J Periodont Res 2016; 51: 103–111 All rights reserved

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd JOURNAL OF PERIODONTAL RESEARCH doi:10.1111/jre.12288

Expression of antiPorphyromonas gingivalis peptidylarginine deiminase immunoglobulin G and peptidylarginine deiminase-4 in patients with rheumatoid arthritis and periodontitis

A. Shimada1, T. Kobayashi1,2, S. Ito3, M. Okada1, A. Murasawa3, K. Nakazono3, H. Yoshie1 1 Division of Periodontology, Department of Oral Biological Science, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan, 2General Dentistry and Clinical Education Unit, Niigata University Medical and Dental Hospital, Niigata, Japan and 3Niigata Rheumatic Center, Shibata, Japan

Shimada A, Kobayashi T, Ito S, Okada M, Murasawa A, Nakazono K, Yoshie H. Expression of anti-Porphyromonas gingivalis peptidylarginine deiminase immunoglobulin G and peptidylarginine deiminase-4 in patients with rheumatoid arthritis and periodontitis. J Periodont Res 2016; 51: 103–111. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Background and Objective: Autoimmunity against citrullinated proteins through peptidylarginine deiminase (PAD) may be involved in the pathophysiology of rheumatoid arthritis (RA). The present study evaluated the serum levels of antibodies to citrullinated proteins and to Porphyromonas gingivalis PAD (PPAD), and the endogenous expression of PAD-4, in individuals with and without RA, as well as before and after periodontal treatment. Material and Methods: The study participants consisted of 52 patients with RA (RA group) and 26 age-, gender- and smoking status-matched healthy controls (non-RA group). Of the 52 patients, 26 were randomly assigned to receive oral hygiene instruction and supragingival scaling (RA subgroup). After periodontal and rheumatologic assessments, the serum levels of anti-cyclic citrullinated peptide (CCP) immunoglobulin G (IgG), anti-PPAD IgG and PAD-4 were determined using ELISA. Results: The serum levels of anti-CCP IgG and anti-PPAD IgG were significantly higher in the RA group than in the non-RA group (p < 0.001 and p = 0.03). A significant, positive correlation was observed between the serum levels of anti-PPAD IgG and anti-CCP IgG (p = 0.04), but not between the serum levels of PAD-4 and anti-CCP IgG. Multiple logistic regression analyses revealed a significant association between anti-PPAD IgG responses and RA after adjustment for age, gender and smoking (p = 0.004). Supragingival scaling significantly improved the periodontal condition and disease activity of RA (p < 0.05), but failed to decrease the serum levels of anti-CCP IgG, anti-PPAD IgG and PAD-4 after 2 mo of treatment.

Tetsuo Kobayashi, DDS, PhD, Division of Periodontology, Department of Oral Biological Science, Niigata University Graduate School of Medical and Dental Sciences, 2-5274 Gakkocho-dori, Chuo-ku, Niigata 951-8514, Japan Tel: (+81) 25-227-2870 Fax: (+81) 25-227-0808 e-mail: [email protected] Key words: peptidylarginine deiminase;

periodontitis; Porphyromonas gingivalis; rheumatoid arthritis Accepted for publication April 1, 2015

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Conclusion: These results might suggest an association between anti-PPAD IgG and anti-CCP IgG responses, implicating a role for PPAD in protein citrullination in patients with RA and periodontitis.

