, Brazil) was administered on day -21 consisting in a total of 0.1 ml emulsion injected at multiple locations at the tail base with a needle (300 μg of type-II collagen in Incomplete Freund’s Adjuvant (IFA—F5506–10 mL; Sigma-Aldrich, São Paulo, São Paulo, Brazil). The second dose, booster dose, was administered subcutaneously on day -14 (injection containing 100 ug of CII in IFA). The second immunization can increase the incidence and severity of arthritis. The third immunization occurred at day -7 through two injections of CFA (1mg/ml) (0.2 ml total): one in the paw subcutaneously (0.1 ml), and the other at the knee joint via intra-articular (0.1 mL). This protocol was based on the studies of Lohman et al. [23] and Sitônio et al. [24], associating with CII/IFA emulsion and CFA immunizations.

Rat periodontitis model.

To induce experimental periodontitis, one of the mandibular first molars of each animal was randomly assigned to receive a cotton ligature (Corrente Algodão 10, Coats Corrente, São Paulo, São Paulo, Brazil) in a cervical position knotted submarginally. The ligatures were kept in position in order to allow biofilm accumulation over 28 days. The contralateral tooth was left unligated so that it could be used as a control. This procedure was performed under general anesthesia by the intramuscular administration of ketamine hydrochloride (0.5 mL/kg) and xylazine hydrochloride (10 mg/kg).

Euthanasia and specimens collecting.

Forty-nine days after the start of the study, the animals were euthanized by CO2 inhalation. The mandibles were excised for morphometric analysis. The buccal gingival tissue from the area surrounding the lower first molar subjected to experimental periodontitis was also collected for the quantification of immune-inflammatory mediators using the Luminex/MAGpix assay. The collected tissues were placed in sterile tubes containing 400μl phosphate buffered saline (PBS) with 0.05% Tween-20. All samples were stored at -20°C until the imumuno-enzymatic assay. After gingival dissection, the mandibles were de-fleshed after immersion in 8% sodium hypochlorite for 4 h. The specimens were washed in running water and immediately dried with compressed air. To distinguish the cementum enamel junction (CEJ), 1% aqueous methylene blue solution (Sigma-Aldrich, St. Louis, MO) was applied for 1 min to the specimens, which were then washed in running water. Then, the specimens were kept in room temperature into identified cases for posterior photography and morphometry.

Measurement of alveolar bone loss.

After mandibles preparation as described above, photographs were taken with a 6.1-megapixel digital camera (EOS 40D; Canon, New York, NY, USA) placed on a tripod to keep the camera parallel to the ground at the minimal focal distance. The specimens were fixed in wax with their occlusal planes parallel to the ground and long axes perpendicular to the camera. Photographs of the buccal aspects were taken from both test and control sides. To validate measurement conversions, all specimens were photographed alongside a millimeter ruler [25, 26]. Alveolar bone loss was assessed on the buccal surface of the lower first molars by measuring the distance of the CEJ from the alveolar bone crest at three equally distant sites. The average alveolar bone height of each tooth was calculated. A single examiner, who was unaware of the experimental data, carried out morphometric measurements of alveolar bone loss. The measurements were performed after intraexaminer calibration by evaluating 10 images not taken for this study that show alveolar bone loss similar to the present study. The examiner took the linear measurements of all photographs twice within 24 hours. The intraclass correlation showed 95.7% reproducibility.

Rheumatoid factor and anti-citrullinated protein antibody serum levels.

Here, 100 μL of blood was collected from the orbital veniplex of rats under anesthesia at T1 (day 0) and at the euthanasia (T2) and the serum samples were stored at −70°C. The level of RF and anti-citrullinated protein antibody in serum aliquots were measured by using the Rat Elisa Kit (MBS720877—monoclonal anti-RF antibody and an RF-HRP conjugate; Mybiosorce, San Diego, California, USA; E-EL-R1431—monoclonal antibody specific to and Avidin-Horseradish Peroxidase (HRP) conjugate -Elabscience, Beijing, China, respectively). The levels of ACCP were also analyzed in gingival tissue. Samples and standards were prepared following the manufacturer’s instructions and analyzed on a fluorescence plate reader.

Immunodetection of pro-inflammatory cytokines.

