A Cross-Sectional and Longitudinal Study to Define Alarmins and A-SAA Variants as Companion Markers in Early Rheumatoid Arthritis

Abstract

Nowadays, in the study of rheumatoid arthritis (RA), more and more interest is directed towards an earlier effective therapeutic intervention and the determination of companion markers for predicting response to therapy with the goal to prevent progressive joint damage, deformities, and functional disability. With the present work, we aimed at quantifying in a cohort of early RA (ERA) patients naïve to DMARD therapy, proteins whose increase was previously found associated with RA: serum amyloid A (A-SAA) and alarmins. Five A-SAA variants (SAA1α, SAA1β, SAA1γ, SAA2α, and SAA2β) but also S100A8 and S100A9 proteins were simultaneously quantified in plasma applying a method based on single targeted bottom-up proteomics LC-MS/MS. First, we compared their expression between ERA (n = 100) and healthy subjects (n = 100), then we focused on their trend by monitoring ERA patients naïve to DMARD treatment, 1 year after starting therapy. Only SAA1α and SAA2α levels were increased in ERA patients, and SAA2α appears to mostly mediate the pathological role of A-SAA. Levels of these variants, together with SAA1β, only decreased under biologic DMARD treatment but not under methotrexate monotherapy. This study highlights the importance to better understand the modulation of expression of these variants in ERA in order to subsequently better characterize their biological function. On the other hand, alarmin expression increased in ERA compared to controls but remained elevated after 12 months of methotrexate or biologic treatment. The work overcomes the concept of considering these proteins as biomarkers for diagnosis, demonstrating that SAA1α, SAA1β, and SAA2α variants but also S100A8 and S100A9 do not respond to all early treatment in ERA and should be rather considered as companion markers useful to improve the follow-up of treatment response and remission state. Moreover, it suggests that earlier use of biologics in addition to methotrexate may be worth considering.

Keywords: A-SAA variants; DMARDs; alarmins; companion markers; early RA.

Figure 1

Classification of ERA patients in good and non-responders according to DAS28-CRP. The 100 patients suffering from ERA were divided in two groups: good (R) and non-responders (NR) according to the variation of DAS28-CRP from T0 and T12 (after 12 months of treatment). When DAS28-CRP ≤ 2.6, the patient is in remission (dotted red line). The mean for each group is indicated with a full red line. Four patients were removed from the analysis because they did not fulfill the criteria for either R or NR.

Figure 2

Quantification of A-SAA by LC-MS/MS and ELISA. (A) Expression of A-SAA variants and total A-SAA in healthy volunteers (controls, CTRL) and ERA patients at time T0 and after 12 months of treatment (T12). (B) Expression of A-SAA variants and total A-SAA in ERA patients sorted in two groups: good (R) and non-responders (NR). Scatter dot plots represent the median with interquartile range. *p-value ≤ 0.05, **p-value ≤ 0.01, ***p-value ≤ 0.001 (Kolmogorov–Smirnov test); ^#p-value ≤ 0.05, ^##p-value ≤ 0.01; ^###p-value ≤ 0.001 (Wilcoxon test).

Figure 3

Quantification of proteins S100A8, S100A9, and calprotectin. (A) Expression of alarmins in controls (CTRL) and ERA patients at time T0 and after 12 months of treatment (T12). (B) Variation of the expression of alarmins in good (R) and non-responders (NR). Scatter dot plots represent the median with interquartile range. *p-value ≤ 0.05, **p-value ≤ 0.01, ***p-value ≤ 0.001 (Kolmogorov–Smirnov test).

Figure 4

Differences in the expression of proteins according to the treatment. Graphs depict the expression of A-SAA variants (A) and alarmins (B) in patients, at T0 and T12, sorted by treatment: methotrexate monotherapy (MTX) or biologics (BIO). Scatter dot plots represent the median with interquartile range. *p-value ≤ 0.05, **p-value ≤ 0.01, ***p-value ≤ 0.001 (Wilcoxon test). R, good responders; NR, non-responders.