Technical Advance: Changes in neutrophil migration patterns upon contact with platelets in a microfluidic assay

Abstract

Neutrophils are traditionally regarded as the "first responders" of the immune system. However, recent observations revealed that platelets often respond earlier to recruit and activate neutrophils within sites of injury and inflammation. Currently, platelet-neutrophil interactions are studied by intravital microscopy. Although such studies provide exceptional, physiologic in vivo data, they are also laborious and have low throughput. To accelerate platelet-neutrophil interaction studies, we have developed and optimized an ex vivo microfluidic platform with which the interactions between platelets and moving neutrophils are measured at single-cell level in precise conditions and with high throughput. With the use of this new assay, we have evaluated changes in neutrophil motility upon direct contact with platelets. Motility changes include longer distances traveled, frequent changes in direction, and faster neutrophil velocities compared with a standard motility response to chemoattractant fMLP. We also found that the neutrophil-platelet direct interactions are transient and mediated by CD62P-CD162 interactions, localized predominantly at the uropod of moving neutrophils. This "crawling," oscillatory neutrophil behavior upon platelet contact is consistent with previous in vivo studies and validates the use of this new test for the exploration of this interactive relationship.

Diagnosis of sepsis from a drop of blood by measurement of spontaneous neutrophil motility in a microfluidic assay

“…Platelet activation has also been widely reported in sepsis 25–27 and has been suggested as a candidate rapid-response element during disease 28 . A previous study by some of the authors of this work demonstrated that activated platelets could induce spontaneous neutrophil motility and oscillatory motility patterns 29 . While no single factor has yet been identified that can accurately stratify sepsis from non-sepsis, in combination, these responses likely contribute to the specific neutrophil spontaneous motility signatures that we capture using the whole blood assay presented here.…”

Regulation of Innate Immune Responses by Platelets

“…In platelet-depleted mice, or when the PSGL-1 interaction with its receptor on neutrophils was blocked, the neutrophil typical crawling was suppressed (42). These results were further validated by a novel ex vivo microfluidic system that allowed a better understanding of the interaction between these two cells types and the importance of the P-selectin and PSGL-1 (P-selectin glycoprotein ligand-1) in that context (48). Curiously, this same interaction also leads to the generation of neutrophil-derived vesicles filled with arachidonic acid, which are promptly internalized by platelets via Mac-1.…”

Diverse signalling of the platelet P2Y1 receptor leads to a dichotomy in platelet function

“…We used fMLP as a platelet chemoattractant here because it has been reported to induce robust platelet chemotaxis and is pertinent to host defence being a constituent of bacterial cell walls, but has not been described to induce platelet aggregation (Czapiga et al, 2005). We have also investigated PINC, akin to in vivo neutrophil intravascular crawling, that has previously been optimized in vitro to investigate interactions between platelets and neutrophils at the single cell level under precise conditions that are platelet P-selectin dependent (Bengtsson et al, 1999;Kornerup et al, 2010;Amison et al, 2015), and using microfluidic assays (Frydman et al, 2017;Rainger et al, 1997). Here, we used MDC as the chemotactic substance, because it has been shown to activate platelets via the activation of CCR4 receptors, and is highly expressed during LPS induced neutrophilic inflammation with no associated thrombosis or changes to haemostasis (Kornerup et al, 2010;Amison et al, 2017b;Clemetson et al, 2000).…”