Neutrophil AKT2 regulates heterotypic cell-cell interactions during vascular inflammation

Abstract

Interactions between platelets, leukocytes, and activated endothelial cells are important during microvascular occlusion; however, the regulatory mechanisms of these heterotypic cell-cell interactions remain unclear. Here, using intravital microscopy to evaluate mice lacking specific isoforms of the serine/threonine kinase AKT and bone marrow chimeras, we found that hematopoietic cell-associated AKT2 is important for neutrophil adhesion and crawling and neutrophil-platelet interactions on activated endothelial cells during TNF-α-induced venular inflammation. Studies with an AKT2-specific inhibitor and cells isolated from WT and Akt KO mice revealed that platelet-and neutrophil-associated AKT2 regulates heterotypic neutrophil-platelet aggregation under shear conditions. In particular, neutrophil AKT2 was critical for membrane translocation of αMβ2 integrin, β2-talin1 interaction, and intracellular Ca 2+ mobilization. We found that the basal phosphorylation levels of AKT isoforms were markedly increased in neutrophils and platelets isolated from patients with sickle cell disease (SCD), an inherited hematological disorder associated with vascular inflammation and occlusion. AKT2 inhibition reduced heterotypic aggregation of neutrophils and platelets isolated from SCD patients and diminished neutrophil adhesion and neutrophil-platelet aggregation in SCD mice, thereby improving blood flow rates. Our results provide evidence that neutrophil AKT2 regulates αMβ2 integrin function and suggest that AKT2 is important for neutrophil recruitment and neutrophil-platelet interactions under thromboinflammatory conditions such as SCD.

Quantitative microfluidic fluorescence microscopy to study vaso-occlusion in sickle cell disease

“…The crawling of neutrophils followed by nucleation of platelets, shown sequentially in the Online Supplementary Movie S5, is similar to observations reported in mice in vivo. 3,4,14 We observed that platelet nucleation on arrested neutrophils in SS blood led to the formation of aggregates which partially occluded the microfluidic channels (Online Supplementary Figure S4). As shown previously in SCD mice in vivo, 3 RBCs were found to be trapped in these aggregates (Online Supplementary Figure S4D-F).…”

“…The capture of activated platelets by adherent neutrophils is believed to play a role in the onset of vasoocclusion 3,4 in the venules of SCD mice. Using the twostep imaging strategy shown in Figure 1B,C, neutrophils were observed to arrest and then crawl in P-selectin, ICAM-1 and IL-8 coated microfluidic channels ( Figure 3A) which enabled nucleation of platelets on top of crawling neutrophils ( Figure 3B).…”

“…1 In vivo imaging in transgenic SCD mice has identified molecular events that may promote vasoocclusion. [2][3][4] However, the relevance of these mechanisms is not completely understood in humans. As non-invasive in vivo imaging in humans is limited by low-resolution, [5][6][7] there is a need for in vitro approaches 8 that can allow visualization of single cell events in flowing human blood.…”

ARQ 092, an orally-available, selective AKT inhibitor, attenuates neutrophil-platelet interactions in sickle cell disease

“…[9][10][11][12] Among several receptors and counter-receptors, the neutrophil-platelet association is primarily mediated by the interaction of neutrophil P-selectin glycoprotein ligand-1 (PSGL-1) and αMb2 integrin with platelet Pselectin and glycoprotein Ibα (GPIbα), respectively. 13 We have shown that AKT2 positively regulates the function of αMb2 integrin and P-selectin during vascular inflammation 12 and that combining hydroxyurea with AKT2 inhibition has immediate benefits in acute vaso-occlusive events and improves survival in SCD mice. 9 Although these results suggest that AKT2 inhibition may be a supplemental therapy for SCD patients with vaso-occlusive crises, no AKT2-specific inhibitor is currently available in the clinic.…”

“…[17][18][19] In neutrophils, which express AKT1 and AKT2, only AKT2 regulates cell migration, NADPH oxidase 2 activation, b2 integrin function, and neutrophil-platelet interactions under inflammatory conditions. 12,20 As a major isoform in endothelial cells, AKT1 modulates the activity of endothelial NO synthase and is involved in angiogenesis, acute inflammation, and atherosclerosis. [21][22][23] Human AKT isoforms share around 98% sequence homology with mouse proteins.…”