Antibodies to Porphyromonas gingivalis Are Associated with Anticitrullinated Protein Antibodies in Patients with Rheumatoid Arthritis and Their Relatives

MATERIALS AND METHODS

Patients, relatives, and controls

Patients with RA were recruited from a Cree and Ojibway NAN population in central Canada. Patients visiting rheumatology clinics in urban (Winnipeg, Saskatoon) and rural (Norway House, St. Theresa’s Point) locations were approached to bring along unaffected relatives who were willing to participate in this study. All study subjects were over 18 years of age. The unaffected population consisted mainly of first-degree relatives (76%) and was composed of siblings and offspring, with the remainder being second-degree relatives (cousins, nieces, nephews). Eighty-five families were included in the study, comprising 82 probands and 205 relatives. All relatives had at least 1 family member with RA, but some relatives did not have their affiliated RA proband(s) included in the study, thus accounting for the larger number of families than probands. Additionally, 47 unrelated healthy controls were recruited from the same populations. Sixty white controls were randomly selected from a serum bank of healthy individuals prior to undergoing vaccination for overseas travel. The control populations were not closely matched for age and sex.

The clinical and serological characteristics of the patients with RA and relatives are described in Table 1. RA was diagnosed according to the American College of Rheumatology criteria²². Clinical assessment of swollen, stiff, and painful joints of all patients and relatives was performed by a rheumatologist at the time of inclusion. Of the relatives, 8 presented with 1 or more swollen joints at inclusion, but did not meet criteria for RA or any other rheumatic disease, and were classified as having undifferentiated arthritis (UA). Analyses were undertaken with and without this small group of individuals with UA, and their inclusion did not affect the outcome or significance of each analysis. These individuals were analyzed as part of the unaffected relatives population. There was no difference in the demographic characteristics between the first-degree and second-degree relatives. In the patients with RA, radiographic erosions in hands and feet were assessed by review of the radiographs and/or radiograph reports, and were categorized as present or absent in each. The 47 unrelated controls had no swollen joints at inclusion and no first-degree relatives with RA.

All participants gave written consent, and the Biomedical Research Ethics Boards of the University of Manitoba and the Band Councils of each rural community approved the protocol.

ACPA antibody isotype testing

Total IgG ACPA seropositivity was assayed by ELISA using a second-generation anti-CCP kit (CCP2; Inova Diagnostics, San Diego, CA, USA). Positive tests were defined as those with values ≥ 20 units, per the manufacturer’s specifications. ACPA isotypes (IgA, IgG1-4, and IgM) were measured in baseline serum samples of patients with RA, healthy relatives, and unrelated healthy controls, using CCP2 plates (Euro-Diagnostica, Arnhem, The Netherlands) and the ELISA mentioned²¹. A successive dilution of 1 reference standard, consisting of a pool of 20 ACPA-positive samples, was used in all plates. Distinct dilutions of this standard (IgA and IgM: 1:12.5; IgG1: 1:400; IgG2: 1:6.25; IgG3 and IgG4: 1:12.5) were defined as containing 1000 arbitrary units per ml (AU/ml).

Determination of cutoff values for ACPA ELISA

The level of specific antibodies in each serum sample was determined using the reference standard curve. Cutoff values for the citrullin-specific responses were calculated in each assay and were defined as 2 SD above the mean concentration of serum samples obtained from 30 healthy White controls. In case the concentrations of several control samples were below the detection limit of the ELISA, the cutoff was calculated as the lowest concentration situated on the ascending region of the standard curve. Borderline samples (with concentrations between 10 AU above and below the cutoff value) were tested at least twice for IgA ACPA and all IgG isotypes. All samples reacting with the citrullinated peptides in fine-specificity assays were retested. Only samples that were positive every time tested were considered positive.

RF measurements

IgM RF values reported throughout the study were determined by nephelometry. IgA RF and IgM RF were also measured by ELISA, using human IgG1 as the capture antigen, and F(ab’)2 fragments of peroxidase-conjugated antihuman IgA or IgM, as described²³. The cutoffs used to assign positivity were based on 60 White controls. The IgM RF values as determined by ELISA were comparable to those obtained by nephelometry.

Humoral immune response against P. gingivalis antigens

Lipopolysaccharides (LPS) of P. gingivalis were isolated by the method described by Darveau and Hancock²⁴. The amount of contaminating proteins was evaluated using a protein assay kit (Biorad Laboratories, Mississauga, ON, Canada) and was < 0.001%.

