ARTHRITIS & RHEUMATOLOGY Vol. 66, No. 5, May 2014, pp 1090–1100 DOI 10.1002/art.38348 © 2014, American College of Rheumatology

Periodontitis and Porphyromonas gingivalis in Patients With Rheumatoid Arthritis Ted R. Mikuls,1 Jeffrey B. Payne,2 Fang Yu,3 Geoffrey M. Thiele,1 Richard J. Reynolds,4 Grant W. Cannon,5 Jeffrey Markt,3 David McGowan,5 Gail S. Kerr,6 Robert S. Redman,6 Andreas Reimold,7 Garth Griffiths,7 Mark Beatty,8 Shawneen M. Gonzalez,2 Debra A. Bergman,1 Bartlett C. Hamilton III,1 Alan R. Erickson,1 Jeremy Sokolove,9 William H. Robinson,9 Clay Walker,10 Fatiha Chandad,11 and James R. O’Dell1 Objective. To examine the degree to which shared risk factors explain the relationship of periodontitis (PD) to rheumatoid arthritis (RA) and to determine the associations of PD and Porphyromonas gingivalis with pathologic and clinical features of RA. Methods. Patients with RA (n ⴝ 287) and patients with osteoarthritis as disease controls (n ⴝ 330) underwent a standardized periodontal examination. The HLA–DRB1 status of all participants was imputed using single-nucleotide polymorphisms from the extended major histocompatibility complex. Circulating anti–

P gingivalis antibodies were measured using an enzymelinked immunosorbent assay, and subgingival plaque was assessed for the presence of P gingivalis using polymerase chain reaction (PCR). Associations of PD with RA were examined using multivariable regression. Results. Presence of PD was more common in patients with RA and patients with anti–citrullinated protein antibody (ACPA)–positive RA (n ⴝ 240; determined using the anti–cyclic citrullinated peptide 2 [anti–CCP-2] test) than in controls (35% and 37%, respectively, versus 26%; P ⴝ 0.022 and P ⴝ 0.006, respectively). There were no differences between RA patients and controls in the levels of anti–P gingivalis or the frequency of P gingivalis positivity by PCR. The anti–P gingivalis findings showed a weak, but statistically significant, association with the findings for both anti–CCP-2 (r ⴝ 0.14, P ⴝ 0.022) and rheumatoid factor (RF) (r ⴝ 0.19, P ⴝ 0.001). Presence of PD was associated with increased swollen joint counts (P ⴝ 0.004), greater disease activity according to the 28-joint Disease Activity Score using C-reactive protein level (P ⴝ 0.045), and higher total Sharp scores of radiographic damage (P ⴝ 0.015), as well as with the presence and levels of anti–CCP-2 (P ⴝ 0.011) and RF (P < 0.001). The expression levels of select ACPAs (including antibodies to citrullinated filaggrin) were higher in patients with subgingival P gingivalis and in those with higher levels of anti–P gingivalis antibodies, irrespective of smoking status. Associations of PD with established seropositive RA were independent of all covariates examined, including evidence of P gingivalis infection. Conclusion. Both PD and P gingivalis appear to shape the autoreactivity of RA. In addition, these results

Supported by the Rheumatology Research Foundation (Disease Targeted Research Initiative grant to Dr. Mikuls, principal investigator). Dr. Mikuls’ work was also supported by the Nebraska Arthritis Outcomes Research Center, the Veterans Affairs Office of Research and Development (VA Merit award), and the National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH. 1 Ted R. Mikuls, MD, MSPH, Geoffrey M. Thiele, PhD, Debra A. Bergman, MPH, Bartlett C. Hamilton III, BS, Alan R. Erickson, MD, James R. O’Dell, MD: Omaha VA Medical Center and University of Nebraska Medical Center, Omaha; 2Jeffrey B. Payne, DDS, MDentSc, Shawneen M. Gonzalez, DDS, MS: University of Nebraska Medical Center, Lincoln; 3Fang Yu, PhD, Jeffrey Markt, DDS: University of Nebraska Medical Center, Omaha; 4Richard J. Reynolds, PhD: University of Alabama at Birmingham; 5Grant W. Cannon, MD, David McGowan, DDS: George Wahlen VA Medical Center, Salt Lake City, Utah; 6Gail S. Kerr, MD, FRCP, Robert S. Redman, DDS, MSD, PhD: Washington DC VA Medical Center, Washington; 7Andreas Reimold, MD, Garth Griffiths, DDS, MS: Dallas VA Medical Center, Dallas, Texas; 8Mark Beatty, DDS, MS: Omaha VA Medical Center, Omaha, Nebraska; 9Jeremy Sokolove, MD, William H. Robinson, MD, PhD: Palo Alto VA Medical Center and Stanford University, Palo Alto, California; 10Clay Walker, PhD: University of Florida, Gainesville; 11Fatiha Chandad, PhD: Universite´ Laval, Que´bec, Que´bec, Canada. Address correspondence to Ted R. Mikuls, MD, MSPH, Department of Medicine, University of Nebraska Medical Center and Omaha VA Medical Center, 986270 Nebraska Medical Center, Omaha, NE 68198-6270. E-mail: [email protected]. Submitted for publication September 3, 2013; accepted in revised form December 31, 2013. 1090

PERIODONTITIS AND P GINGIVALIS IN RA

demonstrate an independent relationship between PD and established seropositive RA. Periodontitis (PD) has emerged as a recognized risk factor in a number of health conditions, including rheumatoid arthritis (RA) (1). Sharing both morphologic and histopathologic similarities with RA (2), PD is an inflammatory disease initiated by bacterial infection, resulting in destruction of the soft and hard tissue and ultimately leading to tooth loss. In addition to shared inflammatory pathways, PD and RA share risk factors for susceptibility and progression, most notably cigarette smoking and, possibly, shared epitope (SE)–containing HLA–DRB1 alleles, the latter being associated with localized aggressive PD (3–10). Although a causal link between these conditions has not been established, several studies have demonstrated an increased prevalence of PD in RA patients compared to controls (11–18). Growing evidence suggests that pathogens associated with PD could play a role in the propagation of RA. Chief among the organisms of interest is Porphyromonas gingivalis (19). P gingivalis is the only known pathogen that expresses peptidylarginine deiminase (PAD). Similar to its human counterpart, P gingivalis PAD catalyzes the citrullination of arginine. This is noteworthy because citrullinated antigens, demonstrated to be present in the periodontium of patients with PD (20), drive adaptive immune responses that are nearly exclusive to RA. The potential role of P gingivalis in the pathogenesis of RA has been elucidated in epidemiologic investigations. Concentrations of circulating antibodies to P gingivalis are associated with the expression of anti–citrullinated protein antibodies (ACPAs) (21– 23), an autoantibody subset that that has been detected in the gingival crevicular fluid of select patients with PD (24). More recently, our group has shown that the presence of antibodies to P gingivalis is associated with the presence of RA-related autoantibodies among individuals at increased risk for disease but who have not yet developed RA (25), underscoring the potential role of this pathogen in RA development. As part of this study, we conducted a large case–control investigation to examine the relationship between PD and RA. We sought to examine the degree to which this relationship is affected by shared genetic and/or environmental factors. We also sought to elucidate the degree to which the relationship of PD to RA may be associated with infection and/or colonization with P gingivalis. By using a rigorously selected group of control subjects, we attempted to mitigate the issues of

