Rheumatic diseases (RDs) are characterized by long disease duration, diverse clinical manifestations, and various prognoses. The diagnosis of these diseases is primarily determined by the clinical manifestations and the presence of specific autoantibodies. In clinical practice, early diagnosis of RDs among some patients is challenging due to the absence of specific autoantibodies. Exploration of new antibodies is essential in the study of RDs. Anti-carbamylated protein antibody (anti-CarPA) is one of the new autoantibodies discovered in recent years. According to some studies, patients with RDs always have higher levels of anti-CarPA than healthy individuals. Indeed, antibodies to anti-CarPA are widely used in rheumatological research in RA and have been demonstrated to be associated with its diagnostic efficiency (1), risk stratification (2, 3), and treatment evaluation of RA patients (4), making it an ideal biomarker.

Carbamylation is a non-enzymatic process by which self-proteins are added to a cyanate group. In this process, lysine is converted to a homo-citrulline in the tertiary structure (5). Several proteins in the body, including albumin, low density lipoprotein, fibrinogen, enolase, 78 kDa glucose regulatory protein, vimentin, and α-1 antitrypsin, could be carbamylated (6–10). Many physiological and pathological processes, such as aging, cataracts, atherosclerosis, chronic kidney disease, and nervous system disorders, are also affected by carbamylated proteins (11). Additionally, some studies have suggested that the positive charge is inhibited after protein carbamylation, changing the interactions among ions on the protein surface. During these processes, the secondary and/or tertiary structure of proteins could be changed, exposing the abnormal region of the protein, thereby producing anti-CarPA (11, 12). Initially described in 2011 among patients with RDs, anti-CarPA could recognize homocitrullinated peptides (13). In subsequent studies, anti-CarPA was found in non-RA RDs, such as systemic lupus erythematosus (SLE), systemic sclerosis (SSC), and primary Sjögren’s syndrome (pSS), with different outcomes (14–20). Studies on anti-CarPA have revealed that it is associated with poor disease outcomes, including increased disease activity, radiographic progression, and mortality in patients with RA (1–3, 21, 22). Recent studies have also linked anti-CarPA with RA associated interstitial lung disease (RA-ILD), which may explain the increased mortality rate among anti-CarPA-positive patients with RA (2, 23).

Rheumatic disease-related interstitial lung disease (RD-ILD) is a common complication in patients with RDs. Depending on the screening method and the sample population, ILD affects 3–70% of patients with RDs. Difficult diagnosis, poor prognosis, and lack of effective treatments made RD-ILD one of the main causes of death in patients with RDs, thus prompting greater efforts to detect the disease as early as possible. However, clinically effective biomarkers for RD-ILD are still lacking, and early detection is problematic. A number of risk factors have been identified in patients with RD-ILD; they include smoking, male gender, higher disease activity, longer disease duration, older age, positive rheumatoid factor (RF), and anticitrullinated protein antibodies (ACPAs) (23, 24). Several biomarkers have also been proposed for RD-ILD screening, but none has been universally accepted. Recent studies have identified anti-CarPA as a potentially useful biomarker for RA-ILD, but other RD-ILDs have not been studied yet. Besides, the clinical significance in RDs other than RA was still unclear. Therefore, we sought to determine the presence of anti-CarPA in Chinese Han patients with RDs and its clinical significance. In this study, serum anti-CarPA levels and the association between anti-CarPA and its clinical significance including RD-ILD were also investigated among a diverse population of patients with RDs.

Beside the level of anti-CarPA, the following clinical information of RD patients was collected directly from the medical record (all of patients were hospitalized): rheumatoid factor (RF), anti-cyclic citrullinated peptide antibodies (CCP), antinuclear antibody (ANA), anti-Smith antibodies (SM),anti-double-stranded DNA antibody (dsDNA), antibodies reactive against the ribonucleoprotein antigens Ro/Sjögren’s syndrome A antigen (SSA), antibodies reactive against the ribonucleoprotein antigens La/Sjögren’s syndrome B antigen (SSB), and myositis-specific autoantibodies (MSAs). RF was detected by immune turbidimetry (0–20KU/L). SSA, SSB, and dsDNA were measured by ELISA with the normal range: SSA (0–20 RU/ml), SSB (0–20 RU/ml), and dsDNA (0–100 IU/ml). ANA was detected by indirect immunofluorescence. CCP and SM were measured by chemiluminescent immunoassay with the normal range: CCP (0–20 U/ml) and SM (0–20 CU). MSAs were detected using line blot techniques.

