Defective Neutrophil Transendothelial Migration and Lateral Motility in ARPC1B Deficiency Under Flow Conditions

Abstract

The actin-related protein (ARP) 2/3 complex, essential for organizing and nucleating branched actin filaments, is required for several cellular immune processes, including cell migration and granule exocytosis. Recently, genetic defects in ARPC1B, a subunit of this complex, were reported. Mutations in ARPC1B result in defective ARP2/3-dependent actin filament branching, leading to a combined immunodeficiency with severe inflammation. In vitro, neutrophils of these patients showed defects in actin polymerization and chemotaxis, whereas adhesion was not altered under static conditions. Here we show that under physiological flow conditions human ARPC1B-deficient neutrophils were able to transmigrate through TNF-α-pre-activated endothelial cells with a decreased efficiency and, once transmigrated, showed definite impairment in subendothelial crawling. Furthermore, severe locomotion and migration defects were observed in a 3D collagen matrix and a perfusable vessel-on-a-chip model. These data illustrate that neutrophils employ ARP2/3-independent steps of adhesion strengthening for transmigration but rely on ARP2/3-dependent modes of migration in a more complex multidimensional environment.

Keywords: ARP2/3 complex; ARPC1B deficiency; inborn error of immunity; neutrophil; neutrophil transmigration; primary immunodeficiency; vessel-on-a-chip.

Figure 1

ARPC1B-deficient neutrophils display impaired subendothelial motility upon transendothelial migration. (A) Neutrophil TEM through TNF-α inflamed HUVECs was investigated upon physiological flow conditions. Neutrophils (green = control; red = patient) rolled over the endothelium, whereupon they firmly adhered (left panel) and transmigrated (right panel). Neutrophils are circular above the endothelium (left panel, arrow) and become polarized (right panel, arrow) under the endothelium. Representative stills are displayed, see also Supplementary Video 1 . Scale bar = 100 µm. (B) The number of firmly adhered ARPC1B-deficient neutrophils was quantified and normalized to control neutrophils (mean ± SD, n = 3). (C) Average time of neutrophils to complete transendothelial migration, starting from firm adherence. Individual cells are depicted. Colors (black, green and red) are corresponding to independent experiments. (D) Transendothelial migratory events were quantified and normalized to the number of firmly adhered neutrophils (mean ± SD, n = 3). (E–G) Subendothelial motility of transmigrated neutrophils, with (E) average velocity of neutrophils (n = 3, individual cells are depicted, colors are corresponding to independent experiments) and cell track analysis of subendothelial neutrophils with (F) representative cell trajectories of control and ARPC1B-deficient neutrophils as indicated lasting for 45 minutes (scale bar = 70 µm) and (G) showing trajectory plots displayed with their origins brought to a common point. Scale bar = 50 µm. Results are representative of 3 independent experiments. The Student t test was used to test statistical significance (*p < 0.05; **p < 0.01; ns, non-significant).

Figure 2

Neutrophil infiltration into 3D tissue matrices is defective in ARPC1B deficiency. (A) Motility tracks of neutrophils (green = control; red = patient) in a collagen-I 3D matrix upon C5a stimulation, see also Supplementary Video 2 . Only control neutrophils show motility tracks as ARPC1B-deficient neutrophils were found to be non-motile. Scale bar = 100 µm. Heat bar = time in minutes. Results are representative of 3 independent experiments. (B) Migration speed of neutrophils in collagen matrix (cells of 3 experiments pooled). (C) Representative images of neutrophil TEM in a perfusable vessel-on-a-chip, see also Supplementary Videos 3 , 4 . Scale bar = 100 µm. Heat bar = distance starting from vessel surface. Results are representative of 6 vessels, 2 fields of view per vessel were analyzed. (D–F) Quantification of neutrophil TEM using vessel-on-a-chip model, with (D) number of neutrophils retrieved in the vessel, (E) number of intraluminal neutrophils (normalized to total number of neutrophils), (F) number of neutrophils infiltrated into subendothelial collagen matrix (normalized to total number of neutrophils), (G) and average migration distance of neutrophils into the vessel. Mean ± SD, results are representative of 6 vessels, 2 fields of view per vessel were analyzed. The Student t test was used to test statistical significance (**p < 0.01; ***p < 0.001; ns, non-significant).