1091

bias or unmeasured confounding that may have had an impact in previous efforts in which healthy volunteers were used as comparators (16–18). Finally, using a multiplex approach, we examined the associations of PD and P gingivalis with autoreactivity to several citrullinated autoantigens that have been implicated in the pathogenesis of RA. PATIENTS AND METHODS Study participants. Patients with RA (cases) and patients with osteoarthritis (OA) (controls) were enrolled from rheumatology, orthopedic, and primary care clinics in 4 US Veterans Affairs Medical Centers and a single academic coordinating center. All cases of RA satisfied the American College of Rheumatology classification criteria for RA (26) (disease onset after age 18 years). On the basis of existing pilot data (12), we enrolled patients with OA as disease controls with the expectation that these patients would share similar sociodemographic characteristics as those of the RA patients. The diagnosis of OA was confirmed through medical record review, based on documentation or imaging results consistent with the presence of degenerative arthritis in the absence of inflammatory arthritis (e.g., lupus, spondylitis, or polymyalgia rheumatica). Additional inclusion criteria included the presence of ⱖ9 posterior teeth. Patients were excluded if any of the following were present: use of tetracyclines or related antibiotics in the previous 6 months; prior use of cyclosporine or phenytoin; or a requirement for antibiotic prophylaxis prior to dental probing. The latter exclusion involved RA patients with a history of any total joint replacement or, for control subjects, having received a total joint replacement within the previous 24 months (27,28). This study was approved by the Institutional Review Board at each participating center, and all study participants provided their written informed consent. Periodontal assessments. Periodontal assessments were completed by a single dentist or periodontist at each site. The periodontal examiners were not provided with information on the arthritis case status of the subjects or with the results of any other clinical or laboratory evaluations prior to the full-mouth periodontal examination. Probing depth and gingival recession measurements were determined at 6 sites per tooth for all teeth (except for the third molars). Prior to study initiation, the findings of the periodontal assessors were calibrated against those of a gold-standard periodontist (JBP), by ensuring that at least 85% of probing depth and gingival recession measurements were within 1 mm. PD was defined a priori according to the criteria proposed by Machtei et al, defined as the presence of a clinical attachment loss of ⱖ6 mm on ⱖ2 teeth, and one or more sites with probing depths of ⱖ5 mm (29). Additional periodontal measurements included bleeding on probing, the presence of supragingival plaque (serving as an indicator of oral hygiene [30]), and missing teeth (of a total of 28; third molars excluded). Subgingival plaque was collected from up to 4 mesiobuccal sites. Following removal of visible supragingival plaque, subgingival plaque samples were collected using a single sterile

1092

endodontic paper point for each site (31). The samples were frozen at ⫺70°C until analyzed. Measures of RA disease activity and severity. ACPAs in the serum were measured using a second-generation anti– cyclic citrullinated peptide (anti–CCP-2) IgG enzyme-linked immunosorbent assay (ELISA) (Diastat; Axis-Shield), in which serum positivity was defined as a level of ⱖ5 units/ml. Both rheumatoid factor (RF) (positivity defined as a titer of ⱖ15 IU/ml) and the levels of high-sensitivity C-reactive protein (hsCRP; in mg/liter) were determined by nephelometry (Siemens Healthcare Diagnostics). Serum samples were also evaluated for 19 specific ACPAs, using a bead-based multiplex antigen array on the BioPlex platform (32), an array that measures disease-specific autoantibody reactivity to multiple citrullinated autoantigens. An ACPA score was calculated as the sum of normalized fluorescence values, divided by 19 (the latter representing the number of specific ACPAs tested). Other disease characteristics assessed included the tender and swollen joint counts (range 0–28 joints) and provider’s and patient’s global assessments of well-being (on 10-cm visual analog scales). The 28-joint Disease Activity Score using CRP level (DAS28-CRP) (33) was also calculated. Standard posteroanterior hand and wrist radiographs were obtained from all patients with RA. The radiographs were scored for the severity of joint damage using the modified Sharp/van der Heijde scoring method (34). All radiographic images were scored by a single investigator (ARE) who was blinded with regard to each patient’s PD status. Bacterial serologic measurements and the detection of P gingivalis in subgingival plaque. Serum concentrations of IgG antibodies to P gingivalis were measured using 2 previously described ELISAs. The first ELISA utilized strain 381 of P gingivalis (American Type Culture Collection) and measured antibody responses to outer membrane antigen (OMA) (25). Prior studies have demonstrated that several strains of P gingivalis, including strain 381, express common OMAs (35). Case–control investigations have further demonstrated higher antibody reactivity among PD cases compared to controls for a majority of the OMAs identified (35,36). The second ELISA approach measured antibody responses to P gingivalis–specific lipopolysaccharide (LPS) (21). Antibodies (of the IgG subtype) to OMA of both Prevotella intermedia (IgG anti-Pi) and Fusobacterium nucleatum were also evaluated (25) as coaggregates with P gingivalis in PD-related biofilms (37). Bacterial antibody concentrations (determined in ␮g/ml) were extrapolated from a standard curve, and then logtransformed for analysis. Nested polymerase chain reaction (PCR) was used for the detection of P gingivalis in subgingival plaque samples (38). Genotyping for the HLA–DRB1 SE. We utilized singlenucleotide polymorphism (SNP)–based imputation of 4-digit HLA–DRB1 alleles, using the web interface HLA-IMP:02, as previously described (39,40). SNP markers across the extended major histocompatibility complex (from 26 Mb to 34 Mb on chromosome 6) that were used for imputation were obtained from samples genotyped on the Illumina ImmunoChip array. Agreement of imputation with direct HLA–DRB1 sequencing was examined among 116 RA patients for whom these data were available as part of a separate study (41). The 2 methods were concordant in defining SE status (positive versus negative) in 108 of the 116 patients (93%; ␬ ⫽ 0.79). Levels of

