IgG Fc galactosylation predicts response to methotrexate in early rheumatoid arthritis

Abstract

Background: Methotrexate (MTX) is the standard first-line therapy in rheumatoid arthritis (RA) with variable clinical efficacy that is difficult to predict. The glycosylation status of immunoglobulin G (IgG) is altered in RA and influenced by MTX treatment. We aimed to further investigate if IgG glycosylation in untreated early RA can predict therapeutic response to MTX.

Methods: We used a shotgun proteomic approach to screen for the Fc glycopeptides in the serum of 12 control subjects and 59 untreated patients with early RA prior to and following MTX initiation. MTX treatment response was defined according to the European League Against Rheumatism at a median of 14 weeks (range 13-15) after treatment initiation. Seropositive patients were defined as those testing positive for anticitrullinated protein antibodies and/or rheumatoid factor at baseline (n = 44). Data analysis was performed using uni- and multivariate statistics.

Results: We could confirm a low abundance of galactosylated glycans in untreated patients with early RA compared with control subjects that was partially restored by MTX treatment. This was more evident among future nonresponders than among responders to MTX treatment. Results were further validated and confirmed by multivariate statistical analysis of the baseline Fc glycan, proteomic, and clinical data. We found that the ratio between the main agalactosylated (FA2) and main mono- and di-galactosylated Fc glycans (FA2G1 and FA2G2) of IgG1 ranked as the most prominent factor distinguishing responders from nonresponders. A low baseline ratio of FA2/[FA2G1 + FA2G2]-IgG1 was associated with nonresponse (OR 5.3 [1.6-17.0]) and was able to discriminate future nonresponders from responders to MTX therapy with a sensitivity of 70% (95% CI 46-88%) and a specificity of 69% (95% CI 52-83%). For seropositive patients (n = 44), this trend was improved with a sensitivity of 73% (95% CI 45-92%) for nonresponse and a specificity of 79% (95% CI 60-92%).

Conclusions: We show that the FA2/[FA2G1 + FA2G2] of IgG1 is a biomarker candidate that is significantly associated with nonresponding patients and has potential value for prediction of MTX clinical response.

Keywords: Biomarker; Complement; Glycosylation; Immunoglobulin; Methotrexate; Rheumatoid arthritis.

Fig. 1

Overview of study recruitment and data collection. ACR American College of Rheumatology, DAS28 Disease Activity Score in 28 joints, EULAR European League Against Rheumatism, MTX Methotrexate

Fig. 2

Distribution of galactosylated immunoglobulin G1 (IgG1)- and IgG2-fragment crystallizable (Fc) glycans. a Quantified glycan structures grouped as agalactosylated, (aGal) and galactosylated (Gal) glycans. Glycan nomenclature is according to Royle et al. [35]. The core oligosaccharide structure comprises a biantennary heptasaccharide moiety (A2). Usually, the first sugar unit (an N-acetylglucosamine) is additionally core-fucosylated (e.g., FA2). The biantennary structure can also be transected by an additional N-acetylglucosamine (FA2B). Furthermore, the outer glucosamine units can be elongated with galactoses (FA2Gn, n = 1 or 2), and the galactoses can be further extended with sialic acids (FA2GnSn, n = 1 or 2). b Distribution of galactosylated IgG1- and IgG2-Fc glycans at baseline in healthy control subjects and in patients with early rheumatoid arthritis (RA). The relative distribution (%) of galactosylated Fc glycans differed significantly (p < 0.05) in healthy control subjects compared with patients with early RA and when comparing patients with good response and no response. Collectively, these data demonstrate a changed galactosylation pattern of Fc glycans in RA, with lower proportions of galactosylated glycans than their agalactosylated counterparts. The effect is most pronounced in IgG1. Following false discovery rate correction, p = 0.001 remains significant. C Complement protein, MR Moderate response

Fig. 3

Multivariate model based on baseline information of the patients prior to methotrexate treatment. a Principal component analysis (PCA) score plot. The majority of patients with no response to treatment (NR, dark circles) cluster together along component 1 (x-axis). This indicates that there are factors at baseline that are characteristic for this patient group within the model. b The orthogonal projections to latent structures discriminant analysis (OPLS-DA) loading plot of the same data reveals which factors these are. The error bars represent the range of each marker’s value (with >95% confidence). Features that are positive correlate with no response, features that are negative correlate with response. Shown are only the features correlating with 95% confidence. For details of the complete model, see Additional file 1: Table S7. C Complement protein, CF Complement factor, FA2 Main agalactosylated Fc glycan, FA2G1 Main monogalactosylated Fc glycan, FA2G2 Main digalactosylated Fc glycan, GR Good response, MR Moderate response, NR No response

Fig. 4

The log[FA2/(FA2G1 + FA2G2)] value of immunoglobulin G1 (IgG1) as a predictive factor for response. a Good (GR) and moderate responders (MR) compared with patients with no response (NR) to methotrexate treatment. Patients are split into seropositive (Pos) and seronegative (Neg) groups. Cutoff −0.045. b ROC curve for distinguishing the responding and nonresponding patient cohorts. c ROC curve for distinguishing the responding and nonresponding patient cohorts when only seropositive patients were included. Following false discovery rate correction, p = 0.001 remains significant. FA2 Main agalactosylated, FA2G1 Main monogalactosylated Fc glycan, FA2G2 Main digalactosylated Fc, NS Not significant