Rheumatoid arthritis (RA) is a systemic autoimmune disease of unknown etiology characterized by synovial inflammation and destruction of cartilage and bone in the joints, which results in functional disability (1). Autoantibodies to anti-cyclic citrullinated peptide (CCP) have been shown to be a specific marker that can be detected years before the onset of RA, and their presence and levels in serum correlate strongly with the severity of RA (2,3). Protein citrullination is carried out by endogenous peptidylarginine deiminases (PADs), enzymes that catalyze the post-translational modification reaction of arginine residues to citrulline (4). Five mammalian PAD family members (PAD-1 to PAD-4 and PAD-6), encoded by five genes clustered on chromosome 1p35-36, have been described and exhibit tissue-specific distribution in most body tissues (4,5). Of these, PAD-4 has been shown as the candidate isotype that is most closely associated with RA, according to genetic and immunohistochemical analyses (6–10). Periodontitis represents a chronic inflammatory disease characterized by local inflammation and destruction of the periodontal tissue and shares many pathologic features with RA (11,12). It has been documented that periodontitis is linked to a biofilm that contains a consortium of oral anaerobic pathogens, including Porphyromonas gingivalis (13). It has been reported that P. gingivalis expresses PAD (known as PPAD) (14), and that PPAD can generate citrullinated host peptides among oral bacterial pathogens (15). The serum levels of anti-PPAD immunoglobulin G (IgG) were elevated in patients with RA compared with patients with periodontitis and healthy control individuals (16). Recently, infection with a PPAD-null mutant was reported to decrease the serum levels of anti-CCP

IgG and the severity of arthritis in an established mouse model (17,18). These observations suggest that infection with P. gingivalis may have a profound impact on the onset and progression of RA via protein citrullination through PPAD. In contrast, recent data indicated that serum levels of anti-PPAD IgG did not correlate with those of anti-CCP IgG (19,20), suggesting that anti-PPAD antibodies were not part of the antiCCP response. In the light of these findings, it is required to evaluate simultaneously the roles of exogenous PPAD and endogenous PAD in protein citrullination in patients with RA, which has not yet been studied. Therefore, the aim of the present study was to compare the serum levels of anti-PPAD IgG and PAD-4 in individuals with and without RA. Furthermore, the effects of nonsurgical periodontal treatment were also evaluated on the same parameters in the present study.

Material and methods

interleukin-6 (IL-6) receptor at the time of the examination. Of the individuals referred to the Periodontics Department of the Niigata University Medical and Dental Hospital, 26 race-, age- and gender-matched control individuals, with no signs of RA and other systemic diseases (21 female subjects and five male subjects; 49– 78 years of age; mean age: 59.9 years) (non-RA group) were included from October 2010 to January 2013. The study was approved by the Institutional Review Board of the Niigata University Faculty of Dentistry (no. 22-R9-10-06, on June 16, 2010) and the Niigata Rheumatic Center (no. 1, on April 26, 2010). Exclusion criteria were as follows: the presence of diabetes mellitus and pregnancy; a history or the presence of any periodontal therapy within the previous 3 mo; and having fewer than 15 teeth. All participants were classified as current-smokers, former-smokers or never-smokers, according to information provided on a standard questionnaire.

Participants

Clinical assessments

Fifty-two Japanese adults with RA (44 female subjects and eight male subjects; 30–82 years of age; mean age: 62.0 years) (RA group), followed at the Niigata Rheumatic Center, Shibata, Japan, were recruited between July 2010 and January 2013 for the study. All patients with RA fulfilled the 1987 revised classification criteria of the American Rheumatism Association (21), as well as the 2010 RA classification criteria of the American College of Rheumatology and European League Against Rheumatism (1). All the patients took corticosteroids, disease-modifying antirheumatic drugs (DMARDs), or nonsteroidal antiinflammatory drugs (NSAIDs), but did not receive any inhibitors of tumor necrosis factor (TNF) and

Clinical periodontal assessments were performed by one trained and calibrated examiner (T.K.) who was masked from the rheumatologic data. The calibration was performed before the study with five volunteer subjects in Niigata University Faculty of Dentistry. Reproducibility of the clinical measurements was calculated using the intra-examiner intraclass correlation coefficient, and a value of 0.90 was obtained for clinical attachment level with a difference of  1 mm. All participants were evaluated clinically in the following measurements: number of teeth present; probing depth; clinical attachment level; supragingival plaque accumulation; and bleeding on probing (BOP). The presence or absence of supragingival plaque and