MIKULS ET AL

agreement were similar among Caucasian RA patients (96% concordance; ␬ ⫽ 0.86) and African American RA patients (89% concordance; ␬ ⫽ 0.72). Comparisons of SE status and multivariable models in which HLA–DRB1 SE was included as a variable were thus limited to Caucasian and African American patients. The following HLA–DRB1 alleles were considered to be SE positive: *0101, *0102, *0104, *0105, *0401, *0404, *0405, *0408, *0409, *1001, *1402, and *1406. Statistical analysis. When a single measure was collected from 1 subject, the 2-sample t-test or Wilcoxon’s rank sum test was used for comparison of continuous data, and the chi-square test was used for comparison of categorical data. When multiple measures (e.g., probing depths) were used in different oral sites in 1 subject, the data were categorized as binary data, and generalized estimating equation models with logit link and compound symmetry variance structure were used for analyses in order to account for within-subject correlations. Case–control differences in bacterial measurements and RA outcomes (limited to RA cases) based on the presence of PD were evaluated. Spearman’s correlation coefficients were used to quantify correlations. Multivariate logistic regression was used to evaluate the association of PD with RA risk among Caucasian and African American subjects for whom imputed HLA–DRB1 SE data were available. Given their relevance (3–10), HLA–DRB1 SE positivity and smoking status (ever versus never) were included in all models. Other confounders included age, sex, race, body mass index (BMI), oral hygiene (supragingival plaque as a surrogate), self-reported diabetes, marital status, patient-reported oral dryness, and education. Stepwise variable selection with a selection criterion significance level of P ⬍ 0.1 for both entry and elimination was used to select the other important risk factors. P values less than 0.05 were considered statistically significant. Significance analysis of microarrays (SAM) (42) was used to analyze a multiplex of specific ACPAs among anti– CCP-2–antibody positive RA patients, in order to identify differences in ACPA profiles associated with PD, smoking, or both. We also compared ACPA scores among current, former, and never smokers stratified by PD status, using one-way analysis of variance. SAM was used to assess whether there were differences in ACPA expression among anti–CCP-2– positive RA cases based on tertiles of anti–P gingivalis OMA antibody positivity or P gingivalis PCR positivity in subgingival samples, accounting for the effects of smoking in stratified analyses. SAM output was sorted based on false discovery rates, in order to identify antigens with the greatest differences in autoantibody reactivity. Cluster hierarchical clustering software (version 3.0) was used to arrange the SAM results according to similarities among autoantibody specificities, and significant ACPA values were displayed as heatmaps using the Java TreeView program (version 1.1.3) (both available at http://rana.lbl.gov/EisenSoftware.htm).

RESULTS Characteristics of the study participants. There were 617 participants, including 287 RA cases and 330 controls. The demographic and clinical characteristics of

PERIODONTITIS AND P GINGIVALIS IN RA

Table 1.

1093

Characteristics of the study participants

Sociodemographic features Age, mean ⫾ SD years Male, % Race, % Caucasian African American Other Education, mean ⫾ SD years Married, % Comorbidity and health factors Body mass index, mean ⫾ SD kg/m2 Smoking status, % Never Former Current Diabetes mellitus, % Hypertension, % Cardiovascular disease, % Osteoporosis, % HLA–DRB1 SE positivity, %*

Total (n ⫽ 617)

Rheumatoid arthritis cases (n ⫽ 287)

Controls (n ⫽ 330)

59 ⫾ 11 62

59 ⫾ 12 63

59 ⫾ 11 60

0.867 0.384

75 20 5 14 ⫾ 2 65

77 17 6 14 ⫾ 2 68

72 23 5 14 ⫾ 2 62

0.174

31 ⫾ 7

30 ⫾ 7

32 ⫾ 7

0.001

46 39 15 22 52 11 13 59

38 43 19 18 45 13 11 76

54 35 11 25 57 10 15 45

P

0.113 0.135

⬍0.001 0.053 0.004 0.259 0.111 ⬍0.001

* Comparison of HLA–DRB1 shared epitope (SE) positivity was limited to Caucasians and African Americans (n ⫽ 530).

the 617 study participants are shown in Table 1. There were no between-group differences in sociodemographic features. Consistent with the known disease epidemiology (43), RA cases were more likely to be smokers and more likely to be HLA–DRB1 SE positive. Control subjects were found to have a higher mean BMI and higher frequency of hypertension when compared to the patients with RA. Periodontal findings. Results from the periodontal assessments are summarized in Table 2. PD was

present in a higher proportion of RA cases compared to controls (35% versus 26%, unadjusted odds ratio [ORunadj] 1.49, 95% confidence interval [95% CI] 1.06– 2.11; P ⫽ 0.022), as well as in a higher proportion of anti–CCP-2–positive RA cases compared to controls (37% versus 26%, ORunadj 1.65, 95% CI 1.15–2.36; P ⫽ 0.006). Furthermore, compared to control subjects, patients with RA and those with anti–CCP-2–positive RA demonstrated a higher percentage of sites with a probing depth of ⱖ5 mm (P ⫽ 0.026 and P ⫽ 0.005, respectively),

Table 2. Periodontal measurements in RA cases, anti–CCP-2 antibody–positive RA cases, and controls* RA (n ⫽ 287)

Periodontitis, % patients No. of missing teeth, mean ⫾ SD (range 0–28) Any plaque present, % patients Plaque, mean ⫾ SD % sites/patient Any bleeding on probing % patients Mean ⫾ SD % sites/patient Any probing depth ⱖ5 mm % patients Mean % sites/patient Any attachment loss ⱖ5 mm % patients Mean % sites/patient

Anti–CCP-2–positive RA (n ⫽ 240)

Controls (n ⫽ 330)

Value

P

Value

P

Value

P

34.8 3.2 ⫾ 3.1 95.3 42.0

0.022 0.877 0.157 0.320

37.1 3.3 ⫾ 3.1 96.7 42.7

0.006 0.838 0.675 0.202

26.4 3.3 ⫾ 3.5 97.3 39.9

Referent Referent Referent Referent

87.1 21.0

0.001 0.383

87.9 21.2

0.005 0.450

94.6 21.0

Referent Referent

51.8 3.6

0.208 0.026

53.8 4.1

0.095 0.005

46.7 2.4

Referent Referent

71.7 9.3

0.833 0.135

73.3 10.0

0.525 0.060

70.9 7.5

Referent Referent

* RA ⫽ rheumatoid arthritis; anti–CCP-2 ⫽ anti–cyclic citrullinated peptide 2 antibody.

1094

MIKULS ET AL

Table 3.

