Human neutrophils swim and phagocytize bacteria

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Controlling T cells spreading, mechanics and activation by micropatterning

International audienceAbstract We designed a strategy, based on a careful examination of the activation capabilities of proteins and antibodies used as substrates for adhering T cells, coupled to protein microstamping to control at the same time the position, shape, spreading, mechanics and activation state of T cells. Once adhered on patterns, we examined the capacities of T cells to be activated with soluble anti CD3, in comparison to T cells adhered to a continuously decorated substrate with the same density of ligands. We show that, in our hand, adhering onto an anti CD45 antibody decorated surface was not affecting T cell calcium fluxes, even adhered on variable size micro-patterns. Aside, we analyzed the T cell mechanics, when spread on pattern or not, using Atomic Force Microscopy indentation. By expressing MEGF10 as a non immune adhesion receptor in T cells we measured the very same spreading area on PLL substrates and Young modulus than non modified cells, immobilized on anti CD45 antibodies, while retaining similar activation capabilities using soluble anti CD3 antibodies or through model APC contacts. We propose that our system is a way to test activation or anergy of T cells with defined adhesion and mechanical characteristics, and may allow to dissect fine details of these mechanisms since it allows to observe homogenized populations in standardized T cell activation assays

Synchronizing atomic force microscopy force mode and fluorescence microscopy in real time for immune cell stimulation and activation studies

International audienceA method is presented for combining atomic force microscopy (AFM) force mode and fluorescence microscopy in order to (a) mechanically stimulate immune cells while recording the subsequent activation under the form of calcium pulses, and (b) observe the mechanical response of a cell upon photoactivation of a small G protein, namely Rac. Using commercial setups and a robust signal coupling the fluorescence excitation light and the cantilever bending, the applied force and activation signals were very easily synchronized. This approach allows to control the entire mechanical history of a single cell up to its activation and response down to a few hundreds of milliseconds, and can be extended with very minimal adaptations to other cellular systems where mechanotransduction is studied, using either purely mechanical stimuli or via a surface bound specific ligand

A Bistable Mechanism Mediated by Integrins Controls Mechanotaxis of Leukocytes

International audienceRecruitment of leukocytes from blood vessels to inflamed zones is guided by biochemical and mechanical stimuli, with mechanisms only partially deciphered. Here, we studied the guidance by flow of primary human effector T lymphocytes crawling on substrates coated with ligands of integrins LFA-1 (aLb2) and VLA-4 (a4b1). We reveal that cells segregate in two populations of opposite orientation for combined adhesion, and show that decisions of orientation rely on a bistable mechanism between LFA-1-mediated upstream and VLA-4-mediated downstream phenotypes. At the molecular level, bistability results from a differential front-rear polarization of both integrins affinity, combined with an inhibiting crosstalk of LFA-1 towards VLA-4. At the cellular level, direction is determined by the passive, flow-mediated orientation of the non-adherent cell parts, the rear uropod for upstream migration and the front lamellipod for downstream migration. This chain of logical events provides a comprehensive mechanism of guiding, from stimuli to cell orientation

Microfluidic device to study flow-free chemotaxis of swimming cells

International audienceMicrofluidic devices have been used in the last two decades to study in vitro cell chemotaxis, but few existing devices generate gradients in flow-free conditions. Flow can bias cell directionality of adherent cells and precludes the study of swimming cells like naïve T lymphocytes, which only migrate in a non-adherent fashion. We developed two devices that create stable, flow-free, diffusion-based gradients and are adapted for adherent and swimming cells. The flow-free environment is achieved by using agarose gel barriers between a central channel with cells and side channels with chemoattractants. These barriers insulate cells from injection/rinsing cycles of chemoattractants, they dampen residual drift across the device, and they allow co-culture of cells without physical interaction, to study contactless paracrine communication. Our devices were used here to investigate neutrophil and naïve T lymphocyte chemotaxis

Leukocyte transmigration and longitudinal forward-thrusting force in a microfluidic Transwell device

International audienceTransmigration of leukocytes across blood vessels walls is a critical step of the immune response. Transwell assays examine transmigration properties in vitro by counting cells passages through a membrane; however, the difficulty of in situ imaging hampers a clear disentanglement of the roles of adhesion, chemokinesis, and chemotaxis. We used here microfluidic Transwells to image the cells' transition from 2D migration on a surface to 3D migration in a confining microchannel and measure cells longitudinal forward-thrusting force in microchannels. Primary human effector T lymphocytes adhering with integrins LFA-1 (a L b 2) had a marked propensity to transmigrate in Transwells without chemotactic cue. Both adhesion and contractility were important to overcome the critical step of nucleus penetration but were remarkably dispensable for 3D migration in smooth microchannels deprived of topographic features. Transmigration in smooth channels was qualitatively consistent with a propulsion by treadmilling of cell envelope and squeezing of cell trailing edge. Stalling conditions of 3D migration were then assessed by imposing pressure drops across microchannels. Without specific adhesion, the cells slid backward with subnanonewton forces, showing that 3D migration under stress is strongly limited by a lack of adhesion and friction with channels. With specific LFA-1 mediated adhesion, stalling occurred at around 3 and 6 nN in 2 Â 4 and 4 Â 4 mm 2 channels, respectively, supporting that stalling of adherent cells was under pressure control rather than force control. The stall pressure of 4 mbar is consistent with the pressure of actin filament polymerization that mediates lamellipod growth. The arrest of adherent cells under stress therefore seems controlled by the compression of the cell leading edge, which perturbs cells front-rear polarization and triggers adhesion failure or polarization reversal. Although stalling assays in microfluidic Transwells do not mimic in vivo transmigration, they provide a powerful tool to scrutinize 2D and 3D migration, barotaxis, and chemotaxis