Anti-P. gingivalis PAD IgG in RA BOP were recorded at four and six sites around each tooth, respectively. Measurements of probing depth and clinical attachment level were conducted using a Williams probe at six sites around each tooth, recorded to the nearest millimeter, and every observation close to 0.5 mm was rounded to the lower whole number. The averaged score for whole-mouth probing depth, clinical attachment level and the number of sites with supragingival plaque and BOP, divided by the total number of sites per mouth and multiplied by 100, were calculated for each subject. The presence of periodontitis was defined as having at least one site with clinical attachment level of ≥ 3 mm, and severity of periodontitis was determined according to the classification of the American Academy of Periodontology (22,23). The disease activity of RA was determined using the Disease Activity Score in 28 joints using C-reactive protein (DAS28-CRP), which is obtained with a formula that takes into account the number of tender and swollen joints (the tender joint count and the swollen joint count, respectively), the patient’s general assessment of their condition scored on a visual analog scale and CRP. The DAS28-CRP has four categories: remission (DAS28-CRP < 2.3); low disease activity (2.3 ≤ DAS28-CRP < 2.7); moderate disease activity (2.7 ≤ DAS28-CRP < 4.1); and high disease activity (4.1 ≤ DAS28-CRP) (24). After the clinical assessments, 26 patients (22 female subjects and four male subjects; 30–82 years of age; mean age: 60.7 years) in the RA group (RA subgroup) were randomly assigned to receive periodontal treatment, including oral hygiene instruction and full-mouth supragingival scaling with ultrasonic instruments without local anesthesia. These periodontal treatments were performed once by one calibrated clinician (M.O.) and were not accompanied by any medications. The clinical reassessments were performed after 2 mo of periodontal treatment, as described above.

Measurements of serum rheumatoid factor, anti-CCP IgG, and CRP, IL-6, TNF-a and PAD-4 levels

Peripheral venous blood samples were obtained by venipuncture from all participants after the clinical assessments. Serum was isolated from the blood by centrifugation at 1500 g for 20 min, and stored at 70°C until used. Serum concentrations of rheumatoid factor (RF) and high-sensitive CRP were determined using a latex particleenhanced and a simple nephelometric method (SRL, Tokyo, Japan). The serum levels of anti-CCP IgG, and of IL-6, TNF-a and PAD-4, were determined using sensitive ELISAs from commercially available kits (anti-CCP IgG: Medical & Biological Laboratories, Aichi, Japan; IL-6 and TNF-a: Quantikine ELISA kit, R&D Systems, Minneapolis, MN, USA; and PAD-4: MyBioSource, San Diego, CA, USA), according to the manufacturer’s instructions. The microtiter plates were read at a wavelength of 450 nm for anti-CCP IgG and PAD-4, or at 490 nm for IL-6 and TNF-a, using an automated microplate reader (Bio-Rad Japan Laboratories, Tokyo, Japan). The lower limits of detection for these measurements were as follows: RF, 1.25 IU/mL; anti-CCP IgG, 0.4 U/ mL; CRP, 0.004 mg/dL; IL-6, 0.016 pg/mL; TNF-a, 0.038 pg/mL; and PAD-4, 0.039 ng/mL. Positivity of RF was defined as a result of > 15 IU/ mL. Measurement levels below the lower limit of detection were recorded as being not determined, and were deleted from the statistical analyses. Measurements of serum anti-PPAD IgG

Serum levels of IgG to PPAD were determined by ELISA, as previously described (16) with minor modification. In brief, a 96-well microtiter plate was coated with 50 lL of PPAD peptide (CLGTDALHC-Cit-THEVADKGC) (0.1 lg/mL) (Sigma Aldrich Japan Genosys, Hokkaido, Japan) in 0.1 M sodium carbonate coating buffer (pH 9.5) (BD Biosciences, San Jose, CA, USA), overnight at 37°C. Wells were washed three times (10 min for each

