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

Abstract

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 β2 integrin ligands, ICAM-1 and fibrinogen or the β1/β2 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.

Cytohesin-2/ARNO: A Novel Bridge Between Cell Migration and Immunoregulation in Synovial Fibroblasts

“…Moreover, ARNO -/mice showed a reduced eosinophilic inflammation and IL-5 expression in an OVA-induced model of rhinitis (54). Finally, ARF6 regulates production of superoxide in neutrophils, as well as degranulation and their chemotaxis, polarization and adhesion (55)(56)(57). Nevertheless, these observations are still rare compared to those relating to its regulation of cytoskeletal functions, particularly protein trafficking, cell migration or recycling of membrane components and thus, we are only starting to understand how ARNO modulates inflammation in different cell types, including immune and non-immune cells.…”

Pathways of integrins in the endo-lysosomal system

“…ARF6-mediated integrin recycling can affect migration capacity of neutrophil granulocytes as well, which may lead to immunodeficiency disorders characterized by returning infections (Gamara et al, 2020). Additionally, α5β1 integrins play a role in lamellipodia formation of macrophages and hence regulates their migration capacities (Veale et al, 2010).…”

Decoding the signaling profile of hematopoietic progenitor kinase 1 (HPK1) in innate immunity: A proteomic approach

“…Proteins involved in β 2 integrin signaling including DAP12, Syk, Rac1, and ARHGEF6 were no longer part of the HPK1 interacting network. The only protein in activated, adherent dHL‐60 HPK1‐EGFP cells with a reported function for neutrophil activation was ADP‐ribosylation factor 6 (Arf6, gene ARF6 ) [26]. Arf6 forms cluster 3 in Mn 2+ ‐activated, adherent dHL‐60 HPK1‐EGFP cells together with CD157, uPAR, and integrin α5.…”

“…Surprisingly, the majority of the identified interacting proteins of HPK1 in neutrophils were not previously described as HPK1 interactors in T and B cells which may explain the different functions of HPK1 in neutrophils versus T and B cells [10,15]. Some of the proteins found to interact with HPK1 in neutrophils, such as CD157, platelet endothelial cell adhesion molecule 1 (PECAM-1), uPAR, and Arf6, have already been reported to regulate neutrophil adhesion, spreading, chemotactic migration, and extravasation as well as postadhesion functions [23,25,26,41]. Recently, HPK1 has emerged as potent target for cancer therapy [42,43].…”