Data analyses were performed using SPSS 26 software for Windows (IBM SPSS Inc., Chicago, USA) and GraphPad Prism 9 for Windows (San Diego, CA, USA). Results from parametric data were expressed as mean SD, and differences between groups were analyzed using a student’s T test. For nonparametric data, results were expressed as median (interquartile range) values, and differences between groups were analyzed using the Mann-Whitney test and Kruskal and Willis test. A correlation analysis between two continuous variables was performed using Spearman’s analysis. Multivariable analysis was then used to compare variables that had a p-value <0.1 by single-variable analysis. For all statistical analyses, p-values <0.05 were considered statistically significant.

Anti-CarPA was shown to be widely distributed among patients with RDs in this study. Past studies have shown that anti-CarPA persists in parts of RDs: Verheul et al. (1) enrolled more than 5,000 patients with RA, and the positive rate of anti-CarPA was 34–53%. Nakabo et al. (14) included 241 patients with SLE in Japan, and the positive rate of anti-CarPA was 54.4%. Two studies from Europe showed that the positive rates of anti-CarPA in patients with pSS were 26.9% and 30%, respectively (15, 18). Riccardi et al. (17) included 448 French patients with SSC, and the positive rate of anti-CarPA was 14%. These studies showed that anti-CarPA were not the specific antibody for RA, which inspired us it may exist in various RDs. Besides, the clinical value of anti-CarPA in different RDs remained unclear, which should be identified whether it related to occurrence and development of some manifestations in RDs. Currently, the research object of previous studies were mainly among Caucasian, but the research on the distribution of anti-CarPA among the Chinese Han population remains insufficient. Therefore, we conducted this study among the Chinese Han population to explore the distribution and clinical significance of anti-CarpA. Our study confirmed the presence of anti-CarpA in several RDs, including RA, SLE, pSS, SSC, PM/DM, UCTD, SpA, and BD. In our study, anti-CarPA was detected in 48.70% of patients with RA, 28.57% of patients with SLE, 31.25% of patients with pSS, 62.26% of patients with PM/DM and 55.00% of patients with UCTD, further confirming and expanding these findings. As described above, preliminary results indicated that anti-CarPA antibodies are broadly distributed and have a low specificity in RDs. According to our observations, the serum titer of anti-CarpA was obviously higher in RA than in other RDs. Furthermore, anti-CarPA was proven to be detectable almost 14 years before RA appeared (36). Anti-CarPA seemed to play a significant role in the diagnosis and prediction of RA, especially among patients with RA-ILD.

In terms of clinical and laboratory variables, anti-CarPA-positive and negative patients were quite different in several aspects. Patients with anti-CarPA positive RA had younger onset ages and longer disease durations, which were consistent with previous observations (3, 22, 37). In spite of these findings, we observed that RA patients with positive anti-CarPA had higher levels of RF and DAS28, which potentially resulted in more severe joint damage (38, 39), and could indicate poor prognosis. A significant increase in ESR and SLEDAI were observed in SLE individuals with positive anti-CarPA in our study. Li et al. (19) observed that anti-CarPA was associated with high disease activity in SLE patients. It was consistent with our results, and suggested that anti-CarPA was associated with disease activity in patients with SLE. Massaro and Ceccarelli (40, 41) confirmed that anti-CarPA was associated with joint damage in patients with SLE. However, our results showed no significant difference between anti-CarPA and arthralgia. It could be accounted for that joint ultrasonography was not a regular examination for SLE patients in our department, so that patients with joint damage in early stage, especially patients with mild symptom, were prone to miss in this study. In the PM/DM group, we observed an association between anti-CarPA and arthralgia (p = 0.034). The most common finding in joint damage of idiopathic inflammatory myopathies (IIM) is active synovitis, which is similar to RA (42). Highly abundant neutrophils in synovium have been observed to be capable of forming neutrophil extracellular traps (NETs) that externalize carbamylated autoantigens to the extracellular space, resulting in an increase in the production of anti-CarPA (43, 44), which in turn causes joint inflammation. Anti-CarPA may play a role in arthritis associated with PM/DM in a manner similar to RA. However, further validation is required. The correlation between anti-CarPA and RA-ILD has been proposed in recent years (23, 45, 46). Our study identified for the first time that serum anti-CarPA was upregulated in various patients with RD-ILD, not only RA but also PM/DM and pSS. This upregulation was associated with the severity of pulmonary fibrosis but not the image classifications of HR-CT. Anti-CarPA may act as a biomarker for predicting RA-ILD, suggesting more serious lung involvement.

