Background

Extracellular vesicles (EVs) are a family of cell-derived membrane-encapsulated particles produced constitutively, but also in response to inflammatory stimuli, or activation of programmed cell death pathways (Yanez-Mo et al., 2015). Carrying functional molecular cargoes, EVs have considerable potential as short- and long-range intercellular communication vehicles. Microvesicles (MVs) are an EV subclass derived directly from the cell membrane that display lineage-specific surface markers enabling identification of the parental cell source. During diseases associated with systemic inflammation, such as sepsis and severe burns injury, circulating levels of platelet- and myeloid cell-derived MVs are increased acutely and associated with clinical severity (Nieuwland et al., 2000; Joop et al., 2001; O'Dea et al., 2016). As these MV subtypes carry pro-inflammatory molecular cargoes, they may play a key role in long-range propagation of organ injury during systemic inflammation. Despite this potential, little is known of MV trafficking within the circulation and their cell/tissue-specific interactions during systemic inflammation.

We previously developed a lipopolysaccharide (LPS)-induced model of sub-clinical endotoxemia to evaluate cell/tissue specific interactions of circulating MVs under systemic inflammatory conditions in vivo. In vitro generated MVs labelled with the fluorescent lipophilic dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine (DiD) were injected intravenously and their cellular uptake quantified in lung, liver, and spleen cell suspensions by flow cytometry (O'Dea et al., 2020). Using in vivo analysis of cell-associated MVs rather than biodistribution methods, which measure total tissue/organ associated MV label (Willekens et al., 2005; Wiklander et al., 2015), enabled us to determine the relative uptake of MVs by different cell populations within and between organs. We found that during systemic inflammation there is a dramatic increase in MV uptake by pulmonary intravascular ‘marginated’ monocytes, resulting in an overall ≥20-fold increase in uptake by the lungs.

To enable more detailed analyses of the enhanced MV-monocyte interactions during systemic inflammation, we developed a novel in vitro model of MV uptake by in vivo derived pulmonary vascular perfusate cells. In vitro analysis of intravascular monocyte functions, including uptake of MVs, is severely constrained in mice due to their low abundance and small volumes of blood; this problem is further exacerbated by withdrawal of monocytes from the circulation by their margination to vascular beds of lungs, liver, and other tissues during systemic inflammation (O'Dea et al., 2005 and 2009; Hamon et al., 2017). Although differentiated monocytes can be obtained from the bone marrow reserve, such preparations are mixed with immature precursors and, moreover, much of the mature monocyte pool is rapidly released into the circulation during inflammation (O'Dea et al., 2009; Shi et al., 2011). Conversely, the pulmonary circulation is a major vascular site for monocyte accumulation, marginated in significant numbers within its vast capillary network under both resting and inflammatory conditions (Doherty and Worthen, 1994; Kuebler and Goetz, 2002). As such, the lungs represent a previously ‘untapped’ source for in vitro evaluation of monocytes and neutrophils, which also accumulate in substantial numbers within the pulmonary circulation during inflammation (van Eeden et al., 1997; Yipp et al., 2017).

Here, we describe in detail our protocol (summarized in Figure 1) for routine recovery of pulmonary vascular cells and evaluation of MV uptake by monocyte subsets and neutrophils, the findings of which were reported recently by us (Figure 6, Figure 7, and Supplementary Figure S2 in O'Dea et al., 2020). In brief, using this method we determined that MV uptake by monocytes was energy-dependent, involved endocytic processes, and could be inhibited in the presence of phosphatidylserine-enriched liposomes, β3 integrin receptor blocking peptide, and scavenger receptor A blockers.