Multivariable associations between periodontitis and RA* Odds ratio (95% confidence interval)

Periodontitis HLA–DRB1 SE positive Ever smoker

All RA

P

Anti–CCP-2– positive RA

P

1.36 (0.89–2.06) 3.95 (2.68–5.83) 1.93 (1.31–2.83)

0.153 ⬍0.001 0.001

1.59 (1.01–2.49) 5.32 (3.44–8.22) 1.97 (1.29–2.99)

0.043 ⬍0.001 0.002

* Additional factors accounted for in multivariable models included age, sex, race/ethnicity, body mass index, self-reported diabetes mellitus, marital status, presence of oral dryness, and education. Multivariable models were limited to participants reporting either Caucasian or African American race/ethnicity. RA ⫽ rheumatoid arthritis; anti–CCP-2 ⫽ anti–cyclic citrullinated peptide 2 antibody; SE ⫽ shared epitope.

with a nonsignificant trend toward a higher proportion of sites with an attachment loss of ⱖ5 mm among anti–CCP-2–positive RA cases compared to controls (P ⫽ 0.060). The association of anti–CCP-2 antibody– positive RA with a higher percentage of sites with probing depths of ⱖ5 mm remained significant after adjustment for a history of ever smoking (P ⫽ 0.027). Other individual periodontal measurements did not differ significantly by group, with the exception that control subjects were more likely than RA patients to exhibit any bleeding on probing, although there were no between-group differences in the mean proportion of sites per subject exhibiting bleeding on probing. After multivariable adjustments, PD remained significantly more frequent among anti–CCP-2 antibody– positive RA cases than among controls (OR 1.59, 95% CI 1.01–2.49; P ⫽ 0.043), an association that was attenuated and not significant when all RA cases (including anti–CCP-2 antibody–negative patients) were evaluated (OR 1.36, 95% CI 0.89–2.06; P ⫽ 0.153) (Table 3). Additional adjustment for the levels of anti–P gingivalis antibodies (both OMA and LPS sequentially) and the presence of subgingival P gingivalis (determined by PCR) did not alter these results. To examine for evidence of residual confounding, we then limited the analyses to subjects who were never smokers. Although the data did not reach statistical significance (given the reduced sample size), associations of PD with RA (OR 1.37, 95% CI 0.65–2.89) and with anti–CCP-2–positive RA (OR 1.65, 95% CI 0.75– 3.63) were similar in the models limited to never smokers. We observed no evidence of interaction of PD with either cigarette smoking or HLA–DRB1 SE positivity. In multivariable analyses that were limited to female subjects (38% of the cohort), the association of PD with RA remained at a similar level (OR 1.39, 95% CI 0.61–3.16),

whereas the association of PD with anti–CCP-2–positive RA appeared to be attenuated (OR 1.16, 95% CI 0.43–3.18). Associations of bacterial serologic measurements with subgingival P gingivalis. Compared to those without PD, patients with PD had higher (log-transformed) levels of circulating antibodies to P gingivalis OMA (mean ⫾ SD 4.20 ⫾ 0.94 units versus 4.57 ⫾ 0.87 units; P ⬍ 0.001) and P gingivalis LPS (mean ⫾ SD 5.28 ⫾ 0.36 units versus 5.36 ⫾ 0.31 units; P ⫽ 0.008), differences that were not seen with P intermedia or F nucleatum antibodies. We observed no differences in the concentrations of anti–P gingivalis or anti–P intermedia antibodies between RA cases and controls. In contrast, concentrations of antibodies to F nucleatum were significantly higher in RA cases (P ⫽ 0.018) and anti–CCP2–positive RA cases (P ⫽ 0.008) compared to controls (mean ⫾ SD 4.37 ⫾ 0.85 units in RA cases and 4.40 ⫾ 0.85 units in anti–CCP-2–positive RA cases versus 4.21 ⫾ 0.87 units in controls). Subgingival colonization with P gingivalis was common, observed in 67% of participants, and was more common in those with PD (84%) than in those without PD (60%) (P ⬍ 0.001). Compared to those subjects without evidence of subgingival colonization, those with detectable P gingivalis by PCR had higher levels of circulating antibodies to P gingivalis OMA (mean ⫾ SD 4.07 ⫾ 0.97 units versus 4.44 ⫾ 0.88 units; P ⬍ 0.001) and P gingivalis LPS (mean ⫾ SD 5.17 ⫾ 0.36 units versus 5.37 ⫾ 0.31 units; P ⬍ 0.001). Although subgingival P gingivalis was more common among those with PD compared to those without PD, there were no case–control differences in the frequency of detectable subgingival P gingivalis. We observed no significant differences in any of the aforementioned bacterial mea-

PERIODONTITIS AND P GINGIVALIS IN RA

Table 4.

1095

RA-related measures based on the presence of periodontitis*

Disease duration, years Swollen joint count (range 0–28) Tender joint count (range 0–28) High-sensitivity CRP, mg/liter Patient’s global assessment score (range 0–10) DAS28-CRP score† Total Sharp score Joint space narrowing Erosions Anti–CCP-2 % of patients positive Concentration, units/ml RF % of patients positive Concentration, IU/ml Medication, % Methotrexate Prednisone Biologic drug

All RA (n ⫽ 287)

RA with periodontitis (n ⫽ 100)

RA without periodontitis (n ⫽ 187)

12.6 ⫾ 9.9 3.6 ⫾ 4.3

12.0 ⫾ 10.0 4.5 ⫾ 4.5

12.9 ⫾ 9.9 3.1 ⫾ 4.1

0.380 0.004

3.2 ⫾ 4.6

3.8 ⫾ 4.9

2.8 ⫾ 4.4

0.069

8.3 ⫾ 21.1 4.2 ⫾ 2.7

10.6 ⫾ 30.8 4.3 ⫾ 2.7

7.1 ⫾ 13.1 4.2 ⫾ 2.6

0.420 0.610

3.2 ⫾ 1.3 19.5 ⫾ 23.1 15.1 ⫾ 17.1 4.4 ⫾ 8.2

3.5 ⫾ 1.4 24.5 ⫾ 27.7 18.6 ⫾ 19.6 5.8 ⫾ 10.6

3.1 ⫾ 1.2 16.9 ⫾ 19.8 13.3 ⫾ 15.3 3.6 ⫾ 6.4

0.045 0.015 0.011 0.289

85 145 ⫾ 128

90 170 ⫾ 128

82 131 ⫾ 126

0.066 0.011

77 256 ⫾ 491

86 390 ⫾ 633

72 185 ⫾ 379

0.006 ⬍0.001

62 30 31

61 33 37

62 28 27

P

0.864 0.359 0.089

* Except where indicated otherwise, values are the mean ⫾ SD. RA ⫽ rheumatoid arthritis; anti–CCP-2 ⫽ anti–cyclic citrullinated peptide 2 antibody; RF ⫽ rheumatoid factor. † P values for the 28-joint Disease Activity Score using C-reactive protein level (DAS28-CRP) were generated by comparing log-transformed values and using a 2-sample t-test. Actual calculated values are shown for each group.

