Molecular signature of methotrexate response among rheumatoid arthritis patients

Abstract

Background: Methotrexate (MTX) is the first line treatment for rheumatoid arthritis (RA), but failure of satisfying treatment response occurs in a significant proportion of patients. Here we present a longitudinal multi-omics study aimed at detecting molecular and cellular processes in peripheral blood associated with a successful methotrexate treatment of rheumatoid arthritis.

Methods: Eighty newly diagnosed patients with RA underwent clinical assessment and donated blood before initiation of MTX, and 3 months into treatment. Flow cytometry was used to describe cell types and presence of activation markers in peripheral blood, the expression of 51 proteins was measured in serum or plasma, and RNA sequencing was performed in peripheral blood mononuclear cells (PBMC). Response to treatment after 3 months was determined using the EULAR response criteria. We assessed the changes in biological phenotypes during treatment, and whether these changes differed between responders and non-responders with regression analysis. By using measurements from baseline, we also tried to find biomarkers of future MTX response or, alternatively, to predict MTX response.

Results: Among the MTX responders, (Good or Moderate according to EULAR treatment response classification, n = 60, 75%), we observed changes in 29 partly overlapping cell types proportions, levels of 13 proteins and expression of 38 genes during treatment. These changes were in most cases suppressions that were stronger among responders compared to non-responders. Within responders to treatment, we observed a suppression of FOXP3 gene expression, reduction of immunoglobulin gene expression and suppression of genes involved in cell proliferation. The proportion of many HLA-DR expressing T-cell populations were suppressed in all patients irrespective of clinical response, and the proportion of many IL21R+ T-cells were reduced exclusively in non-responders. Using only the baseline measurements we could not detect any biomarkers or prediction models that could predict response to MTX.

Conclusion: We conclude that a deep molecular and cellular phenotyping of peripheral blood cells in RA patients treated with methotrexate can reveal previously not recognized differences between responders and non-responders during 3 months of treatment with MTX. This may contribute to the understanding of MTX mode of action and explain non-responsiveness to MTX therapy.

Keywords: flow cytometry; gene expression; methotrexate; plasma proteins; rheumatoid anhritis; transcriptomics; treatment response.

Figure 1

The effect of MTX treatment in non-responders (x-axes) or responders (y-axes) on cell type phenotypes (A), protein expression (B) and gene expression (C). Beta coefficients in A and B, and log₂ (fold changes) in C, of variables that are significantly regulated (FDR ≤ 10%) during MTX treatment in either non-responders, responders, or both. A dotted 45° inclined line indicates the expected parity for individual effects for groups of non-responders and responders to MTX. (A) The changes in cell type phenotypes as measured by flow cytometry. Three different groups of cell phenotypes are indicated by colors: The proportions of HLA-DR+ out of different IL21R+ CD4- T-cell subsets (green), the proportion of HLA-DR+ out of different T-cell subsets (purple), and the proportion of IL21R+ out of several T-cell subsets (red). (B) The changes in protein expression. (C) The change in gene expression, where a group of immunoglobulin genes are indicated by green color.

Figure 2

ROC AUC performance of ML models predicting response based on clinical, flow cytometry, protein, and RNA-seq data in MTX therapies recorded during the baseline patient visit, and a longitudinal difference between the three-month follow-up and baseline. The points in the plots showcase the mean ROC AUC values of the median repeat in each of the ML tasks, along with the corresponding 95% confidence interval of the standard error of the mean (SEM).