Rheumatoid arthritis (RA) is a chronic systemic disease that can lead to local bone erosion and generalized osteoporosis. Rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA) are the two most notable autoantibodies commonly used in diagnosing or classifying RA and providing a variety of clinical and pathophysiological information (1). Patients positive for ACPA and/or RF may be labeled together as having “seropositive” RA (SPRA) and compose approximately 50–80% of the RA population (1). Several studies have indicated that SPRA patients experience greater disease severity in terms of disease activity, functional impairment, increased mortality over time (2, 3), and might show poor response to treatment compared to seronegative RA (SNRA) patients (4). Although both SPRA and SNRA fulfill the 1987 American College of Rheumatology (ACR) revised classification criteria (5) or the 2010 ACR/European League Against Rheumatism (EULAR) classification criteria (6), their clinical manifestations, courses, and prognostic features are distinctive.

It has been reported that the annual rate of bone loss in patients with active RA ranges between 5.5 and 10% (7). Meanwhile, RF and/or ACPA are associated with juxta-articular osteoporosis, erosions, and generalized bone loss (8, 9). Several studies have shown that ACPAs and RF synergize to promote RA-associated inflammation, disease activity, and clinical onset (10, 11) and can be associated with the bone erosion and structural damage seen in RA (12). The combination of ACPA and RF could predict the therapeutic responses to rituximab and abatacept (13). However, the clinical predictive value of each antibody (singly or in combination) on systemic bone loss has not been well studied.

This study aimed to explore long-term bone mineral density (BMD) changes in patients with SPRA and SNRA and investigate the association between antibody number and systemic bone loss in patients with RA.

Our study provides 3-year follow-up results on the impact of seropositivity and the number of autoantibodies on BMD changes in patients with RA. Compared to baseline, SPRA patients experienced a significant decrease in BMD at three measured sites but not in SNRA at TH and L1–4. Regardless of AOT, autoantibodies present could increase the progression of bone loss at all sites. In patients without AOT, SPRA participants had the most obvious bone loss at all sites. Furthermore, we found that patients with higher numbers of antibodies had more systemic bone loss at TH and L1–4, irrespective of taking AOT therapy or not.

It has been well documented that RA patients have a higher risk of osteoporosis and fragility fractures than the general population (18). RA-associated bone loss is not only related to traditional risk factors of osteoporosis (e.g., aging, female) but also to factors related to the disease itself (e.g., disease activity and duration, GC use, and functional disability) (19, 20). Biologics for patients with RA had a protective effect on bone loss. This beneficial effect was also observed in patients who did not exhibit a clinical response (21). However, it is still controversial whether seropositivity affects osteoporosis and BMD changes in RA. Some studies have reported that SNRA patients have a greater chance of developing osteoporosis and lower BMD than SPRA patients (22, 23), but other investigations revealed opposite results (17, 24–26). The aforementioned studies were subjected to a cross-sectional study with a small sample size and no confounding factor adjustment. In terms of the influential factors screened, we conducted a 3-year, longitudinal, observational study with adequate sample size and adjusted the analysis, factoring in confounders. Our study’s results suggest that seropositivity has a detrimental effect on systemic bone loss in RA patients.

Recent investigations have suggested that not only do SPRA patients have distinct genomic backgrounds (27), clinical presentations (28), and treatment responses (29) from SNRA patients, but they also have higher mortality (3). It raises the possibility that SPRA and SNRA are two distinctive disease entities that mediate the different patterns of systemic bone loss seen in these two subtypes of RA (17). Current investigations revealed that RA-related factors and treatment, including baseline DAS28-ESR, ESR, and rate of GC and biologics use, were significantly higher in SPRA than SNRA, which further suggests that these subtypes of RA are different disease entities. RA disease activity (19, 30) and GC use (21, 31) are two of the determinants of RA-related systemic bone loss, suggesting that higher disease activity and greater GC usage are important determinants of greater systemic bone loss and a higher proportion of osteoporosis in SPRA than SNRA in our cohort.

In addition to the indirect effect of different disease patterns on bone loss between SPRA and SNRA, the direct effect of seropositivity on bone loss has been demonstrated. Harre et al. revealed an association between autoantibodies against citrullinated vimentin and serum markers for osteoclast-mediated bone resorption in RA patients (32). RA-associated autoantibodies have recently been found to directly induce differentiation and activation of osteoclasts, which might partly mediate systemic bone loss in RA (33–35). The additive effect of ACPA and RF on the production of the pro-inflammatory cytokine TNF-α, which is among the most potent cytokines to stimulate osteoclastogenesis, has been noted (10, 36, 37). This may explain the antibody-dependent enhancement of systemic bone loss and the concurrence of ACPA and RF in RA patients having the most detrimental effect on BMD in groups I–III. We noticed that more negative regression-adjusted ΔBMD% at TH and L1–4, irrespective of taking AOT therapy, were considered expected factors of the concurrence of RF and ACPA.

Interestingly, we found that not only is substantial bone loss consistent compared to baseline at three measured sites either in SPRA or RA patients with more autoantibodies, but regression-adjusted ΔBMD% over time is also more negative at all sites, especially at TH. This is consistent with previous studies’ findings suggesting that RA patients experienced more bone loss at the hip than the spine (38–40). Orsolini et al. demonstrated that the independent role of ACPA has a negative titer-dependent effect on systemic bone loss, in particular at cortical sites such as the hip (41).

