Proteasome Inhibition Diminishes the Formation of Neutrophil Extracellular Traps and Prevents the Death of Cardiomyocytes in Coculture with Activated Neutrophils during Anoxia-Reoxygenation

Abstract

Objective: Polymorphic mononuclear neutrophils (PMN) are very important cells participating in nonspecific defense of the organism. Among their well-known functions, the formation of neutrophil extracellular traps (NET) is interesting and potentially dangerous for the mechanisms of other cells. Ubiquitin-dependent proteasomal proteolysis is a very important regulator of all cellular activities, but the role of proteasomal proteolysis in NET formation has not been investigated. Methods: We performed experiments with PMN activated to form NET with phorbol 12-myristate 13-acetate (PMA) and the application of a proteasome inhibitor. We also added activated neutrophils to primary culture of isolated rat neonatal cardiomyocytes with or without anoxia-reoxygenation modeling. Results: The data obtained show that proteasomes participate in NET formation and proteasome inhibitors facilitate the blocking of the NET program. The percentage of NET after PMA application was 70.8 ± 7.2 and the proteasome inhibitor decreased this amount to 4.7 ± 0.9%. In coculture with cardiomyocytes during anoxia-reoxygenation, this effect prevented cardiac cell death induced by activated PMN. The stimulation of NET formation by PMA in coculture with isolated cardiomyocytes led to an increase in the number of necrotic cardiomyocytes of up to 33.1 ± 12.9% and a corresponding decrease in living cardiomyocytes to 66.9 ± 12.9%. The number of living cardiomyocytes in coculture after incubation with both PMA and proteasome inhibitor was 76.6 ± 13.3% (p < 0.05), and the number of necrotic cardiomyocytes was 23.4 ± 13.3% (p < 0.05). Conclusion: Proteasome inhibition blocks NET formation and prevents cardiomyocyte necrosis in coculture with activated neutrophils.

Hyperbaric Oxygen Reduces Production of Reactive Oxygen Species in Neutrophils from Polytraumatized Patients Yielding in the Inhibition of p38 MAP Kinase and Downstream Pathways

“…On the basis of these observations, we speculate that PMA-triggered ROS production promotes fast Mcl-1 degradation, probably by the activation of the proteasomal pathway. This hypothesis is supported by a current report showing suppression of PMA-induced NETs formation by proteasome inhibitors [ 58 ]. In fact, HBO treatment did not significantly alter Mcl-1 protein levels after 3 h, neither in control nor in patient neutrophils.…”

Neutrophil hyperactivation correlates with Alzheimer's disease progression

“…48 Increased expression of CD83 transcript was also associated with upregulation of TAP1, involved in the proteolytic steps of major histocompatibility complex class I antigen processing by neutrophils, 49 suggesting that neutrophils from AD-D patients can adapt a function as antigen presenting cell. 57 Finally, we observed an increased expression of PER1 gene (p 5 0.06). 50 Importantly, in AD-D patients, we observed significant increased expression of four genes (SIK1, CRIP2, GRASP, and AK5) involved in regulation of signaling pathways.…”

“…55 Furthermore, upregulation of functions associated with ubiquitination machinery (DTX1, PSMB8) in AD-D patients could result in increased neutrophil death by apoptosis 56 as well as by NETosis in these patients. 57 Finally, we observed an increased expression of PER1 gene (p 5 0.06). PER1 encodes for period circadian protein homolog 1 protein, a key regulator of internal biological clock in neutrophils, that has been involved in the mobilization of young neutrophils from the bone marrow and their aging in the peripheral circulation.…”

Metformin ameliorates BSCB disruption by inhibiting neutrophil infiltration and MMP‐9 expression but not direct TJ proteins expression regulation

“…The granulocytes were then washed with HBSS buffer and diluted in RPMI media with 10% (v/v) heat‐inactivated FBS. The neutrophils were plated in the 6‐well dish with a density of 100,000 cells/cm 2 31. Neutrophils were treated with TNF‐α (100 ng/ml), metformin (1 mM) and compound C (50 μM) for 6 hrs in conventional cell incubator.…”