Methotrexate potentiates oxidative stress-mediated transdifferentiation and metabolic reprogramming of neutrophils

Rheumatoid arthritis (RA) is associated with inflammation, oxidative stress and infiltration of immune cells (~ 90% neutrophils) into inflamed joints. Neutrophils have demonstrated the ability to transdifferentiate into 'neutrophil-dendritic cell hybrids' or N-DCs, acquire antigen presenting properties (HLA-DR/CD80/CD86) and demonstrate enhanced oxidative stress. Classical neutrophils rely mainly on glycolysis, but the bioenergetics of N-DCs remains poorly defined. Accordingly, this study aimed to assess the impact of methotrexate (MTX) on neutrophils with regard to their transdifferentiation and bioenergetics. The ex-vivo effect of MTX on neutrophils sourced from healthy controls was studied (+/- Phorbol 12-myristate 13-acetate, PMA) in terms of CD83⁺, HLA-DR⁺, generation of reactive oxygen species, activation (myeloperoxidase) and apoptotic status (Annexin V, Caspase 3 and Bcl-2) by flow cytometry, while the bioenergetics was assessed using Agilent XFp analyser, along with expression of key metabolic regulatory enzymes by real time PCR. In neutrophils, MTX did not impact on the generation of ROS; however, in a pro-oxidant milieu induced by PMA, MTX demonstrated a synergistic enhancement in the generation of ROS, increased %CD83^high/HLA-DR^high, raised myeloperoxidase, elevation of apoptotic markers (Annexin V, Caspase 3) along with downregulation of Bcl-2. MTX per se did not alter the oxygen consumption rate (OCR), whereas following PMA-induced oxidative stress, MTX enhanced OCR, while glycolytic mechanisms remained unaltered. MTX, the gold standard for treatment of RA did not alter the redox status of neutrophils, but in a PMA-induced pro-oxidant milieu, MTX facilitated transdifferentiation and activation of neutrophils, altered their apoptotic and bioenergetic status, suggestive of an adverse bystander effect.