Cross-talk between Rho GTPases and PI3K in the neutrophil

Abstract

Neutrophils are short-lived, abundant peripheral blood leukocytes that provide a first line of defense against bacterial and fungal infections while also being a key part of the inflammatory response. Chemokines induce neutrophil recruitment to inflammatory sites, where neutrophils perform several diverse functions that are aimed at fighting infections. Neutrophil effector functions are tightly regulated processes that are governed by an array of intracellular signaling pathways and initiated by receptor-ligand binding events. Dysregulated neutrophil activation can result in excessive inflammation and host damage, as is evident in several autoimmune diseases. Rho family small GTPases and agonist-activated phosphoinositide 3-kinases (PI3Ks) represent 2 classes of key regulators of the highly specialized neutrophil. Here we review cross-talk between these important signaling intermediates in the context of neutrophil functions. We include PI3K-dependent activation of Rho family small GTPases and of their guanine nucleotide exchange factors and GTPase activating proteins, as well as Rho GTPase-dependent regulation of PI3K.

Keywords: Cdc42; FcγR; GPCR; NADPH oxidase; PI3K; PtdIns(3,4,5)P; Rac; RhoA; chemotaxis; neutrophil; phagocytosis; polarization.

Figure 1.

Neutrophil function involves numerous processes that are regulated by Rho GTPases. Circulating neutrophils (center) leave the circulation and chemotax along gradients of chemokines/chemoattractants to reach inflammatory sites (top left). In contrast to the round, circulating cells, chemotaxing neutrophils are polarized and characterized by a leading edge with actin-rich lamellipodium (indicated here by a zigzag line) and a trailing end. As professional phagocytes, neutrophils recognize and engulf small pathogens (e.g., bacteria and yeast; left). Phagocytosis involves the formation of a phagocytic cup which closes around the particle, forming the phagosome. Once engulfed, pathogens are killed intracellularly, in a process that uses ROS, antimicrobial peptides and proteases. Killing depends on 2 distinct processes, the assembly and activation of the NADPH oxidase (bottom left), as well as degranulation (bottom right). The NADPH oxidase catalyzes the generation of oxygen radicals, while degranulation ensures delivery of enzymes required for their conversion to biocidal ROS [in particular myeloperoxidase (MPO), a component of primary/azurophil granules; shown here in yellow]. Secondary/specific granules (shown in orange) deliver antimicrobial peptides and proteases into the phagosomes, which also contribute to intracellular killing. Under certain conditions killing occurs extracellularly, for example when pathogens are too large to be engulfed (e.g., parasites or fungal hyphae) or in conditions of sepsis, neutrophils release NETs (right). NETs consist of decondensed chromatin and antimicrobial proteins, and trap and kill pathogens. At the end of their short lives, neutrophils undergo apoptosis (top right). To limit the inflammation generated, they display ‘eat-me’ signals, thus triggering their own uptake by pro-resolution macrophages in a process termed efferocytosis.

Figure 2.

PI3Kβ is activated by phosphopeptide, Gβγ and Rac/Cdc42 in the integrin/immune complex-stimulated neutrophil. (Left) Neutrophil integrin or FcγR ligation causes Src family kinase (SFK)-dependent phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs), triggering the activation of Syk kinase, which in turn recruits the p85 adaptor in an SH2 domain- and phosphotyrosine motif-dependent fashion to activate PI3Kβ. Integrin/FcγR ligation and the PIP₃ also activate Rac/Cdc42 GEFs. (Right) In a paracrine feed-forward loop, PI3Kβ drives LTB4 production. LTB4 triggers activation of its GPCR (BLT1), resulting in release of Gβγ subunits activate PI3Kβ by binding to p110β. GPCR ligation and PIP₃ also activate Rac/Cdc42 GEFs. (Center panel) Rac/Cdc42 GEFs enable GTP-loading of Rac/Cdc42, which can then bind the p110β RBD, to further activate PI3Kβ. Full activation of PI3Kβ requires phosphotyrosine motifs, Gβγ and Rac/Cdc42.