Real-time deformability cytometry reveals sequential contraction and expansion during neutrophil priming

Abstract

It has become increasingly apparent that the biomechanical properties of neutrophils impact on their trafficking through the circulation and in particularly through the pulmonary capillary bed. The retention of polarized or shape‐changed neutrophils in the lungs was recently proposed to contribute to acute respiratory distress syndrome pathogenesis. Accordingly, this study tested the hypothesis that neutrophil priming is coupled to morpho‐rheological (MORE) changes capable of altering cell function. We employ real‐time deformability cytometry (RT‐DC), a recently developed, rapid, and sensitive way to assess the distribution of size, shape, and deformability of thousands of cells within seconds. During RT‐DC analysis, neutrophils can be easily identified within anticoagulated “whole blood” due to their unique granularity and size, thus avoiding the need for further isolation techniques, which affect biomechanical cell properties. Hence, RT‐DC is uniquely suited to describe the kinetics of MORE cell changes. We reveal that, following activation or priming, neutrophils undergo a short period of cell shrinking and stiffening, followed by a phase of cell expansion and softening. In some contexts, neutrophils ultimately recover their un‐primed mechanical phenotype. The mechanism(s) underlying changes in human neutrophil size are shown to be Na+/H+ antiport‐dependent and are predicted to have profound implications for neutrophil movement through the vascular system in health and disease.

The mechanics of myeloid cells

“…RT‐DC revealed that neutrophil priming via several different stimuli induces a common series of phenotypic changes, with an initial phase of contraction and stiffening lasting 1–5 min, followed by a phase of expansion and increased deformability achieved within 15–30 min post‐priming. Neutrophils simultaneously became shape changed in a matter of seconds but returned to their original morphologic phenotype within 2–5 h [Bashant et al., ]. This finding aligns with previous reports of neutrophil “de‐priming” [Kitchen et al., ; Ekpenyong et al., ].…”

“…This finding aligns with previous reports of neutrophil “de‐priming” [Kitchen et al., ; Ekpenyong et al., ]. Thus, high‐throughput DC demonstrated primed neutrophils could be stiffer or softer depending on the time frame at which cells were analysed [Bashant et al., ]. Similarly, in the context of macrophages and dendritic cells, some inflammatory mediators have been reported to stiffen cells while others have the opposite effect [Bufi et al., ].…”

“…A second study employing micropipette aspiration demonstrated that stimulation of monocytes with pro‐inflammatory linoleic acid increases monocyte deformability, and the subsequent retention of these cells in the microcapillary bed may drive deformability‐driven increases in the surface area available for adherence to the endothelium [Rinker et al., ]. Interestingly, and like the mechanokinetics of activated neutrophils [Bashant et al., ], it was reported that immortalised monocytes stiffen and then soften when intracellular free calcium levels are increased [Richelme et al., ]. Other studies showed that monocytes stimulated with inflammatory cytokines are more deformable but less compressible, potentially explaining the above contrasting studies, which did not use techniques capable of analysing compressive and shear deformation separately [Ravetto et al., ].…”

“…This microvasculature is a complex network of particularly narrow capillaries [Hogg and Doerschuk, ]. Cytoskeletal stiffening, as it occurs during the first phase of neutrophil priming [Bashant et al., ], promotes the sequestration of neutrophils in the lung pulmonary microvasculature [Yoshida et al., ]. There is both in vitro and in vivo evidence to indicate that if the priming stimulus is removed, primed neutrophils gradually de‐prime, reverting to a quiescent state resembling unprimed neutrophils.…”

TRPV4—A Missing Link Between Mechanosensation and Immunity

“…Since F-actin has been shown to bind to activated TRPV4, it can be speculated that this priming/depriming occurs as a function of TRPV4 activation secondary to the formation of F-actin rims (24). Given the implications of such mechanical effects due to e.g., changes in neutrophil shape and stiffness not only on neutrophil kinetics through the vascular system but also on their biological responsiveness in health and disease (86), the molecular dissection of the underlying signaling pathways and the potential link to TRPV4 mechanosensation may be of considerable scientific interest and relevance. Shear stress in the vasculature is a result of blood flow velocity, vessel diameter, and blood viscosity and primarily acts on endothelial cells outlining the vessel lumen.…”