imaging-neutrophil-migration-mouse-skin-to-investigate-subcellular

Imaging Neutrophil Migration in the Mouse Skin to Investigate Subcellular Membrane Remodeling Under Physiological Conditions

The study of immune cell recruitment and function in tissues has been a very active field over the last two decades. Neutrophils are among the first ...

National Cancer Institute

UNC-Chapel Hill

Neutrophils are the primary immune cells that respond to inflammation and combat microbial transgression. To thrive, the bacteria residing in their mammalian host have to withstand the antibactericidal responses of neutrophils. We report that enterobactin (Ent), a catecholate siderophore expressed by , inhibited PMA-induced generation of reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) in mouse and human neutrophils. Ent also impaired the degranulation of primary granules and inhibited phagocytosis and bactericidal activity of neutrophils, 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 compared with wild-type neutrophils but was 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 antiradical defense system that neutralizes the immune functions of neutrophils.

Bacterial Siderophores Hijack Neutrophil Functions.

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.

The LTB-BLT1 axis regulates the polarized trafficking of chemoattractant GPCRs during neutrophil chemotaxis.

Inflammatory bowel disease: DCs sense LTB to drive T1 and T17 differentiation.

Neutrophils are the most common leukocyte in human blood and are the first cells to respond to injury and infection. Improper neutrophil chemotaxis can have deleterious effects on human health, including autoimmune diseases, poor innate immune response, and cancer. Therefore, gaining a better understanding of the signaling pathways governing chemotactic responses in these cells is important. One of the main challenges of working with primary human neutrophils is their short lifespan (about 1 day), making genetic manipulations not feasible. PLB-985 cells, which are pluripotent hematopoietic cells that can easily be differentiated to neutrophil-like cells, are amenable to genetic manipulations, including the expression of fluorescently tagged proteins-of-interest (POI) and gene editing using the CRISPR/CAS9 system to delete genes-of-interest (GOI). The use of PLB-985 cells can therefore greatly facilitate our understanding of the molecular mechanisms governing neutrophil biology during chemotaxis and serve as a good system to complement results gained from pharmacological inhibition of primary neutrophils. To better study the role and localization of proteins during chemotaxis, the underagarose assay has become a widely used and quantitative assay for measuring several aspects of chemotaxis. The objective of this chapter is to provide protocols for (1) the generation of genetically altered PLB-985 cell lines, (2) the set-up of an underagarose chemotaxis assay, and (3) the analysis of cell movement in chemotactic gradients from an underagarose experiment.

Genetic manipulation of PLB-985 cells and quantification of chemotaxis using the underagarose assay.

Normal function of the adaptive immune system requires trafficking of T cells between the blood and lymphoid organs. Lymphocyte homing to lymph nodes requires that they cross endothelial barriers present in blood vessels and lymphatics. This multi-step process requires a remodeling of the lymphocyte plasma membrane, which is mediated by the dynamic re-arrangement of the actin cytoskeleton. Pak1 plays a central role in cell morphology, adhesion and migration in various cell types. Here we demonstrate that Pak1 is required for activated CD4 T cell trafficking to lymph nodes. Pak1 deficiency in T cells causes a defect in the transcription of CCR7 and L-selectin, thereby altering lymphocyte trafficking. Additionally, we report an increase in L-selectin shedding in Pak1-deficient T cells, which correlates with a decrease in the recruitment of calmodulin to the cytoplasmic tail of L-selectin during T cell activation. Overall, our findings demonstrate that by regulating the expression of two major lymph node homing molecules, L-selectin and CCR7, Pak1 mediates activated CD4 T cell trafficking.

Pak1 Kinase Promotes Activated T Cell Trafficking by Regulating the Expression of L-Selectin and CCR7.

The eicosanoid leukotriene B4 (LTB4) relays chemotactic signals to direct neutrophil migration to inflamed sites through its receptor BLT1. However, the mechanisms by which the LTB4-BLT1 axis relays chemotactic signals during intravascular neutrophil response to inflammation remain unclear. Here, we report that LTB4 produced by neutrophils acts as an autocrine/paracrine signal to direct the vascular recruitment, arrest, and extravasation of neutrophils in a sterile inflammation model in the mouse footpad. Using intravital subcellular microscopy, we reveal that LTB4 elicits sustained cell polarization and adhesion responses during neutrophil arrest in vivo. Specifically, LTB4 signaling coordinates the dynamic redistribution of non-muscle myosin IIA and β2-integrin, which facilitate neutrophil arrest and extravasation. Notably, we also found that neutrophils shed extracellular vesicles in the vascular lumen and that inhibition of extracellular vesicle release blocks LTB4-mediated autocrine/paracrine signaling required for neutrophil arrest and extravasation. Overall, we uncover a novel complementary mechanism by which LTB4 relays extravasation signals in neutrophils during early inflammation response.

Article	Year
Bacterial Siderophores Hijack Neutrophil Functions. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 28432145	2017
The LTB-BLT1 axis regulates the polarized trafficking of chemoattractant GPCRs during neutrophil chemotaxis. Journal of cell science\| PubMed ID: 30158177	2018
Inflammatory bowel disease: DCs sense LTB to drive T1 and T17 differentiation. Cellular & molecular immunology\| PubMed ID: 30275533	2020
Genetic manipulation of PLB-985 cells and quantification of chemotaxis using the underagarose assay. Methods in cell biology\| PubMed ID: 30616826	2019
Pak1 Kinase Promotes Activated T Cell Trafficking by Regulating the Expression of L-Selectin and CCR7. Frontiers in immunology\| PubMed ID: 30891040	2019
The LTB4-BLT1 axis regulates actomyosin and β2-integrin dynamics during neutrophil extravasation. The Journal of cell biology\| PubMed ID: 32854115	2020

Bhagawat Subramanian

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