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Inflammatory diseases are often characterised by excessive neutrophil infiltration from the blood stream to the site of inflammation, which damages healthy tissue and prevents resolution of inflammation. Development of anti-inflammatory drugs is hindered by lack of in vitro and in vivo models which accurately represent the disease microenvironment. In this study, we used the OrganoPlate to develop a humanized 3D in vitro inflammation-on-a-chip model to recapitulate neutrophil transmigration across the endothelium and subsequent migration through the extracellular matrix (ECM). Human umbilical vein endothelial cells formed confluent vessels against collagen I and geltrex mix, a mix of basement membrane extract and collagen I. TNF-α-stimulation of vessels upregulated inflammatory cytokine expression and promoted neutrophil transmigration. Intriguingly, major differences were found depending on the composition of the ECM. Neutrophils transmigrated in higher number and further in geltrex mix than collagen I, and did not require an N-formyl-methionyl-leucyl-phenylalanine (fMLP) gradient for transmigration. Inhibition of neutrophil proteases inhibited neutrophil transmigration on geltrex mix, but not collagen I. These findings highlight the important role of the ECM in determining cell phenotype and response to inhibitors. Future work could adapt the ECM composition for individual diseases, producing accurate models for drug development., (© 2022. The Author(s).)

Formyl peptide receptor 2 (FPR2) is a G protein-coupled pattern recognition receptor sensing both mitochondrial- and bacterial-derived formylated peptides, including the PSMα toxins secreted by community-associated methicillin-resistant Staphylococcus aureus strains. Similar to many other FPR2 agonistic peptides, nanomolar concentrations of both PSMα2 and PSMα3 activate neutrophils to increase the cytosolic concentration of Ca 2+ and release NADPH oxidase-derived reactive oxygen species. In addition, the PSMα peptides induce FPR2 homologous desensitization, actin polymerization, and neutrophil reactivation through a receptor cross-talk mechanism. However, in contrast to conventional FPR2 agonistic peptides, including the host-derived formyl peptide MCT-ND4, we found that the PSMα peptides lacked the ability to recruit β-arrestin and induce neutrophil chemotaxis, supporting the previous notion that β-arrestin translocation is of importance for cell migration. Despite the lack of β-arrestin recruitment, the PSMα peptides induced an FPR2-dependent ERK1/2 phosphorylation and internalization. Furthermore, structure-activity relationship analysis with PSMα2 derivatives revealed critical roles of the first 3 aa linked to N-fMet as well as the C terminus of PSMα2 in promoting FPR2 to recruit β-arrestin. In summary, our data demonstrate a novel neutrophil activation pattern upon FPR2 sensing of PSMα peptides, signified by the ability to induce increased intracellular Ca 2+ , ERK1/2 phosphorylation, internalization, and NADPH oxidase activity, yet lack of β-arrestin recruitment and neutrophil chemoattraction. These novel features adopted by the PSMα peptides could be of importance for S. aureus virulence and might facilitate identification of new therapeutic strategies for treating S. aureus infections., (Copyright © 2019 by The American Association of Immunologists, Inc.)

FPR2, a member of the family of G protein-coupled receptors (GPCRs), mediates neutrophil migration, a response that has been linked to β-arrestin recruitment. β-Arrestin regulates GPCR endocytosis and can also elicit non-canonical receptor signaling. To determine the poorly understood role of β-arrestin in FPR2 endocytosis and in NADPH-oxidase activation in neutrophils, Barbadin was used as a research tool in this study. Barbadin has been shown to bind the clathrin adaptor protein (AP2) and thereby prevent β-arrestin/AP2 interaction and β-arrestin-mediated GPCR endocytosis. In agreement with this, AP2/β-arrestin interaction induced by an FPR2-specific agonist was inhibited by Barbadin. Unexpectedly, however, Barbadin did not inhibit FPR2 endocytosis, indicating that a mechanism independent of β-arrestin/AP2 interaction may sustain FPR2 endocytosis. This was confirmed by the fact, that FPR2 also underwent agonist-promoted endocytosis in β-arrestin deficient cells, albeit at a diminished level as compared to wild type cells. Dissection of the Barbadin effects on FPR2-mediated neutrophil functions including NADPH-oxidase activation mediated release of reactive oxygen species (ROS) and chemotaxis revealed that Barbadin had no effect on chemotactic migration whereas the release of ROS was potentiated/primed. The effect of Barbadin on ROS production was reversible, independent of β-arrestin recruitment, and similar to that induced by latrunculin A. Taken together, our data demonstrate that endocytic uptake of FPR2 occurs independently of β-arrestin, while Barbadin selectively augments FPR2-mediated ROS production independently of receptor endocytosis. Given that Barbadin binds to AP2 and prevents the AP2/β-arrestin interaction, our results indicate a role for AP2 in FPR2-mediated ROS release from neutrophils. • Barbadin inhibits AP2/β-arrestin interaction induced by an FPR2-specific agonist. • Barbadin does not inhibit FPR2 endocytosis. • Barbadin augments FPR2-mediated ROS production. [ABSTRACT FROM AUTHOR]