surements between cases and controls after the analysis was limited to those with PD. PD and RA disease characteristics. The frequency of HLA–DRB1 SE positivity did not differ between RA patients with PD and those without PD (78% versus 74%; P ⫽ 0.442). Compared to RA patients without PD, those with PD had significantly higher swollen joint counts, DAS28-CRP scores, and radiographic damage scores (Table 4). Likewise, patients with PD were more likely to be RF positive and had

higher concentrations of circulating RF and anti–CCP-2 compared to patients without PD. To examine whether these associations between PD and RA disease characteristics might be mediated by the effects of RA on oral hygiene, we examined whether the proportions of sites with visible supragingival plaque (examined in tertiles) were associated with these RA measures. None of the measures of RA disease activity or severity differed across these groups, with the exception of slightly higher joint space narrowing scores in

Figure 1. Heatmap demonstrating increased levels of several antigen-specific anti–citrullinated protein antibodies (ACPAs; determined using the anti–cyclic citrullinated peptide 2 [anti–CCP-2] test) in anti–CCP-2 antibody–positive rheumatoid arthritis (RA) patients with or without periodontitis (PD) and a history of ever smoking or never smoking. Only those ACPAs with significantly elevated expression are shown.

1096

those in the middle and upper tertiles of proportions of sites with plaque (16.1 and 18.2 Sharp units, respectively versus 11.2 Sharp units; P ⫽ 0.037). Associations of serologic measurements in patients with RA. There was a weak, but significant, correlation between the concentrations of anti–P gingivalis OMA antibodies and the concentrations of anti–CCP-2 antibodies (r ⫽ 0.14, P ⫽ 0.022), RF (r ⫽ 0.19, P ⫽ 0.001), or hsCRP (r ⫽ 0.15, P ⫽ 0.011). We observed similarly weak correlations between the concentrations of anti–P gingivalis LPS antibodies and the concentrations of RF (r ⫽ 0.14, P ⫽ 0.018). However, there were no associations of the concentration of this bacterial LPS antibody or alternative bacterial serologic measurements with the anti–CCP-2 or hsCRP concentrations, nor were there any correlations of alternative bacterial serologic measurements with the RF concentration (r ⬍ 0.1, P ⬎ 0.1). PD, anti–P gingivalis antibodies, and ACPA expression. Based on the observed associations with the anti–CCP-2 concentration (not observed with the alternative bacterial serologic measurements), we subsequently examined the associations of PD and anti– P gingivalis OMA antibody concentrations with specific ACPA subtypes, limiting the analyses to anti–CCP-2– positive RA cases (n ⫽ 240). In all analyses, we accounted for smoking status. Compared to never smokers without PD, we observed minimal differences in ACPA expression in those who were ever smokers or those with PD alone (Figure 1). In contrast, RA patients with both PD and a status of ever smoking demonstrated greater expression of ACPAs targeting several citrullinated antigens, including those derived from clusterin, enolase, filaggrin, and fibrinogen (Figure 1). Among patients with seropositive RA who were ever smokers, higher concentrations of anti–P gingivalis OMA antibodies were similarly associated with increased expression of ACPAs targeting clusterin, enolase, filaggrin, and fibrinogen (see Supplementary Figure 1, available on the Arthritis & Rheumatology web site at http://online library.wiley.com/doi/10.1002/art.38348/abstract). In contrast, among seropositive RA patients who were never smokers, higher concentrations of anti–P gingivalis antibodies were associated with increased expression of ACPAs targeting filaggrin, histone 2A, and vimentin (both recombinant protein and citrulline-containing peptide 58–77) (see Supplementary Figure 1, available on the Arthritis & Rheumatology web site at http:// onlinelibrary.wiley.com/doi/10.1002/art.38348/abstract). Similar associations were observed in anti– CCP-2–positive patients based on the presence of sub-

MIKULS ET AL

Figure 2. The anti–citrullinated protein antibody (ACPA) score was compared among anti–cyclic citrullinated peptide 2 (anti–CCP-2) antibody–positive rheumatoid arthritis (RA) patients with or without periodontitis (PD) who were categorized as either current (C), former (F), or never (N) smokers. Results are the mean ⫾ SD (see Table 1 for distribution of RA patients by smoking status).

gingival P gingivalis (see Supplementary Figure 2, available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/art.38348/ abstract). Among seropositive RA patients with a history of smoking, the presence of subgingival P gingivalis was associated with increased expression of several ACPAs, including autoantibodies recognizing citrullinated forms of clusterin, histone 2B, apolipoprotein E, fibrinogen, and filaggrin. Among anti–CCP-2–positive RA patients who were never smokers, the presence of subgingival P gingivalis was associated with increased expression of ACPAs targeting filaggrin, histone 2B, and apolipoprotein E (see Supplementary Figure 2, available on the Arthritis & Rheumatology web site at http://onlinelibrary. wiley.com/doi/10.1002/art.38348/abstract). In analyses stratified by the presence or absence of PD, current smokers without PD demonstrated a significantly higher ACPA score than did never smokers (P ⫽ 0.043). Moreover, there was a nonsignificant trend toward a higher ACPA score in current smokers without PD than in former smokers (P ⫽ 0.083) (Figure 2). In contrast, among those with PD, there were no observed differences in ACPA score based on smoking status. DISCUSSION Our observations confirm and extend the findings from prior reports (44), demonstrating a higher frequency of PD in the context of RA and, in particular, seropositive RA. In lending support to the idea that this relationship is most relevant to ACPA-positive disease, our results demonstrate that this association is independent of factors that have been previously hypothesized to act as confounders or mediators. Of the covariates examined in this study, cigarette smoking has been speculated to serve as perhaps the single greatest con-