To proceed the immune-enzymatic assay the gingival tissue collected was weighed, then cut into small pieces (1 mm3 to 2 mm3) using scissors and blades [26, 27] and solubilized in PBS to a final concentration of 100 mg tissue/ml. After extraction on a Vortex mixer for 10 min, each sample was centrifuged at 370 g for 5 min, and the supernatant was collected, divided into small portions, and stored at -70°C until use. To avoid protease activity, the entire procedure was carried out at 4°C. The levels of IL-1β, IL-4, IL-6, IL-17 and TNF-α were determined by Luminex/MAGpix assay using commercially available kits (RCYTOMAG-80K; Millipore, Billerica, MA, USA) and following the manufacturers’ instructions. Initially, a 96-well plate was pre-wet with washing buffer, and after discarding the wash buffer, microsphere magnetic beads coated with monoclonal antibodies against the 3 different target analytes were added to the wells. Samples and standards were added to the wells and incubated overnight at 4°C. The wells were washed using a magnetic manifold, and a mixture of biotinylated secondary antibodies was added. After incubation for 1 h, streptavidin conjugated to the fluorescent protein, Rphycoerythrin (streptavidin-RPE), was added to the beads and incubated for 30 min. After washing to remove the unbound reagents, sheath fluid‡‡ was added to the wells, and the beads (minimum of 50 per analyte) were analyzed in the MAGpix™ instrument (MAGpix™ MiraiBio, Alameda, CA, USA). Samples were diluted with the kits’ diluents. The dilution was taken into consideration when calculating the concentration of each substance with a standard curve, prepared using the standard proteins in the kit. The standard curve range used for IL-1β measurement was 2.4–10,000 pg/ml; for IL-4 measurement, 4.9–20,000 pg/ml; for IL-6 7.3–300,000 pg/ml, for TNF-α 2.4 to 10,000 pg/ml; and for IL-17 7.3 to 30,000 pg/Ml.

Paw swelling analysis.

Systemic macroscopic features of arthritis regarding paw swelling (as measured in ml by plethysmometer, Ugo Basile) [2831] were recorded every week until the end of the experiment.

Statistical analysis.

To test the null hypothesis that RA had no influence on alveolar bone loss and cytokine levels, intergroup analysis was performed through a two-way ANOVA test followed by a Tukey test. RF data were analyzed through ANOVA/Bonferroni test. Kruskal-Wallis test was performed for the intergroup analysis of ACCPA and Wilcoxon test for the intragroup comparisons. Plethysmometer values were analyzed by Friedman (intragroup) and Kruskal-Wallis (intergroup) tests. The significance level established for all analyses was 5% [Statistical Analysis System (SAS) 9.3, Cary, NC, USA].

Result

Clinical analysis

The animals did not show any signs of systemic illness (except RA) throughout the study period. The rats also did not lose weight throughout the experimental period. No deaths were observed. Twenty-one days after immunization, joint swelling was observed first in the hind paws, and then joint swelling extended to the forelegs and tail. The RA peak occurred on day 28, with multiple and symmetrical joint swelling and redness. Some rats presented deformity and limited mobility in some joints. The greater paw volume was observed especially at the paw that received the CFA injection. Considering the paw swelling assessed by plethysmometer, efficiency in the RA model could be noted once the RA and RA+EP groups presented an increase in swelling after RA induction (days -7, -14, -21 and 28 days), with a significant difference to the control and EP groups at -7 and 0 days (p<0.05). However, after EP induction (day 0), RA groups showed a trend towards a reduction in paw swelling, while RA+EP maintained its volume, being only RA+EP group, in contrast to the control and EP groups until the end of the study (Fig 2 - Figure A in S1 File). Meanwhile, AR symptoms (joints swelling and deformities) lasted until the end of the experimental period in both RA and RA+EP groups. In addition, at the time of euthanasia, clinical examination revealed signs of gingival inflammation, including color/volume changes and bleeding around the ligated teeth of all groups, with no signs of inflammation at the non-ligated sites (contralateral teeth).

thumbnail
Fig 2. Means a± SD of paw swelling (volume—ml) measured by plethysmometer.

* Indicate significant difference to baseline (-21) and † indicate significant difference between RA and EP+RA [Friedman (intragroup) and Kruskal-Wallis (intergroup) p<0.05]. (CONTROL: n = 10; EP: n = 10; RA: n = 10; EP+RA: n = 10).

https://doi.org/10.1371/journal.pone.0174442.g002

Morphometric results

Intragroup analysis showed significant differences in alveolar bone loss between unligated and ligated teeth in all groups (P < 0.05). Intergroup analysis of morphometric outcomes showed greater periodontal destruction in the EP+RA group when compared with the control group, PE and RA (p <0.05). The EP group also presented higher bone loss values than the control and RA groups (p<0.05). No difference between groups were noted in unligated teeth (p>0.05). The morphometric findings are shown in Table 1 (Table A in S1 File) and Fig 3 illustrates the morphometric findings.

thumbnail
Fig 3.

Representative photographs illustrating the morphometric findings of the groups–A–CONTROL (N = 10): Ligated teeth; B–CONTROL (N = 10): Unligated teeth; C–EP (N = 10): Ligated teeth; D–EP (N = 10): Unligated teeth; E–RA (N = 10): Ligated teeth; F–RA (N = 10): Unligated teeth; G–EP+RA (N = 10): Ligated teeth; H—EP+RA (N = 10): Unligated teeth.

https://doi.org/10.1371/journal.pone.0174442.g003

thumbnail
Table 1. Mean ± SD of alveolar bone loss (millimeters) for ligated and unligated teeth.

