DOI: 10.5281/zenodo.15315211
title: Cleavage and inactivation of poly(ADP-ribose) polymerase in peripheral blood mononuclear cells of patients with acute respiratory distress syndrome

description: The availability of olaparib, as the first clinically approved PARP inhibitor, opens the way for the potential repurposing of this drug for non-oncological indications. Because preclinical data demonstrate that PARP-1 deficiency and PARP inhibitors exert beneficial effects in various models of acute respiratory distress syndrome (ARDS), this disease represents a potential indication for such repurposing efforts. In the current project, we have assessed the functional effect of olaparib in peripheral blood mononuclear cells (PMBCs) isolated from the peripheral blood of patients with ARDS. PBMCs were isolated using Ficoll gradient from human healthy volunteers (N=7) and ARDS patients (N=7). PARP-1 and cleaved PARP (cPARP) expression and PAR polymer levels were evaluated by Western blotting.  Cytokine production was measured in plasma and PBMC supernatants preincubated with olaparib (10 µM) or its vehicle for 1h and challenged with LPS 100 ng/ml for 4h. To assess cellular bioenergetics, as measured by the Seahorse XFe24 flux analyzer, PBMCs were incubated with olaparib (10 µM) or its vehicle for 4h, followed by the addition of H2O2 (250 µM) for 2 h. Proteomic analysis (N=5) was also performed in the above conditions. PBMCs obtained from control subjects presented a leukocyte population with predominance of lymphocytes (41%). In the control samples, full-length PARP-1, cleaved PARP (cPARP) and PAR polymers were detectable, with PARP predominantly present in its full-length physiological form. In PBMCs from ARDS patients on Day 1, the PARylated proteins were more abundant than in the PBMCs of control subjects, while PARP was predominantly present in its cleaved form.  On Day 8 of ARDS, PARylation was lower than what was detected in Day 1, and PARP was present both in cleaved and uncleaved forms. These data are consistent with an early, rapid activation of PARP-1 in ARDS, followed by its proteolytic cleavage. TNF-α and sICAM-1, IL-17, MIP-1α and IL-1β were detectable in plasma samples from ARDS patients while in the healthy control plasma only minimal levels of MCP-1 and sICAM-1 were detected.  Incubation with olaparib had no significant effect on the LPS-induced cytokine responses in PBMCs in vitro. In PBMCs from healthy controls, oxidative stress suppressed the various cellular bioenergetic parameters, and this effect was slightly, but significantly attenuated by olaparib pretreatment. In contrast, in PBMCs isolated from ARDS patients – which exhibited a significant baseline suppression of cellular bioenergetic parameters – exposure to oxidative stress in vitro did not produce any further metabolic suppression, and the PARP inhibitor was without any functional effect. There were marked proteomic modifications in PBMCs after exposure to H2O2, but these were unaffected by olaparib. PCA analysis showed an important separation between the groups (CTR, H2O2 and olaparib + H2O2) when analyzed in relation to mitochondrial proteins. Thus, in PBMCs from ARDS patients, olaparib loses its cytoprotective effect, most likely due to the cleavage and inactivation of PARP-1.