PERIODONTITIS AND P GINGIVALIS IN RA

founder in assessments of this relationship. Estimated to account for ⬃1 in 6 RA cases, the attributable risk of smoking in the development of PD is even greater, accounting for ⬃1 in 2 cases (45). With the use of periodontal surgery as a disease surrogate, which yielded a sensitivity of only 25% for the identification of severe PD (46), separate investigators observed positive trends that were evident only in smokers, a relationship that was absent among never smokers (47). This contrasts with the results from our study in addition to the recent results from a study by Scher and colleagues, both of which included full-mouth periodontal examinations, with the latter showing a significantly higher prevalence of PD among RA patients with new-onset, treatmentnaive disease, the majority of whom had never smoked (17). Data demonstrating an independent association of PD with ACPA-positive RA are particularly timely in light of a recent systematic review demonstrating at least moderate evidence that “common risk factors” might explain the association of these 2 conditions (44). In contrast to our hypothesis, the association of PD with established RA did not appear to be dependent on evidence of prior infection or subgingival colonization with P gingivalis. Although the presence of subgingival bacteria and antibody responses to P gingivalis were higher in those with PD (as expected), we found no difference in either antibody concentrations or presence of subgingival P gingivalis in RA cases compared to controls. These results parallel recent reports, including one study examining patients with new-onset disease (17) and another focused on established RA (18). In the latter study, investigators found no significant differences in IgG anti–P gingivalis OMA antibody concentrations or cultivatable subgingival bacteria in RA cases compared to controls (although case–control differences in IgM overall and both IgM and IgG anti–P gingivalis antibody concentrations were observed among those with severe PD). Using pyrosequencing techniques, Scher and colleagues did not detect any significant case– control differences in the presence of subgingival P gingivalis or in the frequency of circulating bacterial antibodies (17). Unexpectedly, we observed that the levels of circulating antibodies to F nucleatum were significantly higher among RA cases compared to controls, the first time, to our knowledge, that this has been reported. In contrast to P gingivalis, F nucleatum is less virulent and there is less robust evidence of an etiologic link with oral disease, a fact that may explain its lack of association with PD in our study. In experimental PD, coinfection of P gingivalis and F nucleatum yields a synergistic effect

1097

on PD-related soft tissue destruction, as compared to monoinfection with either of the bacteria alone (48). Moreover, recent studies of experimental PD have shown that coinfection attenuates immune responses to P gingivalis while having no effect on immune responses to F nucleatum (49). F nucleatum is known to express a variety of surface adhesins and is speculated to act as a bridging organism in the complex biofilm that characterizes PD, linking early colonizers (often gram-positive anaerobes) to later colonizers (gram-negative anaerobes, including P gingivalis) (49). Whether coaggregation of F nucleatum with P gingivalis or alternative oral pathogens explains the observed association of this particular bacterial species with RA remains to be defined. With evolving technologies that include deep sequencing of bacterial DNA, future studies will be needed to examine the complex subgingival microbiome and its relationship to autoimmunity and RA risk and progression. Our results demonstrate that PD is associated with several measures of RA disease activity. The associations of PD with higher concentrations of RF and anti–CCP-2 autoantibodies are perhaps most noteworthy, given the associations of these measures with poor long-term outcomes in RA (50). Indeed, the presence of PD was associated with increased joint damage, as reflected in higher radiographic scores. Similar to the findings in other studies (18), we observed a weak, but significant, correlation between the anti–P gingivalis OMA antibody concentration and the anti–CCP-2 antibody concentration. These correlations appear to be unique to P gingivalis, as they were not observed with the other bacterial serologic measurements. The possibility that specific bacterial antigens drive this association has yet to be determined. Recent studies have focused on the antigens expressed by P gingivalis, including bacterially expressed PAD (51) and intracellular ␣-enolase (52). A prior investigation showed no associations of PD with ACPA fine specificity (18), although that study examined autoreactivity to only 5 citrullinated peptides. We also found no association of PD alone with ACPA fine specificity among anti–CCP-2–positive RA patients. However, PD and smoking appeared to serve as cofactors in shaping the ACPA specificity, suggesting that there is a synergistic effect in promoting autoantibody reactivity to multiple citrullinated antigens. Using the ACPA score as a surrogate for the magnitude of epitope spreading, we examined associations of smoking status with autoantibody expression in those with and those without PD. Although only the difference between cur-

1098

rent and never smokers reached significance, both former and never smokers demonstrated numerically lower ACPA scores when compared to current smokers, but only in the absence of PD. These data suggest that ACPA responses could be modifiable with smoking cessation, but only if PD is absent. Additional longitudinal studies will be essential in understanding whether interventions focused on smoking cessation and/or PD treatment may alter the course of RA. In addition to the potential effect of PD on ACPA expression, our results suggest that P gingivalis may help to shape the autoantibody specificity in RA independent of smoking status. We observed increased expression of ACPAs among both smokers and nonsmokers who had higher serum concentrations of IgG anti–P gingivalis antibodies. Similarly, we observed overexpression of ACPAs targeting both citrullinated filaggrin and histone in patients with evidence of subgingival P gingivalis, regardless of smoking status. To our knowledge, this is the first time that autoreactivity to these antigens has been examined in the context of RA and PD. These results contrast with those in a study by De Smit et al, in which cultivatable subgingival P gingivalis was found to be associated with higher serum concentrations of ACPAs targeting citrullinated fibrinogen only (18). Although we also observed associations of subgingival P gingivalis with anti–citrullinated fibrinogen antibodies, this was present only in smokers and not in never smokers, suggesting that the impact of smoking may at least partially account for these previously reported findings. Whether the diseased periodontium acts as a reservoir for autoantigens in RA remains to be defined. Filaggrin, ubiquitous in skin epithelium, is also highly expressed in the oral mucosa and its expression is up-regulated with smoking (53). Both PD and P gingivalis have been linked to neutrophil activation (54,55), a possible source of deiminated histone (56). There are limitations to this study. With regard to statistical power, findings from secondary analyses have suggested that there is an attenuation of the association of PD with anti–CCP–positive RA in women, and therefore caution should be taken in extending these results to other populations. Inherent to the case–control design of the study, no causal inferences can be made on the basis of these results alone. While it is possible that PD acts as a risk factor in RA, it is also possible that RA (or a treatment received and discontinued prior to enrollment) influenced the development of PD in this study cohort, perhaps being a reflection of the extraarticular targeting of oral mucosa. Furthermore, given the potential importance of oral pathogens in auto-

MIKULS ET AL

immunity and disease progression, it is essential that future efforts be aimed at cross-validation of these results and that replications of bacterial serologic measurements be undertaken. In summary, these results demonstrate an independent relationship between PD and established ACPA-positive RA. Importantly, these data suggest that PD may act in concert with cigarette smoking to shape the autoantibody reactivity that characterizes RA, with supporting evidence to show that P gingivalis infection may influence disease-specific autoantibody responses independent of cigarette smoking. AUTHOR CONTRIBUTIONS All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Mikuls had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. Mikuls, Payne, Yu, Cannon, Kerr, Griffiths, Robinson, O’Dell. Acquisition of data. Mikuls, Payne, Thiele, Cannon, Markt, McGowan, Kerr, Redman, Reimold, Griffiths, Beatty, Gonzalez, Bergman, Hamilton, Erickson, Sokolove, Walker, Chandad, O’Dell. Analysis and interpretation of data. Mikuls, Payne, Yu, Thiele, Reynolds, Markt, Gonzalez, Sokolove, Robinson, O’Dell.