An association seemed to exist between anti-CarPA and ILD in patients with PM/DM in our study. We found that PM/DM-ILD patients had higher levels of CRP than patients with PM/DM-non-ILD. Accordingly, CRP may be involved in the development and progression of PM/DM-ILD. Gono (47) developed a prediction model termed MCK (MDA5, CRP, and KL-6) to identify patients with PM/DM-ILD at low, moderate, or high risk of mortality, using CRP as a risk factor. In our study, we identified an association between anti-CarPA and PM/DM-ILD and proved a relationship between anti-CarPA and severer RD-ILD. Therefore, we speculate that anti-CarPA participates in PM/DM-ILD development and progression, whereas little evidence exists on how anti-CarPA contributes to PM/DM-ILD. In spite of this finding, anti-CarPA is positively correlated with anti-MDA5 and anti-EJ, while negatively correlated with anti-TIF-γ in patients with PM/DM. Several studies found that anti-MDA5 and anti-EJ were associated with more severe ILD in patients with PM/DM, whereas anti-TIF-γ was related to less lung involvement (48). Previous studies confirmed that NETs could participate in the development of ILD in IIM (49–51). Seto (51) identified that anti-MDA5 promotes the formation of NETs, in turn to induce epithelial cell injury and inflammatory cytokine release. In RA, NETs seem to play a role in the production of anti-CarPA, whereas in ILD, the neutrophils that contribute to the production of NETs increase near the lesion site (43). Anti-CarPA is believed to play an important role in the progression of PM/DM-ILD.

Bergum et al. (18) have confirmed that anti-CarPA was associated with the severity of pSS. Our study interestingly found that PLT was lower in anti-CarPA-negative subgroups of pSS. Wu et al. (52) have observed that patients with pSS without thrombocytopenia were more likely to have ILD. Our observations suggest rather that an association exists between them. Additionally, we showed that levels of anti-CarPA were correlated with pSS-ILD and it could predict pSS-ILD. Furthermore, our study found no significant difference in serum anti-CarPA levels between patients with SSC with and without ILD. In a recent study, Riccardi et al. (17) showed an association between anti-CarPA with “fibrotic subset” in patients with SSC, including patients with diffuse cutaneous subset and/or interstitial lung disease. A study with a large sample size is needed to confirm these findings. The SLE-ILD and UCTD-ILD groups had no association with anti-CarPA, indicating that different pathogenetic mechanisms may be involved.

In this study, we extend the study of anti-CarPA to examine its widespread distribution in RDs among Chinese Han nationals. Moreover, it is the first study to examine the relationship between anti-CarPA and RD-ILD, especially non-RA-ILD. According to our results, anti-CarPA plays an important role in RD-ILD and could be used to identify patients with RDs who are at high risk of developing ILD. This study had two main limitations. First, the relative small number of patients enrolled was the main study limitation. Therefore, further studies on a larger population are mandatory to clarify the prognostic value of anti-CarPA in patients with RDs, especially for patients with RD-ILD. Second, the triage system in our country made it likely that our cohort would enroll patients with more severe diseases. However, a series of analyses have indicated that anti-CarPA is associated with several RDs and linked with ILD in patients with RA, PM/DM, and pSS.

For the first time, we demonstrated the presence of anti-CarPA in a Chinese cohort of patients with RDs, such as RA, SLE, PM/DM, pSS, and UCTD. Based on the results of our study, anti-CarPA may assist in the identification of ILD in patients with RA, PM/DM, and pSS. Further replicative investigations may confirm the pathologic role of anti-CarPA in patients with RDs. Serum anti-CarPA could be detected in patients with RA, PM/DM, pSS, SSC, and UCTD among the Chinese. And it may also assist in identifying ILD in patients with RA, PM/DM, and pSS, which emphasized attention to the lung involvement in anti-CarPA-positive patients.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by Shandong Provincial Hospital validated the study protocol (SZRJJ: NO.2021-438). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

RD and YS participated in the study design, literature review, and performed statistical analysis and presentation of the results and participated in the drafting and review of the manuscript. RD, WX, WZX, and ML collected serum samples. ZM, QY, and LZ participated in the study design, the drafting and review of the manuscript. All authors contributed to the article and approved the submitted version.