Platelet-derived stimulators of non-canonical neutrophil GPCR signaling in NETosis and thrombosis

Thrombo-occlusive disorders such as acute myocardial infarction, ischemic stroke and venous thromboembolism are still the leading cause for morbidity and mortality worldwide. The development and progression of these disorders is driven by a complex interplay of platelets and immune cells such as particularly neutrophils. The intercellular interaction between platelets and neutrophils is able to induce the formation of Neutrophil Extracellular Traps (NETosis), which represents a sequence of strictly regulated cellular and molecular events culminating in the expulsion of decondensed DNA, thus affecting thrombo-inflammation. While platelet-induced NETosis is mainly mediated by means of adhesion molecules such as P-Selectin and integrins with their corresponding receptors, there is evidence that platelet-induced NETosis is crucially dependent on soluble messengers secreted upon platelet activation. Thereby, the platelet-derived high-mobility Group Protein B1 (Hmgb1) and the von-Willebrand-Factor (vWF) are well established soluble mediators of platelet-induced NETosis. Additionally, platelets secrete a plurality of bioactive signaling lipids such as lysophosphatidylcholine (LPC) and lysophosphatidylinositols (LPI), which mediate their function via the GPCRs GPR132 or GPR55, respectively.
Neutrophil GPCRs detect and respond to inflammatory cues at sites of infection or tissue injury. In canonical GPCR signaling, G protein–coupled receptor kinase 2 (GRK2)-mediated β-arrestin binding leads to receptor desensitization. However, β-arrestin can also function as a scaffold for non-canonical signaling complexes, modulating diverse downstream effectors such as extracellular signal-regulated kinase (ERK), the serine/threonine kinase AKT, and the actin cytoskeleton. Notably, NADPH oxidase (NOX)-dependent generation of reactive oxygen species (ROS) and neutrophil chemotaxis are mediated, in particular, via these non-canonical GRK2/β-arrestin pathways.​  While NOX-dependent ROS generation is directly involved in NET formation, chemotactic neutrophil migration plays a crucial role in thrombo-inflammation and tissue remodeling upon myocardial ischemia-reperfusion (I/R) injury. In this context, at least the production of ROS was affected by GRK2 upon stimulation with GPR84 agonists in an actin cytoskeleton-dependent manner, indicating a possible role of non-canonical GPCR signaling in bioactive lipid-induced neutrophil function. Interestingly, the immunomodulatory lipid messenger sphingosine-1-phosphate (S1P) is secreted upon platelet stimulation (Fig. 1A). Neutrophils primarily express the S1P receptor (S1P-R) isoforms S1P-R1-3, which are GPCRs and seem to be differentially regulated dependent on the neutrophil function. Moreover, in agreement with literature, own preliminary data demonstrated an opposing role of the S1P-R isoforms on NETosis (Fig. 1B). Whereas the supernatant of thrombin-activated platelets induced NET formation, the pharmacological inhibition of S1P-R1 by NIBR0213 and S1P-R2 by JTE-013 on neutrophils diminished platelet-induced NETosis. However, pharmacological inhibition of S1P-R3 by TY-52156 on neutrophils resulted in a markedly increase in NET formation, pinpointing a distinct and differentially regulated GPCR signaling in S1P-induced NETosis. More remarkably, exogenous S1P seems to induce F-actin stabilization in neutrophils (Fig. 1C), a process which is known to be mediated by non-canonical β-arrestin signaling. Moreover, the context-dependent regulation of the S1P-R1 isoforms by means of non-canonical GRK2/β-arrestin-induced ERK signaling in endothelial and tumor cells is well established, but a detailed characterization of non-canonical GRK2/β-arrestin signaling during NETosis and subsequent thrombosis is still missing.

​

The objective of the underlying project is the characterization of β-arrestin and GRK2 signaling in neutrophils and NETosis, especially upon activation with platelet-derived bioactive molecules such as S1P. To this end we will investigate

1.) the differential expression of the S1P-R isoforms in neutrophils and during NETosis, characterize

2.) the molecular state of the S1P-R isoforms in neutrophils upon stimulation with supernatant from activated platelets or exogenous S1P, and perform

3.) functional measurements and in vitro NET assays to unravel the role of GRK2/β-arrestin during S1P-induced NETosis.