REFERENCES 1. Pizzo G, Guiglia R, Lo Russo L, Campisi G. Dentistry and internal medicine: from the focal infection theory to the periodontal medicine concept. Eur J Intern Med 2010;21:496–502. 2. Snyderman R, McCarty GA. Analogous mechanism of tissue destruction in rheumatoid arthritis and periodontal disease. In: Genco R, Mergenhagen S, editors. Host-parasite interactions in periodontal diseases. Washington (DC): American Society for Microbiology; 1982. p. 354–62. 3. Albandar JM, Streckfus CF, Adesanya MR, Winn DM. Cigar, pipe, and cigarette smoking as risk factors for periodontal disease and tooth loss. J Periodontol 2000;71:1874–81. 4. Bonfil JJ, Dillier FL, Mercier P, Reviron D, Foti B, Sambuc R, et al. A “case control” study on the role of HLA DR4 in severe periodontitis and rapidly progressive periodontitis: identification of types and subtypes using molecular biology (PCR.SSO). J Clin Periodontol 1999;26:77–84. 5. Calsina G, Ramon JM, Echeverria JJ. Effects of smoking on periodontal tissues. J Clin Periodontol 2002;29:771–6. 6. Criswell LA, Merlino LA, Cerhan JR, Mikuls TR, Mudano AS, Burma M, et al. Cigarette smoking and the risk of rheumatoid arthritis among postmenopausal women: results from the Iowa Women’s Health Study. Am J Med 2002;15:465–71. 7. Gregersen PK, Silver J, Winchester RJ. The shared epitope hypothesis: an approach to understanding the molecular genetics of susceptibility to rheumatoid arthritis. Arthritis Rheum 1987;30: 1205–13. 8. Karlson EW, Lee IM, Cook NR, Manson JE, Buring JE, Hennekens CH. A retrospective cohort study of cigarette smoking and risk of rheumatoid arthritis in female health professionals. Arthritis Rheum 1999;42:910–7. 9. Nepom GT, Nepom BS. Prediction of susceptibility to rheumatoid

PERIODONTITIS AND P GINGIVALIS IN RA

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23. 24.

25.

26.

27.

arthritis by human leukocyte antigen genotyping. Rheum Dis Clin North Am 1992;18:785–92. Wordsworth P, Pile KD, Buckely JD, Lanchbury JS, Ollier B, Lathrop M, et al. HLA heterozygosity contributes to susceptibility to rheumatoid arthritis. Am J Hum Genet 1992;51:585–91. De Pablo P, Dietrich T, McAlindon TE. Association of periodontal disease and tooth loss with rheumatoid arthritis in the US population. J Rheumatol 2008;35:70–6. Dissick A, Redman RS, Jones M, Rangan BV, Reimold A, Griffiths GR, et al. Association of periodontitis with rheumatoid arthritis: a pilot study. J Periodontol 2010;81:223–9. Kasser UR, Gleissner C, Dehne F, Michel A, WillershausenZonnchen B, Bolten WW. Risk for periodontal disease in patients with longstanding rheumatoid arthritis. Arthritis Rheum 1997;40: 2248–51. Malmstrom M, Calonius PE. Teeth loss and the inflammation of teeth-supporting tissues in rheumatoid disease. Scand J Rheumatol 1975;4:49–55. Pischon N, Pischon T, Kroger J, Gulmez E, Kleber BM, Bernimoulin JP, et al. Association among rheumatoid arthritis, oral hygiene, and periodontitis. J Periodontol 2008;79:979–86. Potikuri D, Dannana KC, Kanchinadam S, Agrawal S, Kancharla A, Rajasekhar L, et al. Periodontal disease is significantly higher in non-smoking treatment-naive rheumatoid arthritis patients: results from a case-control study. Ann Rheum Dis 2012;71:1541–4. Scher JU, Ubeda C, Equinda M, Khanin R, Buischi Y, Viale A, et al. Periodontal disease and the oral microbiota in new-onset rheumatoid arthritis. Arthritis Rheum 2012;64:3083–94. De Smit MD, Westra J, Vissink A, Doornbos-van der Meer B, Brouwer E, van Winkelhoff AJ. Periodontitis in established rheumatoid arthritis patients: a cross-sectional clinical, microbiological and serological study. Arthritis Res Ther 2012;14:R222. 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;28:311–8. Nesse W, Westra J, van der Wal JE, Abbas F, Nicholas AP, Vissink A, et al. The periodontium of periodontitis patients contains citrullinated proteins which may play a role in ACPA (anti-citrullinated protein antibody) formation. J Clin Periodontol 2012;39:599–607. Hitchon CA, Chandad F, Ferucci ED, Willemze A, Ioan-Facsinay A, van der Woude D, et al. Antibodies to Porphyromonas gingivalis are associated with anticitrullinated protein antibodies in patients with rheumatoid arthritis and their relatives. J Rheumatol 2010;37:1105–12. Mikuls TR, Payne JB, Reinhardt RA, Thiele GM, Maziarz E, Cannella AC, et al. Antibody responses to Porphyromonas gingivalis (P. gingivalis) in subjects with rheumatoid arthritis and periodontitis. Int Immunopharmacol 2009;9:38–42. Ogrendik M, Kokino S, Ozdemir F, Bird PS, Hamlet S. Serum antibodies to oral anaerobic bacteria in patients with rheumatoid arthritis. MedGenMed 2005;7:2. Harvey GP, Fitzsimmons TR, Dhamarpatni AA, Marchant C, Haynes DR, Bartold PM. Expression of peptidylarginine deiminase-2 and -4, citrullinated proteins and anti-citrullinated protein antibodies in human gingiva. J Periodontal Res 2013;48:252–61. Mikuls TR, Thiele GM, Deane KD, Payne JB, O’Dell JR, Yu F, et al. Porphyromonas gingivalis and disease-related autoantibodies in individuals at increased risk for rheumatoid arthritis. Arthritis Rheum 2012;64:3522–30. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315–24. Little JW, Jacobson JJ, Lockhart PB. The dental treatment of patients with joint replacements: a position paper from the Amer-

1099

28.

29.

30.

31.

32.

33.

34.

35.

36. 37. 38.

39. 40. 41.

42. 43.

