Extracellular vesicles selectively mobilize splenic neutrophils

This editorial refers to ‘Rapid neutrophil mobilization by VCAM-11 endothelial cell-derived extracellular vesicles’, by N. Akbar et al., https://doi.org/10.1093/cvr/cvac012. There is a growing body of evidence that cell–cell communication via extracellular vesicles (EVs) plays an important role in the maintenance of homeostasis in various tissues. EVs are released by almost all cell types under physiological and pathological conditions with the goal of causing functional changes in recipient cells. EVs reflect the state of the parent cells producing the EVs and are known to carry a unique collection of bioactive molecules such as proteins, metabolites, DNA, inflammatory lipids, and micro-RNAs (miRNAs) encapsulated in their lipid bilayer. miRNAs are non-coding RNAs that bind to messenger RNAs (mRNA) of recipient cells and inhibit gene expression at the post-transcriptional level, thereby mediating intercellular communication. Several studies have suggested that exosomal miRNAs can be taken up by recipient cells causing changes in cellular gene expression and function.1 EVs can be detected in various biological fluids, and when produced by the endothelium or circulating immune cells, will disseminate throughout the body affecting distal sites. Endothelial cells are ubiquitously located throughout the circulatory network and form the barrier between blood and surrounding tissues. In case of inflammation or an injury, they are exposed to oxidative stress and inflammatory mediators like tumour necrosis factor-alpha (TNF-α), interleukin 1-beta, and thrombin. Furthermore, it has been shown that endothelial cells release EVs in response to inflammatory stress, triggering changes in phenotype in the recipient cells.2 Endothelial cell–derived EVs (EC-EVs) mediate the communication of stress-related signals, and the content of these EVs can reflect the cellular stress in the cell of origin. EC-EVs have been shown to regulate a multitude of diverse functions in target cells mediating the maintenance of cardiovascular homeostasis or inducing cardiovascular pathologies, such as in myocardial infarction (MI). Due to limited regenerative capacity of the heart, MI results in irreversible myocardial cell loss and functional impairment, eventually leading to heart failure and death. One of the main goals in the treatment of MI is the development of effective strategies to stimulate vascular repair mechanisms to achieve adequate tissue perfusion. Neutrophils are the first-line responders of the immune system and are quickly recruited to the ischaemic region following MI, aiming to clean up dead cell debris. However, the prevailing view is that neutrophils are detrimental in the setting of acute MI (AMI). Neutrophils aggravate inflammation and promote myocardial injury by releasing reactive oxygen species, granular components, and neutrophil extracellular traps.3,4 The circulating neutrophil count positively correlates to infarct size, death, and heart failure development.5,6 Experimental studies have also revealed that either neutrophil depletion or inhibition reduces cardiac injury and infarct size.7 In contrast, some studies have shown that neutrophils are important modulators of healing response after MI, highlighting the difficulty of targeting this cell type.8 In this study, Akbar et al. note that recruitment of neutrophils to the injured myocardium happens as early as 2 h after AMI in mice, whereas a peak in blood chemokine levels likely requiring protein synthesis is observed 24 h after AMI. Hence, there must be an alternate chemokine-independent mechanism responsible for recruitment of neutrophils. This study unravels a novel mechanism in which EVs released from endothelial cells in AMI cause the release of neutrophils from the spleen, a site of extramedullary haematopoiesis. They found that the presence of vascular cell adhesion molecule-1 on the surface of these EC-EVs is crucial for the release of splenic neutrophils into peripheral blood. Moreover, EC-EVs are enriched in miRNA-126-3p and 5p responsible for altering neutrophil transcriptome in the spleen before recruitment to the inflamed myocardium. Using antagomir to silence these miRNAs and preventing their binding to mRNAs resulted in a 12% reduction of infarct size in mice. Ultimately, the translation of this knowledge into clinically feasible therapeutic strategies could potentially aid in the effective treatment of AMI in humans, a leading cause of mortality worldwide (Figure 1). Extracellular vesicles released from endothelial cells in the heart after AMI selectively mobilize activated neutrophils from the spleen. Created with BioRender.com. EVs under pathophysiological conditions represent potential targets for disease treatment. However, a major challenge in therapeutic targeting of EVs is the specificity of targeting the disease-promoting EVs without interfering with the normal physiological processes mediated by host EVs. This raises the question, ‘Are all EVs the same or can different stimuli induce the production of different EVs?’. For example, in 1992, Patel et al.9 exposed endothelium to oxidative stress, which is a likely scenario in AMI, and identified EVs that contained high concentrations of platelet-activating factor (PAF)-like phospholipids, a potent stimulant of neutrophils. While transcriptomics dominates the research landscape, the authors highlight the rapid events that transpire in the inflammatory process, and one should not forget the rapid production of inflammatory lipids ranging from leukotrienes to PAF and also pro-repair lipid mediators such as resolvins. Indeed, it is possible that EV cargo could first be filled with inflammatory lipids due to oxidative stress and later with other molecules due to TNF. An intriguing finding is that the EVs were able to activate and mobilize splenic but not bone marrow–derived neutrophils. This raises the possibility that bone marrow harbours a different neutrophil population than the spleen. While single-cell RNA sequencing has certainly highlighted many different neutrophil subsets simplistically, there is likely an anti-microbial and a repair neutrophil subset. In the case of the spleen, it is possible that anti-microbial/inflammatory neutrophils are released, while repair neutrophils in the bone marrow fail to detect the injury. Interestingly, the spleen has both mature inflammatory neutrophils and less mature neutrophils that may be similar to repair neutrophils in the bone marrow. If this is the case, then the EVs may have mobilized only one subset from the spleen, highlighting the selectivity of EVs. Going forward, bioengineered EVs that encapsulate desired molecules could be a viable treatment option. However, for optimal benefit of such a strategy, it will be important to determine the mechanisms by which the EV cargo is delivered. In a previous study by the same group, the authors showed that EC-EVs are taken up by monocytes in the spleen, which, in turn, enhanced monocyte motility.10 Whether a similar biology is at play for neutrophils remains unclear. While phagocytosis is the dominant pathway of uptake by neutrophils, EVs may deliver their cargo through a route similar to viruses via the escape of endosomal compartments or inhibit the phagolysosome from releasing oxidants and proteases for content degradation. Understanding these molecular processes will almost certainly help boost EVs’ therapeutic potential. In the current study, antagomir for miRNA-126 was delivered 5 and 2 days prior to the induction of AMI in mice. This was necessary as antagomirs take at least 12 h to produce noticeable reductions in miRNA levels. Even though these experiments confirm the relevance of EC-EV-miRNA-126 in orchestrating neutrophil mobilization post-AMI, this will not be useful clinically. As an alternative, engineered EVs bearing selective miRNA that is delivered rapidly may allow neutrophil transcriptomes to be modulated in a way that delays their recruitment and activation, hence delaying cardiac tissue injury. Additionally, EVs expressing neutrophil-targeting peptides on their surface and carrying therapeutic miRNAs that promote pathways of repair and regeneration in neutrophils could be synergistically used. Future research in the field of EVs should investigate EV cell targeting and routes of intracellular trafficking of EVs in target cells to optimally realize the enormous potential of EVs for therapeutic purposes.

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