Generation of recombinant citrullinated and/or carbamylated vimentin isoforms
Tobacco smoke contains pathogenic ingredients, i.e., hydrocyanic acid (OCN-), which is normally rapidly detoxified by transsulfuration forming thiocyanate ions (-SCN). These thiocyanate ions can be oxidized in vivo and subsequently form cyanide in inflammatory loci through the action of hydrogen peroxide plus myeloperoxidase (MPO) from neutrophils or eosinophil peroxidase (EPO). Therefore, we established the procedure for generation of carbamylated vimentin with the nonenzymatic incubation of potassium cyanate (KCN). As shown in supplementary figure S5, nearly quantitative carbamylation (approximately 80 %) of vimentin could be verified by amino acid analysis using MALDI-TOF mass. Furthermore, no additional modifications of other amino acid residues were detectable indicating that only homocitrulline residues were formed from lysine residues after KCN- exposure.
The procedure of vimentin citrullination was established with a very low amount of PAD as described previously . Cross-reaction with the PAD enzyme was excluded as shown by Western blot analysis in supplementary figure S3. To generate a double-modified vimentin isoform, a sequential procedure was established by citrullinating the mutated vimentin in a first step, followed by carbamylation.
The efficiency of each step was verified by SDS-PAGE electrophoresis with coomassie brilliant staining showing a shift to higher molecular weight for the citrullinated isoform and a backward shift after additional carbamylation (supplementary figure S4). To confirm that the migration shift was due to citrullination, blots containing the same samples were analyzed using affinity purified anti-citrulline antibody from our previously study (supplementary figure S2 B) . The results showed that the citrullinated and the citrullinated plus carbamylated isoform cross-react with the purified antibodies. These results are in agreement with the distinct modification positions for citrullination (modified arginine R) and carbamylation (modified lysine K), as illustrated in supplementary figure S6.
Autoantibodies reactivity against RF and anti-CCP2
By using regular assays for the detection of RF and anti-CCP2, frequencies of RF IgM (48.5 %), RF IgA (60.6 %) and RF IgG (45.5 %) were higher than anti-CCP2 (39.4 %) antibodies in patients with early RA. In patients with established RA, prevalence of RF IgM (66.0 %) and RF IgA (73.5 %) was also higher than anti-CCP2 (58.8 %) followed by RF IgG (51.5 %). The most frequently detected standard autoantibody in this cohort was RF IgA (Fig. 1). Of note, higher autoantibody titers were observed in patients with established RA (Fig. 2b) compared to early disease (Fig. 2a).
Autoantibodies reactivity against carbamylated and citrullinated antigens based on the sequence of vimentin
The reactivity against carbamylated and citrullinated vimentin antigens showed a higher prevalence compared to anti-CCP2 and RF in Cuban patients with RA (Fig. 1). Interestingly, patients with early RA showed highest frequencies for anti-carbMCV IgM (90.9 %) and anti-carbVIM IgM (87.9 %) followed by anti-MCV IgG (81.8 %). In patients with established RA, these antibodies were also predominant with the highest prevalence for anti-MCV IgG (88.2 %) followed by anti-carbVIM IgM (83.8 %) and anti-carb MCV IgM (76.5 %).
Patients with RA showed also higher frequencies of IgG autoantibodies against the carbamylated peptide of vimentin HC52, compared to RF IgM, RF IgG and anti-CCP2 (Fig. 1). However, these frequencies were lower than those observed for anti-carbVIM IgM. Thus, reactivity against citrullinated and carbamylated isoforms of vimentin was higher than those observed against modified peptides. Overall, reactivity against newly found citrullinated and carbamylated sequences of vimentin was more frequent with IgM and IgG antibodies than with IgA.