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washing) with 200 lL of phosphatebuffered saline (pH 7.5) containing Tween 20 (0.05%) (Thermo Fisher Scientific Japan, Yokohama, Japan) and blocked with the blocking buffer (BD Biosciences) for 2 h at room temperature. After incubation with 100 lL of serum (1/100 dilution) for 30 min at room temperature, the plates were washed (as described above) and incubated with 100 lL of horseradish peroxidase-conjugated mouse monoclonal antibody to human IgG Fc (1/1000 dilution) (Abcam, Tokyo, Japan) for 1 h at room temperature. After a final wash, bound antibodies were detected with 100 lL of 3,30 ,5,50 tetramethylbenzidine (BD Biosciences) in hydrogen peroxide-buffered solution. The reaction was stopped by the addition of 100 lL of 1 M phosphoric acid, and the absorbance was measured at 450 nm using a microplate reader (BioRad Japan Laboratories). Anti-PPAD IgG levels were expressed as ELISA units (EU). Statistical analyses

Sample size and power calculation tests were performed on the serum citrulline levels (25) before the analysis, and the results revealed that more than 20 patients in each of the two groups would exceed 0.8 statistical power. After evaluating the normality of the distribution using Kolmogorov–Smirnov tests, differences in parameter values between the RA and non-RA groups were assessed using Mann–Whitney U-tests, whereas those before and after periodontal treatment were evaluated using Wilcoxon signed-rank tests. The associations between the serum levels of antiPPAD IgG and PAD-4, and susceptibility to RA, were assessed by multiple logistic regression analysis after adjustment for age, gender and smoking status, using statistical software (SPSS statistics version 21; IBM Japan, Tokyo, Japan). Confounders considered for this multiple logistic regression analysis included subject age, gender (male = 0, female = 1), and smoking status (never-smoker = 0, former-smoker = 1, current-smoker = 2). The Spearman’s rank correlation coef-

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ficient was used to determine the relationship between serum levels of antiPPAD IgG and PAD-4 and DAS28CRP in patients with RA, and between serum levels of anti-PPAD IgG and PAD-4 and serum parameter values in all participants. Statistical significance was accepted at 5% (p < 0.05).

Results Comparison between the RA and non-RA groups

The characteristics of individuals in the RA and non-RA groups are summarized in Table 1. There were no

significant differences between the two groups in any demographic, clinical or laboratory parameter (p > 0.05), except for the number of teeth present and the serum levels of CRP, IL-6 and TNF-a (p < 0.05) (Table 1). In the age, gender-, smoking status-, and periodontal condition-matched cohorts, univariate analyses revealed that the serum levels of anti-CCP IgG and antiPPAD IgG were significantly higher in the RA group than in the non-RA group [RA vs. non-RA: (mean  standard error) 155.35  19.64 U/mL vs. 2.32  0.03 U/mL for anti-CCP, p < 0.001; 1.41  0.12 EU vs. 0.93  0.07 EU for anti-PPAD, p = 0.03) (Fig. 1), but those of PAD-4 were com-

parable between the groups (2.26  0.19 ng/mL vs. 2.66  0.17 ng/mL, p > 0.05) (Fig. 1). A significant, positive correlation was observed between the serum levels of anti-PPAD IgG and anti-CCP IgG (p = 0.04), but not between the serum levels of anti-PPAD IgG and DAS28-CRP (p > 0.05) (Fig. 2). On the other hand, the serum levels of PAD-4 were not associated with those of anti-CCP IgG and DAS28-CRP (p > 0.05) (Fig. 2). Multiple logistic regression analyses revealed a significant association between the serum levels of anti-PPAD IgG and RA, after adjustment for age, gender and smoking status (p = 0.004; odds ratio = 4.59; 95% confidence

Table 1. Demographic, clinical and laboratory characteristics of individuals with and without rheumatoid arthritis (RA) RA subgroup

Parameters Demographic Age (years) Female Smoker: current/former/never Clinical Number of teeth present Percentage of sites with plaque Percentage of sites with BOP Probing depth (mm) Percentage of sites with probing depth ≥ 4 mm Clinical attachment level (mm) Percentage of sites with clinical attachment level ≥ 4 mm Duration of RA (years) DAS28-CRP DAS28-CRP category Remission Low activity Moderate activity High activity Tender joint count Swollen joint count Visual analog scale (mm) RA medication Corticosteroids DMARDs NSAIDs Laboratory RF (IU/mL) RF positive CRP (mg/dL) IL-6 (pg/mL) TNF-a (pg/mL)