https://doi.org/10.1371/journal.pone.0174442.t001

RF and ACCPA levels

In T1 (day 0 –after RA induction and prior to EP induction), the levels of RF were higher in EP+RA and RA groups when compared to control and EP groups (p < 0.05) (Table 2 –Table B in S1 File). However, in the EP+RA group, serum levels of RF increase from T1 to T2 (p < 0.05). Thus, at T2 (day 28 after EP and the time of euthanasia) the EP+RA group presented the highest serum concentration of RF (p<0.05), which was also higher than the RA group (p<0.05). Moreover, the RA group still had higher levels of RF compared to the control and EP groups (p < 0.05).

thumbnail
Table 2. Means ± SD of rheumatoid factor (RF) serum levels (U/ml) measured by ELISA assay.

https://doi.org/10.1371/journal.pone.0174442.t002

Considering serum levels of ACCPA, the EP+RA and RA groups had higher amounts of anti-citrullinated protein antibody than the control and EP groups in T1 (p < 0.05). Interestingly, ACCPA showed a continuous increase over time in both RA and EP+RA, while no significant changes occurred in the control or EP groups. In T2, the control group presented lower values of ACCPA when compared to EP+RA and RA (p < 0.05). (Table 3 –Table C in S1 File). In gingival tissues, collected at the end of the experimental period (T2), the EP+RA group had the highest value of ACCPA (p<0.05), although the EP group presented higher levels than the RA, but not than control groups (p < 0.05) (Table 3 -Table C in S1 File).

thumbnail
Table 3. Means ± SD of anti-citrullinated protein anti-body (ACCPA) serum and gingival levels (U/ml) measured by ELISA assay.

https://doi.org/10.1371/journal.pone.0174442.t003

Gingival tissue interleukin

Multiplex analysis of inflammatory markers in gingival tissues revealed a higher level of IL-1β in the EP+RA, RA and EP groups than the control one (p<0.05). Considering IL-4 concentration, there was no difference among the groups (p>0.05). The levels of IL-6 and TNF-α were higher in the EP+RA and RA groups, when compared with the control group (p < 0.05). Interestingly, the IL-17 amount was higher in the EP+AR and RA groups when compared to the control and EP groups (p<0.05). (Table 4 - Table D in S1 File).

thumbnail
Table 4. Means ± SD of the mean of IL-4, IL-1β, IL-6, IL-17 and TNF-α concentrations (picograms per milliliter) measured by multiplex assay.

https://doi.org/10.1371/journal.pone.0174442.t004

Discussion

There is some evidence about the bidirectional pathway to the development of rheumatoid arthritis and periodontitis, although there is no clear pathway by which it occurs. This study evaluated local and systemic markers in arthritic rats submitted to experimental periodontitis. The results show that, in the presence of RA, periodontal breakdown was higher than PE alone, although it was effective in promoting more bone resorption compared to the control and RA groups. Interestingly, although RA and/or EP modulate the local release of cytokines, the RA, per se, seems to increase the local production of IL-17. However, when RA and PE were simultaneously induced, ACCPA and RF were directly affected by the presence of gingival inflammation, increasing their levels, including the local levels of ACCPA. Additionally, the clinical feature of RA, paw swelling, appear to be worsened by periodontitis.

It has been shown that Th17 profile has a critical role in RA and collagen-induced arthritis pathology [32, 33]. IL-17, a proinflammatory cytokine produced by RA synovium [32], stimulates the macrophages’ production of IL-1 and TNF-a [34] and activates human synoviocytes to produce IL-6, IL-8, GM-CSF, and PGE2 [35, 36]. When endogenous IL-17 was blocked, amelioration of collagen-induced arthritis was observed at the same time as its overexpression enhanced synovial inflammation and joint destruction [33]. In the same way, IL-17 is also related to periodontal tissue destruction. Th17-type response triggers osteoclastogenesis, as well as induction of the receptor activator of nuclear factor-kB ligand (RANKL) [37, 38, 39]. In the present study, greater values of IL-17 was observed only when RA was present, in spite of PE, confirming the major role of arthritis in IL-17 production indirectly collaborating to periodontal breakdown. However, the fact that nor IL-17, neither IL-1β, TNF-a, IL-6 or IL-4 were specifically increased/decreased when both pathologies were induced, and the higher periodontal destruction observed at EP+RA, might be related to other inflammatory mechanisms. Queiroz-Junior et al. [40] associated RA and EP in mice and found enhanced expression of the Th1 immune responses associated with worse RA [41] and EP [42] prognosis. Additionally, the same authors observed a reduction in the expression of transcription factors associated with the control of EP (T regulatory cells—Foxp3), and the Th2 response (GATA-3) [5]. This could help to explain the worst periodontal conditions observed in subjects affected by RA observed in clinical studies [8, 9].

On the other hand, some recent findings have indicated that periodontal disease can worsen the severity of collagen-induced arthritis [16, 19, 43]. De Aquino et al. [16] used a bacteria periodontitis inducing model (using mixed P. nigrescens and P. gingivalis) and showed an exacerbated arthritis, with increased arthritic bone erosion, by the activation of CII-specific T cell response toward the Th17 phenotype without affecting Th1. The authors found that the levels of IL-17 induced by periodontal pathogens in CII-specific T cells was correlated with the intensity of arthritic bone erosion.