Lymphocytes perform reverse adhesive haptotaxis mediated by LFA-1 integrins

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The leukocyte-stiffening property of plasma in early acute respiratory distress syndrome (ARDS) revealed by a microfluidic single-cell study: the role of cytokines and protection with antibodies

International audienceAbstractBackgroundLeukocyte-mediated pulmonary inflammation is a key pathophysiological mechanism involved in acute respiratory distress syndrome (ARDS). Massive sequestration of leukocytes in the pulmonary microvasculature is a major triggering event of the syndrome. We therefore investigated the potential role of leukocyte stiffness and adhesiveness in the sequestration of leukocytes in microvessels. MethodsThis study was based on in vitro microfluidic assays using patient sera. Cell stiffness was assessed by measuring the entry time (ET) of a single cell into a microchannel with a 6 × 9–μm cross-section under a constant pressure drop (ΔP = 160 Pa). Primary neutrophils and monocytes, as well as the monocytic THP-1 cell line, were used. Cellular adhesiveness to human umbilical vein endothelial cells was examined using the laminar flow chamber method. We compared the properties of cells incubated with the sera of healthy volunteers (n = 5), patients presenting with acute cardiogenic pulmonary edema (ACPE; n = 6), and patients with ARDS (n = 22), of whom 13 were classified as having moderate to severe disease and the remaining 9 as having mild disease. ResultsRapid and strong stiffening of primary neutrophils and monocytes was induced within 30 minutes (mean ET >50 seconds) by sera from the ARDS group compared with both the healthy subjects and the ACPE groups (mean ET <1 second) (p < 0.05). Systematic measurements with the THP-1 cell line allowed for the establishment of a strong correlation between stiffening and the severity of respiratory status (mean ET 0.82 ± 0.08 seconds for healthy subjects, 1.6 ± 1.0 seconds for ACPE groups, 10.5 ± 6.1 seconds for mild ARDS, and 20.0 ± 8.1 seconds for moderate to severe ARDS; p < 0.05). Stiffening correlated with the cytokines interleukin IL-1β, IL-8, tumor necrosis factor TNF-α, and IL-10 but not with interferon-γ, transforming growth factor-β, IL-6, or IL-17. Strong stiffening was induced by IL-1β, IL-8, and TNF-α but not by IL-10, and incubations with sera and blocking antibodies against IL-1β, IL-8, or TNF-α significantly diminished the stiffening effect of serum. In contrast, the measurements of integrin expression (CD11b, CD11a, CD18, CD49d) and leukocyte–endothelium adhesion showed a weak and slow response after incubation with the sera of patients with ARDS (several hours), suggesting a lesser role of leukocyte adhesiveness compared with leukocyte stiffness in early ARDS. ConclusionsThe leukocyte stiffening induced by cytokines in the sera of patients might play a role in the sequestration of leukocytes in the lung capillary beds during early ARDS. The inhibition of leukocyte stiffening with blocking antibodies might inspire future therapeutic strategies

Functional Mapping of Adhesiveness on Live Cells Reveals How Guidance Phenotypes Can Emerge From Complex Spatiotemporal Integrin Regulation

International audienceImmune cells have the ubiquitous capability to migrate disregarding the adhesion properties of the environment, which requires a versatile adaptation of their adhesiveness mediated by integrins, a family of specialized adhesion proteins. Each subtype of integrins has several ligands and several affinity states controlled by internal and external stimuli. However, probing cell adhesion properties on live cells without perturbing cell motility is highly challenging, especially in vivo . Here, we developed a novel in vitro method using micron-size beads pulled by flow to functionally probe the local surface adhesiveness of live and motile cells. This method allowed a functional mapping of the adhesiveness mediated by VLA-4 and LFA-1 integrins on the trailing and leading edges of live human T lymphocytes. We show that cell polarization processes enhance integrin-mediated adhesiveness toward cell rear for VLA-4 and cell front for LFA-1. Furthermore, an inhibiting crosstalk of LFA-1 toward VLA-4 and an activating crosstalk of VLA-4 toward LFA-1 were found to modulate cell adhesiveness with a long-distance effect across the cell. These combined signaling processes directly support the bistable model that explains the emergence of the versatile guidance of lymphocyte under flow. Molecularly, Sharpin, an LFA-1 inhibitor in lymphocyte uropod, was found involved in the LFA-1 deadhesion of lymphocytes; however, both Sharpin and Myosin inhibition had a rather modest impact on adhesiveness. Quantitative 3D immunostaining identified high-affinity LFA-1 and VLA-4 densities at around 50 and 100 molecules/μm 2 in basal adherent zones, respectively. Interestingly, a latent adhesiveness of dorsal zones was not grasped by immunostaining but assessed by direct functional assays with beads. The combination of live functional assays, molecular imaging, and genome editing is instrumental to characterizing the spatiotemporal regulation of integrin-mediated adhesiveness at molecular and cell scales, which opens a new perspective to decipher sophisticated phenotypes of motility and guidance