Arf6 regulates energy metabolism in neutrophils.

The small GTPase Arf6 regulates many cellular processes, including cytoskeletal remodeling, receptor endocytosis, and pathogen phagocytosis. Arf6 silencing in neutrophil (PMN)-like cells is well-known to inhibit chemotactic peptide-mediated activation of phospholipase D, the oxidative burst, and ß2 integrin-dependent adhesion. In conditional knockout (cKO) mice, the migration to inflammatory sites of Arf6-deficient PMNs was diminished and associated with reduced cell surface expression of ß2 integrins. In this study we assessed the impact of Arf6 depletion on the functions and gene expression profile of PMNs isolated from the mouse air pouch. Numerous genes involved in response to oxygen levels, erythrocyte and myeloid differentiation, macrophage chemotaxis, response to chemicals, apoptosis, RNA destabilization, endosome organization, and vesicle transport were differentially expressed in PMNs cKO for Arf6. Lpar6 and Lacc-1 were the most up-regulated and down-regulated genes, respectively. The deletion of Arf6 also decreased Lacc-1 protein level in PMNs, and silencing of Arf6 in THP-1 monocytic cells delayed LPS-mediated Lacc-1 expression. We report that fMLP or zymosan-induced glycolysis and oxygen consumption rate were both decreased in air pouch PMNs but not in bone marrow PMNs of Arf6 cKO mice. Reduced oxygen consumption correlated with a decrease in superoxide and ROS production. Deletion of Arf6 in PMNs also reduced phagocytosis and interfered with apoptosis. The data suggest that Arf6 regulates energy metabolism, which may contribute to impaired phagocytosis, ROS production, and apoptosis in PMN-Arf6 cKO. This study provides new information on the functions and the inflammatory pathways influenced by Arf6 in PMNs.

Assessment of Arf6 Deletion in PLB-985 Differentiated in Neutrophil-Like Cells and in Mouse Neutrophils: Impact on Adhesion and Migration.

Chemoattractant sensing, adhesiveness, and migration are critical events underlying the recruitment of neutrophils (PMNs) to sites of inflammation or infection. Defects in leukocyte adhesion or migration result in immunodeficiency disorders characterized by recurrent infections. In this study, we evaluated the role of Arf6 on PMN adhesion in vitro and on migration to inflammatory sites using PMN-Arf6 conditional knockout (cKO) mice. In PMN-like PLB-985 silenced for Arf6 fMLP-mediated adhesion to the ß2 integrin ligands, ICAM-1 and fibrinogen or the ß1/ß2 integrin ligand fibronectin was significantly reduced. Furthermore, overexpression of wild-type Arf6 promoted basal and fMLP-induced adhesion to immobilized integrin ligands, while overexpression of the dominant-negative Arf6 has the opposite effects. Using the Elane-Cre deleting mouse strains, we report that the level of Arf6 deletion in inflammatory PMNs isolated from the dorsal air pouches was stronger when compared to naïve cells isolated from the bone marrow. In PMN-Arf6 cKO mice, the recruitment of PMNs into the dorsal air pouch injected with LPS or the chemoattractant fMLP was significantly diminished. Impaired cell migration correlated with reduced cell surface expression of CD11a and CD11b in Arf6 cKO PMNs. Our results highlight that Arf6 regulates the activity and possibly the recycling of PMN integrins, and this compromises PMN migration to inflammatory sites.

Genetic Approaches for Definitive Diagnosis of Agammaglobulinemia in Consanguineous Families.

Autosomal recessive agammaglobulinemia (ARA) is a primary immunodeficiency characterized by absent peripheral B cells, severe hypogammaglobulinemia, and absent BTK gene mutations. In ARA, mutations occur in genes encoding the pre-B cell receptor (pre-BCR) or downstream signaling proteins. In this work, we used candidate gene and whole-exome sequencing to investigate the molecular basis of ARA in 6 patients from 4 consanguineous North-African families. Sanger sequencing of candidate genes encoding the pre-BCR components (ΙGΗΜ, CD79A, CD79B, IGLL1, and VPREB1) was initially performed and determined the genetic defect in five patients. Two novel mutations in IGHM (p.Val378Alafs*1 and p.Ile184Serfs*21) were identified in three patients from two unrelated kindred and a novel nonsense mutation was identified in CD79A (p.Trp66*) in two siblings from a third kindred. Whole-exome sequencing (WES) was performed on the sixth patient who harbored a homozygous stop mutation at position 407 in the RAG2 gene (p.Glu407*). We concluded that conventional gene sequencing, especially when multiple genes are involved in the defect as is the case in ARA, is costly and time-consuming, resulting in delayed diagnosis that contributes to increased morbidity and mortality. In addition, it fails to identify the involvement of novel and unsuspected gene defects when the phenotype of the patients is atypical. WES has the potential to provide a rapid and more accurate genetic diagnosis in ARA, which is crucial for the treatment of the patients.

Regulators and Effectors of Arf GTPases in Neutrophils.

Polymorphonuclear neutrophils (PMNs) are key innate immune cells that represent the first line of defence against infection. They are the first leukocytes to migrate from the blood to injured or infected sites. This process involves molecular mechanisms that coordinate cell polarization, delivery of receptors, and activation of integrins at the leading edge of migrating PMNs. These phagocytes actively engulf microorganisms or form neutrophil extracellular traps (NETs) to trap and kill pathogens with bactericidal compounds. Association of the NADPH oxidase complex at the phagosomal membrane for production of reactive oxygen species (ROS) and delivery of proteolytic enzymes into the phagosome initiate pathogen killing and removal. G protein-dependent signalling pathways tightly control PMN functions. In this review, we will focus on the small monomeric GTPases of the Arf family and their guanine exchange factors (GEFs) and GTPase activating proteins (GAPs) as components of signalling cascades regulating PMN responses. GEFs and GAPs are multidomain proteins that control cellular events in time and space through interaction with other proteins and lipids inside the cells. The number of Arf GAPs identified in PMNs is expanding, and dissecting their functions will provide important insights into the role of these proteins in PMN physiology.