Kauno regioninis biomedicininių tyrimų etikos komitetas (02.28)

The extracellular vesicle and particle research field exploded with new data over the recent decade, erasing the barriers between the different extracellular vesicle classes and extending the size range of these intercellular communicators up and down from nearly cell-sized apoptotic blebs to several nanometer particles. Exosomes, microvesicles, migrasomes, oncosomes, mitovesicles, exomeres, chromatimeres are smurfing from cell to cell, travelling large distances, ignoring biological barriers and exchanging intracellular material all over the body, between the gut, the airway, the brain and the heart. In this talk, we will overview the role of different extracellular vesicles and particles in physiology and pathology, with a specific focus on vesicular communication between the airway and the brain during viral infections, vesicular crosstalk in the tumour microenvironment and beyond, involvement of extracellular vesicles in neurodegeneration and current trends in cell-derived vesicle application.

Introduction: Viral respiratory infections induce airway cell exosomes containing viral genetic material and inflammation-related inflammatory factors. Exosomes easily cross the blood-brain barrier and can transmit the inflammatory signal to the brain. Our previous study demonstrated that rodent poly I:C-primed airway ELV enter microglial cells in the brain within an hour after intranasal delivery and induce inflammatory changes. This study aimed to investigate the effect of poly I:C-primedhuman bronchial epithelial cell (HBEC) exosomes on human microglial cells. Methods: Exosome-like vesicles (ELV) were isolated from poly I:C-primed HBEC culture medium by polymer precipitation and characterized by particle number/size (Zetasizer), structure (TEM) and specific markers (ELISA). Poly I:C in ELV was monitored by DHR-poly I:C. The internalization of fluorescently-labelled ELV by human microglia (HM) was identified by fluorescent microscopy. Mitochondria and glycolysis of HM were assessed by Seahorse XFp analyzer. Intracellular ROS were determined by DCFDH, mitochondrial superoxide – by MitoSOX™ Red. Inflammasome activation was detected by caspase-1 luminescent assay. Inflammatory gene expression was assessed by RT-PCR, protein expression - by Western Blot. Statistical analysis was performed by SigmaPlot 13.0. Results: Over 95% of the EVs produced by poly I:C primed HBEC were 40-140 nm in diameter, surrounded by a lipid bilayer and contained CD9, CD63, CD81, syntenin-1, Tsg101, but not calnexin and Hsp70. ELV carried poly I:C molecules and were internalized by HM cells within 2 hours of the treatment. Poly I:C-primed HBEC ELV induced suppression of mitochondrial respiration and activation of glycolysis, generation of intracellular and mitochondrial ROS and activation of NLRP3 inflammasome-related caspase-1 followed by IL-1beta and IL-18 secretion, and a significant increase in expression of IL-6, TNF-alfa and PTGS2 genes and proteins. None of the effects was observed after treating HM with not poly I:C-primed HBEC exosomes. Moreover, untreated HBEC exosomes significantly reduced the expression of the inflammatory genes. Summary/Conclusion: In HM, poly I:C-primed HBEC exosomes induce the transition of energy production phenotype from mitochondrial to glycolytic and stimulate inflammatory activation manifested as ROS generation, inflammasome activation and production of pro-inflammatory cytokines. On the contrary, exosomes from the untreated HBEC have an anti-inflammatory effect on HM.