RA group (n = 52)

Before treatment (n = 26)

After treatment (n = 26)

Non-RA group (n = 26)

62.0  1.8 44 (84.6) 0/33/67

60.7  2.7 22 (84.6) 0/31/69

60.7  2.7 22 (84.6) 0/31/69

59.9  1.6 21 (80.8) 0/19/81

22.1  0.7a 55.6  3.2 30.2  3.2 2.9  0.1 22.8  0.3 3.0  0.1 24.9  0.3 12.5  1.7 2.5  0.1

23.6  0.8 60.9  4.2b 36.8  5.0b 3.2  0.1b 30.1  4.4b 3.2  0.2b 33.5  4.7b 12.4  2.6 2.4  0.1b

23.6  0.8 28.1  3.5b 10.9  3.6b 2.7  0.1b 15.0  4.0b 2.8  0.1b 21.3  5.2b 12.4  2.6 2.1  0.1b

25.4  0.4a 53.8  3.2 23.0  2.6 2.8  0.1 25.8  2.9 3.0  0.1 30.8  3.7 NA NA

24 (46.2) 11 (21.1) 14 (26.9) 3 (5.8) 1.0 (0 to +18) 0.0 (0 to +12) 28.3  3.8

12 (46.2) 7 (26.9) 6 (23.1) 1 (3.8) 1.0 (0 to +8) 1.0 (0 to +12) 29.7  5.2

15 (57.7) 9 (34.6) 1 (3.8) 1 (3.8) 1.0 (0 to +6) 1.0 (0 to +4) 30.0  4.6

NA NA NA NA NA NA NA

27 (51.9) 36 (69.2) 12 (23.1)

14 (53.8) 20 (76.9) 7 (26.9)

14 (53.8) 20 (76.9) 7 (26.9)

NA NA NA

78.9  14.6 33 (63.5) 0.44  0.08a 4.87  0.63a 2.25  0.62a

63.0  14.3 18 (69.2) 0.41  0.12 5.35  0.99 1.96  0.66

66.4  16.0 18 (69.2) 0.39  0.12 5.62  1.14 0.87  0.17

NA NA 0.03  0.01a 1.88  0.35a 1.19  0.08a

Values are given as mean  standard error, n (%), % or median (range), and the bold values show the statistical significance. a Significantly different between the RA and non-RA groups (p < 0.05). b Significantly different before and after periodontal treatment in the RA subgroup (p < 0.05). BOP, bleeding on probing; CRP, C-reactive protein; DAS28-CRP, Disease Activity Score in 28 joints using C-reactive protein; DMARDs, disease-modifying antirheumatic drugs; IL6, interleukin-6; NA, not applicable; NSAIDs, nonsteroidal anti-inflammatory drugs; TNF-a, tumor necrosis factor-a; RF, rheumatoid factor.

Anti-P. gingivalis PAD IgG in RA interval: 1.63 to 12.92) (Table 2). A significant, positive correlation was obtained between the serum levels of anti-PPAD IgG and CRP (p = 0.004), and between the serum levels of anti-PPAD IgG and IL-6 (p = 0.003) (Fig. 3). Comparison before and after periodontal treatment in the RA subgroup

The results for the RA subgroup showed that the distribution and dose of RA medication with corticosteroids, DMARDs or NSAIDs remained unchanged, and showed no complications, during the study period. The RA subgroup consisted of individuals with mild (n = 0), moderate (n = 21) and severe (n = 5)

periodontitis (23), and showed a significant difference in probing depth, percentage of sites with probing depth ≥ 4 mm, clinical attachment level and percentage of sites with clinical attachment level ≥ 4 mm compared with the RA patients without periodontal treatment (p < 0.05 for all comparisons). Supragingival scaling significantly decreased the values of periodontal parameters, including the percentage of sites with plaque and BOP, probing depth and the percentage of sites with probing depth ≥ 4 mm, and clinical attachment level and the percentage of sites with clinical attachment level ≥ 4 mm (p < 0.05 for all comparisons) as well as the DAS28-CRP (p < 0.05) (Table 1), but failed to reduce significantly serum levels of anti-CCP IgG,

