Metabolic energy expenditure in human blood long-lived neutrophils reprogrammed with inflammatory cytokines

The development of new analytical tools has revealed the heterogeneity of neutrophils in healthy and disease subjects. Knowledge of this heterogeneity has led to the identification, in healthy individuals, of a minor subset of blood neutrophils that express anti-protease genes characteristic of in vivo long-lived neutrophils, similar to those we previously described in vitro , which are expanded in autoimmune diseases. We can reprogram normal human blood neutrophils in vitro using GM-CSF, TNF, and IL-4, resulting in long-lived (LL) cells with enhanced glycolysis and oxygen consumption. We further report that these LL neutrophils express numerous genes associated with metabolism and mitochondria, including PLPP3 and SLC25A27 . In addition, we confirmed that LL neutrophils express anti-peptidase genes, the most expressed being the PI3 gene, and secrete the peptidase inhibitor elafin and the secretory leukocyte protease inhibitor. Extracellular flux analysis revealed that PI3 -expressing LL neutrophils exhibit enhanced glycolysis and respiration in response to pro-inflammatory cytokines, whereas non-reprogrammed neutrophils remain unresponsive. PI3 -expressing LL neutrophils have a mitochondrial respiration partly driven by pyruvate oxidation, as demonstrated by the use of an inhibitor of mitochondrial pyruvate carrier. In contrast, oxygen consumption in control neutrophils was driven by fatty acid oxidation, as shown by the effect of inhibiting carnitine palmitoyltransferase 1. Thus, the reprogramming of neutrophils with GM-CSF, TNF, and IL-4 into cells capable of producing peptidase inhibitors is associated with an original metabolic phenotype characterized by active mitochondrial pathways. • TNF and GM-CSF enhance neutrophils’ glycolysis and oxygen consumption • Long-lived neutrophils produce the protease inhibitors elafin and SLPI • Long-lived neutrophils are metabolically responsive to TNF and IL-1β • Long-lived neutrophils exhibit mitochondrial respiration partly fueled by pyruvate Summary sentence: Reprogramming of human blood neutrophils by inflammatory cytokines leads to PI3 -expressing neutrophils with an active metabolism following their activation and dependent on mitochondrial respiration, partly driven by pyruvate oxidation.