Antibodies to P. gingivalis LPS and ELISA index calculations

IgG antibodies specific to LPS of P. gingivalis were measured using ELISA. The wells of 96-well flat-bottom microtiter plates were coated in triplicate with LPS of P. gingivalis. After washing and blocking the plates, serum samples were added to individual wells and specific human IgG antibodies were detected with an alkaline phosphatase-conjugated antihuman immunoglobulin. The absorbance was read at 405 nm using an ELISA plate reader. Each assay included a conjugate control, a substrate control, a conjugate-substrate reactivity control, and a serially diluted reference serum control. The results were expressed as an ELISA Index (EI), which was the mean OD405 of a given serum divided by the mean OD405 of the calibrator (reference serum). Positive calibrator and negative assay controls were included in each run to control for intraassay and interassay variation. Values were reported as median (interquartile range; IQR).

Smoking and oral health questionnaires

Subjects answered questionnaires regarding smoking and oral hygiene habits. Smoking was assessed by the duration and the number of smoked cigarettes [i.e., 1 cigarette/day for 3 or more months, at least 5 cigarettes in the past 6 months (current smoker); 1–5 cigarettes/month, 1–5 cigarettes/week, fewer than 10 cigarettes/day, half to 1 pack/day, or more than 1 pack/day). Oral hygiene habits were assessed by asking participants how many times a week they brushed and flossed their teeth, on a scale of never, 1–6 times, 7–14 times, or 15+ times. They also were asked how often they see a dentist, on a scale of never, 1–2 times/year, or 2+ times/year.

Statistical analysis

Statistical analyses were performed using the SPSS (version 14.0) software. Data distribution was tested for normality, and non-normally distributed data reported as median and IQR. Mann-Whitney U or Kruskal-Wallis tests were used to compare age, joint counts, C-reactive protein, and levels of P. gingivalis, ACPA isotypes, and RF. Differences in the distribution of the ACPA isotypes between healthy relatives and patients with RA and associations with dental habits and smoking were calculated using the chi-squared test. The chi-squared test was also used for calculating OR with 95% CI, and p values for the association of SE with ACPA. When a cell contained < 5 individuals, Fisher’s exact 2-tailed p value was calculated. A p value < 0.05 and a 95% CI that excluded the value of 1 were considered significant.

RESULTS

The clinical characteristics of the study subjects are shown in Table 1. The relatives were younger than patients, and less likely to be women. As reported²¹, 91% of patients and 19% of relatives had ACPA of at least 1 isotype, and patients had a greater number of ACPA isotypes and higher ACPA levels. Similar findings were seen using a CCP2 kit that measures only total IgG ACPA (RA 81% vs relatives 5% positive). There were no significant differences between patients and relatives in the presence of SE alleles (81% vs 73%) or in the presence of 2 copies of SE alleles (33% vs 23%), although both tended to be higher in patients. Relatives with 2 SE alleles were more likely to be ACPA-positive (OR 2.5, p = 0.02). HLA-DRB1*0404 and *1402 are the most prevalent SE alleles in this population²⁰.

IgG antibody responses to P. gingivalis LPS were measured in the sera from patients with RA (n = 82), relatives (n = 205), unrelated NAN controls from the same population (n = 47), and non-NAN controls from a serum bank (n = 60). Five patients with RA and 9 relatives had samples that could not be analyzed for anti-P. gingivalis LPS responses, but these individuals did not differ significantly from the remainder of the cohort. As shown in Figure 1, anti-P. gingivalis levels were higher in the patients with RA compared to the other groups combined [median (IQR) 42 (30) vs 33 (19) AU; p = 0.001], but there were no differences between the relatives and the 2 control groups. Patients with RA with and without erosions did not differ in anti-P. gingivalis levels (data not shown).

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Figure 1.

Levels of anti-P. gingivalis ELISA index in patients with RA, relatives, First Nations (NAN) controls, and non-NAN controls. Numbers represent arbitrary units derived from ELISA optical densities.

