ENDOTHELIAL CELL-DERIVED EXTRACELLULAR VESICLES ELICIT NEUTROPHIL DEPLOYMENT FROM THE SPLEEN FOLLOWING ACUTE MYOCARDIAL INFARCTION

Acute myocardial infarction rapidly increases blood neutrophils (<2 hours). Release of neutrophils from bone marrow, in response to chemokine elevation, has been considered their source, but chemokine levels peak up to 24 hours after injury, and after neutrophil elevation. This suggests that additional non chemokine-dependent processes may be involved. Endothelial cell (EC) activation promotes the rapid (<30 minutes) release of extracellular vesicles (EVs), which are enriched in vascular cell adhesion molecule-1 (VCAM-1) and miRNA-126, and are thus a potential mechanism for communicating with remote tissues. Here, we show that injury to the myocardium rapidly mobilises neutrophils from the spleen to peripheral blood and induces their transcriptional activation prior to their arrival at injured tissue. Ischemic myocardium leads to the generation and release of EC-derived-EVs bearing VCAM-1. EC-EV delivery to the spleen alters inflammatory gene and chemokine protein expression, and mobilises neutrophils to peripheral blood. Using CRISPR/Cas9 genome editing we generated VCAM-1-deficient EV and showed that its deletion removed the ability of EC-EV to provoke the mobilisation of neutrophils. Furthermore, inhibition of miRNA-126 in vivo reduced myocardial infarction size in a mouse model. Our findings show a novel mechanism for the rapid mobilisation of neutrophils to peripheral blood from a splenic reserve, independent of classical chemokine signalling, and establish a proof of concept for functional manipulation of EV-communications through genetic alteration of parent cells. One Sentence Summary Extracellular vesicles mediate neutrophil mobilisation from the spleen following acute myocardial infarction. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement NA and RC acknowledge support by research grants from the British Heart Foundation (BHF) Centre of Research Excellence, Oxford (NA and RC: RE/13/1/30181), British Heart Foundation Project Grant (NA and RC: PG/18/53/33895), the Tripartite Immunometabolism Consortium, Novo Nordisk Foundation (RC: NNF15CC0018486), Oxford Biomedical Research Centre (BRC), Nuffield Benefaction for Medicine and the Wellcome Institutional Strategic Support Fund (ISSF) (NA), the National Institutes of Health (NIHR) (R35HL135799,P01HL131478 (KJM) and T32HL098129 (CvS)) and the American Heart Association (19CDA346300066 (CvS)). DRFC and GM acknowledge BBSRC (BB/P006205/1). The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All clinical investigations were conducted in accordance with the Declaration of Helsinki. The Oxfordshire Research Ethics Committee (references 08/H0603/41 and 11/SC/0397) approved human clinical cohort protocols. All patients provided informed written consent for inclusion in the study. LAD ligation model All animal procedures were approved by an ethical review committee at the University of Oxford or NYU Lagone Health. UK experimental interventions were carried out by UK Home Office personal licence holders under the authority of a Home Office project licence. All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes Data Availability RNA Sequencing data are deposited at Gene Expression Omnibus. Pending accession number.