Table 2. Significance of association between serum levels of anti-Porphyromonas gingivalis peptidylarginine deiminase (PPAD) IgG and peptidylarginine deiminase-4 (PAD-4) and rheumatoid arthritis (RA) RA group vs. non-RA group Parameter

OR (95% CI)

pa

Serum anti-PPAD IgG (EU) Serum PAD-4 (ng/mL)

4.59 (1.63–12.92) 0.77 (0.48–1.23)

0.004b 0.28

The present model had a likelihood ratio v2 = 18.65 (degrees of freedom = 5), R2 = 0.21, with 78 observations included. 95% CI, 95% confidence interval; EU, ELISA units; OR, odds ratio. a The p-value was obtained by multiple logistic regression analysis after adjustment for age, gender and smoking status. b Significantly associated with RA (p < 0.05).

Anti-CCP IgG p < 0.001

200 100 0 –100

Non-RA group (n = 26)

PAD-4 NS

5

10

4

8

3

6

2

4 2

1

0

0

RA group (n = 52)

To the best of the authors’ knowledge, this is the first study to evaluate simultaneously the expression levels of anti-PPAD IgG and PAD-4, as well as anti-CCP IgG responses, in patients with RA. The results indicated that the serum levels of antiCCP IgG and anti-PPAD IgG were increased in patients with RA compared with the control individuals who were matched for age, gender, smoking status and periodontal condition, which is consistent with the results of other studies (15,16). A positive correlation was also observed between anti-PPAD IgG and antiCCP IgG responses, which is supported by the findings of other, recent studies (26,27). Moreover, a positive association was found between antiPPAD IgG responses and RA by multiple logistic regression analyses, after adjustment for age, gender and smoking status. These observations suggest an association between anti-

(ng/mL)

(U/mL)

300

Discussion

p = 0.03

ELISA Units (450 nm)

400

anti-PPAD IgG and PAD-4 2 mo after treatment (before treatment vs. after treatment: 183.48  24.97 U/mL vs. 182.43  23.59 U/mL for anti-CCP, p > 0.05; 1.62  0.18 EU vs. 1.56  0.17 EU for anti-PPAD, p > 0.05; and 2.96  0.27 ng/mL vs. 2.23  0.26 ng/mL for PAD-4, p > 0.05) (Fig. 4).

Anti-PPAD IgG

500

107

RA group (n = 52)

Non-RA group (n = 26)

RA group (n = 52)

Non-RA group (n = 26)

Fig. 1. Serum levels of anti-cyclic citrullinated peptide (CCP) immunoglobulin G (IgG), anti-Porphyromonas gingivalis peptidylarginine deiminase (PPAD) IgG and peptidylarginine deiminase-4 (PAD-4) in individuals with and without rheumatoid arthritis (RA). The p values show the significance of differences between the two groups (p < 0.05). NS, no significant difference was observed between the two groups (p > 0.05).

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Anti-PPAD – Anti-CCP r = 0.23, p = 0.04

PAD-4 – Anti-CCP NS 500

Anti-CCP IgG (U/mL)

Anti-CCP IgG (U/mL)

500 400 300 200 100

400 300 200 100 0

0 0

1

5

2

3

4

0

2

3

4

5

PAD-4 (ng/mL)

Anti-PPAD–DAS28CRP NS

PAD-4 – DAS28-CRP NS

5

6

7

6

7

4

DAS28-CRP

4

DAS28-CRP

1

Anti-PPAD IgG (ELISA Units)

3 2

3 2 1

1

0

0 0

1

2

3

4

0

1

2

3

Anti-PPAD IgG (ELISA Units)

4

5

PAD-4 (ng/mL)

Fig. 2. Relevance of serum anti-Porphyromonas gingivalis peptidylarginine deiminase (PPAD) immunoglobulin G (IgG) and peptidylarginine deiminase-4 (PAD-4) levels to serum anti-cyclic citrullinated peptide (CCP) IgG levels and rheumatoid arthritis (RA) activities. A significant, positive correlation was observed between the serum levels of anti-PPAD IgG and anti-CCP IgG. NS, no significant association was observed between the two parameter values (p > 0.05).

