Effects of the PARP Inhibitor Olaparib on the Response of Human Peripheral Blood Leukocytes to Bacterial Challenge or Oxidative Stress

Abstract

Prior studies demonstrate the activation of poly-(ADP-ribose) polymerase 1 (PARP1) in various pathophysiological conditions, including sepsis. We have assessed the effect of olaparib, a clinically used PARP1 inhibitor, on the responses of human peripheral blood leukocytes (PBMCs) obtained from healthy volunteers in response to challenging with live bacteria, bacterial lipopolysaccharide (LPS), or oxidative stress (hydrogen peroxide, H2O2). The viability of PBMCs exposed to olaparib or to the earlier generation PARP inhibitor PJ-34 (0.1–1000 µM) was monitored using Annexin V and 7-aminoactinomycin D. To evaluate the effects of olaparib on the expression of PARP1 and its effects on protein PARylation, PBMCs were stimulated with Staphylococcus aureus with or without olaparib (1–10 μM). Changes in cellular levels of nicotinamide adenine dinucleotide (NAD+) and adenosine triphosphate (ATP), as well as changes in mitochondrial membrane potential (MMP), were measured in PBMCs exposed to H2O2. Bacterial killing was evaluated in PBMCs and polymorphonuclear leukocytes (PMNs) incubated with S. aureus. Cytokine production was measured in supernatants using a cytometric bead array. Reactive oxygen species (ROS), nitric oxide (NO) production, and phagocytic activity of monocytes and neutrophils were measured in whole blood. For ROS and NO production, samples were incubated with heat-killed S. aureus; phagocytic activity was assessed using killed Escherichia coli conjugated to FITC. Olaparib (0.1–100 µM) did not adversely affect lymphocyte viability. Olaparib also did not interfere with PARP1 expression but inhibits S. aureus-induced protein PARylation. In cells challenged with H2O2, olaparib prevented NAD+ and ATP depletion and attenuated mitochondrial membrane depolarization. LPS-induced production of TNF-α, MIP-1α, and IL-10 by PBMCs was also reduced by olaparib. Monocytes and neutrophils displayed significant increases in the production of ROS and NO after stimulation with S. aureus and phagocytic (E. coli) and microbicidal activity, and these responses were not suppressed by olaparib. We conclude that, at clinically relevant concentrations, olaparib exerts cytoprotective effects and modulates inflammatory cytokine production without exerting adverse effects on the cells’ ability to phagocytose or eradicate pathogens. The current data support the concept of repurposing olaparib as a potential experimental therapy for septic shock.

Efficacy of Clinically Used PARP Inhibitors in a Murine Model of Acute Lung Injury

“…Importantly, the repurposing concept is further supported by data presented in the current report showing that these inhibitors do not exacerbate DNA damage after endotoxin challenge. These findings are also in line with other recent data [ 12 , 52 ], demonstrating that olaparib—while providing cytoprotective effects and modulatory effects on the production of cytokines in human mononuclear cells—does not impair the ability of these cells to mount an effective antibacterial immune response.…”

“…We do not suggest that from the clinically used PARP inhibitors, olaparib is the only (or best) agent for repurposing, although in the current model, its efficacy, at least on some parameters, appeared to be superior to the efficacy of rucaparib. Nevertheless, olaparib is the first clinically used PARP inhibitor; arguably, the largest body of clinical information is available with this agent, and also in preclinical studies focusing on various non-oncological conditions, this agent was the one that has been utilized most extensively in preclinical studies focused on PARP inhibitor repurposing [ 9 , 10 , 11 , 12 ].…”

“…The underlying mechanisms are likely multiple: they may involve direct cytoprotective effects, regulation of various inflammatory mediators, and—as a consequence of these—suppression of inflammatory cell infiltration and attenuation of various self-amplifying cycles of cell and tissue injury. These mechanisms have already been characterized extensively in various in vitro and in vivo models using various classes of PARP inhibitors, including clinically approved ones such as olaparib [ 1 , 2 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 ].…”

“…However, clinical translation of this concept was not possible due to the lack of clinically approved PARP inhibitors. With the clinical introduction of several novel, ultrapotent PARP inhibitors (with ovarian and breast cancer being their approved indications), the concept that PARP inhibitors may have potential clinical utility in various non-oncological diseases, including ALI, through drug repurposing has received renewed justification [ 9 , 10 , 11 , 12 , 13 ]. The current project evaluated the effect of the clinical-stage PARP inhibitors olaparib, veliparib, rucaparib, talazoparib, and niraparib in oxidatively stressed cultured epithelial cells and monocytes in vitro and also assessed the efficacy of two selected inhibitors, olaparib and rucaparib in a murine model of ALI in vivo.…”

Mesenchymal stromal cells secretome restores bioenergetic and redox homeostasis in human proximal tubule cells after ischemic injury

“…The integrity of mitochondrial membrane potential (Δψ) was measured using JC-10 (Abcam, ab112134) [ 39 ]. ciPTECs were rinsed once with HBSS and incubated with 50 μl of JC-10 loading solution for 30 min at 37 °C in the dark.…”