We next looked specifically at the association between anti-P. gingivalis responses and ACPA serology in patients with RA and relatives. Levels of ACPA isotypes (IgG1-4, IgA, IgM) were analyzed by ELISA as described. Individuals were considered ACPA-positive if their ELISA levels were above the cutoff threshold established for any of the ACPA isotypes. The data in Figure 2A indicate that anti-P. gingivalis responses were significantly higher in ACPA-positive than in ACPA-negative individuals. In the unaffected relatives group, ACPA-positive individuals had higher anti-P. gingivalis LPS levels [median (IQR) 44 (21) vs 32 (22) AU; p < 0.0001] compared to ACPA-negative relatives. In the group of patients with RA, ACPA-positive individuals had higher anti-P gingivalis levels compared to ACPA-negative patients with RA [median (IQR) 43 (29) vs 25 (28) AU; p = 0.04] even though only 6/82 patients (7%) were ACPA-negative. There was no difference in anti-P. gingivalis levels between ACPA-positive relatives and ACPA-positive patients with RA. Moreover, of the ACPA-positive relatives and patients, there were no significant differences in the anti-P. gingivalis levels for each individual isotype (data not shown). These data indicate that there is an association between humoral immune responses to P. gingivalis and ACPA positivity, and that this association is independent of having RA itself. In contrast, there was no association between anti-P. gingivalis antibody levels and RF, as shown in Figure 2B. Further, as shown in Table 2, after stratifying for ACPA in the IgM RF-positive or IgA RF-negative individuals, the association of P. gingivalis levels with ACPA remained in the relatives, but not in the patients with RA. In contrast, in ACPA-positive individuals there were no associations of IgM or IgA RF with anti-P. gingivalis levels. This supports a primary association between ACPA positivity and P. gingivalis levels.

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Figure 2.

Levels of anti-P. gingivalis in relatives and patients with RA according to the presence of (A) anticitrullinated protein antibodies (ACPA) or (B) rheumatoid factor (RF). Numbers represent arbitrary units derived from ELISA optical densities.

We next examined whether poor oral health habits were associated with immune responses to P. gingivalis, with the presence of ACPA, or with RA. A subset of subjects (56 patients; 141 relatives) answered a detailed questionnaire regarding oral hygiene habits. There were no significant clinical, serological, or anti-P. gingivalis antibody level differences between subjects who did or did not answer the oral hygiene questions. As shown in Table 3, patients with RA were more likely to have dentures (37% vs 15%; p < 0.001) compared to relatives, and visited the dentist less often. Both groups had similar oral hygiene habits, with 44% of patients and 55% of relatives brushing at least once daily and > 60% flossing. There were no associations between oral hygiene habits and ACPA positivity apart from an increased tendency to have dentures.

Since smoking has been associated in studies with both ACPA^5,7 and PD^25,26, we investigated whether there was any association between smoking habits and the humoral immune response to P. gingivalis and to ACPA positivity. The data shown in Table 3 indicate that 75% of patients with RA and 74% of relatives had smoked at least 1 cigarette/day for at least 3 months, while 22% of patients with RA and 23% of relatives smoked more than a half pack/day. On the basis of these similar high smoking rates in both patients with RA and relatives, no clear association could be established between smoking-related measurements and the immune response to P. gingivalis or ACPA positivity. Moreover, no association was seen between smoking, SE (single or 2 copies), and ACPA of any isotype in patients with RA or relatives. There was no association between anti-P. gingivalis levels and SE.

DISCUSSION

There has been a well documented association between RA and PD since the 1950s, although the biological basis for this association has not been clearly elucidated. This epidemiological association was confirmed in the NHANES III study, which suggested an OR 1.82, 95% CI 1.04–3.20¹⁵. It has been hypothesized that this association may be based on the capacity of P. gingivalis, the major etiological agent of periodontitis, to express a PAD, an enzyme responsible for posttranslational citrullination of arginine residues¹³. This enzymatic activity potentially exposes affected individuals to citrullinated antigens, and in the context of the appropriate immunogenetic background, would predispose to the development of ACPA. ACPA are known to be relatively specific for RA, to precede disease onset, and to potentially be involved in the pathogenesis of RA synovitis^27,28.

We investigated whether there was an association between immune responses to the periodontal pathogen P. gingivalis and the presence of RA and/or ACPA. Sera from a cohort of NAN patients with RA and their unaffected relatives who are at risk for disease development²¹ were analyzed for ACPA, RF, and specific IgG antibodies to P. gingivalis LPS. We demonstrate that serum levels of the anti-P. gingivalis LPS were higher in patients with RA compared to their relatives, and to 2 distinct control groups, 1 of which was from the same population. Importantly, the data reveal that the immune responses to P. gingivalis LPS were significantly higher in ACPA-positive than in ACPA-negative individuals, irrespective of whether they had RA.