Another point of view is the potential of periodontal pathogens in modulate “destructive” arthritic markers. In the present study, experimental periodontitis was induced in previously arthritic rats (RA was induced 21 days before). Interestingly, greater amount of ACCPA in the serum and gingival tissue of the EP+RA group than in RA or EP alone (and control group, as well) was seen. This phenomenon could be linked to the presence of periodontal-pathogens in subgingival biofilm [44], once the model was described as a plumicrobial infection [45]. Although the present study has used the ligature-induced periodontitis model and has not performed identification analysis, the microbial profile observed by other authors in the ligature model was similar to that observed in human periodontitis [46, 47]. Through a DNA-DNA checkerboard analysis, a previous study identify microbial profile in subgingival environment after ligature-induced periodontitis and found streptococcus- and Actinomyces-like species in high mean counts, followed by Fusobacterium-, Prevotella nigrescens-, Parvimonas micra-, Aggregatibacter actinomycetemcomitans- and Porphyromonas gingivalis-like species [47]. Another study evaluated the presence and concentrations of Porphyromonas gingivalis, Tannerella forsythia and Aggregatibacter actinomycetemcomitans in the cotton ligatures used to induce periodontitis by real-time PCR and it was possible to identify and quantify all the three species in the analyzed samples [46]. This reaffirm the reliability of ligature model to mimic the human periodontal infection. The Red Complex (Porphyromonas Gingivalis, Treponema denticola and Tannerella forsythia) [47] found in the subgingival plaque, is the main group of bacteria related to periodontitis. Among the three species of red complex, Pg has been related to the pathogenesis of RA and may be important in the disease development processes because of its capacity of citrullinate proteins by releasing peptidylarginie deiminase [14, 15]. Recently, Aggregatibacter actinomycetemcomitans (Aa) was identified as a potencial trigger of autoimmunity in RA. The study observed induced hypercitrullination in host neutrophils by the pore-forming toxin leukotoxin A (LtxA), reproducing membranolytic pathways that occurs in the RA joint, supporting the autoantigen citrullination [48]. The authors also verified that LtxA induced changes in neutrophil morphology with the release of hypercitrullinated proteins. Additionaly, exposure to leukotoxic Aa strains was confirmed in patients with RA and periodontitis and was associated with both anticitrullinated protein antibodies and rheumatoid factor levels [48]. Interestingly, these authors did not find association of Pg with hipercitrullination in neutrophils in periodontitis samples [48]. Our results also showed that the association of periodontitis and rheumatic arthritis increased the serum levels of citrullinated proteins from T1 (the day of EP induction) to T2 (euthanasia). Interestingly, at the end of the experimental period, the levels of ACCPA in the EP group were similar to the groups with RA. This is in accordance with the results of Konig et al. [48], which demonstrated that LtxA from Aa hypercitrullinated neutrophis in similar process of RA joint. Accordingly, the serum levels of rheumatoid factor increased after the association of EP with experimental RA and the amount of this substance was greater in the EP+RA group when compared to the other groups at the end of the experiment. Thé and Ebersole [49] showed that the RF of seropositive patients show a cross-reaction with oral bacterial epitopes. Moreover, Bonagura et al. [50] verified that Pg proteinase is responsible for the epitope development in the RF-Fc (region of an antibody that interacts with cell surface receptors) region, probably due to the capacity of Pg in decompose lysine and arginine [51, 52], once both aminoacids were found in Fc regions of the IgG molecule [53]. Konig et al. [48] verified significant association of anti-LxtA positivity in RA and ACCPA and RF, when compared to anti-LtxA antibody–negative RA. In this vein, it takes clear that Pg and Aa play an important role in the RF production of rheumatoid cells and their increase in arthritic rats after periodontitis induction.

Meanwhile, the discussion of the possible clinical impact of these findings is important. Rheumatoid factor and the anti-citrullinated protein antibody are highly specific and sensitive markers for rheumatoid arthritis and, importantly, they have been associated with poor outcomes of rheumatoid arthritis, such as increased disease activity, radiographic progression and disability [54, 55, 56]. Besides, ACCPA can be detected very early and predict clinical disease outcome [54, 55, 56]. Considering the fact that paw swelling, a clinical aspect of experimental RA, also showed an impact of periodontal disease, our findings could confirm the negative influence of periodontitis on the development and exacerbation of rheumatoid arthritis; this was show in some previous clinical trials [7, 8, 9] and explains how oral conditions jeopardize arthritic conditions.