Demographic, clinical characteristics and autoantibody reactivity of RA patients, depending on the positivity for anti-MCV IgG
Patients with anti-MCV IgG antibodies showed no statistical difference in demographic characteristics compared to the seronegative cohort (Table 1). Nevertheless, clinical phenotypes were different in these two groups of patients with a higher rate of low DAS28 and remission achieved in anti-MCV negative patients (p = 0.0048, OR 0.2, 95 % CI 0.6–0.1)
By comparing the titers of the RF IgM, anti-CCP2 and anti-carbVIM IgM, we observed higher values for anti-MCV IgG positive compared to negative patients (Table 2). Differences were only significant for RF IgM and anti-CCP2 antibodies in both early (p < 0.01) and established (p < 0.05) RA patients. A high coincidence rate of autoantibodies was especially observed for combinations of anti-carbVIM IgM and anti-MCV IgG in patients with early (up to 72.7 %), and established RA (up to 75.0 %). Out of 25 patients tested negative for all isotypes of RF and anti-CCP2, 18 were positive for anti-MCV IgG (Table 2).
From the patients with negative results for anti-CCP2, anti-carbamylated antigens IgG antibodies were detectable in 13.9 and 3.0 % of cases against the protein and peptide, respectively. Concurrent seropositivity for anti-carbVIM IgM, anti-carbMCV IgM, anti-MCV IgG, RF IgM and anti-CCP2 was detectable in 12.0 % of samples from patients with early RA and in 30.0 % of patients with established RA (Table 2). Of note, all anti-MCV seronegative patients were also negative for RF IgM and anti-CCP2 assays in both early and established RA.
In anti-MCV IgM-negative patients, determination of anti-carbVIM IgM identified an additional proportion of 15.2 % of positive early RA cases and 8.8 % of patients with established disease. Combination of these two assays was able to detect autoantibodies in 97 % of patients with early or established RA (Fig. 3). In contrast, combination of anti-CCP2 and anti-carbVIM IgM assay was able to detect autoantibodies at a lower frequency.
Diagnostic value of anti-MCV IgG, anti-carbVIM IgM and anti-CCP2 assays
The diagnostic sensitivity and specificity of each assay using different cutoffs and ROC analysis are shown in Table 3. Optimal cutoff value, based on the highest possible sensitivity and specificity, using YI, was the same as recommended by the manufacturer for anti-MCV assay IgG; for anti-carbVIM IgM cutoffs of 20 and 30 U/mL showed very similar balance of sensitivity and specificity and for anti-CCP2 optimal cutoff was 40 U/mL.
A higher sensitivity was observed by using combination of assays, when positive results were considered for at least one assay anti-carbVIM IgM or anti-MCV IgG (97.0 %). For combination of anti-carbVIM IgM or anti-CCP2, the sensitivity was 91.1 %. Interestingly, specificity observed for combination of anti-carbVIM IgM and anti-CCP2, when positive results were considered for both assays, was higher (99.1 %) than specificity observed for combination of anti-carbVIM IgM and anti-MCV IgG (96.0 %). The highest possible sensitivity and specificity balance using YI was observed for combination of assays, when positive results were considered for at least one of both: anti-carbVIM IgM and anti-CCP2 assays. Of note, the AUC of ROC analysis was superior for anti-carbVIM IgM assay. The reactivity of anti-MCV IgG, anti-carbVIM IgM and anti-CCP2 assays in disease and healthy controls are shown in supplementary table S7.
In patients with established RA, a weak correlation with DAS28 was only observed for anti-CCP2 assay (supplementary table S8). Low DAS28 and remission were more frequently achieved in anti-CCP2-negative patients (p = 0.0091, OR 0.2, 95 % CI 0.7–0.1).
Interestingly, 75.0 % of patients positive for anti-CCP2, 90 % of patients positive for anti-MCV, 80 % of patients positive for anti-carbMCV IgM and 90.0 % of patients positive for anti-carbVIM IgM were smokers. A significant association was only evident for smoking status and presence of anti-CCP2 (p = 0.0216, OR 3.9, 95 % CI 1.3–11.9), for CRP and RF IgM (p = 0.0001, OR = 6.4, 95 % CI 2.5–16.8) as well as anti-MCV IgG (p = 0.0307, OR = 5.3, 95 % CI 1.3–21.3) and anti-CCP2 (p = 0.0000, OR = 5.8, 95 % CI 2.4–14.1).
The Spearman rank correlation test was significant (p < 0.05) for anti-CCP2, anti-MCV IgG as well as anti-carbVIM IgM antibodies and RFIgM in both early and established RA patients. Anti-CCP2, anti-MCV IgG and anti-carbVIM IgM showed positive correlation with ESR in early RA patients (supplementary table S8).