OC 49.4 - Platelet-released mitochondria interact with neutrophils, altering their phenotype and merging with the existing mitochondrial network

Background: Activated platelets produce a heterogenous population of extracellular vesicles packaged with pro-inflammatory and pro-thrombotic molecules. A small subset of these vesicles contains functional mitochondria that actively respire and consume oxygen. 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. Notably, mitochondria are damaged associated molecular patterns which raises the question whether mitochondria vesicles will interact with and affect immune cells.

Aims: To investigate if platelet vesicles containing mitochondria interact with neutrophils thereby changing their phenotype.

Methods: Washed platelets (3x10^8/ml) stained with MitoTracker Orange (20nm) were incubated with TRAP-6 (20µM; 2 hours, 37˚C) to stimulate the production of vesicles. Cell sorting was used to separate mitochondria negative (PMV) and positive (mitoPMV) vesicles. Isolated neutrophils were incubated with sorted vesicles (45 mins, 37˚C), prepared for immunofluorescence or analysed for the expression of CD66b, CD11b and CXCR2 by flow cytometry. Data were analysed using Image J, FlowJo v.10 and GraphPad Prism.

Results: MitoPMVs accounted for 19±1% of the platelet vesicle population, had high levels of P-selectin expression (96±1%, n=8) and interacted with and were internalised by neutrophils. Microscopy revealed mitoPMVs merged with the neutrophil mitochondria network. Furthermore, neutrophils incubated with mitoPMVs, but not those incubated with PMVs, showed significant increases in expression of CD66b (1.5±0.2 fold; n=6, p< 0.05) and CD11b (1.3±0.1; n=6, p< 0.05) with a concurrent reduction in CXCR2 expression (0.7±0.07 fold, n=6, p< 0.05).

Conclusion(s): Mitochondrial transfer has been demonstrated in numerous cells highlighting a mechanism in which mitochondrial function may be augmented. Here we show that mitochondria encapsulated within platelet vesicles are internalised by neutrophils and can merge with existing neutrophils networks. Further work is required to determine the contribution to neutrophil metabolic capacity but may be associated with neutrophils adopting an activated and phagocytic phenotype.