In the meantime, the citrullination of protein is a process that can also influence both diseases. Pg-associated citrullination by peptidylarginine deiminase enhance its capacity of surviving in biofilms [50, 51]. On the other hand, citrullinated residues are generated by deamination of the guanidino group of carboxyterminal arginine residues on a variety of peptides, producing citrulline and free ammonia, by a peptidylarginine deiminase. Ammonia interferes with neutrophil function [57, 58] and protects against acidic cleansing cycles of mouth. Moreover, LtxA from Aa changes neutrophil morphology [48] and this may also interferes with its function. Besides, citrullination alters the activity of the complement system, inactivates epidermal growth factors and induces the production of prostaglandin, E2, with all events enrolled in the periodontal destruction [59, 60, 61, 62]. Thus, the increased ACCPA, not only deteriorate arthritic status, but, once it is increased in gingival tissues, could also affect the periodontal destruction. This is the first study to assess locally produced ACCPA and this innovative finding should be evaluated in the future regarding its effect on periodontal breakdown.

In general, our findings confirmed some previously reported conclusions. Rheumatoid arthritis can worsen periodontal destruction, as well as periodontitis altering some arthritic markers, as shown in the analysis of paw swelling. Only the RA groups (RA+EP and RA) presented differences related to the baseline. Differences was observed among groups presenting RA (RA+EP and RA) and other groups without RA (EP and Control). Interestingly, when EP was associated with RA, the paw swelling remained in progress until the end of the experiment, with a significant difference when compared to EP and control. This result is in agreement with Cantley’s study [19], where the association of arthritis and periodontitis resulted in higher paw swelling over the time when compared to RA alone. Besides, some new findings can highlight and strengthen this bidirectional relationship. ACCPA level increase in gingival tissues also should be considered to explain this interaction, as well as the role of TH17 pathway on both diseases. The evident impact of periodontitis in rheumatoid factor levels also brings new light to this. All of these findings, based on shared patterns, could reinforce the idea of a bidirectional relationship between them.

Thus, with the limits of this study, it can be concluded that rheumatoid arthritis enhances periodontal tissue destruction. RA alone does not enhance bone loss, but increases the concentration of inflammatory cytokines, especially IL-17. Periodontitis increases the serum levels of rheumatoid factor, as well as the gingival tissue concentration of ACCPA, confirming the bidirectional aspect of both diseases.

Supporting information

S1 File. Supporting Information underling the findings.

https://doi.org/10.1371/journal.pone.0174442.s001

(XLSX)

Author Contributions

  1. Conceptualization: MGC FVR SPP RCVC FRC MZC.
  2. Data curation: MGC RCVC MZC.
  3. Formal analysis: MGC RCVC MZC.
  4. Funding acquisition: RCVC MZC.
  5. Investigation: MGC FVR SPP RCVC FRC MZC.
  6. Methodology: MGC SBS FVR SPP RCVC FRC MZC.
  7. Project administration: MGC FVR SPP RCVC FRC MZC.
  8. Resources: MGC FVR SPP RCVC FRC MZC.
  9. Supervision: RCVC MZC.
  10. Validation: MGC RCVC.
  11. Visualization: MGC SBS RCVC MZC.
  12. Writing – original draft: MGC.
  13. Writing – review & editing: MGC RCVC MZC.

