Neutrophils are the primary immune cells that respond to inflammation and combat microbial transgression. In order to thrive, the bacteria residing in their mammalian host have to withstand the anti-bactericidal responses of neutrophils. We report that enterobactin (Ent), a catecholate siderophore expressed by E. coli, inhibited PMA-induced generation of reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) in both mouse and human neutrophils. Ent also impaired the degranulation of primary granules, inhibited phagocytosis and bactericidal activity of neutrophils, but without affecting their migration and chemotaxis. Molecular analysis revealed that Ent can chelate intracellular labile iron that is required for neutrophil oxidative responses. Other siderophores (pyoverdine, ferrichrome, deferoxamine) likewise inhibited ROS and NETs in neutrophils, thus indicating that the chelation of iron may largely explain their inhibitory effects. To counter iron theft by Ent, neutrophils rely on the siderophore-binding protein lipocalin 2 (Lcn2) in a ‘tug-of-war’ for iron. The inhibition of neutrophil ROS and NETs by Ent was augmented in Lcn2-deficient neutrophils when compared to WT neutrophils, but rescued by the exogenous addition of recombinant Lcn2. Taken together, our findings illustrate the novel concept that microbial siderophore’s iron scavenging property may serve as an anti-radical defense system, that neutralizes immune functions of neutrophils.

Directed leukocyte migration is a hallmark of inflammatory immune responses. Leukotrienes are derived from arachidonic acid and represent a class of potent lipid mediators of leukocyte migration. In this review, we summarize the essential steps leading to the production of LTB in leukocytes. We discuss the recent findings on the exosomal packaging and transport of LTB in the context of chemotactic gradients formation and regulation of leukocyte recruitment. We also discuss the dynamic roles of the LTB receptors, BLT1 and BLT2, in mediating chemotactic signaling in leukocytes and contrast them to other structurally related leukotrienes that bind to distinct GPCRs. Finally, we highlight the specific roles of the LTB-BLT1 axis in mediating signal-relay between chemotaxing neutrophils and its potential contribution to a wide variety of inflammatory conditions including tumor progression and metastasis, where LTB is emerging as a key signaling component.

Neutrophils sense and respond to diverse chemotactic cues through G-protein-coupled receptors (GPCRs). However, the precise trafficking dynamics of chemoattractant GPCRs during neutrophil activation and chemotaxis remain unclear. Here, by using small-molecule inhibitors and CRISPR-based knockouts, we establish that two primary chemoattractant GPCRs - formyl peptide receptor 1 (FPR1) and complement component 5a (C5a) receptor 1 (C5aR1) - internalize in a CDC42-actin-dependent manner. Through live-cell imaging, we demonstrate that, upon stimulation, FPR1 rapidly clusters and re-distributes along the plasma membrane to the trailing edge, where it internalizes and is directionally trafficked towards the front of migrating primary human neutrophils. In contrast to FPR1 and C5aR1, the leukotriene B (LTB) receptor (BLT1, also known as LTB4R), which relays LTB signals in response to primary chemoattractants during neutrophil chemotaxis, fails to internalize upon physiological stimulation with LTB, N-formyl-Met-Leu-Phe (fMLF) or C5a. Importantly, we report that blocking the LTB-BLT1 axis or downstream myosin activation enhances the internalization of FPR1 and C5aR1, thus reducing downstream signaling and impairing chemotaxis to primary chemoattractants. The polarized trafficking of chemoattractant GPCRs and its regulation by the BLT1-mediated myosin activation therefore drives persistent chemotactic signaling in neutrophils.This article has an associated First Person interview with the first author of the paper.