PB0732 - Platelet mitochondrial transfer alters neutrophil phenotype and function

Background: Activated platelets produce a heterogenous population of microvesicles packaged with pro-inflammatory and pro-thrombotic molecules. A small subset of these, contain functional mitochondria that actively respire. Interactions between platelet vesicles and cells within the circulation have been described, however to date, mitochondria-containing vesicles have not been investigated as a separate population.

Aims: To investigate how transfer of mitochondria from platelets via microvesicles affects neutrophil phenotype.

Methods: Microvesicles generated from platelets, stained with MitoTracker Orange were separated into two populations: mitochondria positive (mitoPMV) and negative (PMV) microvesicles. These subpopulations were combined with neutrophils to explore the differential effects on activation markers and phagocytosis by flow cytometry. Further, neutrophil extracellular trap (NET) formation was stimulated by lipopolysaccharide (LPS, 25ng/ml) or phorbol 12-myristate 13-acetate (PMA, 200nM) in the presence of the two microvesicle subpopulations and analysed by immunofluorescence. Analysis was performed using ImageJ, FlowJo v.10 and GraphPad Prism.

Results: MitoPMVs readily interacted with and were internalised by neutrophils. Following incubation with mitoPMVs, but not PMVs, neutrophils exhibited an activated phenotype with increases in expression of CD66b (1.5±0.2-fold, p< 0.05), CD11b (1.3±0.1-fold, p< 0.05) and CD192 (1.6±0.2-fold, p< 0.05) with a concurrent reduction in CXCR2 expression (0.7±0.07-fold, p< 0.05). Interestingly, mitoPMVs but not PMVs caused a reduction in neutrophil phagocytic ability (0.4±0.08-fold, p< 0.05).

Formation of NETs by neutrophils in response to 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): Mitochondrial transfer has been demonstrated in numerous cells highlighting mechanisms through which mitochondrial function is augmented or altered. Here we show that neutrophil phenotype and function are altered following the internalisation of mitochondria encapsulated within platelet vesicles. Further work will demonstrate whether these effects are mediated through changes in metabolic function or through other pathways within neutrophils.