References

  1. 1. Tobon GJ, Youinou P, Saraux A. The environment, geo-epidemiology, and autoimmune disease: rheumatoid arthritis. J Autoimmun 2010; 35(1):10e4.
  2. 2. Vossenaar ER, van Venrooij WJ. Citrullinated proteins: sparks that may ignite the fire in rheumatoid arthritis. Arthritis Res Ther 2004; 6(3):107e11.
  3. 3. Kinane DF, Preshaw PM, Loos BG. Working Group 2 of Seventh European Workshop on Periodontology. Host-response: Understanding the cellular and molecular mechanisms of host-microbial interactions—Consensus of the Seventh European Workshop on Periodontology. J Clin Periodontol 2011; 38: 44–48.
  4. 4. Hitchon CA, El-Gabalawy HS. Infection and rheumatoid arthritis: still an open question. Curr Opin Rheumatol 2011; 23(4):352–7. pmid:21532483
  5. 5. Garlet GP, Cardoso CR, Mariano FS, Claudino M, de Assis GF, Campanelli AP, et al. Regulatory T cells attenuate experimental periodontitis progression in mice. J Clin Periodontol 2010; 37(7):591–600. pmid:20642629
  6. 6. Moynagh PN. The NF-kappaB pathway. J Cell Sci. 2005; 15(Pt20):4589–92.
  7. 7. de Pablo P, Dietrich T, McAlindon TE. Association of periodontal disease and tooth loss with rheumatoid arthritis in the US population. J Rheumatol 2008; 5(1):70e6.
  8. 8. Mercado F, Marshall RI, Klestov AC, Bartold PM. Is there a relationship between rheumatoid arthritis and periodontal diseases? J Clin Periodontol 2000; 27(4): 267–272. pmid:10783841
  9. 9. Mercado FB, Marshall RI, Klestov AC, Bartold PM. Relationship between rheumatoid arthritis and periodontitis. J Periodontol 2001; 72(6): 779–787. pmid:11453241
  10. 10. Ribeiro J, Leao A, Novaes AB. Periodontal infection as a possible severity factor for rheumatoid arthritis. J Clin Periodontol 2005; 32(4): 412–416. pmid:15811060
  11. 11. Havemose-Poulsen A, Westergaard J, Stoltze K, Skjødt H, Danneskiold-Samsøe B, Locht H, et al. Periodontal and hematological characteristics associated with aggressive periodontitis, juvenile idiopathic arthritis, and rheumatoid arthritis. J Periodontol 2006; 77(2): 280–288. pmid:16460255
  12. 12. Al-Katma MK, Bissada NF, Bordeaux JM, Sue J, Askari AD. Control of periodontal infection reduces the severity of active rheumatoid arthritis. J Clin Rheumatol 2007; 13(3): 134–137. pmid:17551378
  13. 13. Ortiz P, Bissada NF, Palomo L, Han YW, Al-Zahrani MS, Panneerselvam A, et al. Periodontal therapy reduces the severity of active rheumatoid arthritis in patients treated with or without tumor necrosis factor inhibitors. J Periodontol 2009; 80(4): 535–540. pmid:19335072
  14. 14. Rosenstein ED, Greenwald RA, Kushner LJ, Weissmann G. Hypothesis: the humoral immune response to oral bacteria provides a stimulus for the development of rheumatoid arthritis. Inflammation 2004; 28(6): 311–318. pmid:16245073
  15. 15. Wegner N, Lundberg K, Kinloch A, Fisher B, Malmström V, Feldmann M, et al. Autoimmunity to specific citrullinated proteins gives the first clues to the etiology of rheumatoid arthritis. Immunology Reviews 2010; 2331(1): 34–54.
  16. 16. de Aquino SG, Abdollahi-Roodsaz S, Koenders MI, van de Loo FA, Pruijn GJ, Marijnissen RJ, et al. Periodontal pathogens directly promote autoimmune experimental arthritis by inducing a TLR2- and IL-1-driven Th17 response. J Immunol 2014; 192(9):4103–11. pmid:24683190
  17. 17. Gully N, Bright R, Marino V, Marchant C, Cantley M, Haynes D, et al. Porphyromonas gingivalis peptidylarginine deiminase, a key contributor in the pathogenesis of experimental periodontal disease and experimental arthritis. PLoS One 2014; 9(6):e100838. pmid:24959715
  18. 18. Marchesan JT, Gerow EA, Schaff R, Taut AD, Shin SY, Sugai J, et al. Porphyromonas gingivalis, oral infection exacerbates the development and severity of collagen-induced arthritis. Arthritis Res Ther 2013;15(6): R186. pmid:24456966
  19. 19. Cantley MD, Haynes DR, Marino V, Bartold PM. Pre-existing periodontitis exacerbates experimental arthritis in a mouse model. J. Clin.Periodontol. 2011; 38(6): 532–541. pmid:21434962
  20. 20. Hara Y, Kaneko T, Yoshimura A, Kato I. Serum rheumatoid factor induced by intraperitoneal administration of periodontopathic bacterial lipopolysaccharide in mice. J Periodontal Res. 1996; 31(7):502–7. pmid:8915954
  21. 21. Okada M, Kobayashi T, Ito S, Yokoyama T, Komatsu Y, Abe A, et al. Antibody responses to periodontopathic bacteria in relation to rheumatoid arthritis in Japanese adults. J Periodontol. 2011; 82(10):1433–41. pmid:21342003
  22. 22. Ramamurthy NS, Greenwald RA, Celiker MY, Shi EY. Experimental arthritis in rats induces biomarkers of periodontitis which are ameliorated by gene therapy with tissue inhibitor of matrix metalloproteinases. J Periodontol. 2005;76(2):229–33. pmid:15974846