Mikuls, et al recently compared the levels of anti-P. gingivalis antibodies in patients with RA, PD, and healthy controls²⁹. These data showed that the levels of anti-P. gingivalis antibodies were highest in PD, lowest in controls, and intermediate in RA. This study also showed an association between the levels of anti-P. gingivalis antibodies and levels of IgM and IgG2 ACPA, but not RF, in the patients with RA. Our study is consistent with these data. It should be pointed out that we tested the antibody response to a more restricted P. gingivalis antigen, LPS, while the Mikuls study evaluated antibody responses to a broader antigenic spectrum using lysates of whole organisms.

We present data from a unique cohort of the disease-free relatives of patients with RA who have a high prevalence of ACPA. This allowed us to address a key question: whether antibody responses to P. gingivalis are associated with ACPA outside the context of RA. The data clearly indicate that this is the case. Moreover, this association was specific for ACPA as there was no association with RF. The lack of association between antibody responses to P. gingivalis and RF is also reported in the Mikuls study²⁹. The findings are consistent with the hypothesis that immune responses to P. gingivalis are in some way involved with the breaking of immune tolerance to citrullinated antigens, as indicated by the presence of ACPA. Since P. gingivalis expresses PAD and can potentially citrullinate peptides in vivo, host immune responses to such neoantigens may trigger autoimmunity to endogenous citrullinated antigens through mechanisms such as molecular mimicry and epitope-spreading¹³.

It is currently unknown which peptides or proteins may constitute the most important in vivo antigen for ACPA, but several citrullinated proteins have been reported as possible targets, including citrullinated fibrinogen, vimentin, type II collagen, and α-enolase^30–33. Citrullinated α-enolase peptide 1 (CEP-1), the immunodominant peptide of human α-enolase, has 82% homology with enolase from P. gingivalis, and anti-CEP1 antibodies have been described to be cross-reactive with the bacterial enzyme³⁴. On the basis of these data, it was concluded that bacterial infection with P. gingivalis may play a role in priming the ACPA response. Although antibody responses to CEP-1 were not measured as part of our study, we have used a different α-enolase peptide in our fine specificity studies²¹. This peptide (called C6) was recognized by 14% of NAN patients with RA and none of the relatives, thus precluding a meaningful analysis in the context of our study. Large-scale studies and longitudinal followup of this cohort, to determine which of the ACPA-positive relatives may develop RA, will be required to more clearly elucidate the possible associations between ACPA, antienolase reactivity, and P. gingivalis.

We attempted to determine whether oral hygiene and smoking were associated with RA, ACPA, and with immune responses to P. gingivalis. It is known that smoking and poor oral health habits both increase the risk of PD. Smoking is a risk factor for PD possibly through the effects of nicotine on inflammatory cytokine profiles^35,36 and matrix metalloproteinase activity^37,38 or potentially even through direct effects on P. gingivalis gene expression³⁹. We did find a high prevalence of both smoking and poor oral health habits in the study population based on self-report questionnaire data; however, we could not define a clear association between these factors and the presence of either RA or ACPA. Studies of NAN populations from a similar geographical area have shown a high prevalence of PD that is associated with poor dental hygiene habits⁴⁰. Further studies, including formal dental assessments and sampling, are under way to confirm the presence and extent of PD in this population.

The reported association between immune responses to the oral pathogen P. gingivalis and the presence of ACPA in a population with a high background prevalence of RA-pre-disposing HLA-DRB1 alleles is consistent with a gene-environment interaction that may result in breaking self-tolerance to citrullinated antigens and/or amplification of these autoimmune responses, and ultimately leading to the development of RA. It should be added that the sample size tested in this study precluded an analysis of such as gene-environment interaction. Moreover, the demonstrated association between P. gingivalis and ACPA may reflect a broader association with periodontitis, which was not addressed by systematic oral examinations. While further studies are needed to support this association and to assess longitudinal outcomes in high-risk individuals, we suggest that interventions directed at modulating these environmental risk factors, such as improved oral health and smoking cessation, may play an important role in reducing or delaying the onset of future RA.