Formation of neutrophil extracellular traps in mitochondrial DNA-deficient cells

Abstract

Neutrophil extracellular trap (NET) formation plays an important role in inflammatory diseases. Although it is known that NET formation occurs via NADPH oxidase (NOX) dependent and NOX independent pathways, the detailed mechanism remains unknown. Therefore, in this study, we aimed to elucidate the role of mito chondria in NOX dependent and NOX independent NET formation. We generated mitochondrial DNA deficient cells (ρ 0 cells) by treating HL 60 cells with dideoxycytidine and differentiated them to neutrophil like cells. These neutrophil like ρ 0 cells showed markedly reduced NOX independent NET formation but not NOX dependent NET formation. However, NET associated intracellular histone citrullination was not inhibited in ρ 0 cells. Furthermore, cells membrane disruption in NOX dependent NET formation occurred in a Myeloperoxidase (MPO) and mixed lineage kinase domain like pseudokinase (MLKL) dependent manner; however, cell membrane disruption in NOX independent NET formation partially occurred in an MLKL dependent manner. These results highlight the importance of mitochondria in NOX independent NET formation.

The Small GTPase Cdc42 Negatively Regulates the Formation of Neutrophil Extracellular Traps by Engaging Mitochondria

“…Furthermore, we demonstrated that inhibition of mtROS reduces NET formation in response to Cdc42 inhibition in primary human neutrophils. This data is consistent with observations of Takishita et al showing that mitoTEMPO did not completely suppress NET formation in cells stimulated with the calcium ionophore A23187 and PMA ( 37 ). This group hypothesized that not only mtROS but also mitochondrial signaling is involved in NET formation in response to PMA and the calcium ionophore ( 37 ).…”

“…To investigate the influence of mtROS on NET formation induced by PMA, ionomycin and Cdc42-inhibition, we used the mitochondria-restricted antioxidant mitoTEMPO to specifically inhibit mtROS. MitoTEMPO was described to act mitochondria specific to scavenge superoxide, thereby preventing mitochondrial damage ( 37 , 38 ). We could show that NET formation induced by PMA or ionomycin cannot be dampened by inhibiting mtROS formation ( Figure 3D ).…”

Studying Neutrophil Function in vitro: Cell Models and Environmental Factors

“… 142 However, these data have been contradicted by other studies, which demonstrated that ATRA-dHL-60 cells can also react to CIs and DMSO-dHL-60 cells also to PMA. 143 , 144 …”

“…Importantly, Takishita et al found that the DNA expulsed by dHL-60 cells was genomic rather than mitochondrial, as removal of mitochondrial DNA had no effect on PMA-induced NET release. 143 …”

Multiple Origins of Extracellular DNA Traps

“…These NETs are more resistant to the action of DNases ( 64 ). In the formation of NETs by the independent way of NADPH oxidase, ROS come from the mitochondria and the blockade of MPO does not inhibit the extrusion of NETs, although it is partially dependent on MLKL ( 61 , 65 ). Processes such as vesiculation, DNA decondensation, release of nuclear DNA into the cytoplasm and expulsion of extracellular DNA can be observed in this pathway ( 66 ).…”