  23. 23. Lohman RJ, Cotterell AJ, Barry GD, Liu L, Suen JY, Vesey DA, et al. An antagonist of human protease activated receptor-2 attenuates PAR2 signaling, macrophage activation, mast cell degranulation, and collagen-induced arthritis in rats. FASEB J. 2012;26(7):2877–87. pmid:22467762
  24. 24. Sitônio MM, Carvalho Júnior CH, Campos Ide A, Silva JB, Lima Mdo C, Góes AJ, et al. Anti-inflammatory and anti-arthritic activities of 3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b]pyran-5,6-dione (β-lapachone). Inflamm Res. 2013;62(1):107–13 pmid:23052183
  25. 25. Barrella GE, Suffredini IB, Ribeiro FV, Cirano FR, Pimentel SP. Evaluation of the effect of an organic extract obtained from Ipomoea alba L. on experimental periodontitis in rats. Braz Oral Res. 2012;26(2): 158–164. pmid:22473352
  26. 26. Casati MZ, Algayer C, Cardoso da Cruz, Ribeiro FV, Casarin RC, Pimentel SP, et al. Resveratrol decreases periodontal breakdown and modulates local levels of cytokines during periodontitis in rats. J Periodontol. 2013; 84(10): 58–64.
  27. 27. Pimentel SP, Barrella GE, Casarin RC, Cirano FR, Casati MZ, Foglio MA, et.al. Protective effect of topical Cordia verbenacea in a rat periodontitis model: immune-inflammatory, antibacterial and morphometric assays. BMC Complement Altern Med. 2012; 21:12:224.
  28. 28. Milici AJ, Kudlacz EM, Audoly L, Zwillich S, Changelian P. Cartilage preservation by inhibition of Janus kinase 3 in two rodent models of rheumatoid arthritis. Arthritis Res Ther. 2008;10(1):R14. pmid:18234077
  29. 29. Orhan CE, Önal A, Uyanıkgil Y, Ülker S. Antihyperalgesic and antiallodynic effect of sirolimus in rat model of adjuvant arthritis. Eur J Pharmacol. 2013 Apr 5;705(1–3):35–41. pmid:23461852
  30. 30. Umukoro S, Oluwole OG, Eduviere AT, Adrian OI, Ajayi AM. Jobelyn® exhibited anti-inflammatory, antioxidant, and membrane-stabilizing activities in experimental models. J Basic Clin Physiol Pharmacol. 2015 Sep;26(5):501–8. pmid:26020554
  31. 31. Singh S, Kumar R, Jain H, Gupta YK. Anti-inflammatory and antiarthritic activity of UNIM-301 (a polyherbal unani formulation) in Wistar rats. Pharmacognosy Res. 2015 Apr-Jun;7(2):188–92. pmid:25829793
  32. 32. Chabaud M, Fossiez F, Taupin JL, Miossec P. Enhancing effect of IL-17 on IL-1-induced IL-6 and leukemia inhibitory factor production by rheumatoid arthritis synoviocytes and its regulation by Th2 cytokines. J. Immunol. 1998; 161(1):409. pmid:9647250
  33. 33. Lubberts E, Joosten LA, Oppers B, van den Bersselaar L, Coenen-de Roo CJ, Kolls JK, et al. IL-1-independent role of IL-17 in synovial inflammation and joint destruction duringCollagen-induced arthritis. J. Immunol. 2001; 15(2): 1004–1013.
  34. 34. Jovanovic DV, DiBattista JA, Martel-Pelletier , Jolicoeur FC, He Y, Zhang M, et al. IL-17 stimulates the production and expression of proinflammatory cytokines, IL-1b and TNF-a, by human macrophages. J. Immunol. 1998; 160(7):3513. pmid:9531313
  35. 35. Fossiez F, Djossou O, Chomarat P, Flores-Romo L, Ait-Yahia S, Maat C, et al. T-cell IL-17 induces stromal cells to produce proinflammatory and hematopoietic cytokines. J Exp Med 1996; 183(6):2593. pmid:8676080
  36. 36. Chabaud J.M. Durand N. Buchs, Fossiez F, Page G, Frappart L, et al. Human interleukin-17: a T cell-derived proinflammatory cytokine produced by the rheumatoid synovium. Arthritis Rheum 1999; 42(5): 963–970. pmid:10323452
  37. 37. Dutzan N, Gamonal J, Silva A, Sanz M, Vernal R. Overexpression of forkhead box P3 and its association with receptor activator of nuclear factor-kappa B ligand, interleukin (IL) -17, IL-10 and transforming growth factor-beta during the progression of chronic periodontitis. J Clin Periodontol 2009;36(5):396–403. pmid:19419438
  38. 38. Sato K, Suematsu A, Okamoto K, Yamaguchi A, Morishita Y, Kadono Y, et al. Th17 functions as an osteoclastogenic helper T cell subset that links T cell activation and bone destruction. J Exp Med. 2006; 203(12):2673–2682. pmid:17088434
  39. 39. Mitani A, Niedbala W, Fujimura T, Mogi M, Miyamae S, Higuchi N, et al. Increased expression of interleukin (IL)-35 and IL-17, but not IL-27, in gingival tissues with chronic periodontitis. J Periodontol 2015; 86(2):301–9. pmid:25272982
  40. 40. Queiroz-Junior CM, Madeira MF, Coelho FM. Experimental arthritis exacerbates Aggregatibacter actinomycetemcomitans-induced periodontitis in mice. J Clin Periodontol 2012; 39(7):608–16. pmid:22582749
  41. 41. Firestein GS. Immunologic mechanisms in the pathogenesis of rheumatoid arthritis. J Clin Rheumatol 2005; 11 (3 Suppl):39–44.
