Formation of neutrophil extracellular traps in mitochondrial DNA-deficient cells (1)
NET formation has been reported in cancer, diabetes, and autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. Moreover, NETs bind to platelets, thereby causing vascular damage and arteriosclerosis. Neutrophils can extrude NETs via NOX-independent mechanisms in response to stimulants such as calcium ionophores (e.g., A23187). Mitochondrial dysfunction and oxidative stress in and around mitochondria have been implicated in pathogenetic mechanisms, including inflammation and auto-immune reactions.
For the isolation of infiltrating neutrophils, C57BL/6 mice were intraperitoneally administered 2 ml of 2.98% thioglycollate in PBS. At 4 h after the administration, neutrophils infiltrating the peritoneal cavity were collected using PBS. The isolated neutrophils were washed three times with PBS and then used for experiments.
HL-60 cells were treated with 10 mM A23187 or 10 nM phorbol myristate acetate (PMA) for 3 h, whereas the murine neutrophils were treated with 10 mM A23187 or 1 mM PMA for 3 h. Then, all the cells were treated with 20 U/ml micrococcal nuclease for 20 min at 37°C. The DNA containing supernatants were collected after centrifugation at 200 x g for 8 min at 4°C.
The rate of NETosis was quantified hourly using SpectraMax® (485 nm excitation, 525 nm emission) in the presence of SYTOX green. To calculate the relation of NETosis, fluorescence of the cells with 1% (v/v) Triton X-100 was considered as 100% DNA, and NETosis at each time was showed at the % of total DNA.
Compared to the wild-type mouse neutrophils, the gp91phox KO mouse neutrophils did not release DNA into the extracellular space after PMA stimulation.
MitoTEMPO treatment slightly decreased the A23187- and PMA-induced NET formation in HL-60 cells.
ddC, which prevents mtDNA replication, was used to establish r0 cells in a short period (1 week). HL-60 cells treated with 1 uM ddC for 7 days depleted gene and protein expression of cytochrome C.
With HL-60 cells treatment with 1.25% DMSO for 3 days, CD11b was expressed on day 3 of differentiation in HL-60 cells. Atp6, which is encoded in mtDNA, was markedly decreased in the DMSO-induced neutrophil-like r0cells.
After A23187 stimulation, extracellular DNA release from neutrophillike r0 cells was significantly lower than that from neutrophil-like HL-60 cells. However, there was no difference between the PMA-induced extracellular DNA release in neutrophil-like HL-60 cells and neutrophil-like r0 cells.
A23187-stimulated neutrophil-like HL-60 and r0 cells showed increased expression of PAD4, an enzyme that converts histone arginine residues to citrulline and increased expression of citrullinated H3.
ABAH, an MPO inhibitor, treatment did not suppress A23187-induced NET formation but markedly suppressed PMA-induced NET formation. HClO-generation was observed in both neutrophil-like HL60 and r0 cells after PMA stimulation but not after A23187 stimulation. NSA, an MLKL inhibitor, treatment suppressed both A23187- and PMA-induced NET formation.
