Page 99 - Haematologica May 2020

Platelet vesicles and monocyte interaction
platelets and the substrate.15,53 However, these bonds also exhibit high off rates. Thus, under conditions of shear stress (i.e. blood flow) the rapid formation and dissolution of bonds supports rolling adhesion.15,53 We observed that monocytes bearing GPIbα also rolled on purified VWF. However, on EC bearing VWF, monocytes were rapidly activated, which is consistent with our previous observa- tions on the activity of EC derived stimuli such as C-C chemokine ligand 2 (CCL2).15
notypical changes induced by binding and internalisation of PEV by monocytes. In this study we did not observe major changes in integrin expression as a marker of activa- tion. However, we do not exclude changes in monocyte activation and/or function relevant to vascular disease over longer periods of interaction.
The paradigm discussed above may provide a novel thrombo-inflammatory mechanism for the continuous low levels of monocyte delivery in chronic inflammatory conditions such as atherosclerosis. However, our Golden Hour data suggest that acquisition of this pathway of monocyte recruitment could also lead to the clearance of monocytes from the blood during acute and severe trau- ma. Indeed, in this injured patient cohort, PEV counts increase in the circulation57 and monocytes rapidly acquired CD41-derived from PEV (1 hour after trauma) which we believe led to their clearance from the circula- tion, as frequency of CD41+ monocyte numbers are lower 4 hours after trauma. This may be due to clearance by the reticulo-EC system, or alternatively by the expedited recruitment to damaged and inflamed tissues, or indeed a combination of both. Whatever the pathway of their removal from the circulation, we speculate that the rapid clearance of immune cells from the circulation may exac- erbate cell turnover and result in immune suppression and the increased risk of septic complications. In addition, monocytes bearing pro-coagulant PEV could also con- tribute to the initiation and propagation of disseminated intravascular coagulation (DIC) which is a potential and serious complication of traumatic injury.27
In conclusion, we believe that this new paradigm for leukocyte recruitment is an important step in understand- ing the contribution of platelets to thrombo-inflammatory pathology. By acquiring GPIbα in the circulation, mono- cytes may be provided with a means of interacting with the vessel wall, which is ordinarily restricted to platelets during haemostasis. In chronic diseases such as athero- sclerosis, this process may occur with a low frequency over protracted periods of time. Nevertheless, the dynam- ic nature of PEV-monocyte interaction demonstrated in this study implies that such routes of thrombo-inflamma- tion may be major contributors to pathology.
Funding
This work was supported by a British Heart Foundation (BHF) programme grant (RG/12/7/29693 to GER), a BHF Chair (CH/03/003 to SPW), a Royal Society Dorothy Hodgkin research fellowship (DH160044 to MC) and a BHF studentship (FS/14/42/30956, GER). This work was also supported by grants from the NIHR Surgical Reconstruction Microbiology Research Centre (SRMRC) (JL, JH) and the Scar Free founda- tion (JL, PH). The NIHR-SRMRC is a partnership between University Hospitals Birmingham NHS Foundation Trust, the University of Birmingham and the Royal Centre for Defence Medicine. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. AB is supported by British Heart Foundation Senior Basic Science Research Fellowship (FS/19/30/34173). This work was also supported by a BHF Accelerator Award (AA/18/2/34218).
Trogocytosis is the phenomenon by which lymphocytes extract surface molecules from antigen presenting cells through transfer of plasma membrane at the immunologi- cal synapse.54 This process has been observed for T, B and NK cells and neutrophils54,55 and is a fast and efficient means of transferring molecules involved in the regulation of immune functions.54 We cannot completely exclude that monocytes in whole blood do not bind whole platelets and acquire GPIbα and CD41 via a trogocytosis like process, although a synapse like structure has not been reported in this context to our knowledge. However, both trogocytosis and PEV accumulation by monocytes require activation dependent cytoskeletal rearrangement to achieve the trans- fer of membrane cargo that alters the function of the recip- ient cells. Thus, the processes may not be unrelated in their mechanisms of initiation and progression. However, trogo- cytosis does appear to be specific to the immunological synapse or related structures.54,56 Here however, we have shown that purified labelled PEV bind to isolated mono- cytes or monocytes in whole blood with the same dynam- ics as agonist stimulated systems. This clearly demon- strates that a trogocytosis like process is not required for accumulation of PEV once they have been generated by platelet activation (Figure 3). Our colleagues have also shown that PEV levels increase dramatically after trauma and thus are likely to be the source of GPIbα found on leukocytes in trauma patients.57
Using intravital microscopy we observed GPIbα-depen- dent recruitment of PEV-treated monocytes to the vascu- lature. Interestingly, the short-lived adhesive interactions, here termed ‘transient rolling’ which did not result in pro- longed monocyte localisation and activation at the vessel wall, have previously been shown to have physiological roles. Thus, under steady-state conditions (non-inflamed), circulating platelets expressing GPIbα are able to interact transiently with sinusoidal Kupffer cells in the liver via sur- face-expressed VWF, interactions which are important for host defence, as they facilitate uptake and disposal of bac- teria by liver resident macrophages (Kupffer cells).58 In the context of CVD, we showed that induction of pulmonary inflammation with pollution nanoparticles, a known risk factor for thrombo-inflammatory disease associated with atherosclerosis,59 induced the formation of circulating monocyte-PEV aggregates. Moreover, such aggregates showed a significantly enhanced capacity to bind to the artery wall in the ApoE-/- mouse after induction of disease by feeding a high fat western diet. Thus, we propose that the transfer of platelet cargo to monocytes by PEV can contribute to the progression of plaque formation by pro- moting the recruitment of inflammatory monocytes. It would be interesting to investigate the functional and phe-
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