Background
The aetiological association between genes and environmental factors in the development of rheumatoid arthritis (RA) is well-established today, with specific
HLA-DRB1 alleles, termed the shared epitope (SE), as the major genetic contributor [
1], and smoking as the main environmental risk factor [
2]. Moreover, there is a strong biological interaction between smoking and SE, specifically in anti-citrullinated protein antibody (ACPA)-positive RA [
3]. Importantly though, current knowledge does not explain the whole risk of developing RA and there has been an extensive search for additional environmental factors predisposing individuals to RA.
The link between an infectious agent and the development of RA has long been discussed, and three decades ago researchers reported that patients with RA have higher frequency and increased antibody titres against Epstein-Barr virus (EBV) compared to controls [
4,
5]. Since then, a number of studies have addressed the possible role of viral infections in the aetiopathogenesis of RA. In addition to EBV [
6], other viruses have been implicated, including human parvovirus B19 (B19) [
7,
8], and cytomegalovirus (CMV) [
9].
EBV and CMV belong to the human herpes virus family. Primary infection is often asymptomatic, but may cause severe morbidity, even mortality, in immunocompromised patients. Infection by parvovirus B19 may cause erythema infectiosum (fifth disease), predominantly affecting children. In adults, B19 often causes self-limiting acute symmetric polyarthritis [
10], with symptoms disappearing within a few weeks, although arthralgia may persist for months, even years, in about 20%, usually women [
11], suggesting that polyarthritis caused by B19 may progress to RA.
Whereas a number of serological studies have identified higher antibody levels against these viruses and/or increased antibody frequency in RA compared to controls [
4,
5,
7,
12], other studies have not replicated these findings [
13‐
15]. In addition to serological evidence, viral DNA has been detected in the synovium and bone marrow of patients with RA [
7,
12‐
23], and studies also report on the presence of viral proteins in the joints of patients with RA [
7,
16,
17]. However, other studies have had conflicting results [
24‐
26].
Most studies performed to date, on the association between viral infections and RA development, have used small cohorts and often lack well-matched control groups. With this in mind, it seems difficult to draw any conclusions from the current literature, which presents contradictory results. Furthermore, studies during the past decade have highlighted the importance of autoimmunity to citrullinated proteins, when investigating genetic and environmental risk factors for RA, and such studies have not been conducted when it comes to viral infections.
Using the large population-based Swedish Epidemiological Investigation of Rheumatoid Arthritis (EIRA) case-control study, we had a unique opportunity to analyse anti-viral antibody levels in relation to RA, autoantibodies, genetic risk factors, smoking habits and clinical parameters. Hence, in the present study we have investigated the associations of EBV, B19 and CMV viruses with ACPA-positive and ACPA-negative disease, in relation to SE, smoking status, disease activity score in 28 joints (DAS28) and C-reactive protein (CRP) levels by measuring anti-viral IgG in sera from 990 patients with RA (cases) and 700 matched controls.
Discussion
In this study, we have investigated a possible association between infection by common viruses and the development of RA. To our knowledge, this is the largest epidemiological study conducted to date, where the prevalence of anti-EBV, anti-CMV and anti-B19 antibodies was investigated in a population-based RA case-control cohort. For the first time, these viruses have been examined in the context of ACPA, and genetic (HLA-DRB1 SE) and environmental (smoking) risk factors, which today are known to contribute to disease development only in subsets of RA.
The frequencies of anti-viral antibodies were high in EIRA, and rather similar when comparing patients with ACPA-positive RA, patients with ACPA-negative RA and controls. We detected no significant differences in anti-EBV or anti-CMV IgG, while the prevalence of anti-B19 antibody-positive individuals was somewhat higher in ACPA-positive RA compared to controls. Interestingly, we found significant associations between low anti-EBV and low anti-B19 IgG levels and ACPA-positive RA. Our data are thus in agreement with a number of previous reports [
12,
14,
15], yet contradictory to others [
4,
5,
13]. Explanations for these discrepant results could be: (1) differences in the number of study participants, where low numbers could result in insufficient statistical power and skewed data; (2) differences in storage and processing of biological material, which may affect the antibody detection assay; (3) differences in study populations, in age, ethnicity and/or disease duration; and/or (4) differences in anti-rheumatic treatments, where immune modulatory and anti-inflammatory treatments could have negative effects on antibody titres, by altering the activity of plasma cells [
31,
32].
We have used the well-characterized EIRA cohort, with 990 patients with early RA and 700 matched controls in our study, providing proper controls and high statistical power. In addition, all patients in the EIRA cohort were DMARD-naïve, and serum samples were stored at minus 80 °C until examined, minimizing potential negative effects on antibody titres. We have measured anti-viral IgG levels, and used these as surrogate markers for viral infections. Others have focused on the presence of viral DNA [
7,
13,
17‐
19,
21,
22] or viral proteins [
7,
16,
20,
24] instead, or they have used other types of assays to detect anti-viral antibodies [
4,
5,
12,
14,
21].