In contrast, results from Bugatti’s analysis indicate that, despite inflammation suppression, spine BMD is sensitive to systemic bone loss in ACPA-positive early RA patients during the first 2 years after treatment onset (42). A Swedish ACPA-positive sub-cohort study demonstrated a trend in reduced BMD at the spine and hip during the first 2 years of treatment (43). Based on the past studies’ findings (38–43), we hypothesized that ACPA and/or RF’s osteoclastogenesis effect on cortical and cancellous bones are different, and the longitudinal assessments of BMD variations in RA are complicated. Further work will hopefully clarify this controversial concern.

The FRAX ^® was launched in 2008 to allow health providers to estimate individual 10-year probabilities of fragility fractures (44); it is a free, reliable, and validated tool that is used globally. The current investigation found a significantly higher FRAX score (hip) in SPRA participants regardless of the presence of a single or double-positive antibody. Despite higher FRAX score and greater bone loss in SPRA patients, no significant difference was observed in previous fractures and new incident fractures over time between SPRA and SNRA, and in groups I–III. This suggests that more RA patients and a longer period of observation are needed to determine whether RA seropositivity also has a detrimental effect on incident fracture.

Osteoporosis is characterized by low bone mass, increased bone loss, and micro-architectural disruption, resulting in bone fragility. Based on epidemiological data, an operational definition of osteoporosis was defined as BMD lower than −2.5 SD below the peak bone mass in young adult white women (15, 16). This reference standard can be used to compare the results between studies and assess the accuracy of novel diagnostic tools. In clinical application, the focus lies more on bone quantity which refers to bone mass or density, whereas new technologies target the assessment of bone strength and bone quality information. The current study demonstrated that ACPA and RF were associated with BMD change. Whether these autoantibodies can affect bone strength or bone quality in RA patients warrants further investigation.

As the current study is a real-world investigation, we did not exclude participants who received AOT during the observation period to explore the interaction between autoantibodies and AOT in terms of bone protective effects. Among participants with AOT, there was significant bone loss at FN and TH in SPRA and at TH in patients with higher numbers of antibodies. Respective of antibodies, AOT had a better protective effect against loss of bone density in all sites, especially in the spine. This result echoes Pazianas et al.’s finding that bisphosphonates have a more pronounced effect on trabecular bone than on cortical bone (45).

The strengths of our study are as follows. As a real-world investigation, we thoroughly documented the reported clinical variables that may be associated with osteoporosis or fracture in RA patients to avoid the possible factors related to BMD changes that were not observed in previous investigations. Our initial investigation revealed a significant difference in the distribution of age between SNRA and SPRA groups. As age and sex are two of the most important determinants of BMD, we performed a 1:2 matching for age and sex to exclude the confounding effects of age and sex, which had not been done in previous investigations. Moreover, to adjust for confounding factors of bone loss, we used multiple regression analysis to establish a model for predicting BMD changes. We also explored the additive effects of ACPA and RF on systemic bone loss in RA, which has not been investigated before. Because of the 3-year observation period, we were able to investigate the link between autoantibodies’ presence and BMD at baseline and determine the impact of these autoantibodies on BMD changes over time.

This study had several limitations. The monocentric observational design used to diagnose RA patients allowed us to conclude our data about associations but not about causal relationships. Our patients did not represent an inception cohort; we presented the data at the time of study initiation, not at the time of RA diagnosis. Some of the measurements, including 25(OH) vitamin D and parathyroid hormone, were performed at baseline only (data not shown); serial measurements might be more suitable for exploring these associations. We did not check bone markers throughout the study, which hinders our understanding of the cohort’s pathogenesis of antibodies on systemic bone loss. Finally, RA-associated antibodies have been associated with the occurrence and progression of bone erosions and periarticular bone loss as well as a decreased BMD. Systemic bone loss might increase susceptibility to focal bone erosions in RA patients. We did not collect the erosion scores such as the vdH Sharp score or Larsen score initially and at the 3-year follow-up. Further study is needed to clarify the relationship between autoantibodies to bone density and focal bone damage.

Compared to SNRA patients, SPRA patients had a higher prevalence of osteoporosis, consistent bone loss at all sites, and a higher 10-year probability of hip fracture. We also found that more autoantibodies in RA were associated with more detrimental effects on BMD. We, therefore, suggest that more attention should be paid to osteoporosis and systemic bone loss in SPRA patients, especially double antibody-positive patients. AOT had a better protective effect against BMD loss irrespective of autoantibodies. As for fragility fractures, further investigations are needed to explore the association between RA seropositivity and future fractures in the long term.

The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

The studies involving human participants were reviewed and approved by the Local Institutional Review Board of Chang Gung Memorial Hospital approved the study (104-3530B, 201901054B0). The patients/participants provided their written informed consent to participate in this study.

S-FY, J-FC, and T-TC were responsible for analysis and data interpretation. S-FY and T-TC were responsible for scientific writing. All authors read and approved the final manuscript and were involved with study conceptualization, design, and reporting.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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