Modulation of Receptor Signaling and Functional Selectivity in Neutrophils

Abstract

Neutrophils are important effector cells of the innate immune system and in the regulation of inflammation. Many of their functions, such as chemotactic migration, secretion of granule constituents and activation of the oxygen radical-producing NADPH-oxidase, are regulated by cell surface receptors. The formyl peptide receptors (FPRs), the ATP receptor (P2Y2R) and the receptor for platelet activating factor (PAFR) belong to the large family of G-protein coupled receptors (GPCRs) and, amongst other receptors, enable neutrophils to sense and respond to host- and pathogen-derived danger signals. Therefore, any regulatory imbalance in GPCR signaling can potentially contribute to the development of severe infections or autoimmune/inflammatory diseases. The work presented in this thesis is focused on basic GPCR-signaling mechanisms in human neutrophils with the aim to generate new knowledge that could be of value for future GPCR-based drug development. To answer the scientific questions raised, numerous cell-biology-based experimental methods were applied, including measurements of neutrophil intracellular calcium release, superoxide production, degranulation, cell migration and cytoskeleton-mediated receptor regulation. The functional responses triggered by GPCRs expressed by neutrophils can be modulated in various ways at the level of receptors/ligand interaction, in dependence of other GPCRs, as well as at the signaling level. Both FPR2 and P2Y2R have been shown to be able to exert functional selective signaling through distinct regulatory mechanisms. An FPR2-specific synthetic lipopeptide allosteric modulator was identified as a biased agonist that does not induce recruitment of β-arrestin or chemotactic migration and exhibits oppositional efficacies for direct FPR2 activation and receptor cross-talk-mediated signaling. Functional selectivity liked to the P2Y2R is not related to biased agonism but instead emerges from an endogenous actin cytoskeleton-dependent regulatory mechanism which selectively inhibits the signals that lead to the generation of oxygen radicals, while leaving other signaling pathways unaffected. In conclusion, this thesis adds new knowledge to the field of neutrophil receptor biology and provides novel insights into the modulation of basic GPCR signaling mechanisms with intend to contribute to strategies for future drug design and treatment of inflammatory disorders and disease.