title: Metabolic regulation of human neutrophils in rheumatoid arthritis
Creators: name: Fresneda Alarcon, Michele
Publisher: name: University of Liverpool Repository
Year: 2024

description: Background: Rheumatoid Arthritis (RA) is a progressive inflammatory disease affecting over 400,000 people in the UK. RA causes joint swelling, pain and stiffness, and if not treated quickly can deform and destroy joints, severely decreasing quality of life. Neutrophils are a type of immune cell that normally protect us from infection. However, inappropriate neutrophil activation in RA induces rapid release of tissue-damaging proteases and reactive oxygen species (ROS) directly onto joint tissue, causing inflammation and damage to cartilage. In addition, secretion of inflammatory cytokines by neutrophils, and exposure of autoantigens on neutrophil extracellular traps (NETs), drives inflammation and activates other immune cells. Recent discoveries have shed new light on how cellular metabolism is regulated during inflammation and inflammatory disease (immunometabolism). Metabolism is regulated and altered in hypoxic tissues, such as the joint, and when cells migrate from the blood to inflamed tissues. Metabolic regulation may be as important as (and indeed a direct controller) of gene expression during inflammation and inflammatory disease.
Methods: We investigated regulation of metabolism in neutrophils from people with RA using NMR metabolomics to characterise the metabolic and functional changes that take place in neutrophils during in vitro and in vivo activation. Then we coupled these findings with gene and protein data to identify the functional effect of key metabolites and metabolic pathways on healthy control (HC) and RA neutrophils. The functional effect on ROS production, oxygen consumption rate, NET release and apoptosis of neutrophils were then
assessed with targeted metabolic inhibitors and measured using luminol-enhanced chemiluminescence, Seahorse XF, sytox green fluorescence and flow cytometry respectively.
Results: The intracellular metabolome of RA neutrophil is different from HC neutrophils at
baseline with altered energy metabolites including ATP, ADP and Lactic acid (adj.
p-value&lt;0.05). Treatment with therapeutic JAK inhibitors did not affect neutrophil metabolic
profile. Transcriptomics analysis highlighted genes related to glycolytic regulation, PFKFB3,
PFKFB4 and PFKFB2 as being significantly different and highly expressed in RA (adj.
p-value&lt;0.05). Whilst neutrophil functions were not affected by inhibition of
glyconeogenesis (with CP-91149), gluconeogenesis (with MB05032) and glutaminolysis
(with BPTES), neutrophil ROS and NET production was significantly decreased in the
presence of inhibitors of glycolysis (2-DG which targets hexokinase, and 3PO which targets
PFKFB3) in RA and HC neutrophils (p&lt;0.05). RA neutrophils expressed higher levels of the glucose transporter (SLC2A3 gene, GLUT3 protein) and did not require an extracellular source of glucose (in culture media) to retain pathogenic ROS and NET functions. HC
neutrophils were unable to produce NETs in the absence of extracellular glucose. 
Conclusion: The metabolome of RA neutrophils is significantly different compared to HC neutrophils. This is characterised by a metabolic adaptation to low glucose environments. RA neutrophils rely on glycolysis to fuel pathogenic neutrophil functions. These are dependent
on the activation of PFKFB3 which is a rate-limiting isoenzyme in central glycolysis, and which may represent a novel target for the development of new therapies to treat immune-mediated inflammatory conditions like RA which are characterised by pathogenic neutrophil activation.