PB0878 - Platelet-derived mitochondria promote neutrophil activation and NETosis

Background: Circulating platelet microvesicles in both physiological and pathological conditions present as heterogenous populations. In both instances, subsets of these microvesicle populations contain actively respiring mitochondria. To date, the influences of these mitochondria-containing microvesicles on other circulating cells remain largely unknown

Aims: To investigate how mitochondria-containing platelet microvesicles affect neutrophil function

Methods: Platelet microvesicles were generated from washed platelets and stained with MitoTracker Orange. Mitochondria positive (mitoPMV) and negative (PMV) microvesicles were subsequently isolated by cell sorting and then either used to study neutrophil extracellular trap (NET) formation or analysed for activation markers by flow cytometry. To assess NET formation, neutrophils were incubated with either lipopolysaccharide (LPS, 25ng/ml) or phorbol 12-myristate 13-acetate (PMA, 200nM) in the presence of sorted populations of microvesicles and then analysed by immunofluorescence. Data were analysed using Image J, FlowJo v.10 and GraphPad Prism.

Results: Mitochondria-containing microvesicles had high P-selectin expression, interacted with neutrophils and were internalised. Following incubation with mitoPMVs, but not with PMVs, neutrophils exhibited an activated phenotype with increases in expression of the surface markers CD192 (1.6±0.2-fold, p< 0.05), CD66b (1.5±0.2 -old, p< 0.05), and CD11b (1.3±0.1-fold, p< 0.05) and a reduction in expression of CXCR2 (0.7±0.07-fold, p< 0.05). NET formation in response to both LPS and PMA was enhanced in the presence of mitoPMVs (LPS-stimulated DNA Area, 107±21µm² vs. 238±6µm²; PMA-stimulated DNA Area, 137±23µm² vs. 240±41µm²; neutrophils vs. neutrophils + mitoPMVs; p< 0.05).

Conclusion(s): Platelet-derived mitochondria can interact with neutrophils, causing their subsequent activation. Further, platelet-derived mitochondria enhance NETosis, consistent with their capacity to act as damaged associated molecular patterns. Further work is required to determine the influence of platelet-derived mitochondria on neutrophils, but data presented here indicate they enhance neutrophil activation and inflammatory function.