Enhanced Recognition and Internalisation of Microvesicles by Lung‐Marginated, Ly‐6Chigh Monocytes During Endotoxaemia
Authors: Tan Ying Ying , O'Dea Kieran Patrick , Soni Sanooj , Shah Sneh , Patel Brijesh Vipinchandra , Takata Masao 
Published: November 2023

BackgroundCirculating microvesicles (MVs) have been implicated as long‐range mediators of vascular inflammation, but their precise role in the pathophysiology of sepsis is unclear. MV recognition and uptake by cells within the vasculature will be critical in determining their role in systemic inflammation. We recently demonstrated that during subclinical endotoxaemia in mice, clearance of circulating MVs is partially re‐directed from the hepatic to pulmonary vasculature through preferential uptake by lung‐marginated Ly‐6Chigh subset monocytes (1). In this study, we investigated the mechanisms responsible for preferential MV uptake by Ly‐6Chigh monocytes in vitro using cells harvested by perfusion from the pulmonary vasculature.MethodsLungs of untreated or low‐dose LPS (20ng, i.v. 2 h) pre‐treated C57BL/6 mice were perfused via the pulmonary artery with PBS‐EDTA buffer to obtain marginated leukocytes. MVs derived from J774A.1 macrophages by ATP stimulation were labelled with the fluorescent membrane‐intercalating dye, DiD, and then co‐incubated with washed lung perfusate cells for up to 1 h. MV uptake was quantified by flow cytometry as cell‐associated DiD mean fluorescence intensity (MFI) and visualised by fluorescence microscopy, using surface marker antibody staining to identify cell subpopulations.ResultsAs previously found in vivo, preferential uptake of MVs was observed in Ly‐6Chigh monocytes (MFI 433±61.1) compared to other leukocyte subpopulations in perfusates from LPS pre‐treated mice, including Ly‐6Clow monocytes (MFI 8.8±6.1), and was substantially enhanced compared to Ly‐6Chigh monocytes from untreated mice (MFI 1.4±3.6). In Ly‐6Chigh monocytes from LPS pre‐treated mice, cell‐associated DiD fluorescence was unaffected by trypsin treatment (MFI: 92.8±16.7% of control), sensitive to low temperature (4°C) incubation (MFI: 2.9±1.1% of control) and partially inhibited by cytochalasin D (MFI: 48.5±9.7% of control). These results, together with imaging experiments, indicated an active internalisation process. MV uptake was significantly reduced by treatments with annexin V (56.2±12.3% of control, p<0.05) and an integrin blocking RGDS peptide (60.9±10.8% of control, p<0.05) suggesting involvement of a phosphatidylserine (PS)‐integrin receptor‐dependent mechanism. Higher expression of the αvβ3 integrin on Ly‐6Chigh monocytes (anti‐αv antibody MFI: 37.9±2.9 Ly‐6Chigh vs. 29.9±7.5 Ly‐6Clow; anti‐β3 antibody: 55.3±4.9 Ly‐6Chigh vs. 12.0±1.8 Ly‐6Clow) suggested that this difference may contribute to the observed preferential MV uptake by Ly‐6Chigh monocytes.ConclusionThese findings demonstrate preferential uptake of MVs by lung‐marginated Ly‐6Chigh monocytes subset under in vitro conditions, and suggest a central role for PS‐αvβ3‐based recognition and/or internalisation of MVs. MV uptake within the pulmonary microvasculature by Ly‐6Chigh monocytes could have significant implications for acute lung injury during endotoxaemia.Support or Funding InformationFunded by the Chelsea and Westminster Health Charity