The observation of lower anti-EBV and anti-B19 IgG levels in ACPA-positive RA could point towards an inability to mount a proper antibody response against pathogens in this subset of RA. However, generally lower antibody response to pathogens is, to our knowledge, not a specific feature of ACPA-positive RA. On the contrary, we have recently shown elevated antibody levels to the oral pathogen
Porphyromonas gingivalis in patients with ACPA-positive RA, compared to patients with ACPA-negative RA and controls [
33]. The use of corticosteroids could have a negative effect on serum IgG levels [
31], and although our study only included newly diagnosed DMARD-naïve patients, we have no information on the potential use of corticosteroids before RA diagnosis. Approximately 30% of all patients in the EIRA cohort are put on prednisolone when they are diagnosed [
29]. Importantly though, ACPA-positive and ACPA-negative patients are clinically very similar at this time point, and prednisolone was not more frequently administered to ACPA-positive patients compared to ACPA-negative in the EIRA cohort (personal communication, Dr Saedis Seavarsdottir). Hence, we find it unlikely that the potential use of cortisone prior to this time point would differ between ACPA-positive and ACPA-negative patients, suggesting that corticosteroids could not explain the lower anti-EBV and anti-B19 IgG levels detected in the ACPA-positive subset in our study.
Low anti-viral IgG levels could reflect low viral replication, and low viral load [
34]. On the other hand, one could speculate that low anti-viral antibody levels may instead indicate an insufficient anti-viral antibody response, with poor viral control, allowing for increased viral replication, resulting in higher viral load [
35]. With this interpretation of data, our observation of an interaction between low anti-EBV and low anti-B19 antibody levels and
HLA-DRB1 SE would support a number of other studies that have linked EBV and B19 to SE and RA. Balandraud and colleagues, for example, have shown that patients with RA expressing
HLA-DRB1*0404 (i.e. SE positive) have higher EBV viral DNA load in peripheral blood mononuclear cells (PBMCs) than patients expressing
HLA-DRB1*07 (i.e. SE negative) [
35], although the difference was not statistically significant (
p = 0.08). In another study, the frequencies of
HLA-DRB1 (*01, *04 and *07) alleles were shown to be significantly higher in individuals with symptomatic acute B19 infection than in healthy controls [
36]. Moreover, Saal et al. showed that SE-positive individuals with EBV DNA detected in the synovial membrane had a higher risk of developing RA than individuals who were negative for one or both of these variables [
19], and Chen et al. demonstrated synergistic effects between
HLA-DRB1*04 and parvovirus B19 infection in RA susceptibility [
12]. Collectively, these reports suggest that EBV and B19 may constitute environmental triggers for the development of RA in genetically predisposed individuals.
A number of studies have also suggested the possibility of autoimmunity arising as a result of molecular mimicry after EBV or B19 infection [
37‐
39]. Anti-VP1 IgG (antibodies directed against B19 structural protein) for example, have been shown to cross-react with type II collagen, a major component in hyaline cartilage, and a target of autoantibodies in RA [
40]. Moreover, antibodies to citrullinated EBV peptides have been described in RA [
41‐
44], and Rossi et al. showed that ACPA from patients with RA have higher affinity for citrullinated autoantigens (in this case histone citrullinated peptide 1), than for citrullinated peptides derived from EBV proteins (i.e. exogenous antigens) [
45], suggesting that these antibodies are initially produced against exogenous antigen and later selected and expanded by autoantigens.
Recent studies have also shown that EBV can persist in self-reactive memory B cells, thereby favouring the survival of pathogenic autoreactive B cells [
46,
47]. In addition, macrophages and lymphocytes infected by B19 provide another possible mechanism by which viruses could contribute to autoimmunity, through the continuous secretion of pro-inflammatory cytokines resulting in polyclonal B cell activation and proliferation of synovial B cells [
7,
48‐
50].
A weakness of our study is that we have only analysed anti-viral IgG levels, which most probably reflect infections occurring in childhood. We have not analysed anti-viral IgM or presence of viral DNA, which could have given us information about acute infections/re-activation of the viruses. Moreover, whether low anti-viral antibody levels reflect low viral load, or the opposite, high viral load, is not clear, and may differ from virus to virus. In order to dissect the role of common viruses in RA aetiology in detail, and the relationship between viral load and the anti-viral immune response, in the context of SE and ACPA, longitudinal studies should be performed, in which viral DNA and anti-viral IgM is measured in addition to anti-viral IgG.
Acknowledgements
We thank patients and controls, research nurses and clinicians, and the EIRA study group for their important contributions; Professor Lars Klareskog, for support and scientific discussions, and for establishing the EIRA cohort together with Professor Lars Alfredsson; scientists previously involved in collecting data for the EIRA database, specifically: Associate Professor Leonid Padyukov, Dr Patrik Stolt, Professor Johan Askling, Dr Saedis Saevarsdottir, and Dr Helga Westerlind; Dr Martina Johannesson, for support in supervising; and Professor Tomas Olsson and Professor Kristina Broliden, for scientific input and discussions.