44. 45.

ican Academy of Oral Medicine. J Am Dent Assoc 2010;141: 667–71. Watters W III, Rethman MP, Hanson NB, Abt E, Anderson PA, Carroll KC, et al. Prevention of orthopaedic implant infection in patients undergoing dental procedures. J Am Acad Orthop Surg 2013;21:180–9. Machtei EE, Christersson LA, Grossi SG, Dunford R, Zambon JJ, Genco RJ. Clinical criteria for the definition of “established periodontitis”. J Periodontol 1992;63:206–14. Htoon HM, Peng LL, Huak CY. Assessment criteria for compliance with oral hygiene: application of ROC analysis. Oral Health Prev Dent 2007;5:83–8. Walker C, Puumala S, Golub LM, Stoner JA, Reinhardt RA, Lee HM, et al. Subantimicrobial dose doxycycline effects on osteopenic bone loss: microbiologic results. J Periodontol 2007;78:1590–601. Hueber W, Tomooka BH, Zhao X, Kidd BA, Drijfhout JW, Fries JF, et al. Proteomic analysis of secreted proteins in early rheumatoid arthritis: anti-citrulline autoreactivity is associated with up regulation of proinflammatory cytokines. Ann Rheum Dis 2007; 66:712–9. Prevoo ML, van ’t Hof MA, Kuper HH, van Leeuwen MA, van de Putte LB, van Riel PL. Modified disease activity scores that include twenty-eight–joint counts: development and validation in a prospective longitudinal study of patients with rheumatoid arthritis. Arthritis Rheum 1995;38:44–8. Van der Heijde D, Dankert T, Nieman F, Rau R, Boers M. Reliability and sensitivity to change of a simplification of the Sharp/van der Heijde radiological assessment in rheumatoid arthritis. Rheumatology (Oxford) 1999;38:941–7. Curtis MA, Slaney JM, Carman RJ, Johnson NW. Identification of the major surface protein antigens of Porphyromonas gingivalis using IgG antibody reactivity of periodontal case-control serum. Oral Microbiol Immunol 1991;6:321–6. Bohnstedt S, Cullinan MP, Ford PJ, Palmer JE, Leishman SJ, Westerman B, et al. High antibody levels to P. gingivalis in cardiovascular disease. J Dent Res 2010;89:938–42. Dzink JL, Socransky SS, Haffajee AD. The predominant cultivable microbiota of active and inactive lesions of destructive periodontal diseases. J Clin Periodontol 1988;15:316–23. Maeda H, Fujimoto C, Haruki Y, Maeda T, Kokeguchi S, Petelin M, et al. Quantitative real-time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQ gene and total bacteria. FEMS Immunol Med Microbiol 2003;39:81–6. Dilthey AT, Moutsianas L, Leslie S, McVean G. HLA*IMP—an integrated framework for imputing classical HLA alleles from SNP genotypes. Bioinformatics 2011;27:968–72. Leslie S, Donnelly P, McVean G. A statistical method for predicting classical HLA alleles from SNP data. Am J Hum Genet 2008;82:48–56. Mikuls TR, Gould KA, Bynote KK, Yu F, Levan TD, Thiele GM, et al. Anticitrullinated protein antibody (ACPA) in rheumatoid arthritis: influence of an interaction between HLA-DRB1 shared epitope and a deletion polymorphism in glutathione s-transferase in a cross-sectional study. Arthritis Res Ther 2010;12:R213. Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 2001;98:5116–21. Kallberg H, Ding B, Padyukov L, Bengtsson C, Ronnelid J, Klareskog L, et al. Smoking is a major preventable risk factor for rheumatoid arthritis: estimations of risks after various exposures to cigarette smoke. Ann Rheum Dis 2011;70:508–11. Kaur S, White S, Bartold PM. Periodontal disease and rheumatoid arthritis: a systematic review. J Dent Res 2013;92:399–408. Tomar SL, Asma S. Smoking-attributable periodontitis in the United States: findings from NHANES III. National Health and Nutrition Examination Survey. J Periodontol 2000;71:743–51.

1100

46. Genco RJ, Falkner KL, Grossi S, Dunford R, Trevisan M. Validity of self-reported measures for surveillance of periodontal disease in two western New York population-based studies. J Periodontol 2007;78:1439–54. 47. Arkema EV, Karlson EW, Costenbader KH. A prospective study of periodontal disease and risk of rheumatoid arthritis. J Rheumatol 2010;37:1800–4. 48. Polak D, Wilensky A, Shapira L, Halabi A, Goldstein D, Weiss EI, et al. Mouse model of experimental periodontitis induced by Porphyromonas gingivalis/Fusobacterium nucleatum infection: bone loss and host response. J Clin Periodontol 2009;36:406–10. 49. Polak D, Shapira L, Weiss EI, Houri-Haddad Y. The role of coaggregation between Porphyromonas gingivalis and Fusobacterium nucleatum on the host response to mixed infection. J Clin Periodontol 2012;39:617–25. 50. Miriovsky BJ, Michaud K, Thiele GM, O’Dell J, Cannon GW, Kerr G, et al. Anti-CCP antibody and rheumatoid factor concentrations predict greater disease burden in men with rheumatoid arthritis. Ann Rheum Dis 2010;69:1292–7. 51. Quirke AM, Lugli EB, Wegner N, Hamilton BC, Charles P, Chowdhury M, et al. Heightened immune response to autocitrullinated Porphyromonas gingivalis peptidylarginine deiminase: a

MIKULS ET AL

52.

53.

54.

55. 56.

potential mechanism for breaching immunologic tolerance in rheumatoid arthritis. Ann Rheum Dis 2014;73:263–9. Wegner N, Wait R, Sroka A, Eick S, Nguyen KA, Lundberg K, et al. Peptidylarginine deiminase from Porphyromonas gingivalis citrullinates human fibrinogen and ␣-enolase: implications for autoimmunity in rheumatoid arthritis. Arthritis Rheum 2010;62: 2662–72. Reno F, Rocchetti V, Migliario M, Rizzi M, Cannas M. Chronic exposure to cigarette smoke increases matrix metalloproteinases and filaggrin mRNA expression in oral keratinocytes: role of nicotine stimulation. Oral Oncol 2011;47:827–30. Delbosc S, Alsac JM, Journe C, Louedec L, Castier Y, BonnaureMallet M, et al. Porphyromonas gingivalis participates in pathogenesis of human abdominal aortic aneurysm by neutrophil activation: proof of concept in rats. PLoS One 2011;6:e18679. Scott DA, Krauss J. Neutrophils in periodontal inflammation. Front Oral Biol 2012;15:56–83. Dwivedi N, Upadhyay J, Neeli I, Khan S, Pattanaik D, Myers L, et al. Felty’s syndrome autoantibodies bind to deiminated histones and neutrophil extracellular chromatin traps. Arthritis Rheum 2012;64:982–92.