IL-12/15/18-induced cell death and mitochondrial dynamics of human NK cells

Natural killer (NK) cells are lymphocytes with potent antitumor functions and, consequently, several NK cell-based strategies have been developed for cancer immunotherapy. A remarkable therapeutic approach is the adoptive transfer of NK cells stimulated with IL-12, IL-15 and IL-18. This cytokine stimulation endows NK cells with properties that resemble immunological memory and, for this reason, they are known as cytokine-induced memory-like (CIML) NK cells. Very promising results have been reported in clinical trials and yet, there are still unknown aspects of CIML NK cells. Here, we have conducted a preliminary study of their mitochondrial dynamics. Our results show that upon IL-12/15/18 stimulation the viability of NK cells decreased and an increment in mitochondrial superoxide levels was observed. In addition, we found that mitochondria appeared slightly elongated and their cristae density decreased following IL-12/15/18 stimulation, possibly in a process mediated by the low levels of optic atrophy type 1 (OPA1) protein. Interestingly, although mitophagy was slightly impaired, an increase in autophagic flux was observed, which might explain the reduced viability and the accumulation of unfit mitochondria. Our findings could be of relevance in order to design new strategies intended to improve the mitochondrial fitness of IL-12/15/18-stimulated NK cells with the aim of improving their therapeutic efficacy

A Ca 2+ -Dependent Mechanism Boosting Glycolysis and OXPHOS by Activating Aralar-Malate-Aspartate Shuttle, upon Neuronal Stimulation

International audienceCalcium is an important second messenger regulating a bioenergetic response to the workloads triggered by neuronal activation. In embryonic mouse cortical neurons using glucose as only fuel, activation by NMDA elicits a strong workload (ATP demand)-dependent on Na + and Ca 2+ entry, and stimulates glucose uptake, glycolysis, pyruvate and lactate production, and oxidative phosphorylation (OXPHOS) in a Ca 2+ -dependent way. We find that Ca 2+ upregulation of glycolysis, pyruvate levels, and respiration, but not glucose uptake, all depend on Aralar/AGC1/Slc25a12, the mitochondrial aspartate-glutamate carrier, component of the malate-aspartate shuttle (MAS). MAS activation increases glycolysis, pyruvate production, and respiration, a process inhibited in the presence of BAPTA-AM, suggesting that the Ca 2+ binding motifs in Aralar may be involved in the activation. Mitochondrial calcium uniporter (MCU) silencing had no effect, indicating that none of these processes required MCU-dependent mitochondrial Ca 2+ uptake. The neuronal respiratory response to carbachol was also dependent on Aralar, but not on MCU. We find that mouse cortical neurons are endowed with a constitutive ER-to-mitochondria Ca 2+ flow maintaining basal cell bioenergetics in which ryanodine receptors, RyR2, rather than InsP 3 R, are responsible for Ca 2+ release, and in which MCU does not participate. The results reveal that, in neurons using glucose, MCU does not participate in OXPHOS regulation under basal or stimulated conditions, while Aralar-MAS appears as the major Ca 2+ -dependent pathway tuning simultaneously glycolysis and OXPHOS to neuronal activation. SIGNIFICANCE STATEMENT Neuronal activation increases cell workload to restore ion gradients altered by activation. Ca 2+ is involved in matching increased workload with ATP production, but the mechanisms are still unknown. We find that glycolysis, pyruvate production, and neuronal respiration are stimulated on neuronal activation in a Ca 2+ -dependent way, independently of effects of Ca 2+ as workload inducer. Mitochondrial calcium uniporter (MCU) does not play a relevant role in Ca 2+ stimulated pyruvate production and oxygen consumption as both are unchanged in MCU silenced neurons. However, Ca 2+ stimulation is blunt in the absence of Aralar, a Ca 2+ -binding mitochondrial carrier component of Malate-Aspartate Shuttle (MAS). The results suggest that Ca 2+ -regulated Aralar-MAS activation upregulates glycolysis and pyruvate production, which fuels mitochondrial respiration, through regulation of cytosolic NAD + /NADH ratio