IFNγ Augments the Oxygen Consumption Rate and Metabolism in Monocytes Partially Through P2X7

Interferon-gamma (IFNγ) is a pleiotropic cytokine crucial for immune responses to intracellular pathogens. IFNγ causes transcriptional changes that lead to increased production of reactive oxygen species (ROS) which promote microbial killing. IFNγ increases the oxygen consumption rate (OCR), ROS production, as well as NAD salvage, all through transcription regulation by STAT1. The ATP-gated, purinergic pore P2X7 has been identified as an ion channel that allows for the influx of Ca2+ and Na+ and the efflux of K+, as well as playing a role in the processing and release of proinflammatory cytokines in macrophages. P2X7 appears to play a role in IFNγ-induced metabolic reprogramming in monocytes as well as the increase in ROS production. We used a Seahorse Extracellular Flux analyzer to measure oxygen consumption (as an indicator of ROS production), medium acidification, and kinetics in human monocytes in response to IFNγ or inhibitors before and after PMA activation. We used small molecule inhibitors to block NAD salvage and P2X7 pathways. We also used flow cytometry to measure protein expression, using anti-P2X7 antibodies. IFNγ activation led to doubling of expression level of the purinergic pore P2X7 on monocytes as shown by flow cytometry. Inhibition of the STAT1-dependent nicotinamide phosphoribosyltransferase (NAMPT) by daporinad led to a reduction of OCR by 67%. Addition of NAD to NAMPT blocked cells circumvented the blockade and restored normal OCR. Interestingly, inhibition of P2X7 by AZ-11645373 in the setting of NADcomplemented NAMPT blockade led to ~50% reduction of OCR. In the absence of NAMPT inhibition, P2X7 blockade alone leads to about 15% reduction in OCR; in the setting of IFNγ-stimulated OCR, the reduction was 25%. P2X7 expression increases in monocytes following IFNγ-activation, and its patency facilitates NAD-dependent OCR. In the setting of NAMPT blockade, P2X7 plays an important role in trafficking NAD or its metabolites into cells and allowing oxygen consumption following IFNγ stimulation. Blockade of P2X7 leads to reduced OCR, which diminished even further upon IFNγ stimulation and NAMPT blockade. The ability to augment OCR through P2X7 may contribute to control of intracellular pathogens.