Low levels of antibodies against common viruses associate with anti-citrullinated protein antibody-positive rheumatoid arthritis; implications for disease aetiology

This study is based on 990 patients with RA and 700 age-matched, gender-matched and residential-area-matched controls from the EIRA cohort. Incident cases were diagnosed according to the 1987 American College of Rheumatology criteria [27]. Participants donated blood at inclusion (at the time of diagnosis of RA) and filled in a questionnaire on lifestyle/environment. Patients had not been treated with disease-modifying anti-rheumatic drugs (DMARDs) prior to this time point. Details of EIRA are described elsewhere [2]. Information on ACPA, smoking, genetics and clinical data were retrieved from the EIRA database, and has described before [2, 3, 28, 29]. Briefly, EIRA participants were categorised as ever-smokers (current and former smokers) or never-smokers, and genotyped for HLA-DRB1 alleles (HLA-DRB1*01 (except DRB1*0103), *04 and *10 were classified as SE). ACPA was measured as anti-CCP2 IgG using Immunoscan CCPlus® (Euro-Diagnostica AB, Malmö, Sweden). Clinical data (DAS28 and CRP) were captured by linking EIRA with the Swedish rheumatology register, where clinical data are registered as part of standard care [29]. The study population is described in Table 1.

Anti-CMV IgG was analysed using a chemiluminescent microparticle immunoassay (Architect, Abbott Diagnostics, Wiesbaden, Germany). Antibodies against EBV viral capsid antigen, and parvovirus B19, were measured by enzyme-linked immunosorbent assay (ELISA) kits (Biorad, Dreieich, Germany, and Biotrin International, Dublin, Ireland, respectively). All immunoassays were performed and interpreted according to the manufacturers' instructions, including setting of the cutoff values. The cutoff for positivity was ≥ 6.0 arbitrary units (AU)/ml for anti-CMV IgG, and > 1.0 index value for both anti-EBV IgG and anti-B19 IgG. Anti-CMV and anti-EBV antibodies were analysed in 987 patients with RA and 700 controls, while anti-B19 antibodies were analysed in 979 patients with RA and 692 controls.

Levels of IgM rheumatoid factor (RF) were analysed in all 990 patients with RA from the EIRA cohort, using the EliA immunoassay on the Phadia 2500 system (Phadia GmbH, Freiburg, Germany), according to the manufacturer's instructions.

Statistical differences in anti-viral antibody frequencies between different RA subsets and controls were determined using Fisher’s exact test and the chi-squared test. The Mann-Whitney U test was used to examine anti-viral IgG levels. Linear regression was used to determine the correlation between anti-viral antibody levels and DAS28/CRP, age and RF. Unconditional logistic regression was used to calculate odds ratios (OR) with 95% confidence intervals (CI) for the association between high/low anti-viral antibody levels and different RA subsets. Quartiles of anti-viral antibody levels were used, and the highest quartile (25%) was compared to the lower three quartiles (75%). Cutoffs were based on anti-viral IgG levels among controls. Analyses were adjusted for age, gender and residential area. The attributable proportion (AP) due to additive interaction was evaluated between smoking, SE and virus exposure, regarding the risk of ACPA-positive RA, with 95% CI, as previously described [30]. Analyses were performed using GraphPad PRISM 6 and SAS version 9.3. P values <0.05 were considered statistically significant.

In order to determine whether infection with the common viruses CMV, EBV and parvovirus B19 was more frequent in patients with RA than in non-RA subjects, we measured anti-viral antibodies in serum from 990 patients with RA and 700 matched controls, using presence of anti-viral antibodies as surrogate markers for viral infection. We detected high frequencies of anti-viral antibody-positive individuals in the EIRA cohort, with no statistical differences between patients with RA and controls in the prevalence of any of the viruses. The prevalence of anti-EBV IgG was 98.3% and 97.0%, in patients with RA and controls, respectively; anti-B19 IgG was detected in 75.8% of patients with RA and in 72.8% of controls; and anti-CMV IgG was found in 72.2% of patients with RA and in 75.9% of controls. When dividing patients with RA into ACPA-positive and ACPA-negative subsets, there was a marginally higher frequency of anti-B19 IgG-positive individuals in ACPA-positive RA compared to controls (77.6% versus 72.8%, respectively, p = 0.0466) (Fig. 1a). When looking at the combination of anti-EBV, anti-B19 and anti-CMV antibodies we found no major differences between patients with RA and controls, or between patients with ACPA-positive and ACPA-negative RA. Triple positivity was most common (55–57%), followed by double positivity (34–36%), while only 8–11% were single positive, with EBV being the most common reactivity. Less than 1% was triple negative (Additional file 1: Figure S1).

