Low Dose Methotrexate has Divergent Effects on Cycling and Resting Human Hematopoietic Stem and Progenitor Cells

Abstract

Low dose methotrexate (LD-MTX) remains the gold standard in rheumatoid arthritis (RA) therapy. Multiple mechanisms on a variety of immune cells contribute to the anti-inflammatory effects of LD-MTX. Inflammatory signaling is deeply implicated in hematopoiesis by regulating hematopoietic stem and progenitor cell (HSPC) fate decisions; raising the question of whether HSPC are also modulated by LD-MTX. This is the first study to characterize the effects of LD-MTX on HSPC. CD34+ HSPC were isolated from healthy donors non-mobilized peripheral blood. Resting and/or cycling HSPCs were treated with LD-MTX [dose equivalent to that used in RA patients]. Flow cytometry was performed to assess HSPC viability, cell cycle, surface abundance of reduced folate carrier 1 (RFC1), proliferation, reactive oxygen species (ROS) levels, DNA double-strand breaks, p38 activation, and CD34+ subpopulations. HSPC clonogenicity was tested in colony-forming cell assays. Our results indicate that in cycling HSPC, membrane RFC1 is upregulated and, following LD-MTX treatment, they accumulate more intracellular MTX than resting HSPC. In cycling HSPC, LD-MTX inhibits HSPC expansion by promoting S-phase cell-cycle arrest, increases intracellular HSPC ROS levels and DNA damage, and reduces HSPC viability. Those effects involve the activation of the p38 MAPK pathway and are rescued by folinic acid. The effects of LD-MTX are more evident in CD34+CD38High progenitors. In non-cycling HSPC, LD-MTX also reduces the proliferative response while preserving their clonogenicity. In summary, HSPC uptake LD-MTX differentially according to their cycling state. In turn, LD-MTX results in reduced proliferation and the preservation of HSPC clonogenicity. Study Highlight QuestionsO_ST_ABSWhat is the current knowledge on the topic?C_ST_ABSO_LILow dose-methotrexate (LD-MTX) regulates the function of key cells involved in rheumatoid arthritis (RA) pathogenesis (e.g. T cells, macrophages, neutrophils, endothelial cells and fibroblast-like synoviocytes) and through the activation of multiple pathways contribute to the suppression of inflammation in RA. C_LIO_LIInflammatory signaling impacts hematopoiesis by regulating hematopoietic stem and progenitor cells (i.e. HSPC, CD34+ cells). C_LI What question did this study address?O_LIDoes LD-MTX modulate key functional properties of HSPC subpopulations (i.e. resting and/or cycling HSPCs) C_LI What does this study add to our knowledge?O_LIHSPC uptake LD-MTX differentially according to their cycling state. C_LIO_LICycling HSPC upregulate membrane RFC1 and uptake more MTX than resting HSPC with activation of the p38 MAPK pathway. This leads to inhibition of HSPC expansion, increased intracellular ROS levels and DNA damage, and reduced HSPC viability. C_LIO_LILD-MTX preserves the clonogenicity of non-cycling HSPC. C_LI How might this change clinical pharmacology or translational science?O_LIWe describe a novel mechanism of action of LD-MTX that extends its therapeutic effects from mature immune cells to the modulation of hematopoiesis. C_LI