Concentration of antibodies against Porphyromonas gingivalis is increased before the onset of symptoms of rheumatoid arthritis

Rheumatoid arthritis (RA), a complex chronic inflammatory disease with a worldwide prevalence of 0.5–1.0 % [1], is characterized by production of anti-citrullinated protein/peptide antibodies (ACPA) in the majority of patients and persistent inflammation in the synovial tissue of the joints leading to destruction of cartilage and bone [2–4]. The aetiology of RA remains unknown, although a complex interplay exists between genetic and environmental factors [5, 6]. Chronic periodontitis (PD), which is the world’s commonest inflammatory disease often resulting in destruction of alveolar bone and tooth loss, has been suggested as an environmental determinant for the occurrence and severity of RA [7–9].

Elucidation of the potential aetiological link between PD and RA has progressed [8, 10, 11] and a number of studies have identified similarities between these two diseases that possibly explain the epidemiological association. Both PD and RA display systemic markers of inflammation (e.g. C-reactive protein and pro-inflammatory cytokines) [6, 12, 13], and an association with HLA-DRB1 alleles [14] and smoking [15, 16] has been described for both RA and PD. Furthermore, citrullinated proteins have been detected in both rheumatoid joints and inflamed gingival tissue, as well as in other tissues in relation to inflammatory conditions [17–19]. However, an association between PD and established RA could not be confirmed in one of our recent publications [20].

Moreover, data from one of our other studies suggest that the oral pathogen Porphyromonas gingivalis, rather than PD, may be linked to the development of RA [21]. P. gingivalis is a common periodontal pathogen associated with PD [22, 23], and is the only prokaryote known to express an endogenous peptidylarginine deiminase enzyme (P.PAD), a virulence factor capable of citrullinating human and bacterial proteins, including auto-citrullination [24–26]. P.PAD interacts with another major virulence factor, arginine gingipainB (RgpB), an arginine-specific extra-cellular protease expressed on the surface of the bacterial outer membrane [24, 25]. RgpB is essential for P. gingivalis to citrullinate peptides; that is, only after degradation by RgpB can P.PAD convert peptidylarginine into peptidylcitrulline [24, 26].

It is hypothesized that citrullination by P. gingivalis causes a chronic exposure of citrullinated peptides in the inflamed periodontium, possibly leading to a break of immune tolerance in genetically susceptible individuals and subsequent production of ACPA. Epitope spreading, induced by molecular mimicry and cross-reactivity with citrullinated epitopes exposed in the joint, could lead to progression of chronic RA [27–29]. ACPA appear many years before the onset of RA [30, 31], suggesting that the initial immune dysregulation occurs long before symptoms of RA develop, outside the joints, potentially at mucosal sites such as the gingival tissue [17, 27, 32]. Citrullinated P.PAD has been demonstrated to be a target of the ACPA response [25] and we recently demonstrated that elevated anti-RgpB antibody levels have a stronger association with RA than smoking [21], identifying P. gingivalis as a potential mechanistic link between PD and RA. Importantly, in the same study we could also show that anti-RgpB IgG levels were significantly increased in sera from PD patients compared with periodontally healthy individuals, supporting anti-RgpB IgG as a surrogate marker for oral infection by P. gingivalis.

This study investigated whether raised anti-P. gingivalis antibody levels precede onset of symptoms of RA and the ACPA response in order to elucidate the role of P. gingivalis as a potential trigger of autoimmunity and the development of RA. Importantly, because we have no data on periodontal status in our cohorts, we have only focused on P. gingivalis, not on PD, in the present study. Consequently, we analysed the antibody response to RgpB and CPP3, a citrullinated peptide derived from P.PAD, in blood samples collected prior to the onset of symptoms and at the diagnosis of RA. Identifying a potential mechanism capable of breaking immunological tolerance at the earliest stage of disease would provide an insight into the aetiopathology of RA, and could indicate new clinical therapies and interventions.

In the present study we investigated the role of the oral pathogen P. gingivalis in the development of RA, by focusing on the anti-P. gingivalis antibody response in RA patients prior to onset of symptoms. Our recent data show elevated antibody levels against the potent P. gingivalis virulence factor arginine gingipainB in patients with RA, especially in ACPA-positive RA [21]. We have also shown that these antibodies are clearly elevated in patients with PD, compared with periodontally healthy individuals, demonstrating that anti-RgpB IgG probably represents a good surrogate marker for P. gingivalis infection, which has been associated with PD [22, 23]. With the present study, we report increased concentrations of these antibodies in a subset of individuals years before onset of symptoms of RA. Consistent with Quirke et al.’s data in RA [25], concentrations of anti-CPP3 antibodies directed against, a synthetic cyclic citrullinated peptide  derived from another P. gingivalis-specific virulence factor, P.PAD, were also increased in a subset of both pre-symptomatic individuals and RA patients, compared with controls.