Anti-PPAD – CRP r = 0.33, p = 0.004

Anti-PPAD – IL-6 r = 0.34, p = 0.003 3

2

1

0

TNF-α (pg/mL)

20

IL-6 (pg/mL)

CRP (mg/dL)

3

Anti-PPAD – TNF-α NS

15 10 5 0

0

1

2

3

Anti-PPAD IgG (ELISA Units)

4

2

1

0 0

1

2

3

Anti-PPAD IgG (ELISA Units)

4

0

1

2

3

4

Anti-PPAD IgG (ELISA Units)

Fig. 3. Correlation of serum anti-Porphyromonas gingivalis peptidylarginine deiminase (PPAD) immunoglobulin G (IgG) to serum C-reactive protein (CRP), interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a) levels. A significant, positive correlation was observed between the serum levels of anti-PPAD IgG and CRP, and between the serum levels of anti-PPAD IgG and IL-6. NS, no significant association was observed between the two parameter values (p > 0.05).

Anti-P. gingivalis PAD IgG in RA

Anti-CCP IgG

Anti-PPAD IgG

NS

(U/mL)

300 200 100 0 –100

NS

5

10

4

8

3

6

(ng/mL)

ELISA Units (450 nm)

400

2 1

After treatment (n = 26)

4 2

0

Before treatment (n = 26)

PAD-4

NS

500

109

0

Before treatment (n = 26)

After treatment (n = 26)

Before treatment (n = 26)

After treatment (n = 26)

Fig. 4. Serum levels of anti-cyclic citrullinated peptide (CCP) immunoglobulin G (IgG) anti-Porphyromonas gingivalis peptidylarginine deiminase (PPAD) IgG and peptidylarginine deiminase-4 (PAD-4) in individuals with rheumatoid arthritis (RA), before and after periodontal treatment. NS, no significant difference was observed before and after periodontal treatment (p > 0.05).

PPAD IgG and anti-CCP IgG responses, implicating a role of PPAD in protein citrullination in relation to the pathogenesis of RA. Furthermore, a positive correlation was found between the serum levels of antiPPAD IgG and those of CRP and IL-6, which suggests a role of antiPPAD IgG responses in systemic inflammation. However, it cannot be concluded from these findings alone whether PPAD contributes to susceptibility to RA. To confirm and extend the observations obtained from the present study, additional studies are required to evaluate the role of PPAD as well as of other P. gingivalis antigens, such as arginine and lysine gingipains, in protein citrullination, with a larger number of patients and controls. In contrast, the results showed that the serum levels of PAD-4 were comparable between individuals with and without RA. The concentrations of PAD-4 in sera from patients with RA in the present study were similar to those in another study (28), which developed two novel ELISAs for detection of PAD-4. However, the control individuals in the present study exhibited increased levels of PAD-4, which may reflect relatively more systemic and local inflammatory conditions compared with the periodontally healthy individuals. Recently, it has been demonstrated

that citrullinated proteins and PAD-4 were detected in the inflamed gingival tissue (29). Therefore, it is conceivable that the levels of PAD-4 expressed in the periodontal tissue were increased with periodontal inflammation, which may contribute to systemic inflammatory burden. However, the control individuals with periodontitis showed little anti-CCP IgG responses in the present study, which is different from the results of another study (29). The discrepancy between the two studies might be partially explained by differences in the ELISA methodology, in the study cohort or in the time of sample collection. The limitation of the present study was the failure to determine anti-CCP IgG, anti-PPAD IgG and PAD-4 levels in gingival tissue and gingival crevicular fluid, which would be necessary to clarify whether PPAD and PAD4 can be responsible for the onset and progression of RA, directly or indirectly. The present study is the first to evaluate the effects of periodontal treatment on anti-CCP IgG and anti-PPAD IgG responses and expression of PAD4. The results indicated an improvement in the disease activity of RA, as well as in the levels of periodontal inflammation and destruction after 2 mo of periodontal treatment. These findings are in accordance with the results of other studies, which evaluated the effects of periodontal treat-

