Assessment of Poly(ADP-ribose) Polymerase1 (PARP1) expression and activity in cells purified from blood and milk of dairy cattle

Abstract

Poly(ADP-ribosyl)ation (PAR) is a post-translational protein modification catalysed by enzyme member of the poly(ADP-ribose) polymerases (PARPs) family. The activation of several PARPs is triggered by DNA strand breakage and the main PARP enzyme involved in this process is PARP1. Besides its involvement in DNA repair, PARP1 is involved in several cellular processes including transcription, epigenetics, chromatin re-modelling as well as in the maintenance of genomic stability. Moreover, several studies in human and animal models showed PARP1 activation in various inflammatory disorders. The aims of the study were (1) to characterize PARP1 expression in bovine peripheral blood mononuclear cells (PBMC) and (2) to evaluate PAR levels as a potential inflammatory marker in cells isolated from blood and milk samples following different types of infection, including mastitis. Our results show that (i) bovine PBMC express PARP1; (ii) lymphocytes exhibit higher expression of PARP1 than monocytes; (iii) PARP1 and PAR levels were higher in circulating PBMCs of infected cows; (iv) PAR levels were higher in cells isolated from milk with higher Somatic Cell Counts (SCC > 100,000 cells/mL) than in cells from milk with low SCCs. In conclusion, these findings suggest that PARP1 is activated during mastitis, which may prove to be a useful biomarker of mastitis.

New Insights into the Significance of PARP-1 Activation: Flow Cytometric Detection of Poly(ADP-Ribose) as a Marker of Bovine Intramammary Infection

“…Moreover, the characterization of milk cell populations showed that quarters with high SCC exhibit an increase in phagocytes (neutrophils and macrophages). Considering that macrophages are important players in the inflammatory response, we proposed that the high PAR level found in milk with high SCC could be connected to PARP-1 activation in the increased proportion of the macrophages population [9]. According to these findings, PAR could be considered as an inflammatory mediator produced by both lymphocytes and monocytes/macrophages.…”

“…Research on the involvement of PARP-1 in bovine intramammary infection is limited. We have recently reported that unhealthy udders with high SCC show changes in milk somatic cell composition and increased intracellular poly(ADP-ribose) levels [9]. More recently, we showed that the conventional flow cytometry analysis applied to milk samples is a useful tool to detect new immunological markers as potential indicators of udder health [10].…”

PAR level mediates the link between ROS and inflammatory response in patients with type 2 diabetes mellitus

“…To assess the concentration of PAR, nuclear extracts were prepared from freshly collected PBMC samples. PAR quantification was conducted using the HT PARP in vitro Pharmacodynamic Assay II kit from Trevigen, following the manufacturer's protocol [ 36 ]. Chemiluminescent signals were recorded using the Victor X light plate reader from PerkinElmer Inc.…”