The Macrophage Reprogramming Ability of Antifolates Reveals Soluble CD14 as a Potential Biomarker for Methotrexate Response in Rheumatoid Arthritis

Sara Fuentelsaz-Romero,Mónica Torres Torresano,Celia Barrio-Alonso,Raquel García Campos,Laura Nuño,Ana Triguero-Martínez,Ittai B Muller,Rosario García-Vicuña,María-Eugenia Miranda-Carús,Gerrit Jansen,Amaya Puig-Kröger,Alejandro Villalba,Isidoro González-Álvaro

doi:10.3389/fimmu.2021.776879

Abstract

The identification of “trained immunity/tolerance” in myeloid cells has changed our perception of the performance of monocytes and macrophages during inflammatory and immune responses. Pemetrexed (PMX) and methotrexate (MTX) are blockers of the one-carbon metabolism (OCM) and commonly used therapeutic agents in cancer and rheumatoid arthritis (RA). We have previously showed that MTX promotes trained immunity in human macrophages. In the present manuscript, we have assessed the anti-inflammatory effects of PMX and MTX and found that OCM blockers alter the functional and gene expression profile of human macrophages and that OCM blockade reprograms macrophages towards a state of lipopolysaccharide (LPS) tolerance at the signaling and functional levels. Moreover, OCM blockade reduced macrophage LPS responsiveness by impairing the expression of membrane-bound and soluble CD14 (sCD14). The therapeutic relevance of these results was later confirmed in early RA patients, as MTX-responder RA patients exhibit lower sCD14 serum levels, with baseline sCD14 levels predicting MTX response. As a whole, our results demonstrate that OCM is a metabolic circuit that critically mediates the acquisition of innate immune tolerance and positions sCD14 as a valuable tool to predict MTX response in RA patients.

Highlights

Folates are one-carbon donors in biosynthetic pathways like de novo synthesis of purines and thymidylate, amino acid metabolism, and DNA methylation [1, 2]
Comparison of the gene signatures of granulocyte-macrophage colony-stimulating factor (GM-CSF)-primed monocyte-derived macrophages in the absence (GM-MØ) or presence (PMX-GM-MØ) of PMX (50 nM) (Figure 1A) revealed a large number of significant transcriptional differences (Figure 1B) and that the gene profile of PMX-GM-MØ was enriched in terms like “HALLMARK_INFLAMMATORY_RESPONSE” and “TNFA_VIA_NFkB_SIGNALING” gene set enrichment analysis (GSEA) gene sets [31] (Figures 1C, I), supporting the notion that the continuous presence of PMX determines the acquisition of a more proinflammatory profile
We describe the impact of One-carbon metabolism (OCM) on the functional and gene expression profile of GM-CSF-primed human monocyte-derived macrophages (GM-MØ)

Summary

Introduction

Folates are one-carbon donors in biosynthetic pathways like de novo synthesis of purines and thymidylate, amino acid metabolism, and DNA methylation [1, 2]. Antifolates [including methotrexate (MTX) and pemetrexed (PMX)] are potent inhibitors of folate-dependent enzymes engaged in nucleotide synthesis and inhibit DNA replication [2]. PMX is transported into the cells via the reduced folate carrier (RFC; SLC19A1) and, unlike MTX, is an excellent substrate for the proton-coupled folate transporter (PCFT; SLC46A1) [7]. PMX is an efficient substrate for the enzyme folylpoly-g glutamate synthetase (FPGS) that catalyzes the addition of multiple glutamyl residues to the g-carboxyl on the terminal glutamate of both folates and antifolates upon entry into the cell [8]. PMX polyglutamylation enhances both its intracellular retention and its inhibitory potency towards thymidylate synthase (TS) and glycinamide ribonucleotide formyltransferase (GARFT) [8]

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