ment on severity of RA and serum inflammatory markers in patients with RA (30–33). Nevertheless, the data showed that the serum levels of antiCCP IgG, anti-PPAD IgG and PAD-4 were similar before and after periodontal treatment. The observations of no changes in the anti-PPAD IgG responses in the present study are different from the results of a previous study (25), indicating that supragingival scaling reduced the serum levels of antibodies to P. gingivalis sonicated extracts and hemin binding protein 35 in patients with RA (before periodontal treatment vs. after periodontal treatment: 12.6  2.5 EU vs. 10.6  2.2 EU for P. gingivalis sonicated extracts, p = 0.01; 0.94  0.07 EU vs. 0.90  0.08 EU for P. gingivalis hemin binding protein 35, p = 0.03). These inconsistent results might be partially explained by differences in the epitope that recognizes and binds P. gingivalis antigen molecules in the antibodies used for the ELISA. Another possible explanation relates to the differences in the serum antibody responses against P. gingivalis antigens. The results also indicated no changes in serum levels of CRP, IL-6 and TNF-a before and after periodontal treatment. These observations are different from the results of previous and other studies, which examined the effects of scaling and root planing on clinical and biochemical parameters in patients with periodonti-

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tis (26,34,35) or in patients with RA (33). The failure to detect a significant decrease in the inflammatory responses in the present study might be partly a consequence of the lack of subgingival instrumentation, which may have resulted in residual putative periodontal pathogens and subgingival inflammation. In addition, individuals in the RA subgroup under periodontal treatment did not have very severe periodontitis, which may be a possible reason for the failure to detect changes in the serum levels of inflammatory markers. The RA subgroup, randomly selected, showed a difference in the periodontal condition compared with the RA patients without periodontal treatment. It would be necessary to select carefully the RA patients with moderate to advanced periodontitis to understand, in more detail, the effects of periodontal treatment on both periodontal and rheumatologic outcomes. Caution is necessary when interpreting the data of the present study because all patients with RA were on drug treatment, which may affect serum anti-CCP IgG and anti-PPAD IgG responses and periodontal conditions. It has been suggested that individuals who received medication of DMARDs, corticosteroid or NSAIDs are at relatively low risk of periodontitis (36–38). Individuals receiving corticosteroid therapy showed lower levels of gingival inflammation than the control individuals (36). Patients with RA and undergoing treatment with DMARDs and NSAIDs were less likely to have periodontal destruction than the control patients (37,38). Additionally, the beneficial effects of DMARDs, corticosteroids and NSAIDs on the condition of RA have been documented (39–41). However, there is little information on the relationship between these drug treatments and serum anti-CCP IgG and anti-PPAD IgG responses. Another limitation relates to the variability of clinical and serum data, which may be influenced by the potential diurnal variation. For improvement of the reliability of data, it might be necessary to perform the clinical assessment and sampling at the same

appointment time throughout the study period. In summary, the results of the present study might suggest an association between serum anti-PPAD IgG and anti-CCP IgG responses, implying a role for PPAD in protein citrullination in patients with RA and periodontitis. A larger group of patients needs to be followed up.

Acknowledgements The present study was supported by Grant-in Aid for Scientific Research A (no. 25253104) and C (nos 22592309 and 26463130) from the Japan Society for the Promotion of Science, 5-3-1 Kojimachi, Chiyodaku, Tokyo 102-0083, Japan. The authors declare no conflict of interest related to this study.

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Expression of anti-Porphyromonas gingivalis peptidylarginine deiminase immunoglobulin G and peptidylarginine deiminase-4 in patients with rheumatoid arthritis and periodontitis.

Autoimmunity against citrullinated proteins through peptidylarginine deiminase (PAD) may be involved in the pathophysiology of rheumatoid arthritis (R...
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