In line with our previous findings in RA patients, the association between the development of RA in pre-symptomatic individuals and the anti-RgpB antibody response was not dependent on smoking habits or presence of HLA-DRB1 SE or the PTPN22 T-variant [21]. Our data on lower anti-RgpB IgG levels in RA patients who were ever smokers or current smokers compared with non-smokers were also in line with a number of previous reports showing lower anti-P. gingivalis antibody levels in smokers compared with non-smokers [21, 39, 40]. One explanation for the observed trend of lower anti-RgpB antibody levels in RA patients compared with pre-symptomatic individuals (Figs. 1a and 2a) could therefore potentially be the higher frequency of smokers among RA patients (67.2 %), compared with pre-symptomatic individuals (64 %). Furthermore, as we recently showed for the anti-RgpB IgG response in RA, the HLA-DRB1 SE in combination with anti-CPP3 IgG reveals a stronger association with RA and with being pre-symptomatic than HLA-DRB1 SE alone. The same effect occurred when combining smoking with anti-CPP3 IgG positivity, although only in RA patients, not in pre-symptomatic individuals.

In this study, stratification of data into ACPA-positive and ACPA-negative sub-groups was not possible due to the limited number of individuals. However, a weak correlation between the concentration of anti-RgpB antibodies and that of the “classical” ACPA, measured as anti-CEP-1 antibodies, could be observed. Notably, ACPA (anti-CEP-1, anti-cFibβ36-52 and anti-cfilaggrin antibodies) were analysed by the ISAC multiplex assay, which is only a semi-quantitative method [37], and thus are not completely comparable with results from the ELISA used for measuring anti-RgpB IgG.

No relationship was detected between anti-RgpB and anti-CPP3 antibodies. This was unexpected considering the origin of both RgpB and CPP3 as P. gingivalis-specific antigens, and our interpretation of these two antibodies as surrogate markers for a P. gingivalis infection. Although the concentrations of anti-CPP3 antibodies, like anti-RgpB antibodies, were significantly increased in both RA patients and pre-symptomatic individuals compared with controls, the frequency of anti-CPP3 antibodies was only significantly increased in RA patients. Moreover, the anti-RgpB antibody response was elevated (compared with controls) several years earlier (12 years) than the anti-CPP3 antibody response (8 years). The anti-CPP3 antibody response was similar to the “classical” ACPA response; that is, there was no reactivity to the arginine-containing control peptide RPP3, the majority of anti-CPP3 antibody-positive cases were also anti-CCP2 antibody positive and the anti-CPP3 antibody response (both concentrations and the accumulated frequency of positive samples) increased gradually during the pre-dating time until symptom onset [37]. Taken together, these data may suggest that the anti-CPP3 antibody response, rather than being P. gingivalis specific, simply belongs to the generic ACPA response, or rather represents cross-reactivity with another citrullinated antigen. The low frequency of these antibodies (8 % in RA) could point to this. Moreover, in-vivo auto-citrullination by P.PAD has been debated [39], and the CPP3/RPP3 peptide—with its internal rather than C-terminal citrulline residue—may not represent an in-vivo-generated antigen. Still, the CPP3/RPP3 peptide sequence is bacterial derived and does not correspond to any human protein sequence.

Results recently published by Fisher et al. [41] conflict with ours in that they were unable to identify associations between anti-RgpB or anti-CPP3 IgG and pre RA. The two studies differ in several aspects that may explain the discrepant results: their study was based on a smaller study population (n = 103) than ours (n = 251, with 422 samples), with participants from a number of different southern European countries whilst our study subjects were recruited from a geographically defined area in northern Sweden; and not all of the pre-symptomatic individuals in Fisher et al.’s study were confirmed to develop RA, as they were in our study [41]. Additionally, the periodontal microbiota has been shown to vary between countries [42] and bacterial strain diversity has been described previously for P. gingivalis [43]. Altogether, these differences could contribute to the variability in the results of these studies.

Supporting our results, Mikuls et al. [44] reported increased concentrations of anti-P. gingivalis antibodies in high-risk individuals compared with controls. Also in accordance with our data is a study by de Smit et al. [45], in which elevated anti-P. gingivalis antibody levels were observed in arthralgia patients with RF or ACPA positivity compared with controls. However, anti-P. gingivalis antibody levels in de Smit et al.’s study were not higher in arthralgia individuals who developed RA compared with those who did not. This was not possible to evaluate in our study [45].

We believe this to be the largest population-based study analysing the anti-P. gingivalis antibody response in individuals before onset of symptoms of RA performed to date. Our study has limitations: the samples analysed were from different population surveys, and were not collected on a regular basis; no information regarding periodontal status or treatment was available, hence there was an inability to set a cut-off value for the anti-RgpB antibodies response; and it was not possible to investigate the relationship between PD and the development of RA. Also, data on the presence of P. gingivalis DNA were not available, and analysis of the anti-RgpB IgG and the anti-CPP3 IgG responses in relation to the presence of the bacteria was consequently not possible. However, as in our previous study [21], the anti-RgpB IgG response was interpreted as a surrogate marker for a P. gingivalis infection, past or present, whilst our data suggest that the anti-CPP3 antibody response, which follows the “classical” ACPA response, should be considered ACPA specific rather than a P. gingivalis-specific antibody.

Our data demonstrate that antibodies against P. gingivalis are significantly increased in patients with RA compared with controls, and that these antibodies are detectable years before the onset of symptoms, supporting an aetiological role for P. gingivalis in the development of RA. Studies on larger cohorts with samples collected on a regular basis are needed for a deeper understanding of the relationship between P. gingivalis, anti-P. gingivalis antibodies, ACPA and the development of RA.