Since we found no major differences in anti-viral antibody prevalence between groups, we proceeded to compare antibody levels. When including all individuals in these analyses, irrespective of anti-viral antibody status, only anti-EBV IgG levels differed significantly, with lower antibody levels detected in patients with RA compared to controls (p = 0.0155), specifically in ACPA-positive RA (p = 0.0112) (Fig. 1a). When focusing the analysis on anti-viral antibody-positive individuals only, both anti-EBV and anti-B19 IgG levels were significantly lower in ACPA-positive RA compared to controls (p = 0.0026 and p = 0.0086, respectively) (Fig. 1b). Anti-B19 antibody levels were also significantly lower in ACPA-positive RA compared to ACPA-negative RA (p = 0.0082). In order to address the possibility that RF, which is more commonly detected in ACPA-positive RA than in ACPA-negative RA, was interfering with the detection of anti-viral antibodies and thereby caused lower anti-EBV and anti-B19 IgG levels in the ACPA-positive subset, we analysed the levels of IgM RF in relation to all three anti-viral antibody levels in all EIRA RA samples. We found no correlation, hence conclude that RF did not affect anti-viral antibody levels (Additional file 2: Figure S2).

Due to the high frequency of seropositivity for all three anti-viral antibodies, further analyses based on seropositivity versus seronegativity would be non-informative. Instead, we analysed quartiles of anti-viral antibody levels and compared the highest quartile (25%) with the lower three quartiles (75%), in order to investigate whether high levels of anti-viral antibodies were associated with RA and/or different subsets of RA. Logistic regression analysis revealed that high levels of anti-viral antibodies did not increase the risk of RA. If anything, the opposite was observed. High levels of anti-viral antibodies were inversely associated with RA, with statistically significant odds ratios for both anti-EBV (OR = 0.7; 95% CI 0.5–0.9) and anti-B19 IgG (OR = 0.7; 95% CI 0.6–0.9). The significant associations were only confined to the ACPA-positive subset, with odds ratios of 0.6 (95% CI 0.5–0.8) for both EBV and B19 (Table 2). There was no association between ACPA-negative disease and any of the anti-viral antibodies investigated.

In addition, we analysed different combinations of high or low anti-viral antibody levels in RA (ACPA-positive and ACPA-negative) and controls. In line with the data presented in Table 2, this analysis clearly illustrated that patients with ACPA-positive RA differed from controls, and from patients with ACPA-negative disease (Additional file 3: Figure S3). Having low antibody levels to all viruses was the most common combination (44% in controls; 46% in ACPA-negative RA; 53% in ACPA-positive RA), followed by low antibody levels to two viruses (i.e. high antibody levels to one virus - B19 in controls and ACPA-negative RA, and CMV in ACPA-positive RA).

We next analysed the associations and interactions of anti-viral antibody levels with two well-established risk factors for ACPA-positive RA, the HLA-DRB1 SE alleles and smoking. Also here, we compared the highest quartile (25%) of antibody levels with the lower three quartiles (75%). Since high levels of anti-viral antibodies seemed to be protective against ACPA-positive RA, while presence of smoking and SE are well-known risk factors, the referent groups in these analyses were negative for SE and/or smoking, but positive for the highest quartile of anti-viral antibody levels. There was no significant association of low anti-EBV or low anti-CMV antibody levels with ACPA-positive RA in the absence of smoking (Table 3). However, low levels of anti-B19 antibodies were - independent of smoking - associated with ACPA-positive RA (OR = 2.3; 95% CI 1.3–4.1). In the presence of smoking, the odds ratio for this specific association increased to 4.4 (95% CI 2.5–7.7). No signs of interactions, calculated as the attributable proportion (AP) due to additive interaction, were identified between smoking and low levels of any of the anti-viral antibodies.

Analyses of HLA-DRB1 SE in the context of anti-viral antibodies identified significant interaction between SE and low levels of both anti-EBV and anti-B19 antibodies, with an AP due to the interaction of 0.5 (95% CI 0.3–0.7) and 0.3 (95% CI 0.1–0.5), respectively (Table 4). Low levels of anti-B19 antibodies were also independently (of HLA-DRB1 SE) associated with ACPA-positive RA. Low anti-CMV antibody levels were not associated with ACPA-positive RA and there was no interaction with SE.

In order to better clarify the role of SE in relation to lower anti-viral antibody levels, as seen in ACPA-positive RA, we also compared anti-EBV and anti-B19 IgG levels in SE-positive and SE-negative subsets of patients with ACPA-positive RA, patients with ACPA-negative RA and controls. Within each subgroup, anti-viral antibody levels did not differ significantly based on SE status (Fig. 2), hence SE does not seem to directly influence these specific anti-viral antibody levels. Within the SE-positive subset, anti-B19 and anti-EBV IgG levels were significantly lower in RA and ACPA-positive RA (and also in ACPA-negative RA for EBV) compared to controls, while anti-B19 IgG levels were significantly lower in ACPA-positive RA compared to ACPA-negative RA, when comparisons were made within the SE-negative subset.

Finally, we examined whether there was correlation between anti-viral antibody levels and baseline clinical characteristics, specifically inflammation (measured as CRP) and disease activity (measured as the DAS28), and we found no correlation for any virus, in any subset of RA (Additional file 4: Figure S4). Age, on the other hand, correlated weakly with lower anti-B19 IgG levels and higher anti-CMV IgG levels, in both patients with RA and controls (Additional file 5: Figure S5).