  42. 42. Garlet GP, Cardoso CR, Campanelli AP, Garlet TP, Avila-Campos MJ, Cunha FQ, et al. The essential role of INF-gamma in the control of lethal Aggregatibacter actinomycetemcomitans infection in mice. Microbes Infect 2008; 10(5): 489–96. pmid:18403243
  43. 43. Bartold PM, Marino V, Cantley M, Haynes DR. Effect of Porphyromonas gingivalis-induced inflammation on the development of rheumatoid arthritis. J Clin Periodontol 2010; 37(5): 405–411. pmid:20507365
  44. 44. Duarte PM, Tezolin KR, Figueiredo LC, Feres M, Bastos MF. Microbial profile of ligature-induced periodontitis in rats. Arch Oral Biol 2010; 55(2):142–7. pmid:19931851
  45. 45. Listgarten MA. Pathogenesis of periodontitis. J Clin Periodontol 1986;13:418–430. pmid:3522650
  46. 46. Cirano FR, Casarin RCV, Ribeiro FR, Casati MZ, Pimentel SP, Taiete T, et al. Effect of Resveratrol on periodontal pathogens during experimental periodontitis in rats. Braz. Oral Res. 2016;30(1):e128. pmid:27901209
  47. 47. Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL Jr. Microbial complexes in subgingival plaque. J Clin Periodontol. 1998 Feb;25(2):134–44. pmid:9495612
  48. 48. Konig MF, Abusleme L, Reinholdt J, Palmer RJ, Teles RP, Sampson K, et al. Aggregatibacter actinomycetemcomitans-induced hypercitrullination links periodontal infection to autoimmunity in rheumatoid arthritis. Sci Transl Med. 2016; 14;8(369):369ra176.
  49. 49. Thé J, Ebersole JL. Rheumatoid factor from periodontitis patients crossreacts with epitopes on oral bacteria. Oral Dis 1996; 2(4): 253–262. pmid:9171508
  50. 50. Bonagura VR, Artandi SE, Davidson A, Randen I, Agostino N, Thompson K, et al. Mapping studies reveal unique epitopes on IgG recognized by rheumatoid arthritis-derived monoclonal rheumatoid factors. J Immunol 1993; 151(7): 3840–3852. pmid:7690818
  51. 51. Bedi GS. Purification and characterization of lysine- and arginine-specific gingivain proteases from Porphyromonas gingivalis. Prep Biochem. 1994; 24(3–4): 251–61. pmid:7831206
  52. 52. Pike R, McGraw W, Potempa J, Travis J. Lysine and arginine specific proteinases from Porphyromonas gingivalis. Isolation, characterization, and evidence for the existence of complexes with hemagglutinins. J Biol Chem 1994; 269(1): 406–411. pmid:8276827
  53. 53. Bonagura VR, Artandi SE, Davidson A, et al. Mapping studies reveal unique epitopes on IgG recognized by rheumatoid arthritis-derived monoclonal rheumatoid factors. J Immunol 1993; 151(7): 3840–3852. pmid:7690818
  54. 54. Liao F, Li Z, Wang Y, Shi B, Gong Z, Cheng X. Porphyromonas gingivalis may play an important role in the pathogenesis of periodontitis-associated rheumatoid arthritis. Med Hypotheses 2009; 72(6): 732–5. pmid:19246161
  55. 55. Vander Cruyssen B, Peene I, Cantaert T, Hoffman IE, De Rycke L, Veys EM, et.al. Anti-citrullinated protein/peptide antibodies (ACPA) in rheumatoid arthritis: specificity and relation with rheumatoid factor. Autoimmun Rev 2005; 4(7):468–74. pmid:16137613
  56. 56. Humphreys JH, van Nies JA, Chipping J, Marshall T, van der Helm-van Mil AH, Symmons DP, et al. Rheumatoid factor and anti-citrullinated protein antibody positivity, but not level, are associated with increased mortality in patients with rheumatoid arthritis: results from two large independent cohorts. Arthritis Res Ther 2014; 16(6):483. pmid:25471696
  57. 57. Niederman R, Brunkhorst B, Smith S, Weinreb RN, Ryder MI. Ammonia as a potential mediator of adult human periodontal infection: inhibition of neutrophil function. Arch Oral Biol 1990; 35(Supp):205S–9S.
  58. 58. Shawcross DL, Wright GAK, Stadlbauer V, Hodges SJ, Davies NA, Wheeler-Jones C, et al. Ammonia impairs neutrophil phagocytic function in liver disease. Hepatol Baltim Md 2008; 48(4):1202–12.
  59. 59. Shirai H, Mokrab Y, Mizuguchi K. The guanidino-group modifying enzymes: structural basis for their diversity and commonality. Proteins 2006; 64(4):1010–23. pmid:16779844
  60. 60. McGraw WT, Potempa J, Farley D, Travis J. Purification, characterization, and sequence analysis of a potential virulence factor from Porphyromonas gingivalis, peptidylarginine deiminase. Infect Immun 1999; 67(7):3248–56. pmid:10377098
  61. 61. Marquis RE, Bender GR, Murray DR, Wong A. Arginine deiminase system and bacterial adaptation to acid environments. Appl Environ Microbiol 1987; 53(1):198–200. pmid:3103530
  62. 62. Casiano-Colón A, Marquis RE. Role of the arginine deiminase system in protecting oral bacteria and an enzymatic basis for acid tolerance. Appl Environ Microbiol. 1988; 54(6):1318–24. pmid:2843090